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WO2004106291A1 - Haloethyl urea compounds and the use thereof to attenuate, inhibit or prevent cancer cell migration - Google Patents

Haloethyl urea compounds and the use thereof to attenuate, inhibit or prevent cancer cell migration Download PDF

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Publication number
WO2004106291A1
WO2004106291A1 PCT/CA2004/000771 CA2004000771W WO2004106291A1 WO 2004106291 A1 WO2004106291 A1 WO 2004106291A1 CA 2004000771 W CA2004000771 W CA 2004000771W WO 2004106291 A1 WO2004106291 A1 WO 2004106291A1
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Prior art keywords
carcinoma
substituted
aryl
alkyl
nrr
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PCT/CA2004/000771
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French (fr)
Inventor
Rene C. Gaudreault
Original Assignee
Imotep Inc.
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Publication date
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Priority to CA002568607A priority Critical patent/CA2568607A1/en
Publication of WO2004106291A1 publication Critical patent/WO2004106291A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/08—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D263/16—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D263/28—Nitrogen atoms not forming part of a nitro radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/30—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by halogen atoms, or by nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/32—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/40—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/42—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/45—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups at least one of the singly-bound nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom, e.g. N-acylaminosulfonamides
    • C07C311/47—Y being a hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated

Definitions

  • the present invention pertains to the field of therapeutics for diseases or disorders related to cancer cell migration, in particular to therapeutically active haloethyl urea derivatives and their use to inhibit cancer cell migration.
  • a cancer is a malignant tumour of potentially unlimited growth. It is primarily the pathogenic replication (a loss of normal regulatory control) of various given types of cells found in the human body. By select mutation resulting from a primary lesion, the DNA of a cancer cell evolves and converts the cell into an autonomous system. Invasion and metastasis are the most insidious and life-threatening aspects of cancer.
  • Metastasis of the primary tumour produces secondary tumours and disseminated cancer.
  • Therapy for metastasis currently relies on a combination of early diagnosis and aggressive treatment, which may include radiotherapy, chemotherapy or hormone therapy.
  • the high mortality rate for many cancers indicates that improvements are needed in the prevention and treatment of metastasis.
  • WO 97/00956 describes the use of an antibody raised against an adhesion protein on endothelial and muscle cells for inhibiting tumour metastasis.
  • U.S. Patent No. 6,015,893 describes oligonucleoside compounds useful in inhibiting the expression of focal adhesion kinase protein.
  • U.S. Patent No. 5,700,830 describes a method for inhibiting the adherence between cancerous cells and noncancerous structures in a mammal, comprising the administration to the mammal of a nitric oxide-releasing compound.
  • Urea-based compounds have been described for diverse indications, including as herbicides (U.S. Patent No. 3,885,954), as prophylactics against gastrointestinal and cardiovascular disorders (U.S. Patent No. 4,707,478), as anti-parasitic agents (U.S. Patent No. 4,707,478), as anti-athersclerotic agents (U.S. Patent No. 4,623,662), as treatments for gastrointestinal, spasmolytic and ulcerogenic disorders (U.S. Patent No. 4,304,786) and as anti-cancer agents (for example, U.S. Patent Nos. 3,968,249; 4,973,675 and 4,803,223).
  • a class of l-aryl-3-(2- chloroethyl)urea derivatives have been described as anti-cancer agents (U.S. Patent Nos. 5,530,026 and 5,750,547, and International Patent Application WO 00/61546) and as ⁇ -tubulin inhibitors (International Patent Application WO 01/447504).
  • An object of the present invention is to provide novel halo urea compounds for the inhibition of cancer cell motility/migration.
  • compound of formula I there is provided compound of formula I:
  • X is F, CI, Br or I
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
  • B is substituted with one or more substituents selected from the group of (Ci- ⁇ ) alkyl, (Ca-Ci ⁇ ) alkenyl, (C 2 -C 16 ) alkynyl, aryl, -O-(C ⁇ -C 16 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, -O-aryl, -O-CH 2 -aryl, -S-(C ⁇ -C 16 )alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C ⁇ 6 ) alkynyl, -S- aryl, -S-CH 2 -aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8 ) cycl
  • a method of attenuating, inhibiting or preventing cancer cell migration in a mammal comprising administering an effective amount of a compound of formula I.
  • Figure 1 presents a graph illustrating the ability of the compound 1 (A); compound 5 (B); l-(4-t- butyl-phenyl)-3-ethyl urea (tBEU) (C) and cDDP (D) to inhibit tumour cell growth in a dose and time dependent manner.
  • Figure 2 depicts the microtubule depolymerization and cytoskeleton disruption induced by compounds of the invention.
  • Figure 3 presents a Western Blot illustrating the generation of an alkylated form of ⁇ -tubulin by compound 1 and compound 5.
  • Figure 4 presents a Western Blot illustrating the generation of an alkylated form of ⁇ -tubulin by compounds of the invention.
  • Figure 5 presents graphs illustrating the ability of compounds of the invention to inhibit MDA- MB-231 cancer cell migration in the wound assay.
  • Figure 6 presents graphs illustrating the ability of the compound I (A); compound 5 (B); l-(4-t- butyl-phenyl)-3-ethyl urea (tBEU) (C) to inhibit HT1080 cell migration.
  • Figure 7 presents graphs illustrating the ability of the compound 1 (A); compound 5 (B); l-(4-t- butyl-phenyl)-3-ethyl urea (tBEU) (C) and cDDP to impede the growth of two unrelated tumour cell lines in the chick chorioallantoic membrane (CAM) assay.
  • Figure 8 presents graphs illustrating the ability of compound 1, 5, 2 and 30 to impede the growth of CS1 cell line in the chick chorioallantoic membrane (CAM) assay
  • Figure 9 presents a graph illustrating the inhibition of carcinoma cell growth in Balb/c mice.
  • Figure 10 presents a graph illustrating that extracellular matrices and serum do not protect tumour cells against CEU toxiity in clonogenic assay.
  • the invention provides for the use of haloethyl urea derivatives of Formula I for attenuating, inhibiting or preventing cancer cell motility /migration.
  • cancer cell migration is a key feature of cancer cell invasion and metastasis.
  • the invention provides the use of the compounds of the invention to inhibit cancer progression and/or in the prevention of cancer metastasis.
  • halogen refers to fluorine, bromine, chlorine, and iodine atoms.
  • hydroxyl refers to the group -OH.
  • thiol or “mercapto” refers to the group -SH, and -S(O) 0- 2.
  • lower alkyl refers to a straight chain or branched, or cyclic, alkyl group of 1 to 16 carbon atoms. This term is further exemplified by such groups as methyl, ethyl, 72-propyl, i- propyl, 72-butyl, t-butyl, 1-butyl (or 2-methylpropyl), cyclopropylmethyl, z ' -amyl, n-amyl, hexyl and the like.
  • substituted lower alkyl refers to lower alkyl as just described including one or more groups such as hydroxyl, thiol, alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl, substituted cycloalkyl, heterocycle, cycloheteroalkyl, substituted cycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy, hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkyl cycloheteroalkyl, cyano. These groups may be attached to any carbon atom of the lower alkyl moiety.
  • lower alkenyl refers to a straight chain or branched hydrocarbon of 2 to 16 carbon atoms having at least one carbon to carbon double bond.
  • substituted lower alkenyl refers to lower alkenyl as just described including one or more groups such as hydroxyl, thiol, alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl, substituted cycloalkyl, heterocycle, cycloheteroalkyl, substituted cycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy, hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl, alkenyl, alkynyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkyl cycloheteroalkyl, cyano. These groups may be attached to any carbon atom to produce a stable compound.
  • lower alkynyl refers to a straight chain or branched hydrocarbon of 2 to 16 carbon atoms having at least one carbon to carbon triple bond.
  • substituted lower alkynyl refers to lower alkynyl as just described including one or more groups such as hydroxyl, thiol, alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl, substituted cycloalkyl, heterocycle, cycloheteroalkyl, substituted cycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy, hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl, alkenyl, alkynyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkyl cycloheteroalkyl, cyano. These groups may be attached to any carbon atom to produce a stable compound.
  • alkyl alkenyl refers to a group -R-CR-CR'"R"", where R is lower alkyl, or substituted lower alkyl, R', R", R"" are each independently selected from hydrogen, halogen, lower alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, hetaryl, or substituted hetaryl as defined below.
  • alkyl alkynyl refers to a group -R-C ⁇ CR where R is lower alkyl or substituted lower alkyl, R is hydrogen, lower alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, hetaryl, or substituted hetaryl as defined below.
  • alkoxy refers to the group -OR, where R is lower alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heteroalkyl, heteroarylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, or substituted cycloheteroalkyl as defined below.
  • acyl refers to groups -C(O)R, where R is hydrogen, lower alkyl substituted lower alkyl, aryl, substituted aryl.
  • aryloxy refers to groups -OAr, where Ar is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl group as defined below.
  • amino refers to the group NRR', where R and R may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, cycloalkyl, or substituted hetaryl as defined below or acyl.
  • amido or “amide” refers to the group -C(O)NRR', where R and R may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, substituted hetaryl as defined below.
  • Carboxyl refers to the group -C(O)OR, where R may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, substituted hetaryl and the like as defined.
  • aryl refers to an aromatic carbocyclic group having at least one aromatic ring (e.g., phenyl or biphenyl) or multiple condensed rings in which at least one ring is aromatic, (e.g., 1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl, 9-fluorenyl etc.).
  • substituted aryl refers to aryl optionally substituted with one or more functional groups, e.g., halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido or cyano.
  • functional groups e.g., halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alky
  • heterocycle refers to a saturated, unsaturated, or aromatic carbocyclic group having a single ring (e.g., morpholino, pyridyl or furyl) or multiple condensed rings (e.g., naphthpyridyl, quinoxalyl, quinolinyl, indolizinyl, indanyl or benzo[b]thienyl) and having at least one hetero atom, such as N, O or S, within the ring.
  • a single ring e.g., morpholino, pyridyl or furyl
  • multiple condensed rings e.g., naphthpyridyl, quinoxalyl, quinolinyl, indolizinyl, indanyl or benzo[b]thienyl
  • hetero atom such as N, O or S
  • substituted heterocycle refers to heterocycle optionally substituted with, halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido or cyano and the like.
  • heteroaryl or “hetar” refer to a heterocycle in which at least one heterocyclic ring is aromatic.
  • substituted heteroaryl refers to a heterocycle optionally mono or poly substituted with one or more functional groups, e.g., halogen, hydroxyl, thiol, lower allcyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido or cyano and the like.
  • functional groups e.g., halogen, hydroxyl, thiol, lower allcyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, al
  • aralkyl refers to the group -R-Ar where Ar is an aryl group and R is lower alkyl or substituted lower alkyl group.
  • Aryl groups can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl, alkoxy, alkyl thio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
  • heteroalkyl refers to the group -R-Het where Het is a heterocycle group and R is a lower alkyl group.
  • Heteroalkyl groups can optionally be unsubstituted or substituted with e.g., halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
  • heteroarylalkyl refers to the group -R-HetAr where HetAr is an heteroaryl group and R lower alkyl or substituted loweralkyl.
  • Heteroarylalkyl groups can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl, substituted lower alkyl, alkoxy, alkylthio, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
  • cycloalkyl refers to a cyclic or polycyclic alkyl group containing 3 to 15 carbon.
  • these may be multiple condensed rings in which one of the distal rings may be aromatic (e.g. tetrahydronaphthalene, etc.).
  • substituted cycloalkyl refers to a cycloalkyl group comprising one or more substituents with, e.g halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido or cyano and the like.
  • cycloheteroalkyl refers to a cycloalkyl group wherein one or more of the ring carbon atoms is replaced with a heteroatom (e.g., N, O, S or P).
  • substituted cycloheteroalkyl refers to a cycloheteroalkyl group as herein defined which contains one or more substituents, such as halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
  • substituents such as halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
  • alkyl cycloalkyl refers to the group -R-cycloalkyl where cycloalkyl is a cycloalkyl group and R is a lower alkyl or substituted lower alkyl.
  • Cycloalkyl groups can optionally be unsubstituted or substituted with e.g. halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
  • treatment refers to improvement in the recipient's status as well as prophylaxis.
  • the improvement can be subjective or objective and related to features such as symptoms or signs of the disease or condition being treated. Prevention of deterioration of the recipient's status is also encompassed by the term.
  • ameliorate or “amelioration” includes the arrest, prevention, decrease, or improvement in one or more the symptoms, signs, and features of the disease being treated, both temporary and long-term.
  • the compounds according to the instant invention include compounds of the following general formula:
  • X is F, CI, Br or I
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
  • B is substituted with one or more substituents selected from the group of (Ci-Ci ⁇ ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, aryl, -O-(C ⁇ -C ⁇ 6 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, -O- aryl, -O-CH 2 -aryl, -S-(C ⁇ -C ⁇ 6 )alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S-aryl, -S-CH 2 - aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8
  • the compound of the invention is one in which RI and R2 are each independently selected from H, (C ⁇ -C 6 ) alkyl and ( -C 6 ) alkoxy.
  • B is phenyl, substituted with one or more substituents indepnednetly selected from the list as shown above.
  • B is phenyl substituted with halo, -CN, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR, -OR, (C C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl , wherein said alkyl, alkenyl and alkynyl are optionally substituted with -CN, -C(O)R, - C(O)OR*, -OC(O)R, -C(O)NR'R', -OR', wherein R and R' are as defined above.
  • X is F, CI, Br or I
  • RI and R2 are as defined above, and
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; and substituted with at least one substituent selected from, (C;[-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C2-C 16 ) alkynyl, -O ⁇ d-C ⁇ ) alkyl, -O-(C2-C 16 ) alkenyl, -O-(C2-C 16 ) alkynyl, aryl, substituted aryl, -O- aryl, -O-CH 2 -aryl, -S-(C !
  • alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO 2 , -NR'R, -OH, -OR', -O-aryl, -OC(O)R', -OC(S)R, -C(O)R,
  • substituents of the compounds of formula (I) are as follows: X is F, CI, Br or I; RI and R2 are as defined above, and
  • B is phenyl substituted with at least one substituent selected from, (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, -O-(d-C 16 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -d 6 ) alkynyl, aryl, substituted aryl, -O-aryl, -O-CH 2 -aryl, -S-(d-C 16 ) alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S-aryl, -S-CH 2 -aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8
  • alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO 2 , -NR'R, -OH, -OR', -O-aryl, -OC(O)R*, -OC(S)R', -C(O)R,
  • X is F, CI, Br or I
  • RI and R2 are as defined above, and
  • B is phenyl substituted with at least one group selected from, (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl,
  • alkenyl, alkynyl, -O-alkyl, -O-alkenyl and -O-alkynyl are each independently substituted with at least one group selected from halo, -CN, -OH, -OR, -O-aryl, -OC(O)R, -C(O)R, -
  • R and R are as defined above.
  • X is F CI, Br or I
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; and substituted with at least one substituent selected from (d-C 16 ) alkyl, (C 2 -C 1 ) alkenyl, (C2-C 16 ) alkynyl, -O-(d-C 16 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, aryl, substituted aryl, -O- aryl, -O-CH 2 -aryl, -S-(d-C 16 ) alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S-aryl, -S-CH 2 - aryl, (C 3 -C 8 ) cycloalkyl
  • alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO 2 , -NR'R, -OH, -OR, -O-aryl, -OC(O)R, -OC(S)R, -C(O)R',
  • the compounds of formula (I) include the compounds of formula (II):
  • X is F CI, Br or I
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
  • B is substituted with one or more substituents selected from the group of (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, aryl, -O-(d-C 16 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, -O-aryl, -O-CH 2 -aryl, -S-(C C 16 )alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S- aryl, -S-CH 2 -aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8 ) cycloalky
  • X is CI or Br.
  • X is F, CI, Br or I
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine;
  • alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO 2 , -NR'R, -OH, -OR', -O-aryl, -OC(O)R, -OC(S)R, -C(O)R",
  • X is F, CI, Br or I
  • B is phenyl substituted with at least one substituent selected from (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, -O-(d-C 16 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, aryl, substituted aryl, -O-aryl, -O-CH 2 -aryl, -S-(d-C 16 ) alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S-aryl, -S-CH 2 -aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8
  • alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO 2 , -NR'R, -OH, -OR, -O-aryl, -OC(O)R, -OC(S)R, -C(O)R',
  • substituents of the of the compounds of formula (I) are as follows: X is F, CI, Br or I;
  • B is phenyl substituted with at least one group selected from (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl,
  • alkenyl, alkynyl, -O-alkyl, -O-alkenyl and -O-alkynyl are each independently substituted with at least one group selected from halo, -CN, -OH, -OR, -O-aryl, -OC(O)R, -C(O)R, -
  • R and R are as defined above.
  • X is F, CI, Br or I
  • B is substituted with at least one group selected from aryl, -O-aryl, -O-CH 2 -aryl and halo.
  • X is F, CL Br or l
  • B is substituted with at least one substituent selected from the group of (d-C 16 ) alkyl, (d-C 16 ) alkynyl or -O-alkyl; wherein: said alkyl is substituted with at least one substituent selected from the group of-CN, -O-alkyl, -
  • alkyny and -O-alkyl are substituted with at least one substituent selected from -CN, -
  • R is as defined above.
  • X is F, CL Br or l
  • B is substituted with at least one group selected from -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O) 2 OR, -S(O)NRR, -S(O)ONRR, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR; wherein R is as defined above.
  • the compounds of formula (I) include the compounds of formula (III):
  • X is F, CI, Br or I
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
  • B is substituted with one or more substituents selected from the group of (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C ⁇ 6 ) alkynyl, aryl, -O-(d-C 16 ) allcyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, -O- aryl, -O-CH 2 -aryl, -S-(d-d 6 )alkyl, -S-(C 2 -C 16 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S-aryl, -S-CH 2 - aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8 )
  • substitutents of formula (I) are as follows:
  • X is F CI, Br or I
  • RI and R2 are each independently selected from the group of H, (d-C 6 ) alkyl, (C ⁇ -C 6 ) hydroxy allcyl, (d-C 6 ) alkoxy, (C 3 -C 7 ) cycloalkyl, halo substituted (d-C 6 ) alkyl, halo di- substituted (d-C 6 ) alkyl, halo tri-substituted (d-C 6 ) alkyl and halo;
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
  • B is substituted with one or more substituents selected from the group of (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, aryl, -O-(d-C 16 ) alkyl, -O-(C 2 -C 16 ) alkenyl, -O-(C 2 -C 16 ) alkynyl, -O- aryl, -O-CH 2 -aryl, -S-(d-C 16 )alkyl, -S-(C 2 -C ⁇ 6 ) alkenyl, -S-(C 2 -C 16 ) alkynyl, -S-aryl, -S-CH 2 - aryl, (C 3 -C 8 ) cycloalkyl, -O-(C 3 -C 8 ) cycloalkyl, -S-(C 3 -C 8 ) cycl
  • substituents of the compounds of formula (I) are as follows: X is F, CI, Br or I;
  • RI and R2 are each independently selected from the group of H, (C ⁇ -C 6 ) alkyl, (d-C 6 ) hydroxy alkyl, (d-C 6 ) alkoxy, (C 3 -C 7 ) cycloalkyl, halo substituted (d-C 6 ) alkyl, halo di-substituted (d- C 6 ) alkyl, halo tri-substituted (C ⁇ -C 6 ) alkyl and halo;
  • B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; and substituted with at least one substituent selected from (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, -O-(d-d 6 ) alkyl, -O-(C 2 -C 16 ) alkenyl,
  • alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO 2 , -NR'R, -OH, -OR', -O-aryl, -OC(O)R, -OC(S)R', -C(O)R,
  • X is F, C Br or l
  • RI and R2 are each independently selected from the group of H, (C ⁇ -C 6 ) alkyl, (d-C 6 ) hydroxy alkyl, (C ⁇ -C 6 ) alkoxy, (C -C 7 ) cycloalkyl, halo substituted (d-C 6 ) alkyl, halo di-substituted (Ci-
  • B is phenyl substituted with at least one group selected from (d-C 16 ) alkyl, (C 2 -C 16 ) alkenyl,
  • alkenyl, alkynyl, -O-alkyl, -O-alkenyl and -O-alkynyl are each independently substituted with at least one group selected from halo, -CN, -OH, -OR', -O-aryl, -OC(O)R', -C(O)R', -
  • R and R' are as defined above.
  • the compounds of formula (I) include the compounds of formula (IV):
  • R3 is selected from the group of H, R, -halo, OR, -SR, -NRR, -ONRR, -NO 2 , -CN, -C(O)R, -C(S)R,-OC(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -C(S)NR(SR), -CH(CN) 2 , -CH[C(O)R] 2 , -CH[C(S)R] 2 , -CH[C(O)OR] 2 , -CH[C(O)OR] 2 , -CH
  • R3 is selected from halo, -CN, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR, -OR, (d-C 16 ) alkyl, (C 2 -C 16 ) allcenyl, (C -C 16 ) alkynyl, wherein said alkyl, alkenyl and alkynyl are optionally substituted with -CN, -C(O)R, - C(O)OR, -OC(O)R, -C(O)NR'R', -OR', wherein R and R are as defined above.
  • the compounds of formula (I) include the compounds of formula (V):
  • R3 is selected from the group of H, R, -halo, OR, -SR, -NRR, -ONRR, -NO 2 , -CN, -C(O)R, -C(S)R,-OC(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -C(S)NR(SR), -CH(CN) 2 , -CH[C(O)R] 2 , -CH[C(S)R] 2 , -CH[C(O)OR] 2 , -CH[C(O)OR] 2 , -CH
  • R3 is selected from halo, -CN, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR, -OR, (C C ⁇ 6 ) alkyl, (C 2 -C 16 ) alkenyl, (C 2 -C 16 ) alkynyl, wherein said alkyl, alkenyl and alkynyl are optionally substituted with -CN, -C(O)R, - C(O)OR,
  • the compounds according to formula (I) include those listed below: l-(2-Chloro-ethyl)-3-772-tolyl-urea; l-(2-Chloro-ethyl)-3-(3-ethyl-phenyl)-urea; l-(2-Chloro-ethyl)-3-(3-methoxy-phenyl)-urea; l-(2-Chloro-ethyl)-3-[4-(4-hydroxy-butyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-[4-(3-hydroxy-propyl)- ⁇ henyl]-urea; l-(2-Chloro-ethyl)-3-(3-iodo-phenyl)-urea; l-(2-Chloro-ethyl)-3-[4-(5-hydroxy-pentyl)-phenyl]-urea;
  • Acetic acid 4- ⁇ 3-[3-(2-chloro-ethyl)-ureido]-phenyl ⁇ -butyl ester;
  • Compounds of Formula I wherein X is Br or I may undergo rearrangement to provide a rearrangement product. Such rearrangement products are considered to be within the scope of the present invention.
  • the present invention contemplates the compounds of Fonnula I wherein X is Br or I as a form of pro-drugs, for which both the pro-drug form and the rearrangement product may have activity in inhibiting proliferation of cancer cells.
  • this invention includes the pharmaceutically acceptable salts of the compounds defined by Formula I, II, and III.
  • a compound of this invention can possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of organic and inorganic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt refers to salts of the compounds of the above formula which are substantially non-toxic to living organisms.
  • Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts.
  • Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as -toluenesulfonic acid, methanesulfonic acid, oxalic acid, 7-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, phosphoric acid, and the like
  • organic acids such as -toluenesulfonic acid, methanesulfonic acid, oxalic acid, 7-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
  • salts examples include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenyl
  • the pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid and methanesulfonic acid.
  • Salts of amine groups may also comprise quarternary ammonium salts wherein the amino nitrogen carries a suitable organic group such as an alkyl, alkenyl, alkynyl, or aralkyl moiety.
  • Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like.
  • bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like, hi one embodiment, the base addition salt is a potassium or sodium salt.
  • any salt of this invention is usually not of a critical nature, as long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.
  • This invention further encompasses the pharmaceutically acceptable solvates of the compounds of Formula I, II or III. Many of these compounds can combine with solvents such as water, methanol, ethanol and acetonitrile to form pharmaceutically acceptable solvates such as the corresponding hydrate, methanolate, ethanolate and acetonitrilate.
  • the compounds of the present invention have multiple asymmetric (chiral) centers. As a consequence of these chiral centers, the compounds of the present invention occur as racemates, mixtures of enantiomers and as individual enantiomers, as well as diastereomers and mixtures of diastereomers. All asymmetric forms, individual isomers and combinations thereof, are within the scope of the present invention.
  • R and S are used herein as commonly used in organic chemistry to denote the absolute configuration of a chiral center, according to the Cahn-higold-Prelog system.
  • the stereochemical descriptor R rectus
  • S sinister
  • R rectus
  • S sinister
  • the priority of groups is decided using sequence rules as described by Cahn et al, Angew. Chem., 78, 413-447, 1966 and Prelog, V. and Helmchen, G.; Angew. Chem. Int. Ed. Eng, 21, 567-583, 1982).
  • the older D-L system is also used in this document to denote relative configuration, especially with reference to amino acids and amino acid derivatives.
  • a Fischer projection of the compound is oriented so that carbon-1 of the parent chain is at the top.
  • D is used to represent the relative configuration of the isomer in which the functional (determining) group is on the right side of the carbon atom at the chiral center and "L", that of the isomer in which it is on the left.
  • the stereochemistry of the Formula I, II and III compounds may be critical to their potency as agonists or antagonists.
  • the relative stereochemistry is preferably established early during synthesis, which avoids stereoisomer separation problems later in the process. Subsequent synthetic steps then employ stereospecific procedures so as to maintain the preferred configuration.
  • Non-toxic metabolically labile esters and amides of compounds of Formula I, II or III are ester or amide derivatives that are hydrolyzed in vivo to afford said compounds of Formula I, II or III and a pharmaceutically acceptable alcohol or amine.
  • Examples of metabolically labile esters include esters formed with (C ⁇ -C 6 ) alkanols in which the alkanol moiety may be optionally substituted by a (d-C 16 ) alkoxy group, for example, methanol, ethanol, propanol and methoxyethanol.
  • Examples of metabolically labile amides include amides formed with amines such as methylamine.
  • the compounds of Formula I can be used for attenuating, inhibiting or preventing cancer cell migration in a mammal in need of such therapy.
  • the compounds of the invention are used to attenuate, inhibit or prevent cancer cell migration.
  • the compounds can be used alone or they can be used as part of a multi-drug regimen in combination with known therapeutics.
  • cancers include, but are not limited to leukaemia, carcinomas, adenocarcinomas, melanomas and sarcomas.
  • Carcinomas, adenocarcinomas and sarcomas are also frequently referred to as "solid tumours," examples of commonly occurring solid tumours include, but are not limited to, cancer of the brain, breast, cervix, colon, head and neck, kidney, lung, ovary, pancreas, prostate, stomach and uterus, non- small cell lung cancer and colorectal cancer.
  • leukaemia refers broadly to progressive, malignant diseases of the blood-forming organs. Leukaemia is typically characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow but can also refer to malignant diseases of other blood cells such as erythroleukaemia, which affects immature red blood cells. Leukaemia is generally clinically classified on the basis of (1) the duration and character of the disease - acute or chronic; (2) the type of cell involved - myeloid (myelogenous), lymphoid (lymphogenous) or monocytic, and (3) the increase or non-increase in the number of abnormal cells in the blood - leukaemic or aleukaemic (subleukaemic).
  • Leukaemia includes, for example, acute nonlymphocytic leukaemia, chronic lymphocytic leukaemia, acute granulocytic leukaemia, chronic granulocytic leukaemia, acute promyelocytic leukaemia, adult T-cell leukaemia, aleukaemic leukaemia, aleukocythemic leukaemia, basophylic leukaemia, blast cell leukaemia, bovine leukaemia, chronic myelocytic leukaemia, leukaemia cutis, embryonal leukaemia, eosinophilic leukaemia, Gross' leukaemia, hairy-cell leukaemia, hemoblastic leukaemia, hemocytoblastic leukaemia, histiocytic leukaemia, stem cell leukaemia, acute monocytic leukaemia, leukopenic leukaemia, lymphatic leukaemia, lymphoblastic leukaemia, lymphocytic leuk
  • tumour generally refers to a tumour which originates in connective tissue, such as muscle, bone, cartilage or fat, and is made up of a substance like embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • Sarcomas include soft tissue sarcomas, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumour sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented haemorrhagic
  • melanoma is taken to mean a tumour arising from the melanocytic system of the skin and other organs.
  • Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colorectal carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex
  • carcinomas that originate in cells that make organs which have glandular (secretory) properties or that originate in cells that line hollow viscera, such as the gastrointestinal tract or bronchial epithelia. Examples include, but are not limited to, adenocarcinomas of the breast, lung, pancreas and prostate.
  • Additional cancers encompassed by the present invention include, for example, Hodgkin's Disease, Non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumours, primary brain tumours, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, gliomas, testicular cancer, thyroid cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, mesothelioma and medulloblastoma.
  • the compounds of Formula I can be used for attenuating, inhibiting or preventing cancer cell proliferation in a mammal in need of such therapy.
  • the novel compounds described by Formula I of the invention are used to attenuate, inhibit or prevent cancer cell proliferation.
  • the compounds can be used alone or they can be used as part of a multi-drug regimen in combination with known therapeutics.
  • the therapeutic compounds of Formula I are capable of attenuating, inhibiting, or preventing cancer cell migration in vivo.
  • compounds within Formula I will demonstrate different activities in their ability to attenuate, inhibit, or prevent cancer cell migration and to treat the diseases associated with such migration.
  • the ability of the compounds to attenuate, inhibit, or prevent cancer cell migration can be initially determined in vitro if desired.
  • the present invention thus contemplates a preliminary in vitro screening step to further characterize compounds suitable for incorporation into the therapeutic compositions.
  • a number of standard tests to determine the ability of a compound to attenuate, inhibit, or prevent cancer cell migration are known in the art and can be employed to test the compounds of Formula I. Exemplary procedures are described herein.
  • Candidate compounds of Fonnula I can be tested in vitro and in vivo to determine their activity in inhibiting cancer cell migration and metastasis formation.
  • the ability of a compound to inhibit migration of neopalstic cells can be assessed in vitro using standard cell migration assays.
  • assays are conducted in multi-well plates, the wells of the plate being separated by a suitable membrane into top and bottom sections.
  • the membrane can be coated with an appropriate compound, the selection of which is dependent on the type of cell being assessed and can be readily determined by one skilled in the art. Examples include collagen, gelatine or Matrigel.
  • chemo-attractant such as soluble fibronectin, EGM-2, IL-8, ⁇ -FGF, ⁇ -FGF and the like
  • chemo-attractant such as soluble fibronectin, EGM-2, IL-8, ⁇ -FGF, ⁇ -FGF and the like
  • An aliquot of the test cells together with the test compound is added to the upper chamber, typically various dilutions of the test compound are tested. After a suitable incubation time, the membrane is rinsed, fixed and stained. The cells on the upper side of the membrane are wiped off, and then randomly selected fields on the bottom side are counted.
  • Neoplastic cell migration can also be assessed in vitro using the wound healing assay described by Alper et al. (J Natl Cancer hist. 2001 93(18):1375-84) and Tamura et al. (Science 1998;280:1614-7). Briefly, a wound is created in a cell monolayer by the gentle removal of the attached cells. The migration of the cells into the wound is then observed at different time points and in the absence or presence of a test compound to determine the effect of the test compound on cell migration.
  • compositions may be desirable to determine the ability of the compositions to inhibit cell migration of certain specific cancer cell lines, for example drug- resistant or highly metastatic cell lines and that appropriate cell lines can be selected accordingly.
  • Suitable neoplastic cell lines are available from the American Type Culture Collection (ATCC), which currently provides 950 cancer cell lines, and other commercial sources, hi one embodiment of the present invention, in vitro testing of the candidate compounds is conducted in a human cancer cell-line.
  • ATCC American Type Culture Collection
  • suitable human cancer cell-lines for in vitro testing of the compounds of the present invention include, but are not limited to, breast cancer cell-lines MCF- 7, t47D and MDA-MB-23, colon cancer cell-lines CaCo and LoVo, ovarian cancer cell-line SKOV3, prostate cancer cell-line DU-145, chronic myeloid leukaemia cell-line K562 and bladder cancer cell-line T24.
  • Toxicity of the candidate compounds can also be initially assessed in vitro using standard techniques.
  • human primary fibroblasts can be treated in vitro with a compound of Formula I.
  • Cells are then tested at different time points following treatment for their viability using a standard viability assay, such as the assays described above or the trypan-blue exclusion assay.
  • Cells can also be assayed for their ability to synthesize DNA, for example, using a thymidine incorporation assay, and for changes in cell cycle dynamics, for example, using a standard cell sorting assay in conjunction with a fluorocytometer cell sorter (FACS).
  • FACS fluorocytometer cell sorter
  • the ability of the candidate compounds to inhibit tumour growth or metastasis in vivo can be detennined in an appropriate animal model using standard techniques known in the art (see, for example, Enna, et al, Current Protocols in Pharmacology, J. Wiley & Sons, hie, New York, NY).
  • xenograft models in which a human tumour has been implanted into an animal.
  • xenograft models of human cancer include, but are not limited to, human solid tumour xenografts in mice, implanted by sub-cutaneous injection and used in tumour growth assays; human solid tumour isografts in mice, implanted by fat pad injection and used in tumour growth assays; experimental models of lymphoma and leukaemia in mice, used in survival assays, and experimental models of lung metastasis in mice.
  • xenograft models of human cancer include, but are not limited to, human solid tumour xenografts in mice, implanted by sub-cutaneous injection and used in tumour growth assays; human solid tumour isografts in mice, implanted by fat pad injection and used in tumour growth assays; experimental models of lymphoma and leukaemia in mice, used in survival assays, and experimental models of lung metastasis in mice.
  • the candidate compounds can be tested in vivo on solid tumours using mice that are subcutaneously grafted bilaterally with 30 to 60 mg of a tumour fragment, or implanted with an appropriate number of cancer cells, on day 0.
  • Subcutaneous xenografts metastasize infrequently and seldom invade adjacent tissue, therefore, rate of tumour growth or delay of significant tumour growth are the endpoints used in this model.
  • the animals bearing tumours are mixed before being subjected to the various treatments and controls. In the case of treatment of advanced tumours, tumours are allowed to develop to the desired size, animals having insufficiently developed tumours being eliminated.
  • the selected animals are distributed at random to undergo the treatments and controls. Suitable controls will be dependent on the actual compound being tested and may include untreated animals.
  • Chemotherapy generally begins from 1 to 22 days after grafting, depending on the type of tumour, and the animals are observed every day.
  • the compounds can be administered prior to tumour implantation, for example, about 7 days prior.
  • the compounds of the present invention can be administered to the ammals, for example, orally, by i.p. injection or bolus infusion.
  • the different animal groups are weighed about 3 or 4 times a week until the maximum weight loss is attained, after which the groups are weighed at least once a week until the end of the trial.
  • tumours are measured about 2 or 3 times a week until the tumour reaches a pre-determined size and / or weight, or until the animal dies if this occurs before the tumour reaches the predetermined size / weight.
  • the animals are then sacrificed and the tissue histology, size and / or proliferation of the tumour assessed.
  • Orthotopic xenograft models are an alternative to subcutaneous models and may more accurately reflect the cancer development process.
  • tumour cells are implanted at the site of the organ of origin and develop internally. Daily evaluation of the size of the tumours is thus more difficult than in a subcutaneous model.
  • GFP green fluorescent protein
  • a recently developed technique using green fluorescent protein (GFP) expressing tumours in non-invasive whole-body imaging can help to address this issue (Yang and al, Proc. Nat. Aca. Sci, (2000), pp 1206-1211). This technique utilises human or murine tumours that stably express very high levels of the Aqueora vitoria green fluorescent protein.
  • the GFP expressing tumours can be visualised by means of externally placed video detectors, allowing for monitoring of details of tumour growth, angiogenesis and metastatic spread.
  • the use of this model thus allows for simultaneous monitoring od several features associated with tumour progression and has high preclinical and clinical relevance.
  • the ability of the candidate compound to inhibit formation of a solid tumour can also be assessed in the chick chorioallantoic membrane (CAM) assay using published protocols (Brooks et al, in Methods in Molecular Biology, Vol. 129, pp. 257-269 (2000), ed. A.R. Howlett, Humana Press Inc., Totowa, NJ).
  • CAM chick chorioallantoic membrane
  • the animals are grafted with a particular number of cells, and the anti-tumour activity is detennined by the increase in the survival time of the treated mice relative to the controls.
  • compositions of the present invention can be employed.
  • the animals are treated before or after injection or implantation of the neoplastic cells into the animal.
  • the spread of the neoplastic cells from the site of injection for example spread to the lungs and/or lymphoid nodes, is then monitored over a suitable period of time by standard techniques.
  • LLCl Lewis lung
  • the cells are administered intravenously to normal non- immune-compromised mice thus allowing for immediate dissemination of cancerous cells. Treatment can be initiated several days before injection of the LLCl cells in order to observe a preventive effect or immediately after injection of the cells in order to observe an attenuating effect. After about 14 days, the mice are sacrificed, the lungs removed and fixed and the number and size of lung tumours determined. The intravenous route of administration for the LLCl cells in this model allows for rapid evaluation of treatments.
  • LLCl cells are injected subcutaneously to allow the growth of a primary tumour, which is then surgically removed once a certain size is obtained. Following removal of the primary tumour, treatment is initiated for about 14 days, after which the animals are sacrificed and tumours counted as in the intravenous model. The primary tumour is removed in this model is recommended as it can be metastasis-suppressing.
  • In vivo toxic effects of the compounds of Formula I can be evaluated by standard techniques, for example, by measuring their effect on animal body weight during treatment and by performing haematological profiles and liver enzyme analysis after the animal has been sacrificed.
  • Table 1 Examples of xenograft models of human cancer
  • the compounds of the invention can be submitted to other standard tests, such as cytotoxicity tests, stability tests, bioavailability tests and the like.
  • the compounds of the invention will need to meet certain criteria in order to be suitable for human use and to meet regulatory requirements.
  • standard in vitro and in vivo tests can be conducted to determine information about the metabolism and pharmacokinetic (PK) of the compound which can be used to design human clinical trials.
  • PK pharmacokinetic
  • the safety profile of the compounds can be determined in at least two non-human species and then the compounds will progress into Clinical Trials in order to ftirther evaluate their efficacy in attenuating the metastasis of tumours and to obtain regulatory approval for therapeutic use.
  • Clinical Trials progress through phases of testing, which are identified as Phases I, II, III, and IV.
  • Phases I, II, III, and IV In vitro and in vivo information about the metabolism and pharmacokinetic (PK) of the compounds determined from pre-clinical studies facilitates the design of initial Phase I and Phase II clinical studies.
  • Phase I clinical trials are normally performed in healthy human volunteers or in advanced cancer patients.These studies are conducted to investigate the safety, tolerability and PK of the compositions and to help design Phase II studies, for example, in terms of appropriate doses, routes of administration, administration protocols. Phase I studies could incorporate pharmacodynamic assays to evaluate proof of principle in inhibition of target inhumans. An adequate pharmacodynamic endpoint would be to determine the inhibitory activity measured from the plasma of healthy volunteers.
  • Phase I studies allow the selection of safe dose levels for Phase II studies.
  • An important factor in the protocol design of the Phase II studies is the adequate recruitment of the patient population to be studied based on stringent selection criteria defining the demographics (age, race and sex) of the study, the previous medical history of the patient, the type of cancer and stage of its development as well as any previous cancer treatment history. The latter factor can be important when the composition is intended as an adjuvant to first line therapy rather than a treatment to refractory disease.
  • a protocol for Phase II studies typically specifies baseline data that can be used to characterise the population, to evaluate the success of randomization in achieving balance of important prognostic factors, and to allow for consideration of adjusted analyses. Staging of the cancers of interest
  • Staging of the cancer being investigated can be important and, when possible, patients should be recruited such that the cancer stage is as homogeneous as possible across the population to facilitate statistical analysis and interpretation of the data.
  • methods and criteria for staging of a cancer vary depending on the particular cancer being investigated.
  • Clinical biomarkers can be defined as follows (Atkinson A et al: Clin. Pharmacol. Ther. 69, 89-95 (2001):
  • Biomarker a characteristic that is objectively measured and evaluated as an indicator of normal biological process, pathogenic process, or pharmacological response to a therapeutic intervention.
  • Clinical endpoint a characteristic or variable that reflects how a patient feels or functions, or how long a patient survives.
  • Surrogate endpoint biomarker intended to substitute for a clinical endpoint.
  • a clinical investigator uses epidemiological, therapeutic, pathophysiological, or other scientific evidence to select a surrogate endpoint that is expected to predict benefit, harm or the lack of benefit or harm.
  • the FDA defines a surrogate endpoint, or marker, as a laboratory measurement or physical sign that is used in therapeutic trials as a substitute for a clinically meaningful endpoint that is a direct measure of how a patient feels, functions or survive and is expected to predict the effect of the therapy.
  • Phase III trials focus on determining how the compound compares to the standard, or most widely accepted, treatment.
  • patients are randomly assigned to one of two or more "arms".
  • one arm will receive the standard treatment (control group) and the other arm will be treated with the compound (investigational group).
  • Phase IV trials can be used to further evaluate the long-term safety and effectiveness of the compound. Phase IV trials are less common than Phase I, II and III trials and would take place after the compound has been approved for standard use.
  • urea moiety is achieved using the 4-dimethylaminopyridine-catalyzed reaction of the relevant 4-alkylaniline (VI) with di-tert-butyldicarbonate in dichloromethane followed by the trapping of the in situ generated isocyanate with the appropriate (R)- or ( ⁇ S)-2-aminoalcohol (see Knolker, et al, Synlett (1996) 502-504).
  • This procedure ensures a racemization-free synthesis of urea under mild conditions and circumvents side reactions such as the formation of symmetrical disubstituted urea (see Knolker, et al, Synlett (1997) 925-928).
  • the present invention provides methods of treating diseases characterized by neoplastic cell migration in a mammal comprising administering an effective amount of one or more compounds of Formula I, or non-toxic metabolically-labile esters or amides thereof, or pharmaceutically acceptable salts thereof.
  • the compounds of the present invention are typically formulated prior to administration.
  • the present invention thus provides pharmaceutical compositions comprising one or more compounds of Formula I and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the pharmaceutical compositions are prepared by known procedures using well-known and readily available ingredients.
  • Pharmaceutical compositions comprising one or more compounds of Formula I in combination with one or more known cancer chemotherapeutics are also contemplated by the present invention.
  • Compounds of the general Formula I or pharmaceutical compositions comprising the compounds may be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles, hi the usual course of therapy, the active compound is incorporated into an acceptable vehicle to form a composition for topical administration to the affected area, such as hydropohobic or hydrophilic creams or lotions, or into a form suitable for oral, rectal or parenteral administration, such as syrups, elixirs, tablets, troches, lozenges, hard or soft capsules, pills, suppositiories, oily or aqueous suspensions, dispersible powders or granules, emulsions, injectables, or solutions.
  • parenteral as used herein includes subcutaneous injections, intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal injection or infusion techniques.
  • compositions intended for oral use may be prepared in either solid or fluid unit dosage forms.
  • Fluid unit dosage form can be prepared according to procedures known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • An elixir is prepared by using a hydroalcoholic (e.g., ethanol) vehicle with suitable sweeteners such as sugar and saccharin, together with an aromatic flavoring agent.
  • Suspensions can be prepared with an aqueous vehicle with the aid of a suspending agent such as acacia, tragacanth, methylcellulose and the like.
  • Solid fonnulations such as tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate: granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc and other conventional ingredients such as dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, methylcellulose, and functionally similar materials.
  • inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate
  • granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil.
  • Aqueous suspensions contain active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gxun tragacanth and gum acacia: dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example hepta-decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbit
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or fl-propyl- /j-hydroxy benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or fl-propyl- /j-hydroxy benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol,
  • compositions of the invention may also be in the form of oil-in- water emulsions.
  • the oil phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally- occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or a suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution, hi addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Adjuvants such as local anaesthetics, preservatives and buffering agents can also be included in the injectable solution or suspension.
  • the compound(s) of the general Formula I may be administered, together or separately, in the form of suppositories for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient include cocoa butter and polyethylene glycols.
  • the compounds can be formulated into solutions, suspensions, and ointments appropriate for use in the eye (see, for example, Mitra (ed.), (1993) Ophthalmic Drug Delivery Systems, Marcel Dekker, Inc., New York, N.Y.; Havener, (1983) Ocular Pharmacology, CN. Mosby Co., St. Louis).
  • compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams & Wilkins, Philidelphia, PA (2000).
  • therapeutic compounds are administered systemically to patients, for example, by bolus injection or continuous infusion into a patient's bloodstream.
  • one or more compounds of Formula I are administered systemically to a patient in need of therapy.
  • the compounds can be administered prior to, or after, administration of the chemotherapeutic agents, or they can be administered concomitantly.
  • the one or more chemotherapeutic may be administered systemically, for example, by bolus injection or continuous infusion, or it may be administered orally.
  • the dosage to be administered is not subject to defined limits, but it will usually be an effective amount. It will usually be the equivalent, on a molar basis of the pharmacologically active free form produced from a dosage formulation upon the metabolic release of the active free drug to achieve its desired pharmacological and physiological effects.
  • the compositions may be formulated in a unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Examples of ranges for the compound(s) in each dosage unit are from about 0.05 to about 100 mg, or more usually, from about 1.0 to about 30 mg.
  • Daily dosages of the compounds of the present invention will typically fall within the range of about 0.01 to about 100 mg/kg of body weight, in single or divided dose.
  • the actual amount of the compound(s) to be administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.
  • the above dosage range is given by way of example only and is not intended to limit the scope of the invention in any way. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing harmful side effects, for example, by first dividing the larger dose into several smaller doses for administration throughout the day.
  • kits containing the compounds of the invention for use in the inhibition of cancer cell migration in a mammal in need of such therapy.
  • Individual components of the kit would be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects , approval by the agency of manufacture, use or sale for human administration.
  • the liquid solution can be an aqueous solution, for example a sterile aqueous solution.
  • the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the composition may be administered to a patient or applied to and mixed with the other components of the kit.
  • kits of the invention may also be provided in dried or lyophilised form and the kit can additionally contain a suitable solvent for reconstitution of the lyophilised components.
  • the kits of the invention also may comprise an instrument for assisting with the administration of the composition to a patient.
  • Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
  • EtOAc ethyl acetate
  • THF tetrahydrofuran
  • EtOH ethanol
  • TLC thin layer chromatography
  • GC gas chromatography
  • HPLC high pressure liquid chromatography
  • m-CPBA m-chloroperbenzoic acid
  • Et 2 O diethyl ether
  • DMSO dimethyl sulfoxide
  • DBU l,8-diazabicyclo-[5.4.0]undec-7-ene
  • MTBE methyl t-butyl ether
  • FDMS field desorption mass spectrometry.
  • This compound was prepared according to the process of Example 1, except that 4-cyclohexyl aniline was used instead of -t-butyl aniline.
  • the final product was recrystallized from THF/hexane to obtain 82% yield.
  • This compound was prepared according to the process of Example 1, except that 4-heptylaniline was used instead of ⁇ -t-butyl aniline.
  • the final product was recrystallized from THF/hexane to obtain 93% Yield.
  • This compound was prepared according to the process of Example 1, except that 4-iodoaniline was used instead of -t-butyl aniline.
  • the final product was recrystallized from THF/hexane. Yield 60%
  • This compound was prepared according to the process of Example 1, except that 4- benzyloxyaniline was used instead of ⁇ -t-butyl aniline.
  • This compound was prepared according to the process of Example 1, except that 4- hydroxyaniline was used instead of ⁇ -t-butyl aniline. The final product was recrystallized from
  • This compound was prepared according to the process of Example 1, except that 3'- aminoacetanilide was used instead of ⁇ -t-butyl aniline.
  • the final product was recrystallized from ethyl acetate/methanol/hexane. Yield 46%.
  • This compound was prepared according to the process of Example 1, except that 3-amino-N- butylbenzenesulfonmide was used instead of ⁇ -t-butyl aniline.
  • the final product was recrystallized from from ethanol/water. Yield 50%.
  • This compound was prepared according to the process of Example 1, except that 3-(l- hydroxyethyl) aniline was used instead of ⁇ -t-butyl aniline.
  • Acetic acid 5-(3-tert-butoxycarbonylamino-phenyl)-pentyl ester (88 mg, 0.27 mmol) was dissolved in a mixture of trifluoroacetic acid (4.5 mL) and water (0.5 mL) and the solution was stirred for 10 min at room temperature. The solvents were evaporated and the residue was taken up in ethyl acetate. The organic phase was washed with a saturated solution of NaHCO (twice), brine, dried over Na 2 SO and concentrated in vacuo to give acetic acid 5-(3-aminophenyl)pentyl ester (36 mg, 60%).
  • 6-(3-aminophenoxy)hexanoic acid ethyl ester was then reacted with 2-Chloroethyl isocyanate as described in examples 1-12 to obtain compound 19. Purified by flash chromatography (hexane/ethyl acetate 3/2). Yield 85%.
  • 2-(hept-l-ynyl)aniline (628 mg, 3.35 mmol) was dissolved in ethanol (20 mL) and placed in a hydrogenation bottle with 10% Pd/C (88 mg). The bottle was filled with 40 psi of hydrogen and shaken for 4 h. The product was filtered through Celite, and the solvent was removed in vacuo to obtain 2-heptanylaniline. The aniline was used without any further purification (602 mg, %)
  • the purification technique was flash chromatography on silica gel : Hexane / AcOEt (70 / 30).
  • 5-(3-nitrophenyl)pent-4-yn-l-ol (1.0 Eq, 0.970 mmol, 0.200 g) in Pd / C 10% (0.05 Eq, 0.047 mmol, 0.050 g), H 2 (38 PSI), ETOH (10 mL) was hydrogenated as described in Example 32 to yield 5-(3-aminophenyl)pentan-l-ol.
  • the product was purified by flash chromatography on silica gel : Hexane / AcOEt (70 / 30).
  • 3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 5-Hexyn-l-ol (2.58 Eq, 20.670 mmol, 2.030 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh 3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K 2 CO 3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H 2 O (10 mL) as described in Example 32.
  • 6-(3-nitrophenyl)hex-5-yn-l-ol (1.0 Eq, 0.958 mmol, 0.210 g) was hydrogenated with Pd / C 10% (0.05 Eq, 0.047 mmol, 0.050 g), H 2 (38 PSI), ETOH (10 mL) as described in Example 32.
  • 3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 3-Butyn-l-ol (2.58 Eq, 20.670 mmol, 1.449 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh 3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K 2 CO 3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H 2 O (10 mL) as described in Example 32.
  • l-(2-chloroethyl)-3-(3-hydroxyphenyl)urea (1MO-365) was obtained as described in example 39.
  • 3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 4-pentyn-l-ol (2.67 Eq, 21.400 mmol, 1.800 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh 3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K 2 CO3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H 2 O (10 mL) as described in Example 32. The solvent was removed and the residue was purified by flash chromatography on silica gel: hexane / AcOEt (70 / 30).
  • 3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 3-Butyn-l-ol (2.58 Eq, 20.670 mmol, 1.449 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh 3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K 2 CO3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H 2 O (10 mL) as described in Example 32. The solvent was removed and the residue was purified by flash chromatography on silica gel: Hexane / AcOEt (65 / 35).
  • 3-nitrobenzylalcohol (1.0 Eq, 1.959 mmol, 0.300 g) was mixed with triethylamine (3.0 Eq, 5.880 mmol, 0.595 g), acetic anhydride (3.0 Eq, 5.880 mmol, 0.600 g) and 4-py ⁇ olidinopyridine (0.02 Eq, 0.039 mmol, 0.006 g) at room temperature. The solvent was removed and the product was purified by flash chromatography on silica gel: hexane / AcOEt (75 / 25).
  • 6-(3-nitro ⁇ henyl)hex-5-yn-l-ol (1.0 Eq, 1.140 mmol, 0.255 g) was mixed with triethylamine (3.0 Eq, 3.420 mmol, 0.346 g), acetic anhydride (3.0 Eq, 3.420 mmol, 0.349 g) and 4- pyrrolidmopyridine (0.02 Eq, 0.023 mmol, 0.003 g) ) at room temperature. The solvent was removed and the product was purified by flash chromatography on silica gel: Hexane / AcOEt (75 / 25).
  • 6-(3-aminophenyl)hex-5-ynyl acetate (1.0 Eq, 0.643 mmol, 0.168 g) in Pd / C 10% (0.07 Eq, 0.047 mmol, 0.050 g), H 2 (38 PSI), and ETOH (10 mL) was hydrogenated and purified as described in Example 32. The solvent was removed and the product was purified by flash cliromatography on silica gel: dichloromethane / MeOH (99 / 1).
  • Example 61 l-(2-Chloro-ethyl)-3-[3-(5-hydroxy-pent-l-ynyl)-phenyl]-urea (58);
  • Example 62 3-[3-(2-Chloro-ethyl)-ureido]-benzoic acid ethyl ester (59);
  • Example 63 l-(2-Chloro-ethyl)-3-[3-(5-methoxy-pentyl)-phenyl]-urea (60);
  • Example 64 ⁇ 3-[3-(2-Chloro-ethyl)-ureido]-phenyl ⁇ -acetic acid (61);
  • Example 66 l-(2-Chloro-ethyl)-3-(3-heptyl-phenyl)-urea (63);
  • Example 68 Pentanedioic acid mono- ⁇ 3-[3-(2-chloro-ethyl)-ureido]-phenyl ⁇ ester (65);
  • Example 70 3-[3-(2-Chloro-ethyl)-ureido]-benzoic acid (67);
  • Example 72 l-(2-Chloro-ethyl)-3-(3-hexyl-phenyl)-urea (69);
  • Example 74 6- ⁇ 3-[3-(2-Chloro-ethyl)-ureido]-phenyl ⁇ -hexanoic acid ethyl ester (71);
  • Example 78 l-(2-Chloro-ethyl)-3-[3-(4-hydroxy-but-l-ynyl)-phenyl]-urea (75);
  • Example 80 3-[3-(2-Chloro-ethyl)-ureido]-phenyl ⁇ -acetic acid ethyl ester (77);
  • Example 81 Acetic acid 3- ⁇ 3-[3-(2-chloro-ethyl)-ureido]-phenyl ⁇ -propyl ester (78).
  • the compounds can be assayed initially for their ability to inhibit cell growth (i.e. their cytotoxicity) in vitro using standard techniques, hi general, cells of a specific test cell line are grown to an appropriate density (e.g. approximately 1 x 10 4 ) and the candidate compound is added. After an appropriate incubation time (for example 48 to 74 hours), cell survival is assessed, for example, by using the resazurin reduction test (see Fields & Lancaster (1993) Am. Biotechnol. Lab. 11:48-50; O'Brien et al, (2000) Eur. J. Biochem. 267:5421-5426 and U.S. Patent No.
  • the candidate compounds can also be tested in vitro for their ability to inhibit anchorage- independent growth of tumour cells.
  • Anchorage-independent growth is known in the art to be a good indicator of tumourigenicity. In general, anchorage-independent growth is assessed by plating cells from an appropriate cancer cell-line onto soft agar and determining the number of colonies formed after an appropriate incubation period. Growth of cells treated with the compound can then be compared with that of cells treated with an appropriate control (as described above).
  • Cells were grown in an appropriate medium. Cell growth inhibition was assessed using a modified Alamar Blue assay as described by Lancaster et al. (U.S. Patent No. 5,501,959). For the proliferating state, cells were seeded in 96-well plates and preincubated for 24h (or 72 hours for the quiescent state). After addition fresh medium containing increasing concentrations of the candidate compound, cells were incubated at 37°C for 48 hours. The culture medium was removed, cells were washed and contacted with a resazurin solution. Cell survival was calculated from fluorescence (excitation, 485 mn; emission, 590 nm) measured with a FL 600 Reader (BioTek Instruments). Cell growth inhibition was expressed as the dose of drug required to inhibit cell growth by 50%) (GI 50 ). Values are the means of at least three independent detenninations.
  • GI5 0 is the dose required to inhibit cell growth by 50%. Different values obtained from different synthetic batches of compound.
  • EXAMPLE 84 MDA-MB-231 Cell Growth Inhibition by Compounds of the Invention
  • the human tumour cell line MDA-MB-231 was grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. Cells were inoculated into 96 well microtiter plates in 100 ⁇ L at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of the cell line. After cell inoculation, the microtiter plates was incubated at 37° C, 5 % CO 2 , and 100 % relative humidity for 24 h prior to addition of experimental drugs.
  • TCA trichloro acetic acid
  • the plates were incubated for an additional 48 h at 37°C, 5 % CO2, and 100 %> relative humidity.
  • the assay was terminated by the addition of cold TCA.
  • Cells were fixed in situ by the gentle addition of 50 ⁇ l of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded, and the plates were washed five times with tap water.
  • Sulforhodamine B (SRB) solution 100 ⁇ L) at 0,1% (w/v) in 1 % acetic acid (v/v) was added to each well, and plates were incubated for 10 minutes at room temperature.
  • GI 50 is the dose required to inhibit cell growth by 50%.
  • EXAMPLE 85 Inhibition of tumour cell growth by the compounds of the instant invention in a dose- and time-dependent manner.
  • M21 and HT1080 tumour cell lines were inoculated into 96 well tissue culture plates in 100 ⁇ L containing 2 X 10 3 cells and were incubated at 37 °C. After 24 h, freshly solubilized drugs in DMSO were diluted in fresh medium. Aliquots of 100 ⁇ l containing escalating concentration of drugs (0.3 ⁇ M to 100 ⁇ M; A) compound 1, B) compound 2, C) tBEU and D) cDDP) were added to the appropriate microtiter wells already containing 100 ⁇ l of culture medium. The cells were incubated for different period of time ranging from 3 h to 48 h.
  • Bound stain was solubilized with 10 mM trizma base, and the absorbance was read using a ⁇ Quant Universal Microplate Specfrophotometer (Biotek, Winooski, VT) at 585 nm. The results were compared with those of a control reference plate fixed on the freatment day and the growth inhibition percentage was calculated for each drug contact period. The growth inhibition percentage is expressed as the mean of triplicates for each drug contact period, compared with those of a control reference plate fixed on the day of the treatment.
  • the cytotoxicity of the test compounds was compared with that of a classical and strong alkylating agent, namely cisplatin (cDDP).
  • cDDP cisplatin
  • the antimicrotubule agents colchicine, vinblastin and paclitaxel were also tested in this assay but was found not cytotoxic until they reach 48 h of exposure (data not shown). When they were in contact for less than 6 h with either cell lines, virtually none of the tested compounds showed inhibition of tumour cell growth and proliferation. However, as the time of contact between the test compounds and tumour cells was increased from 6 to 48 h, the comparable GI 50 of compound 1 and 5 markedly shifted to the left hand-side of the graph ( Figure 1 A and B) and this was shown in the low micromolar range for all tumour cell lines tested.
  • M21 cells were seeded at lxlO 5 cells in in 35-mm Petri dishes and incubated for 16 h at 37 °C.
  • Cells were treated for 24h with either 100 ⁇ M of compounds 1 and 5 or tBEU or classical antimicrotubule agents (50 ⁇ M of cisplatin (cDDP), 25 ⁇ M of colchicine (COL), 5 ⁇ M of vinblastine (VINB) or 50 ⁇ M of paclitaxel (TAX)).
  • cDDP cisplatin
  • COL colchicine
  • VINB vinblastine
  • TAX paclitaxel
  • the cells were penneabilized and blocked with 0.1 % saponin and 3 % (w/v) BSA in PBS, during 1 h at 37 °C.
  • the cells were then further incubated during 1 h at 37 °C with anti-tubulin (clone TUB2.1, that is specific to ⁇ -tubulin and does not cross-react with other-tubulin isoforms; Sigma-Aldrich; St-Louis, MO) (1: 200) in 0.1 % saponin and 3 % BSA in PBS.
  • anti-tubulin clone TUB2.1, that is specific to ⁇ -tubulin and does not cross-react with other-tubulin isoforms; Sigma-Aldrich; St-Louis, MO
  • the cells were washed three times with PBS containing 0.05 % of Tween 20 and incubated 1 h at 37 °C in blocking buffer containing anti-mouse IgG Alexa-488 (1 : 1000), DAPI (2.5 ⁇ g/ml in PBS) to stain nuclei and Rhodamine-labeled phalloidin (1 : 600) to stain the actin cytoskeleton.
  • the observations were made using a Nikon Eclipse E800 microscope (Tokyo, Japan) equipped with a 40X objective. Images were captured as 16 bit TIFF files with a Hamamatsu ORCA ER cooled (-20°C) digital camera (Photonics Management Management Corp., Bridgewater, N.J.) driven by SimplePCI AIC software (Compix Inc. C Imaging systems, Pennsylvania). Representative fields are shown from three separate experiments.
  • Paclitaxel stabilizes the microtubules, thus inhibiting their depolymerization, whereas the others rather blocking their polymerization, inducing therefore a depolymerization phenotype ( Figure 2).
  • the compound 1 effect on ⁇ -tubulin was indeed drastically different from what was observed after paclitaxel treatment, not as severe as vinblastine, but rather similar to that observed after colchicine cell exposure.
  • the bioisosteric derivative compound 5 showed a similar microtubule dissolution activity as compound 1.
  • tBEU did not exhibit any effect on the microtubule network nor did affected the filamentous structure of actin.
  • EXAMPLE 87 Generation of an alkylated form of ⁇ -tubulin by compound 1 and compound 5.
  • M21 cells or MDA-MB-231 cells were washed in ice-cold PBS, pooled and then solubilized in buffer containing 62.5 mM Tris, pH 6.8, 2 % SDS, 6 M urea, 10 % glycerol, 0.00125 % bromophenol blue, and 720 mM ⁇ - mercaptoethanol.
  • the cell extracts were boiled for 5 min, separated on 10 % SDS-PAGE electrophoresis gel and transfe ⁇ ed onto nitrocellulose membrane.
  • the membranes were blocked for 1 h at 37 °C with 5 % (w/v) milk in Tris buffered saline (TBS) containing 0.1 % Tween 20 (TBST) and then incubated, 1 h at 37 °C, with the appropriate antibody diluted in 5 % milk in TBST.
  • TBS Tris buffered saline
  • TBST Tween 20
  • the apparition of an additional immunoreactive band of ⁇ -tubulin was evaluated with the monoclonal anti-tubulin antibody (1: 500).
  • Membranes were incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1: 2500) (Amersham Canada, Oakville, Canada) diluted in 5 % milk in TBST, 1 h at room temperature, followed by chemiluminescent detection, using an enhanced chemoluminescence (ECL) detection kit (Amersham Pharmacia Biotech).
  • a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1: 2500) (Amersham Canada, Oakville, Canada) diluted in 5 % milk in TBST, 1 h at room temperature, followed by chemiluminescent detection, using an enhanced chemoluminescence (ECL) detection kit (Amersham Pharmacia Biotech).
  • ECL enhanced chemoluminescence
  • EXAMPLE 88 Generation of an alkylated form of ⁇ -tubulin by exemplary compounds 1, (R)24; S(24); 28; 5; (R)23; 81, 82, 83 and 84 of the invention
  • Human breast carcinoma cell line, MDA-MB-231 was grown in RPMI 1640 medium supplemented with 10% bovine calf serum iron supplemented (Hyclone, Road Logan, Utah) in a humidified atmosphere at 37°C in 5% CO 2 . After trypsinization with 1 mL trypsin- EDTA, cells were seeded in 12 well-plates. Each well received around 220 000 cells. After an incubation of 24 hours, the culture medium was removed and drugs, diluted in fresh medium, added.
  • Each of the ten drugs was dissblved in dimethyl sulfoxide to yield a 40mM stock solution. An aliquot of stock solution is mixed with the culture to a final volume concentration of 30 ⁇ M. Eight incubation times for each drug was performed, i.e. 0, 1, 2, 4, 8, 12, 24 and 48 hours. After each incubation time, the medium of each well is collected. 1 mL of cold PBS is added to each well. Remaining cells are scratched from the well surface, centrifugated and washed anew in 500 ⁇ L of cold PBS. Both cell aliquots are mixed.
  • Cell suspension 100 ⁇ L from the cell suspension was pipetted and used to determine cell concentration with NaOH/DO280. The remaining cell solution was centrifugated to obtain a pellet. The pellet is brought to a concentration of 60 000 cells/35 ⁇ L in a solution Laemmli IX + 5 % Beta-mercaptoethanol, resuspended, sonicated 5 seconds, boiled 5 minutes and then centrifugated. SDS-PAGE Analysis and Immunoblotting of /3-tubulin. Samples (60 000 cells) were analyzed by 10% SDS-PAGE. Gels were transferred to a nitrocellulose membrane.
  • Membranes were then incubated with PBSMT [PBS (pH 7.4), 5% fat-free dry milk, and 0.1% Tween-20]. After Ponceau staining, the membrane is immersed with a solution of 1:500 monoclonal anti-/3-tubulin (clone TUB 2.1, Sigma) for 1 h at room temperature. Membranes were washed with PBSMT and incubated with 1:2500 peroxidase-conjugated antimouse imnrunoglobulin (Amersham Canada, Oakville, Canada) in PBSMT for 30 min. Detection of the immunoblot was carried out with the ECL Western blotting detection reagent kit (Amersham Canada, Oakville, Canada).
  • EXAMPLE 89 Generation of an alkylated form of ⁇ -tubulin by exemplary compounds 1; (R)24; (S)24; (R) 85; (S) 85, 5; (R)23; (S)23; (R) 86, (S) 86 of the invention (II)
  • Cell suspension 100 ⁇ L from the cell suspension was pipetted and used to determine cell concenfration with NaOH/DO280. The remaining cell solution was centrifugated to obtain a pellet. The pellet is brought to a concentration of 60 000 cells/35 ⁇ L in a solution Laemmli IX + 5 % Beta-mercaptoethanol, resuspended, sonicated 5 seconds, boiled 5 minutes and then centrifugated.
  • Membranes were washed with PBSMT and incubated with 1:2500 peroxidase-conjugated antimouse immunoglobulin (Amersham Canada, Oakville, Canada) in PBSMT for 30 min. Detection of the immunoblot was carried out with the ECL Western blotting detection reagent kit (Amersham Canada, Oakville, Canada).
  • EXAMPLE 90 Generation of an alkylated form of ⁇ -tubulin by exemplary compounds 53; 57; 30; 63, 31, 28, 66, 47, 49, 68, 69 and 87 of the invention
  • Each of the ten drugs was dissolved in dimethyl sulfoxide to yield a 40mM stock solution. An aliquot of stock solution is mixed with the culture to a final volume concentration of 5 ⁇ M. Eight incubation times for each drug was performed, i.e. 2, 7, 17, 25 and 48 hours. After each incubation time, the medium of each well is collected. 1 mL of cold PBS is added to each well. Remaining cells are scratched from the well surface, centrifugated and washed anew in 500 ⁇ L of cold PBS. Both cell aliquots are mixed.
  • Cell suspension 100 ⁇ L from the cell suspension was pipetted and used to determine cell concentration with NaOH/DO280. The remaining cell solution was centrifugated to obtain a pellet. The pellet is brought to a concentration of 100 000 cells/25 ⁇ L in a solution Laemmli IX + 5 % Beta-mercaptoethanol, resuspended, sonicated 5 seconds, boiled 5 minutes and then centrifugated.
  • MDA-MB-231 cells were grown in DMEM plus 5% FBS (Hyclone, Road Logan, Utah) and L-glutamine without antibiotics. Cells were seeded at 3xl0 5 cells in 6 well plate and grown to confluence. 16 hours prior the experiments, the different drugs were diluted in fresh medium and added to the wells. For a series of measurements, the concentrations selected were 0,5, 3 and 5 ⁇ M. For the other series of measurements, the concentrations selected were 0,25, 0,5 and 1 ⁇ M. The following day, the tip of a cell scraper was used to scratch the bottom of each well, producing a "wound" of roughly 1000 ⁇ m.
  • the medium was washed to remove the detached cells, and two pictures per well were taken at 0, 3, 6, 8 and 24 hours. By superimposing a hemacytometer grid over the picture, the distance between the two fronts was measured at numerous positions. Migration velocity was determined by a graph of the "wound" front distance over time.
  • the assay was repeated at least twice for each drug, at each concentration. For IMO- 236, the concentration at 3 and 5 ⁇ M were too toxic for the cells.
  • EXAMPLE 92 Inhibition of HT1080 tumour cell migration by compounds 1 and 5.
  • the chemotaxis motility of HT1080 was assessed using Boyden chambers. Briefly, the underside of TranswellTM migration chamber membranes (8.0- mm pore size) were coated with collagen IV as described previously (filardo et al., J. Cell Biol. 130:441-450 (1995); Kle ke et al. J. Cell Biol. 127:859-866 (1994) and as modified by Petitclerc et al. (Petitclerc et al. J. Biol. Chem. 275:8051-8061 (2000)).
  • the cells were pre-incubated or not for 16 h with escalating concentrations of different compounds of the invention, then they were added to the top of a collagen IN coated TranswellTM membrane (8.0 mm pore size), separating the lower and upper part of a Boyden chamber in the presence of same drugs. Soluble fibronectin (25 ⁇ g/ml) was added to the lower chamber to induce chemotaxis.
  • the cells were allowed to migrate for 4-6 h at 37 °C, fixed and stained for quatification. The number of migrating cells per well were counted. The results expressed the mean ⁇ s.e. of triplicates.
  • EXAMPLE 93 The compounds of the instant invention impede the growth of two unrelated tumour cell lines in the chick chorioallantoic membrane (CAM) assay.
  • CAM chick chorioallantoic membrane
  • Human HT1080 fibrosarcoma and hamster CS1 melanoma cell lines were used to assess the antitumoural activity of CEUs in the chick chorioallantoic assay (Petitclerc et al. J. Biol. Chem. 275:8051-9061 (2000); Kim et al. Cell 94:353-362 (1998); Lyu et al. Int. J. Cancer 77:257-263 (1998)).
  • day-0 fertilized chicken eggs were purchased from Couvoirs Victoriaville (Victoriaville, QC, Canada).
  • the eggs were incubated for 10 days in a Pro-FI egg incubator fitted with an automatic egg turner, before being transferred to a RoU-X static incubator for the rest of the incubation time (incubators were purchased from Lyon Electric, Chula Vista, San Diego).
  • the eggs were kept at 37 °C in a 60 % humidity atmosphere for the whole incubation period.
  • a hobby drill (Dremel; Racine, WI)
  • a hole was drilled on the side of the egg, and a negative pressure was applied to create a new air sac.
  • a window was opened on this new air sac and was covered with transparent adhesive tape to prevent contamination.
  • HT1080 3.5 x 10 5 cells/egg
  • CS1 5 x 10 6 cells/egg
  • the tested drugs were injected intravenously in 10-12 eggs in a small volume (100 ⁇ l).
  • the eggs were incubated until day, at which time the embryos were euthanized at 4 °C, followed by decapitation. Tumours were collected, pictures were taken to illustrate the different groups and the tumour-wet weights were recorded. In all experiments, the number of dead embryos from the different groups was monitored for any sign of toxicity.
  • Panel A and B of Figure 7 show that incubation of CS1 -derived tumours on the CAM with compound 1 or compound 5 resulted in a significant dose-dependent reduction of the tumours size, as observed also with cDDP (Figure 7D). Moreover, both test compounds also inhibited the formation of HT1080 tumour mass in the same concentration range (data not shown). It is noteworthy that the non-alkylating homologue tBEU failed to influence the growth of CS1 tumours ( Figure 7C), supporting the assumption that the antitumoural effect of compound 1 and compound 5 was dependent on their alkylating activity. In the same experimental settings, only 10 ⁇ g/egg of cDDP was sufficient to inhibit tumour cell growth.
  • EXAMPLE 94 Compounds 1, 2, 5 and 30 impede the growth of CS1 tumour cell line in the chick chorioallantoic membrane (CAM) assay.
  • EXAMPLE 95 Clonogenic survival of M21 cells on extracellular matrices (ECMs).
  • tumour microenvironment could modulate the tumour cells' ability to resist to chemotoxic agents such as cDDP, triggering a pro-survival signal through integrins (Hazlehurst et al. Oncogene 19:4319-4327 (2000); Rintoul et al. Clin. Sci. (Lond) 102:417-424 (2002): Damiano et al. Blood 93:1658-1667 (1999).
  • M21 skin melanoma cells were plated (1 X 10 cells in 100-n ⁇ m Petri dishes) on different ECMs and challenged with test compounds of the instant invention or the strong alkylating agent cDDP, as previously described (Deschesnes et al. Mol. Biol. Cell. 12:1569-1582 (2001)). Briefly, native and heat-denatured type IN collagen (50 ⁇ g/ml), fibronectin (25 ⁇ g/ml) and fibrin (50 ⁇ g/ml) were used to coat non-tissue culture plates ( ⁇ unc). After washes with serum-free DMEM, cells were plated in serum-containing or serum-free media on the different matrices for 16 h.
  • native and heat-denatured type IN collagen 50 ⁇ g/ml
  • fibronectin 25 ⁇ g/ml
  • fibrin 50 ⁇ g/ml

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Abstract

The present invention provides haloethyl urea compounds as described in Formula (I) and their use as therapeutic agent in the attenuation, inhibition, or prevention of cancer cell migration and cancer cell proliferation.

Description

HALOETHYL UREA COMPOUNDS AND THE USE THEREOF TO ATTENUATE, INHIBIT OR PREVENT CANCER CELL MIGRATION
FIELD OF INVENTION
The present invention pertains to the field of therapeutics for diseases or disorders related to cancer cell migration, in particular to therapeutically active haloethyl urea derivatives and their use to inhibit cancer cell migration.
BACKGROUND OF THE INVENTION
A cancer is a malignant tumour of potentially unlimited growth. It is primarily the pathogenic replication (a loss of normal regulatory control) of various given types of cells found in the human body. By select mutation resulting from a primary lesion, the DNA of a cancer cell evolves and converts the cell into an autonomous system. Invasion and metastasis are the most insidious and life-threatening aspects of cancer.
Metastasis of the primary tumour produces secondary tumours and disseminated cancer. Therapy for metastasis currently relies on a combination of early diagnosis and aggressive treatment, which may include radiotherapy, chemotherapy or hormone therapy. The high mortality rate for many cancers indicates that improvements are needed in the prevention and treatment of metastasis.
A number of other approaches that inhibit other aspects of metastasis, such as adhesion, proteolysis, migration, have been developed. For example, WO 97/00956 describes the use of an antibody raised against an adhesion protein on endothelial and muscle cells for inhibiting tumour metastasis. U.S. Patent No. 6,015,893 describes oligonucleoside compounds useful in inhibiting the expression of focal adhesion kinase protein. While U.S. Patent No. 5,700,830 describes a method for inhibiting the adherence between cancerous cells and noncancerous structures in a mammal, comprising the administration to the mammal of a nitric oxide-releasing compound.
Urea-based compounds have been described for diverse indications, including as herbicides (U.S. Patent No. 3,885,954), as prophylactics against gastrointestinal and cardiovascular disorders (U.S. Patent No. 4,707,478), as anti-parasitic agents (U.S. Patent No. 4,707,478), as anti-athersclerotic agents (U.S. Patent No. 4,623,662), as treatments for gastrointestinal, spasmolytic and ulcerogenic disorders (U.S. Patent No. 4,304,786) and as anti-cancer agents (for example, U.S. Patent Nos. 3,968,249; 4,973,675 and 4,803,223). A class of l-aryl-3-(2- chloroethyl)urea derivatives have been described as anti-cancer agents (U.S. Patent Nos. 5,530,026 and 5,750,547, and International Patent Application WO 00/61546) and as β-tubulin inhibitors (International Patent Application WO 01/447504).
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.
SUMMARY OF THE INVENTION
An object of the present invention is to provide novel halo urea compounds for the inhibition of cancer cell motility/migration. In accordance with one aspect of the invention, there is provided compound of formula I:
Figure imgf000003_0001
or a pharmaceutically acceptable salt thereof, wherein:
X is F, CI, Br or I;
RI and R2 are each independently selected from the group of H, -R, -halo, -OR, -SR, -NRR, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NRR, -C(S)NRR, -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, or Rl and R2 when taken together form =O, =S or a C3-C6 spiro group;
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
B is substituted with one or more substituents selected from the group of (Ci- β) alkyl, (Ca-Ciβ) alkenyl, (C2-C16) alkynyl, aryl, -O-(Cι-C16) alkyl, -O-(C2-C16) alkenyl, -O-(C2-C16) alkynyl, -O-aryl, -O-CH2-aryl, -S-(Cι-C16)alkyl, -S-(C2-C16) alkenyl, -S-(C2-Cι6) alkynyl, -S- aryl, -S-CH2-aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -halo, -NRR, -ONRR -NO2, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, - SC(O)R, -SC(S)R, -OC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), - C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, - CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, - S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: each R is independently selected from -H, (CrC16) alkyl, substituted ( - δ) alkyl, (C2- C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cycloalkyl, aryl or substituted aryl; the alkyl, alkenyl, alkynyl and aryl are optionally substituted with one or more substituents independently selected from the group of -halo, trihalomethyl, -R', -OR, -SR', -NR , -NO2, - CN, -OC(O)R, -OC(S)R, -SC(O)R, -SC(S)R -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NR'R', -C(S)NR'R', -NR'C(O)R' and -NRC(O)OR; the cycloalkyl is optionally substituted with one or more substituents independently selected from the group of R', -halo, OR', -SR', -NRR', -ONRR', -NO2, -CN, -C(O)R', -C(S)R', - OC(O)R', -SC(O)R', -SC(S)R', -OC(S)R', -C(O)OR', -C(S)OR', -C(O)SR', -C(S)SR', - C(O)NRR', -C(S)NRR', -C(O)NR'(OR -C(S)NR'
Figure imgf000004_0001
-C(S)NR'(SR , - CH(CN)2, -CH[C(O)R]2, -CH[C(S)R']2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, - CH[C(S)SR]2, -NR'C(O)R', -NR'C(O)OR', -S(O)-R', -S(O)OR', -S(O)2OR', -S(O)NRR', - S(O)ONRR', and each R' is independently selected from the group of -H, ( -C^) alkyl, substituted ( -Ciδ) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C -C16) alkynyl, (C -C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl.
hi accordance with another aspect of the present invention there is provided a method of attenuating, inhibiting or preventing cancer cell migration in a mammal comprising administering an effective amount of a compound of formula I.
In accordance with another aspect of the present invention there is provided a method of attenuating, inhibiting or preventing cancer cell proliferation in a mammal in need of such therapy.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 presents a graph illustrating the ability of the compound 1 (A); compound 5 (B); l-(4-t- butyl-phenyl)-3-ethyl urea (tBEU) (C) and cDDP (D) to inhibit tumour cell growth in a dose and time dependent manner.
Figure 2 depicts the microtubule depolymerization and cytoskeleton disruption induced by compounds of the invention.
Figure 3 presents a Western Blot illustrating the generation of an alkylated form of β-tubulin by compound 1 and compound 5.
Figure 4 presents a Western Blot illustrating the generation of an alkylated form of β-tubulin by compounds of the invention.
Figure 5 presents graphs illustrating the ability of compounds of the invention to inhibit MDA- MB-231 cancer cell migration in the wound assay. Figure 6 presents graphs illustrating the ability of the compound I (A); compound 5 (B); l-(4-t- butyl-phenyl)-3-ethyl urea (tBEU) (C) to inhibit HT1080 cell migration.
Figure 7 presents graphs illustrating the ability of the compound 1 (A); compound 5 (B); l-(4-t- butyl-phenyl)-3-ethyl urea (tBEU) (C) and cDDP to impede the growth of two unrelated tumour cell lines in the chick chorioallantoic membrane (CAM) assay.
Figure 8 presents graphs illustrating the ability of compound 1, 5, 2 and 30 to impede the growth of CS1 cell line in the chick chorioallantoic membrane (CAM) assay
Figure 9 presents a graph illustrating the inhibition of carcinoma cell growth in Balb/c mice.
Figure 10 presents a graph illustrating that extracellular matrices and serum do not protect tumour cells against CEU toxiity in clonogenic assay.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides for the use of haloethyl urea derivatives of Formula I for attenuating, inhibiting or preventing cancer cell motility /migration. As is known in the art, cancer cell migration is a key feature of cancer cell invasion and metastasis. Thus, in one embodiment of the invention provides the use of the compounds of the invention to inhibit cancer progression and/or in the prevention of cancer metastasis.
Definitions
The terms and abbreviations used in the instant examples have their normal meanings unless otherwise designated. For example "°C" refers to degrees Celsius; "N" refers to normal or normality; "mmol" refers to millimole or millimoles; "μM" refers to micromole or micromoles; "g" refers to gram or grams; "mL" means milliliter or milliliters; "M" refers to molar or molarity; "p-" refers to para, "MS" refers to mass spectrometry; "IR" refers to infrared spectroscopy; and "NMR" refers to nuclear magnetic resonance spectroscopy.
The terms are defined as follows:
The term "halogen" refers to fluorine, bromine, chlorine, and iodine atoms.
The term "hydroxyl" refers to the group -OH.
The term "thiol" or "mercapto" refers to the group -SH, and -S(O)0-2.
The term "lower alkyl" refers to a straight chain or branched, or cyclic, alkyl group of 1 to 16 carbon atoms. This term is further exemplified by such groups as methyl, ethyl, 72-propyl, i- propyl, 72-butyl, t-butyl, 1-butyl (or 2-methylpropyl), cyclopropylmethyl, z'-amyl, n-amyl, hexyl and the like.
The term "substituted lower alkyl" refers to lower alkyl as just described including one or more groups such as hydroxyl, thiol, alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl, substituted cycloalkyl, heterocycle, cycloheteroalkyl, substituted cycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy, hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkyl cycloheteroalkyl, cyano. These groups may be attached to any carbon atom of the lower alkyl moiety.
The term "lower alkenyl" refers to a straight chain or branched hydrocarbon of 2 to 16 carbon atoms having at least one carbon to carbon double bond.
The term "substituted lower alkenyl" refers to lower alkenyl as just described including one or more groups such as hydroxyl, thiol, alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl, substituted cycloalkyl, heterocycle, cycloheteroalkyl, substituted cycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy, hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl, alkenyl, alkynyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkyl cycloheteroalkyl, cyano. These groups may be attached to any carbon atom to produce a stable compound.
The term "lower alkynyl" refers to a straight chain or branched hydrocarbon of 2 to 16 carbon atoms having at least one carbon to carbon triple bond.
The term "substituted lower alkynyl" refers to lower alkynyl as just described including one or more groups such as hydroxyl, thiol, alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl, substituted cycloalkyl, heterocycle, cycloheteroalkyl, substituted cycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy, hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl, alkenyl, alkynyl, alkyl alkenyl, alkyl alkynyl, alkyl cycloalkyl, alkyl cycloheteroalkyl, cyano. These groups may be attached to any carbon atom to produce a stable compound.
The term "alkyl alkenyl" refers to a group -R-CR-CR'"R"", where R is lower alkyl, or substituted lower alkyl, R', R", R"" are each independently selected from hydrogen, halogen, lower alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, hetaryl, or substituted hetaryl as defined below.
The term "alkyl alkynyl" refers to a group -R-C≡CR where R is lower alkyl or substituted lower alkyl, R is hydrogen, lower alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, hetaryl, or substituted hetaryl as defined below.
The term "alkoxy" refers to the group -OR, where R is lower alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heteroalkyl, heteroarylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, or substituted cycloheteroalkyl as defined below.
The term "alkylthio" denotes the group -SR, -S(O)n=ι-2 -R, where R is lower alkyl, substituted lower alkyl, aryl, substituted aryl aralkyl or substituted aralkyl as defined below.
The term "acyl" refers to groups -C(O)R, where R is hydrogen, lower alkyl substituted lower alkyl, aryl, substituted aryl. The term "aryloxy" refers to groups -OAr, where Ar is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl group as defined below.
The term "amino" refers to the group NRR', where R and R may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, cycloalkyl, or substituted hetaryl as defined below or acyl.
The term "amido" or "amide" refers to the group -C(O)NRR', where R and R may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, substituted hetaryl as defined below.
The term "carboxyl" refers to the group -C(O)OR, where R may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, substituted hetaryl and the like as defined.
The terms "aryl" or "Ar" refer to an aromatic carbocyclic group having at least one aromatic ring (e.g., phenyl or biphenyl) or multiple condensed rings in which at least one ring is aromatic, (e.g., 1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl, 9-fluorenyl etc.).
The term "substituted aryl" refers to aryl optionally substituted with one or more functional groups, e.g., halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido or cyano.
The term "heterocycle" refers to a saturated, unsaturated, or aromatic carbocyclic group having a single ring (e.g., morpholino, pyridyl or furyl) or multiple condensed rings (e.g., naphthpyridyl, quinoxalyl, quinolinyl, indolizinyl, indanyl or benzo[b]thienyl) and having at least one hetero atom, such as N, O or S, within the ring. The term "substituted heterocycle" refers to heterocycle optionally substituted with, halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido or cyano and the like.
The terms "heteroaryl" or "hetar" refer to a heterocycle in which at least one heterocyclic ring is aromatic.
The term "substituted heteroaryl" refers to a heterocycle optionally mono or poly substituted with one or more functional groups, e.g., halogen, hydroxyl, thiol, lower allcyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido or cyano and the like.
The term "aralkyl" refers to the group -R-Ar where Ar is an aryl group and R is lower alkyl or substituted lower alkyl group. Aryl groups can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl, alkoxy, alkyl thio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
The term "heteroalkyl" refers to the group -R-Het where Het is a heterocycle group and R is a lower alkyl group. Heteroalkyl groups can optionally be unsubstituted or substituted with e.g., halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like. The term "heteroarylalkyl" refers to the group -R-HetAr where HetAr is an heteroaryl group and R lower alkyl or substituted loweralkyl. Heteroarylalkyl groups can optionally be unsubstituted or substituted with, e.g., halogen, lower alkyl, substituted lower alkyl, alkoxy, alkylthio, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
The term "cycloalkyl" refers to a cyclic or polycyclic alkyl group containing 3 to 15 carbon. For polycyclic groups, these may be multiple condensed rings in which one of the distal rings may be aromatic (e.g. tetrahydronaphthalene, etc.).
The term "substituted cycloalkyl" refers to a cycloalkyl group comprising one or more substituents with, e.g halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, trifluoromethyl, alkenyl, alkenyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, sulfamido or cyano and the like.
The term "cycloheteroalkyl" refers to a cycloalkyl group wherein one or more of the ring carbon atoms is replaced with a heteroatom (e.g., N, O, S or P).
The term "substituted cycloheteroalkyl" refers to a cycloheteroalkyl group as herein defined which contains one or more substituents, such as halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.
The term "alkyl cycloalkyl" refers to the group -R-cycloalkyl where cycloalkyl is a cycloalkyl group and R is a lower alkyl or substituted lower alkyl. Cycloalkyl groups can optionally be unsubstituted or substituted with e.g. halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like. The terms "treatment", "therapy" and the like refer to improvement in the recipient's status as well as prophylaxis. The improvement can be subjective or objective and related to features such as symptoms or signs of the disease or condition being treated. Prevention of deterioration of the recipient's status is also encompassed by the term.
The term "ameliorate" or "amelioration" includes the arrest, prevention, decrease, or improvement in one or more the symptoms, signs, and features of the disease being treated, both temporary and long-term.
The compounds according to the instant invention include compounds of the following general formula:
Figure imgf000012_0001
or a pharmaceutically acceptable salt thereof, wherein:
X is F, CI, Br or I;
RI and R2 are each independently selected from the group of H, -R, -halo, -OR, -SR, -NRR, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NRR, -C(S)NRR, -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, or Rl and R2 when taken together form = , =S or a C -C6 spiro group;
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
B is substituted with one or more substituents selected from the group of (Ci-Ciβ) alkyl, (C2-C16) alkenyl, (C2-C16) alkynyl, aryl, -O-(Cι-Cι6) alkyl, -O-(C2-C16) alkenyl, -O-(C2-C16) alkynyl, -O- aryl, -O-CH2-aryl, -S-(Cι-Cι6)alkyl, -S-(C2-C16) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2- aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -halo, -NRR, -ONRR - NO2, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -OC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, - S(O)NRR, -S(O)ONRR; wherein: each R is independently selected from -H, (CrC^) alkyl, substituted (d-C16) alkyl, (C2- C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cycloalkyl, aryl or substituted aryl; the alkyl, alkenyl, alkynyl and aryl are optionally substituted with one or more substituents independently selected from the group of -halo, trihalomethyl, -R, -OR', -SR, -NR'R, -NO2, - CN, -OC(O)R, -OC(S)R, -SC(O)R, -SC(S)R -C(O)R, -C(S)R, -C(O)OR, -C(S)OR', -C(O)SR', -C(S)SR', -C(O)NR'R, -C(S)NR'R', -NR'C(O)R' and -NR*C(O)OR; the cycloalkyl is optionally substituted with one or more substituents independently selected from the group of R', -halo, OR', -SR', -NRR', -ONRR', -NO2, -CN, -C(O)R', -C(S)R', - OC(O)R', -SC(O)R', -SC(S)R', -OC(S)R', -C(O)OR', -C(S)OR', -C(O)SR', -C(S)SR', - C(O)NRR', -C(S)NRR', -C(O)NR'(OR'), -C(S)NR' -(OR'), -C(O)NR'(SR'), -C(S)NR'(SR'), - CH(CN)2, -CH[C(O)R']2, -CH[C(S)R 2, -CH[C(O)OR -CH[C(S)OR 2, -CH[C(O)SR]2, - CH[C(S)SR]2, -NR'C(O)R', -NR'C(O)OR', -S(O)-R', -S(O)OR', -S(O)2OR', -S(O)NRR', - S(O)ONRR', and each R' is independently selected from the group of -H, (CrC16) alkyl, substituted (C\- C16) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C -C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl.
In one embodiment, the compound of the invention is one in which RI and R2 are each independently selected from H, (Cι-C6) alkyl and ( -C6) alkoxy.
In a specific embodiment in the compound of formula (I) B is phenyl, substituted with one or more substituents indepnednetly selected from the list as shown above.
In another embodiment in the compound of formula (I), B is phenyl substituted with halo, -CN, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR, -OR, (C C16) alkyl, (C2-C16) alkenyl, (C2-C16) alkynyl , wherein said alkyl, alkenyl and alkynyl are optionally substituted with -CN, -C(O)R, - C(O)OR*, -OC(O)R, -C(O)NR'R', -OR', wherein R and R' are as defined above.
h another embodiment the substituents of the compounds of formula (I) are as follows:
X is F, CI, Br or I;
RI and R2 are as defined above, and
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; and substituted with at least one substituent selected from, (C;[-C16) alkyl, (C2-C16) alkenyl, (C2-C16) alkynyl, -O^d-C^) alkyl, -O-(C2-C16) alkenyl, -O-(C2-C16) alkynyl, aryl, substituted aryl, -O- aryl, -O-CH2-aryl, -S-(C!-C16) alkyl, -S-(C2-d6) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2- aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -ONRR, -C(O)R, -C(S)R
-C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -OC(S)R, -C(O)NRR, -
C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2,
-CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2,
-NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: said alkyl is substituted with at least one substituent selected from the group of halo, -CN, -NO2,
-NR'R', -O-alkyl, -O-alkenyl, -O-alkynyl, -O-aryl, -OC(O)R, -OC(S)R', -C(O)R, -C(S)R, -
C(O)NR*R' and -C(S)NR'R'; said alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO2, -NR'R, -OH, -OR', -O-aryl, -OC(O)R', -OC(S)R, -C(O)R,
-C(S)R, -C(O)OR, -C(O)NRR' and -C(S)NR'R'; said -O-alkenyl, -O-alkynyl, -S-alkenyl, -S-alkynyl, cycloalkyl, -O-cycloalkyl are are each optionally and independently substituted with at least one group selected from halo, -CN, -NO2, -
NR'R', -OH, -OR', -O-aryl, -OC(O)R, -OC(S)R, -C(O)R, -C(S)R, -C(O)OR, -C(O)NR'R and -
C(S)NR'R, and wherein R, R' and aryl are as defined above and their substituents are as defined above.
In another embodiment the substituents of the compounds of formula (I) are as follows: X is F, CI, Br or I; RI and R2 are as defined above, and
B is phenyl substituted with at least one substituent selected from, (d-C16) alkyl, (C2-C16) alkenyl, (C2-C16) alkynyl, -O-(d-C16) alkyl, -O-(C2-C16) alkenyl, -O-(C2-d6) alkynyl, aryl, substituted aryl, -O-aryl, -O-CH2-aryl, -S-(d-C16) alkyl, -S-(C2-C16) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2-aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -
ONRR, -C(O)R, -C(S)R -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R,
-OC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -
CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -
CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -
S(O)ONRR; wherein: said alkyl is substituted with at least one substituent selected from the group of halo, -CN, -NO2,
-NR'R*, -O-alkyl, -O-alkenyl, -O-alkynyl, -O-aryl, -OC(O)R, -OC(S)R', -C(O)R', -C(S)R, -
C(O)NR'R' and -C(S)NR'R'; said alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO2, -NR'R, -OH, -OR', -O-aryl, -OC(O)R*, -OC(S)R', -C(O)R,
-C(S)R, -C(O)OR, -C(O)NR'R* and -C(S)NR'R; said -O-alkenyl, -O-alkynyl, -S-alkenyl, -S-alkynyl, cycloalkyl, -O-cycloalkyl are are each optionally and independently substituted with at least one group selected from halo, -CN, -NO2, -
NR'R*, -OH, -OR', -O-aryl, -OC(O)R', -OC(S)R', -C(O)R, -C(S)R, -C(O)OR, -C(O)NR'R and -
C(S)NR'R, and wherein R, R' and aryl are as defined above and their substituents are as defined above.
hi another embodiment the substituents of the of the compounds of formula (I) are as follows:
X is F, CI, Br or I;
RI and R2 are as defined above, and
B is phenyl substituted with at least one group selected from, (d-C16) alkyl, (C2-C16) alkenyl,
(C2-Cι6) alkynyl, -O-(Cι-C16) alkyl, -O-(C2-C16) alkenyl, -O-(C2-Cι6) alkynyl, -aryl, -O-aryl, -O-
CH2-aryl, -OC(O)R, -C(O)R, -C(O)OR, -C(O)NR'R, -NRC(O)R, -NRC(O)OR, -S(O)-R, -
S(O)OR, -S(O)NRR, -S(O)ONRR; wherem: said alkyl is substituted with at least one group selected from halo, -CN, -O-alkyl, -O-alkenyl,
-O-alkynyl, -O-aryl, -OC(O)R, -C(O)R, and-C(O)NR'R': said alkenyl, alkynyl, -O-alkyl, -O-alkenyl and -O-alkynyl are each independently substituted with at least one group selected from halo, -CN, -OH, -OR, -O-aryl, -OC(O)R, -C(O)R, -
C(O)OR and -C(O)NR'R, and
R and R are as defined above.
In another embodiment, the substituents of the of the compounds of formula (I) are as follows:
X is F CI, Br or I;
RI and R2 are each independently selected from the group of H, (d-C6) alkyl, (d-C6) hydroxy alkyl, or RI and R2 when taken together form =O;
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; and substituted with at least one substituent selected from (d-C16) alkyl, (C2-C1 ) alkenyl, (C2-C16) alkynyl, -O-(d-C16) alkyl, -O-(C2-C16) alkenyl, -O-(C2-C16) alkynyl, aryl, substituted aryl, -O- aryl, -O-CH2-aryl, -S-(d-C16) alkyl, -S-(C2-C16) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2- aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -ONRR, -C(O)R, -C(S)R
-C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -OC(S)R, -C(O)NRR, -
C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2,
-CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2,
-NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: said alkyl is substituted with at least one substituent selected from the group of halo, -CN, -NO2,
-NR'R', -O-alkyl, -O-alkenyl, -O-alkynyl, -O-aryl, -OC(O)R, -OC(S)R, -C(O)R, -C(S)R, -
C(O)NR'R and -C(S)NR'R; said alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO2, -NR'R, -OH, -OR, -O-aryl, -OC(O)R, -OC(S)R, -C(O)R',
-C(S)R, -C(O)OR, -C(O)NR'R' and -C(S)NR'R'; said -O-alkenyl, -O-alkynyl, -S-alkenyl, -S-alkynyl, cycloalkyl, -O-cycloalkyl are are each optionally and independently substituted with at least one group selected from halo, -CN, -NO2, -
NR'R', -OH, -OR', -O-aryl, -OC(O)R, -OC(S)R, -C(O)R, -C(S)R, -C(O)OR, -C(O)NR'R and -
C(S)NR'R', and wherein R, R and aryl are as defined above and their substituents are as defined above.
In another embodiment, the compounds of formula (I) include the compounds of formula (II):
Figure imgf000017_0001
or a pharmaceutically acceptable salt thereof, wherein:
X is F CI, Br or I;
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
B is substituted with one or more substituents selected from the group of (d-C16) alkyl, (C2-C16) alkenyl, (C2-C16) alkynyl, aryl, -O-(d-C16) alkyl, -O-(C2-C16) alkenyl, -O-(C2-C16) alkynyl, -O-aryl, -O-CH2-aryl, -S-(C C16)alkyl, -S-(C2-C16) alkenyl, -S-(C2-C16) alkynyl, -S- aryl, -S-CH2-aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -halo, -NRR, -ONRR -NO2, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, - SC(O)R, -SC(S)R, -OC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), - C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, - CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, - S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: each R is independently selected from -H, (d-C16) alkyl, substituted (Cι-C16) alkyl, (C2- C16) alkenyl, substituted (C2-d6) alkenyl, (C2-C16) alkynyl, substituted (d-dδ) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cycloalkyl, aryl or substituted aryl; the alkyl, alkenyl, alkynyl and aryl are optionally substituted with one or more substituents independently selected from the group of -halo, trihalomethyl, -R, -OR', -SR, - NR'R', -NO2, -CN, -OC(O)R', -OC(S)R, -SC(O)R, -SC(S)R -C(O)R', -C(S)R, -C(O)OR, - C(S)OR, -C(O)SR, -C(S)SR, -C(O)NR'R', -C(S)NR'R', -NR'C(O)R and -NR*C(O)OR; the cycloalkyl is optionally substituted with one or more substituents independently selected from the group of R', -halo, OR', -SR', -NRR', -ONRR', -NO2, -CN, -C(O)R', -C(S)R', -OC(O)R', -SC(O)R', -SC(S)R', -OC(S)R', -C(O)OR', -C(S)OR', -C(O)SR', -C(S)SR', - C(O)NRR', -C(S)NRR', -C(O)NR'(OR'), -C(S)NR' -(OR , -C(O)NR'(SR'), -C(S)NR'(SR'), - CH(CN)2, -CHJO^R'h, -CH[C(S)R']2, -CH[C(O)OR']2, -CH[C(S)OR]2, -CH[C(O)SR']2, - CH[C(S)SR12, -NR'C(O)R', -NR'C(O)OR', -S(O)-R', -S(O)OR', -S(O)2OR', -S(O)NRR', - S(O)ONRR', and each R' is independently selected from the group of -H, (d-C16) alkyl, substituted (d-Cι6) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-d6) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl. In a specific embodiment in the compound of formula (II) B is phenyl, substituted with one or more substituents indepnednetly selected from the list as shown above.
In another embodiment, in the compounds of formula II, X is CI or Br.
hi another embodiment the substituents of the compounds of formula (II) are as follows:
X is F, CI, Br or I;
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; and
.substituted with at least one substituent selected from (d-C16) alkyl, (C2-Cι6) alkenyl, (C2-C16) alkynyl, -O-(d-d6) alkyl, -O-(C2-C16) alkenyl, -O-(C2-C16) alkynyl, aryl, substituted aryl, -O- aryl, -O-CH2-aryl, -S-(C C16) alkyl, -S-(C2-C16) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2- aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -ONRR, -C(O)R, -C(S)R
-C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -OC(S)R, -C(O)NRR, -
C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2,
-CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2,
-NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: said alkyl is substituted with at least one substituent selected from the group of halo, -CN, -NO2,
-NR'R, -O-alkyl, -O-alkenyl, -O-alkynyl, -O-aryl, -OC(O)R', -OC(S)R, -C(O)R*, -C(S)R, -
C(O)NRR and -C(S)NR'R'; said alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO2, -NR'R, -OH, -OR', -O-aryl, -OC(O)R, -OC(S)R, -C(O)R",
-C(S)R, -C(O)OR, -C(O)NR'R and -C(S)NR*R; said -O-alkenyl, -O-alkynyl, -S-alkenyl, -S-alkynyl, cycloalkyl, -O-cycloalkyl are are each optionally and independently substituted with at least one group selected from halo, -CN, -NO2, - NR'R, -OH, -OR, -O-aryl, -OC(O)R, -OC(S)R, -C(O)R', -C(S)R', -C(O)OR, -C(O)NR'R and - C(S)NRR, and wherein R, R and aryl are as defined above and their substituents are as defined above.
hi another embodiment the substituents of the compounds of formula (I) are as follows:
X is F, CI, Br or I;
B is phenyl substituted with at least one substituent selected from (d-C16) alkyl, (C2-C16) alkenyl, (C2-C16) alkynyl, -O-(d-C16) alkyl, -O-(C2-C16) alkenyl, -O-(C2-C16) alkynyl, aryl, substituted aryl, -O-aryl, -O-CH2-aryl, -S-(d-C16) alkyl, -S-(C2-C16) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2-aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -
ONRR, -C(O)R, -C(S)R -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R,
-OC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -
CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -
CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -
S(O)ONRR; wherein: said alkyl is substituted with at least one substituent selected from the group of halo, -CN, -NO2,
-NR'R, -O-alkyl, -O-alkenyl, -O-alkynyl, -O-aryl, -OC(O)R, -OC(S)R, -C(O)R, -C(S)R, -
C(O)NR'R* and -C(S)NR'R; said alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO2, -NR'R, -OH, -OR, -O-aryl, -OC(O)R, -OC(S)R, -C(O)R',
-C(S)R, -C(O)OR, -C(O)NR*R and -C(S)NR'R; said -O-alkenyl, -O-alkynyl, -S-alkenyl, -S-alkynyl, cycloalkyl, -O-cycloalkyl are are each optionally and independently substituted with at least one group selected from halo, -CN, -NO , -
NR'R, -OH, -OR', -O-aryl, -OC(O)R, -OC(S)R, -C(O)R, -C(S)R, -C(O)OR, -C(O)NR'R and -
C(S)NR'R, and wherein R, R' and aryl are as defined above and their substituents are as defined above.
In another embodiment the substituents of the of the compounds of formula (I) are as follows: X is F, CI, Br or I;
B is phenyl substituted with at least one group selected from (d-C16) alkyl, (C2-C16) alkenyl,
(C2-C16) alkynyl, -O-(d-C16) alkyl, -O-(C2-C16) alkenyl, -O-(C2-C16) alkynyl, -aryl, -O-aryl, -O-
CH2-aryl, -OC(O)R, -C(O)R, -C(O)OR, -C(O)NR'R, -NRC(O)R, -NRC(O)OR, -S(O)-R, -
S(O)OR, -S(O)NRR, -S(O)ONRR; wherein: said alkyl is substituted with at least one group selected from halo, -CN, -O-alkyl, -O-alkenyl,
-O-alkynyl, -O-aryl, -OC(O)R, -C(O)R, and -C(O)NR'R: said alkenyl, alkynyl, -O-alkyl, -O-alkenyl and -O-alkynyl are each independently substituted with at least one group selected from halo, -CN, -OH, -OR, -O-aryl, -OC(O)R, -C(O)R, -
C(O)OR* and -C(O)NR'R', and
R and R are as defined above.
In another embodiment the substituents of the of the compounds of formula (II) are as follows:
X is F, CI, Br or I; and
B is substituted with at least one group selected from aryl, -O-aryl, -O-CH2-aryl and halo.
In another embodiment the substituents of the of the compounds of formula (IT) are as follows:
X is F, CL Br or l;
B is substituted with at least one substituent selected from the group of (d-C16) alkyl, (d-C16) alkynyl or -O-alkyl; wherein: said alkyl is substituted with at least one substituent selected from the group of-CN, -O-alkyl, -
OC(O)R, -C(O)R, , -C(O)NR'R or halo; said alkyny and -O-alkyl are are substituted with at least one substituent selected from -CN, -
OH, -O-alkyl, -OC(O)R, -C(O)R, -C(O)OR', -C(O)NR'R or halo; and
R is as defined above.
In another embodiment the substituents of the of the compounds of formula (II) are as follows:
X is F, CL Br or l;
B is substituted with at least one group selected from -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR; wherein R is as defined above.
In another embodiment, the compounds of formula (I) include the compounds of formula (III):
Figure imgf000021_0001
or a pharmaceutically acceptable salt thereof, wherein:
X is F, CI, Br or I; B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
B is substituted with one or more substituents selected from the group of (d-C16) alkyl, (C2-C16) alkenyl, (C2-Cι6) alkynyl, aryl, -O-(d-C16) allcyl, -O-(C2-C16) alkenyl, -O-(C2-C16) alkynyl, -O- aryl, -O-CH2-aryl, -S-(d-d6)alkyl, -S-(C2-C16) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2- aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -halo, -NRR, -ONRR - NO2, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -OC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: each R is independently selected from -H, (d-C16) alkyl, substituted (d-C16) alkyl, (C - C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cycloalkyl, aryl or substituted aryl; the allcyl, alkenyl, alkynyl and aryl are optionally substituted with one or more substituents independently selected from the group of -halo, trihalomethyl, -R, -OR', -SR, -NR'R, -NO2, - CN, -OC(O)R, -OC(S)R, -SC(O)R, -SC(S)R -C(O)R, -C(S)R, -C(O)OR', -C(S)OR, -C(O)SR, -C(S)SR', -C(O)NR'R', -C(S)NR'R', -NR*C(O)R' and -NR'C(O)OR; the cycloalkyl is optionally substituted with one or more substituents independently selected from the group of R', -halo, OR', -SR', -NRR', -ONRR', -NO2, -CN, -C(O)R', -C(S)R', - OC(O)R', -SC(O)R', -SC(S)R', -OC(S)R', -C(O)OR', -C(S)OR', -C(O)SR', -C(S)SR', - C(O)NRR', -C(S)NRR', -C(O)NR'(OR'), -C(S)NR' -(OR'), -C(O)NR'(SR'), -C(S)NR'(SR'), - CH(CN)2, -CH[C(O)R]2, -CH[C(S)R -CH[C(O)OR]2, -CH[C(S)OR12, -CH[C(O)SR]2, - CH[C(S)SR']2, -NR'C(O)R', -NR'C(O)OR', -S(O)-R', -S(O)OR', -S(O)2OR', -S(O)NRR', - S(O)ONRR', and each R' is independently selected from the group of -H, (d-C16) alkyl, substituted (d-C1 ) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C -C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl.
hi a specific embodiment in the compound of formula (III) B is phenyl, substituted with one or more substituents indepnednetly selected from the list as shown above.
In another specific embodiment, the substitutents of formula (I) are as follows:
Figure imgf000022_0001
X is F CI, Br or I;
RI and R2 are each independently selected from the group of H, (d-C6) alkyl, (Cι-C6) hydroxy allcyl, (d-C6) alkoxy, (C3-C7) cycloalkyl, halo substituted (d-C6) alkyl, halo di- substituted (d-C6) alkyl, halo tri-substituted (d-C6) alkyl and halo;
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
B is substituted with one or more substituents selected from the group of (d-C16) alkyl, (C2-C16) alkenyl, (C2-C16) alkynyl, aryl, -O-(d-C16) alkyl, -O-(C2-C16) alkenyl, -O-(C2-C16) alkynyl, -O- aryl, -O-CH2-aryl, -S-(d-C16)alkyl, -S-(C2-Cι6) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2- aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -halo, -NRR, -ONRR, -NO2, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -OC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: each R is independently selected from -H, (d-C16) alkyl, substituted (d-dό) alkyl, (C2- C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cycloalkyl, aryl or substituted aryl; the alkyl, alkenyl, alkynyl and aryl are optionally substituted with one or more substituents independently selected from the group of -halo, trihalomethyl, -R, -OR', -SR, -NR'R', -NO2, - CN, -OC(O)R, -OC(S)R, -SC(O)R, -SC(S)R -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NR'R', -C(S)NR'R, -NR'C(O)R and -NR'C(O)OR; the cycloalkyl is optionally substituted with one or more substituents independently selected from the group of R', -halo, OR', -SR', -NRR', -ONRR', -NO2, -CN, -C(O)R', -C(S)R', - OC(O)R', -SC(O)R', -SC(S)R', -OC(S)R', -C(O)OR', -C(S)OR', -C(O)SR', -C(S)SR', - C(O)NRR', -C(S)NRR', -C(O)NR'(OR'), -C(S)NR' -(OR'), -C(O)NR'(SR'), -C(S)NR'(SR'), - CH(CN)2, -CH[C(O)R]2, -CH[C(S)R']2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR 2, - CH[C(S)SR'J2, -NR'C(O)R', -NR'C(O)OR', -S(O)-R', -S(O)OR', -S(O)2OR', -S(O)NRR', - S(O)ONRR', and each R' is independently selected from the group of -H, (Cι-Cι6) alkyl, substituted (d-C16) alkyl, (C2-C16) alkenyl, substituted (C2-Cι6) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl.
hi another specific embodiment the substituents of the compounds of formula (I) are as follows: X is F, CI, Br or I;
RI and R2 are each independently selected from the group of H, (Cι-C6) alkyl, (d-C6) hydroxy alkyl, (d-C6) alkoxy, (C3-C7) cycloalkyl, halo substituted (d-C6) alkyl, halo di-substituted (d- C6) alkyl, halo tri-substituted (Cι-C6) alkyl and halo; B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; and substituted with at least one substituent selected from (d-C16) alkyl, (C2-C16) alkenyl, (C2-C16) alkynyl, -O-(d-d6) alkyl, -O-(C2-C16) alkenyl, -O-(C2-C16) alkynyl, aryl, substituted aryl, -O- aryl, -O-CH2-aryl, -S-(C1-C16) alkyl, -S-(C2-C16) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2- aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -ONRR, -C(O)R, -C(S)R
-C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -OC(S)R, -C(O)NRR, -
C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2,
-CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2,
-NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: said alkyl is substituted with at least one substituent selected from the group of halo, -CN, -NO2,
-NR'R', -O-alkyl, -O-alkenyl, -O-alkynyl, -O-aryl, -OC(O)R, -OC(S)R, -C(O)R', -C(S)R*, -
C(O)NR'R and-C(S)NR'R; said alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO2, -NR'R, -OH, -OR', -O-aryl, -OC(O)R, -OC(S)R', -C(O)R,
-C(S)R, -C(O)OR, -C(O)NR'R' and -C(S)NR'R'; said -O-alkenyl, -O-alkynyl, -S-alkenyl, -S-alkynyl, cycloalkyl, -O-cycloalkyl are are each optionally and independently substituted with at least one group selected from halo, -CN, -NO2, -
NR'R, -OH, -OR, -O-aryl, -OC(O)R, -OC(S)R, -C(O)R, -C(S)R, -C(O)OR', -C(O)NR'R and -
C(S)NR'R, and wherein R, R and aryl are as defined above and their substituents are as defined above.
In another embodiment, the substituents of the compounds of formula (I) are as follows:
X is F, C Br or l;
RI and R2 are each independently selected from the group of H, (Cι-C6) alkyl, (d-C6) hydroxy alkyl, (Cι-C6) alkoxy, (C -C7) cycloalkyl, halo substituted (d-C6) alkyl, halo di-substituted (Ci-
C6) alkyl, halo tri-substituted (d-C6) alkyl and halo,
B is phenyl substituted with at least one group selected from (d-C16) alkyl, (C2-C16) alkenyl,
(CVCiβ) alkynyl, -O-(d-C16) alkyl, -O-(C -C16) alkenyl, -O-(C2-C16) alkynyl, -aryl, -O-aryl, -O-
CH2-aryl, -OC(O)R, -C(O)R, -C(O)OR, -C(O)NR'R, -NRC(O)R, -NRC(O)OR, -S(O)-R, -
S(O)OR, -S(O)NRR, -S(O)ONRR; wherein: said alkyl is substituted with at least one group selected from halo, -CN, -O-alkyl, -O-alkenyl,
-O-alkynyl, -O-aryl, -OC(O)R, -C(O)R, and -C(O)NR'R: said alkenyl, alkynyl, -O-alkyl, -O-alkenyl and -O-alkynyl are each independently substituted with at least one group selected from halo, -CN, -OH, -OR', -O-aryl, -OC(O)R', -C(O)R', -
C(O)OR and -C(O)NR'R, and
R and R' are as defined above.
In an another embodiment, the compounds of formula (I) include the compounds of formula (IV):
Figure imgf000025_0001
or a pharmaceutically acceptable salt thereof, wherein:
R3 is selected from the group of H, R, -halo, OR, -SR, -NRR, -ONRR, -NO2, -CN, -C(O)R, -C(S)R,-OC(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; each R is independently selected from -H, (d-C16) alkyl, substituted (d-C1 ) alkyl, (C2- C16) alkenyl, substituted (C2-Cι6) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl; the alkyl, alkenyl, alkynyl and aryl substituents are each independently selected from the group of -halo, trilialomethyl, -R, -OR', -SR, -NR'R', -NO2, -CN, -OC(O)R, -OC(S)R, - SC(O)R, -SC(S)R* -C(O)R, -C(S)R, -C(O)OR', -C(S)OR, -C(O)SR, -C(S)SR', -C(O)NR'R', -C(S)NR'R*, -NR*C(O)R, -NRC(O)OR and aryl; the cycloalkyl substituents are each independently selected from the group of H, R, -halo, OR, -SR, -NRR, -ONRR -NO2, -CN, -C(O)R, -C(S)R, -OC(O)R, -SC(O)R, -SC(S)R, -OC(S)R, - C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), - C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, - CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, - S(O)2OR, -S(O)NRR, -S(O)ONRR', and each R' is independently selected from the group -H, (d-C16) alkyl, substituted (d-C16) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (d-Cg) cyloalkyl, aryl or substituted aryl.
In another embodiment in the compoundof formula (IV), R3 is selected from halo, -CN, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR, -OR, (d-C16) alkyl, (C2-C16) allcenyl, (C -C16) alkynyl, wherein said alkyl, alkenyl and alkynyl are optionally substituted with -CN, -C(O)R, - C(O)OR, -OC(O)R, -C(O)NR'R', -OR', wherein R and R are as defined above.
In an another embodiment, the compounds of formula (I) include the compounds of formula (V):
Figure imgf000026_0001
or a pharmaceutically acceptable salt thereof, wherein:
R3 is selected from the group of H, R, -halo, OR, -SR, -NRR, -ONRR, -NO2, -CN, -C(O)R, -C(S)R,-OC(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; each R is independently selected from -H, (d-C16) alkyl, substituted (d-C16) alkyl, (C2- Cι6) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl; the aryl substituents are each independently selected from the group of -halo, trihalomethyl, -R, -OR', -SR, -NR'R, -NO2, -CN, -C(O)R, -C(S)R, -OC(O)R, -OC(S)R*, -SC(O)R, -SC(S)R, -C(O)OR', -C(S)OR', -C(O)SR', -C(S)SR, -C(O)NR'R, -C(S)NR'R', -NR'C(O)R', -NR*C(O)OR'; the alkyl, allcenyl and alkynyl substituents are each independently selected from the group of -halo, trihalomethyl, -R, -OR', -SR, -NR'R', -NO2, -CN, -OC(O)R, -OC(S)R, -SC(O)R, -SC(S)R -C(O)R, -C(S)R', -C(O)OR', -C(S)OR', -C(O)SR, -C(S)SR, -C(O)NR'R, -C(S)NR'R', -NR'C(O)R, -NR'C(O)OR and aryl; the cycloalkyl substituents are each independently selected from the group of H, R, -halo, OR, -SR, -NRR, -ONRR -NO2, -CN, -C(O)R, -C(S)R, -OC(O)R, -SC(O)R, -SC(S)R, -OC(S)R, - C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), - C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, - CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, - S(O)2OR, -S(O)NRR, -S(O)ONRR', and each R' is independently selected from the group -H, (d-C16) alkyl, substituted (d-C16) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C -C16) alkynyl, substituted (C2-Cι6) alkynyl, (C3-C8) cycloalkyl, substituted (C -C8) cyloalkyl, aryl or substituted aryl.
In another embodiment in the compoundof formula (V), R3 is selected from halo, -CN, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR, -OR, (C Cι6) alkyl, (C2-C16) alkenyl, (C2-C16) alkynyl, wherein said alkyl, alkenyl and alkynyl are optionally substituted with -CN, -C(O)R, - C(O)OR,
-OC(O)R, -C(O)NR'R, -OR', wherein R and R are as defined above.
In an illustrative embodiment, the compounds according to formula (I) include those listed below:
4-iodo-3-(2-chloro-ethyl)-urea; l-(2-Chloro-ethyl)-3-(4-iodo-phenyl)-urea; l-(2-Chloro-ethyl)-3-(4-phenoxy-phenyl)-urea; l-(4-Benzyloxy-phenyl)-3-(2-chloro-ethyl)-urea; l-(2-Chloro-ethyl)-3-[4-(2-methoxy-ethyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-[4-(4-ethoxy-butyl)-phenyl]-urea; l-Biphenyl-4-yl-3-(2-chloro-ethyl)-urea;
1 -(2-Chloro-acetyl)-3 -(4-iodo-phenyl)-urea; l-(2-Chloro-ethyl)-3-[4-(4-fluoro-butyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-[3-(5-hydroxy-pent-l-ynyl)-phenyl]-urea;
Acetic acid 5- {4-[3-(2-chloro-ethyl)-ureido]-phenyl} -pentyl ester; n- {3-[3-(2-Chloro-ethyl)-ureido]-phenyl} -acetamide; n-Butyl-4-(5-chloro- 1 ,3 -dimethyl-2-oxo-pentyl)-benzenesulfonamide;
1 -(2-Chloro-ethyl)-3 - [3 -( 1 -hydroxy-ethyl)-phenyl] -urea; ? l-(2-Bromo-ethyl)-3-(3-iodo-phenyl)-urea;
3-[3-(2-Bromo-ethyl)-ureido]-benzoic acid ethyl ester; l-(4-n-hexyl-phenyl)-3-(2-chloro-l-methyl-ethyl)urea;
1 -(4-iodo-phenyl)-3-(2-bromo- 1 -methyl-ethyl)urea;
(R) 1 -(4-iodo-phenyl)-3-(2-bromo- 1 -methyl-ethyl)urea;
(S) 1 -(4-iodo-ρhenyl)-3-(2-bromo- 1 -methyl-ethyl)urea.
In another illustrative embodiment, the compounds according to formula (I) include those listed below:
1 -(4-tert-butylphenyl)-3 -(2-chloroethyl)urea;
1 -(2-chloroethyl)-3 -(4-cyclohexylphenyl)urea; l-(2-chloroethyl)-3-(4-hepthylphenyl)urea; l-(2-chloroethyl)-3-(4-iodophenyl)urea; l-(2-chloroethyl)-3-(4-phenoxyphenyl)urea; l-(4-benzyloxyphenyl)-3-(2-chloroethyl)urea; l-(biphenyl-4-yl)-3-(2-chloroethyl)urea; l-(2-chloroethyl)-3-(4-hydroxyphenyl)urea;
N-{3-[3-(2-chloroethyl)ureido]phenyl} acetamide;
N-Butyl-3-[3-(2-chloroethyl)ureido]benzenesulfonamide;
1 -(2-chloroethyl)-3-[3-(l -hydroxyethyl)phenyl]urea; l-(2-chloroethyl)3-[4-(2-methoxyethyl)phenyl)urea; l-(2-chloroethyl)-3-[4-(4-ethoxybutyl)phenyl)urea; l-(2-chloroethyl)-3-[4-(4-fluorobutyl)phenyl]urea;
1 -(2-chloroethyl)-3-[3-(5-hydroxypent- 1 -ynyl)phenyl)urea;
1 -(2-chloro ethyl)-3 -[3 -(5 -hydroxypentyl)phenyl)urea;
Acetic acid 5-{4-[3-(2-chloroethyl)ureido]phenyl}pentyl ester;
6-{3-[3-(2-chloroethyl)ureido]phenoxy}hexanoic acid ethyl ester; l-(2-chloroethyl)-2-(2-heptylphenyl)urea;
1 -(2-Chloroacetyl)-3 -(4-iodophenyl)urea; l-(2-chloroacetyl)-3-[3-(5-hydroxypentyl)phenyl]urea;
(R)- 1 -(2-chloro- 1 -methylethyl)-3 -(4-iodophenyl)urea;
(S)- 1 -(2-chloro- 1 -methylethyl)-3 -(4-iodophenyl)urea;
(R)- 1 -(4-tert-Butylphenyl)-3 -(2-chloro- 1 -methylethyl)urea;
(S)- 1 -(4-tert-Butylphenyl)-3-(2-chloro- 1 -methylethyl)urea;
1 -(4-tert-Butylphenyl)-3 -(2-chloro- 1 , 1 -dimethylethyl)urea;
1 -(2-Bromoethyl)-3 -(3 -iodophenyl)urea;
4-tert-Butylphenyl(4,5-dihydrooxazol-2-yl)amine;
hi another illustrative embodiment, the compounds according to formula (I) include those listed below: l-(2-Chloro-ethyl)-3-772-tolyl-urea; l-(2-Chloro-ethyl)-3-(3-ethyl-phenyl)-urea; l-(2-Chloro-ethyl)-3-(3-methoxy-phenyl)-urea; l-(2-Chloro-ethyl)-3-[4-(4-hydroxy-butyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-[4-(3-hydroxy-propyl)-ρhenyl]-urea; l-(2-Chloro-ethyl)-3-(3-iodo-phenyl)-urea; l-(2-Chloro-ethyl)-3-[4-(5-hydroxy-pentyl)-phenyl]-urea;
1 -(2-Chloro-ethyl)-3-[3-(5-hydroxy-pent- 1 -ynyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-[3-(5-hydroxy-pentyl)-phenyl]-urea;
3-[3-(2-Chloro-ethyl)-ureido]-benzoic acid ethyl ester; l-(2-Chloro-ethyl)-3-[3-(5-methoxy-pentyl)-phenyl]-urea; {3-[3-(2-Chloro-ethyl)-ureido]-phenyl} -acetic acid; l-(2-Bromo-ethyl)-3-[3-(5-hydroxy-pentyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-(3-heρtyl-ρhenyl)-urea; l-(2-Chloro-ethyl)-3-[3-(6-hydroxy-hexyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-[3-(4-hydroxy-butyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-[3-(3-hydroxy-propyl)-phenyl]-urea;
5-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-pentanoic acid amide; l-(3-Bromo-phenyl)-3-(2-chloro-ethyl)-urea; l-(2-Chloro-ethyl)-3-(3-chloro-phenyl)-urea;
1 -(2-Chloro-ethyl)-3 -(3 -hydroxy-phenyl)-urea;
Acetic acid 3-[3-(2-chloro-ethyl)-ureido]-phenyl ester; l-(2-Chloro-ethyl)-3-(3-hydroxymethyl-phenyl)-urea;
Acetic acid 5- {3-[3-(2-chloro-ethyl)-ureido]-phenyl} -pentyl ester;
Acetic acid 4-{3-[3-(2-chloro-ethyl)-ureido]-phenyl}-butyl ester;
Acetic acid 3-[3-(2-chloro-ethyl)-ureido]-benzyl ester;
Acetic acid 3- {3-[3-(2-chloro-ethyl)-ureido]-phenyl} -propyl ester; l-(2-Chloro-ethyl)-3-[3-(2-hydroxy-ethyl)-phenyl]-urea;
Acetic acid 2-{3-[3-(2-chloro-ethyl)-ureido]-phenyl}-ethyl ester;
Acetic acid 6-{3-[3-(2-chloro-ethyl)-ureido]-phenyl}-hexyl ester;
Pentanedioic acid mono-{3-[3-(2-chloro-ethyl)-ureido]-phenyl} ester; l-(2-Chloro-ethyl)-3-[3-(7-hydroxy-heptyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-(3-cyano-phenyl)-urea;
3-[3-(2-Chloro-ethyl)-ureido]-benzoic acid; l-(2-Chloro-ethyl)-3-[3-(3-methoxy-propyl)-phenyl]-urea; l-(3-pentyl-phenyl)-3-(2-chloro-ethyl)-urea;
5-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-pentanoic acid ethyl ester;
5- {3-[3-(2-Chloro-ethyl)-ureido]-phenyl} -pentanoic acid;
5-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-pentanoic acid methyl ester;
1 -(2-Chloro-ethyl)-3 -(3 -hexyl-phenyl)-urea; l-(2-Chloro-ethyl)-3-(3-hexyl-phenyl)-urea;
6-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-hexanoic acid ethyl ester; 6- {3 - [3 -(2-Chloro-ethyl)-ureido] -phenyl} -hexanoic acid; 6-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-hexanoic acid methyl ester; 3-[3-(2-Chloro-ethyι)-ureido]-benzoic acid methyl ester; l-(2-Chloro-ethyl)-3-[3-(4-hydroxy-but-l-ynyl)-ρhenyl]-urea; 1 -(2-Chloro-ethyl)-3 -[3 -(3-hydroxy-prop- 1 -ynyl)-phenyl] -urea; l-(2-Chloro-ethyl)-3-(3-cyanomethyl-phenyl)-urea; 2-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-acetamide; 3-[3-(2-Chloro-ethyl)-ureido]-phenyl} -acetic acid ethyl ester; Acetic acid 3-{3-[3-(2-chloro-ethyl)-ureido]-phenyl}-propyl ester.
In another illustrative embodiment, the compounds according to formula (I) include those listed below:
4-iodo-l-[3-(2-chloroethyl)ureido]benzene;
4-tert-butyl- 1 -[3-(2-chloroethyl)ureido]benzene;
4-sec-butyl- 1 -[3-(2-chloroethyl)ureido]benzene;
4-isoproρyl- 1 -[3-(2-chloroethyl)ureido]benzene;
4-(p-[3 -(2-chloroethyl)ureido] phenyl)butanol;
4-pentyl-l-[3-(2-chloroethyl)ureido] benzene;
4-hexyl- 1 -[3-(2-chloroethyl)ureido] benzene;
4-cyclohexyl- 1 -[3-(2-chloroethyl)ureido] benzene;
4-heptyl- 1 -[3-(2-chloroethyl)ureido] benzene;
4-octyl- 1 -[3-(2-chloroethyl)ureido) benzene;
4-decyl- 1 -[3-(2-chloroethyl)ureido) benzene;
4-dodecyl-l-[3-(2-chloroethyl)ureido] benzene;
3-ethoxy-l-[3-(2-chloroethyl)ureido] benzene;
4-pentoxy-l-[3-(2-chloroethyl)ureido] benzene;
4-hexyloxy- 1 -[3-(2-chloroethyl)ureido] benzene;
2,4,6-trimethyl-l-[3-(2-chloroethyl)ureido] benzene;
2-ethyl-l-[3-(2-chloroethyl)ureido] benzene;
2,4-diethyl-l-[3-(2-chloroethyl)ureido] benzene;
2-propyl-l-[3-(2-chloroethyl)ureido] benzene; 2,6-diisopropyl-l-[3-(2-chloroethyl)ureido] benzene;
2-isopropyl-6-methyl-l-[3-(2-chloroethyl)ureido] benzene;
2,5-ditert butyl-l-[3-(2-chloroethyl)ureido] benzene;
2-methoxy-5-methyl-l-[3-(2-chloroethyl)ureido] benzene;
2-methoxy-6-methyl-l-[3-(2-chloroethyl)ureido] benzene;
2-methyl-5-methoxy- 1 -[3-(2-chloroethyl)ureido] benzene;
2-methyl-4-methoxy- 1 -[3-(2-chloroethyl)ureido] benzene;
2-ethoxy- 1 -[3-(2-chloroethyl)ureido] benzene;
4-ethoxy- 1 -(3 -(2-chloroethyl)ureido] benzene;
4-butoxy- 1 -(3-(2-chloroethyl)ureido] benzene;
1 ,4-di[3-(2-chloroethyl)ureido] benzene;
2-cyano-l -[3-(2-chloroethyl)ureido] benzene;
4-(3-hydroxypropyl)- 1 -[3-(2-chloroethyl)ureido] benzene;
4-(2-hydroxybutyl)-l-[3-(2-chloroethyl)ureido] benzene;
4-(4-(2-butyrate ethanoic acid))-l-[3-(2-chloroethyl)ureido] benzene;
4-(3-(2-propylate ethanoic acid))-l-[3-(2-chloroethyl)ureido] benzene;
4-ethylthio- 1 -[3-(2-chloroethyl)ureido] benzene;
2- [3 -(2-chloroethyl)ureido ] fluorene;
5-[3-(2-chloroethyl)ureido] indane;
6-[3-(2-chloroethyl)ureido] indazole;
5-[3-(2-chloroethyl)ureido] 4,6-dimethyl pyridine;
5-[3-(2-chloroethyl)ureido] indole; l-(4-tert-Butyl-phenyl)-3-(2-chloro-ethyl)-urea; l-(2-Chloro-ethyl)-3-(4-cyclohexyl-phenyl)-urea; l-(2-Chloro-ethyl)-3-(4-heptyl-phenyl)-urea; l-(2-Chloro-ethyl)-3-[3-(5-hydroxy-pentyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-(4-methoxy-phenyl)-urea; l-(2-Chloro-ethyl)-3-(4-iodo-phenyl)-urea; l-(2-Chloro-ethyl)-3-(4-phenoxy-phenyl)-urea;
1 -(4-Benzyloxy-phenyl)-3 -(2-chloro-ethyl)-urea; l-(2-Chloro-ethyl)-3-[4-(2-methoxy-ethyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-[4-(4-ethoxy-butyl)-phenyl]-urea; l-Biphenyl-4-yl-3-(2-chloro-ethyl)-urea; l-(2-Chloro-acetyl)-3-(4-iodo-phenyl)-urea; l-(2-Chloro-ethyl)-3-[4-(4-fluoro-butyl)-phenyl]-urea;
1 -(2-Chloro-ethyl)-3-[3-(5-hydroxy-pent- 1 -ynyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-(4-hydroxy-phenyl)-urea;
Acetic acid 5-{4-[3-(2-chloro-ethyl)-ureido]-phenyl}-pentyl ester;
N-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-acetamide;
N-Butyl-4-(5-chloro-l,3-dimethyl-2-oxo-pentyl)-benzenesulfonamide; l-(2-Chloro-ethyl)-3-[3-(l-hydroxy-etlιyl)-phenyl]-urea; l-(2-Chloro-ethyl)-3-[3-(l-hydroxy-ethyl)-phenyl]-urea;
1 -(2-Chloro-ethyl)-3 -(2-heptyl-phenyl)-urea; l-(2-Chloro-acetyl)-3-[3-(5-hydroxy-pentyl)-phenyl]-urea;
R- 1 -(4-tert-Butyl-phenyl)-3-(2-chloro- 1 -methyl-ethyl)-urea;
S-1 -(4-tert-Butyl-phenyl)-3-(2-chloro- 1 -methyl-ethyl)-urea;
1 -(4-tert-Butyl-phenyl)-3 -(2-chloro- 1 , 1 -dimethyl-ethyl)-urea;
1 -(2-Bromo-ethyl)-3 -(3 -iodo-phenyl)-urea;
3-[3-(2-Bromo-ethyl)-ureido]-benzoic acid ethyl ester.
Compounds of Formula I wherein X is Br or I may undergo rearrangement to provide a rearrangement product. Such rearrangement products are considered to be within the scope of the present invention. Thus, the present invention contemplates the compounds of Fonnula I wherein X is Br or I as a form of pro-drugs, for which both the pro-drug form and the rearrangement product may have activity in inhibiting proliferation of cancer cells.
As noted supra, this invention includes the pharmaceutically acceptable salts of the compounds defined by Formula I, II, and III. A compound of this invention can possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of organic and inorganic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" as used herein, refers to salts of the compounds of the above formula which are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts.
Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as -toluenesulfonic acid, methanesulfonic acid, oxalic acid, 7-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such pharmaceutically acceptable salts are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, napththalene-2-sulfonate, mandelate and the like.
hi one embodiment of the invention, the pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid and methanesulfonic acid.
Salts of amine groups may also comprise quarternary ammonium salts wherein the amino nitrogen carries a suitable organic group such as an alkyl, alkenyl, alkynyl, or aralkyl moiety.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like, hi one embodiment, the base addition salt is a potassium or sodium salt.
It should be recognized that the particular counterion forming a part of any salt of this invention is usually not of a critical nature, as long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.
This invention further encompasses the pharmaceutically acceptable solvates of the compounds of Formula I, II or III. Many of these compounds can combine with solvents such as water, methanol, ethanol and acetonitrile to form pharmaceutically acceptable solvates such as the corresponding hydrate, methanolate, ethanolate and acetonitrilate.
The compounds of the present invention have multiple asymmetric (chiral) centers. As a consequence of these chiral centers, the compounds of the present invention occur as racemates, mixtures of enantiomers and as individual enantiomers, as well as diastereomers and mixtures of diastereomers. All asymmetric forms, individual isomers and combinations thereof, are within the scope of the present invention.
The prefixes "R" and "S" are used herein as commonly used in organic chemistry to denote the absolute configuration of a chiral center, according to the Cahn-higold-Prelog system. The stereochemical descriptor R (rectus) refers to that configuration of a chiral center with a clockwise relationship of groups tracing the path from highest to second-lowest priorities when viewed from the side opposite to that of the lowest priority group. The stereochemical descriptor S (sinister) refers to that configuration of a chiral center with a counterclockwise relationship of groups tracing the path from highest to second-lowest priority when viewed from the side opposite to the lowest priority group. The priority of groups is decided using sequence rules as described by Cahn et al, Angew. Chem., 78, 413-447, 1966 and Prelog, V. and Helmchen, G.; Angew. Chem. Int. Ed. Eng, 21, 567-583, 1982). In addition to the R,S system used to designate the absolute configuration of a chiral center, the older D-L system is also used in this document to denote relative configuration, especially with reference to amino acids and amino acid derivatives. In this system a Fischer projection of the compound is oriented so that carbon-1 of the parent chain is at the top. The prefix "D" is used to represent the relative configuration of the isomer in which the functional (determining) group is on the right side of the carbon atom at the chiral center and "L", that of the isomer in which it is on the left.
As would be expected, the stereochemistry of the Formula I, II and III compounds may be critical to their potency as agonists or antagonists. The relative stereochemistry is preferably established early during synthesis, which avoids stereoisomer separation problems later in the process. Subsequent synthetic steps then employ stereospecific procedures so as to maintain the preferred configuration.
Non-toxic metabolically labile esters and amides of compounds of Formula I, II or III are ester or amide derivatives that are hydrolyzed in vivo to afford said compounds of Formula I, II or III and a pharmaceutically acceptable alcohol or amine. Examples of metabolically labile esters include esters formed with (Cι-C6) alkanols in which the alkanol moiety may be optionally substituted by a (d-C16) alkoxy group, for example, methanol, ethanol, propanol and methoxyethanol. Examples of metabolically labile amides include amides formed with amines such as methylamine.
Therapeutic Uses of Compounds of Formula I
The compounds of Formula I can be used for attenuating, inhibiting or preventing cancer cell migration in a mammal in need of such therapy. In one embodiment of the invention, the compounds of the invention are used to attenuate, inhibit or prevent cancer cell migration. The compounds can be used alone or they can be used as part of a multi-drug regimen in combination with known therapeutics.
The migration of cancer cells from a wide variety of cancers may be attenuated, inhibited or prevented by use of the compounds of the invention. Examples of cancers include, but are not limited to leukaemia, carcinomas, adenocarcinomas, melanomas and sarcomas. Carcinomas, adenocarcinomas and sarcomas are also frequently referred to as "solid tumours," examples of commonly occurring solid tumours include, but are not limited to, cancer of the brain, breast, cervix, colon, head and neck, kidney, lung, ovary, pancreas, prostate, stomach and uterus, non- small cell lung cancer and colorectal cancer.
The term "leukaemia" refers broadly to progressive, malignant diseases of the blood-forming organs. Leukaemia is typically characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow but can also refer to malignant diseases of other blood cells such as erythroleukaemia, which affects immature red blood cells. Leukaemia is generally clinically classified on the basis of (1) the duration and character of the disease - acute or chronic; (2) the type of cell involved - myeloid (myelogenous), lymphoid (lymphogenous) or monocytic, and (3) the increase or non-increase in the number of abnormal cells in the blood - leukaemic or aleukaemic (subleukaemic). Leukaemia includes, for example, acute nonlymphocytic leukaemia, chronic lymphocytic leukaemia, acute granulocytic leukaemia, chronic granulocytic leukaemia, acute promyelocytic leukaemia, adult T-cell leukaemia, aleukaemic leukaemia, aleukocythemic leukaemia, basophylic leukaemia, blast cell leukaemia, bovine leukaemia, chronic myelocytic leukaemia, leukaemia cutis, embryonal leukaemia, eosinophilic leukaemia, Gross' leukaemia, hairy-cell leukaemia, hemoblastic leukaemia, hemocytoblastic leukaemia, histiocytic leukaemia, stem cell leukaemia, acute monocytic leukaemia, leukopenic leukaemia, lymphatic leukaemia, lymphoblastic leukaemia, lymphocytic leukaemia, lymphogenous leukaemia, lymphoid leukaemia, lymphosarcoma cell leukaemia, mast cell leukaemia, megakaryocytic leukaemia, micromyeloblastic leukaemia, monocytic leukaemia, myeloblastic leukaemia, myelocytic leukaemia, myeloid granulocytic leukaemia, myelomonocytic leukaemia, Naegeli leukaemia, plasma cell leukaemia, plasmacytic leukaemia, promyelocytic leukaemia, Rieder cell leukaemia, Schilling's leukaemia, stem cell leukaemia, subleukaemic leukaemia, and undifferentiated cell leukaemia.
The term "sarcoma" generally refers to a tumour which originates in connective tissue, such as muscle, bone, cartilage or fat, and is made up of a substance like embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas include soft tissue sarcomas, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumour sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented haemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.
The term "melanoma" is taken to mean a tumour arising from the melanocytic system of the skin and other organs. Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.
The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colorectal carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, haematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, non-small cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.
The term "carcinoma" also encompasses adenocarcinomas. Adenocarcinomas are carcinomas that originate in cells that make organs which have glandular (secretory) properties or that originate in cells that line hollow viscera, such as the gastrointestinal tract or bronchial epithelia. Examples include, but are not limited to, adenocarcinomas of the breast, lung, pancreas and prostate.
Additional cancers encompassed by the present invention include, for example, Hodgkin's Disease, Non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumours, primary brain tumours, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, gliomas, testicular cancer, thyroid cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, mesothelioma and medulloblastoma.
The compounds of Formula I can be used for attenuating, inhibiting or preventing cancer cell proliferation in a mammal in need of such therapy. In one embodiment of the invention, the novel compounds described by Formula I of the invention are used to attenuate, inhibit or prevent cancer cell proliferation. The compounds can be used alone or they can be used as part of a multi-drug regimen in combination with known therapeutics.
Efficacy of the Therapeutic Compounds
In accordance with the present invention, the therapeutic compounds of Formula I are capable of attenuating, inhibiting, or preventing cancer cell migration in vivo. One skilled in the art will appreciate that compounds within Formula I will demonstrate different activities in their ability to attenuate, inhibit, or prevent cancer cell migration and to treat the diseases associated with such migration. The ability of the compounds to attenuate, inhibit, or prevent cancer cell migration can be initially determined in vitro if desired. The present invention thus contemplates a preliminary in vitro screening step to further characterize compounds suitable for incorporation into the therapeutic compositions. A number of standard tests to determine the ability of a compound to attenuate, inhibit, or prevent cancer cell migration are known in the art and can be employed to test the compounds of Formula I. Exemplary procedures are described herein.
Functional Assays
Candidate compounds of Fonnula I can be tested in vitro and in vivo to determine their activity in inhibiting cancer cell migration and metastasis formation.
1. In vitro Testing
Inhibition of Cell Migration Assays
In general, the ability of a compound to inhibit migration of neopalstic cells can be assessed in vitro using standard cell migration assays. Typically, such assays are conducted in multi-well plates, the wells of the plate being separated by a suitable membrane into top and bottom sections. The membrane can be coated with an appropriate compound, the selection of which is dependent on the type of cell being assessed and can be readily determined by one skilled in the art. Examples include collagen, gelatine or Matrigel. An appropriate chemo-attractant, such as soluble fibronectin, EGM-2, IL-8, α-FGF, β-FGF and the like, is added to the bottom chamber as a chemo-attractant An aliquot of the test cells together with the test compound is added to the upper chamber, typically various dilutions of the test compound are tested. After a suitable incubation time, the membrane is rinsed, fixed and stained. The cells on the upper side of the membrane are wiped off, and then randomly selected fields on the bottom side are counted.
Neoplastic cell migration can also be assessed in vitro using the wound healing assay described by Alper et al. (J Natl Cancer hist. 2001 93(18):1375-84) and Tamura et al. (Science 1998;280:1614-7). Briefly, a wound is created in a cell monolayer by the gentle removal of the attached cells. The migration of the cells into the wound is then observed at different time points and in the absence or presence of a test compound to determine the effect of the test compound on cell migration.
One skilled in the art will appreciate that it may be desirable to determine the ability of the compositions to inhibit cell migration of certain specific cancer cell lines, for example drug- resistant or highly metastatic cell lines and that appropriate cell lines can be selected accordingly.
A variety of cancer cell-lines suitable for testing the candidate compounds are known in the art. Suitable neoplastic cell lines are available from the American Type Culture Collection (ATCC), which currently provides 950 cancer cell lines, and other commercial sources, hi one embodiment of the present invention, in vitro testing of the candidate compounds is conducted in a human cancer cell-line. Examples of suitable human cancer cell-lines for in vitro testing of the compounds of the present invention include, but are not limited to, breast cancer cell-lines MCF- 7, t47D and MDA-MB-23, colon cancer cell-lines CaCo and LoVo, ovarian cancer cell-line SKOV3, prostate cancer cell-line DU-145, chronic myeloid leukaemia cell-line K562 and bladder cancer cell-line T24.
Toxicity of the candidate compounds can also be initially assessed in vitro using standard techniques. For example, human primary fibroblasts can be treated in vitro with a compound of Formula I. Cells are then tested at different time points following treatment for their viability using a standard viability assay, such as the assays described above or the trypan-blue exclusion assay. Cells can also be assayed for their ability to synthesize DNA, for example, using a thymidine incorporation assay, and for changes in cell cycle dynamics, for example, using a standard cell sorting assay in conjunction with a fluorocytometer cell sorter (FACS).
II. In vivo Testing
The ability of the candidate compounds to inhibit tumour growth or metastasis in vivo can be detennined in an appropriate animal model using standard techniques known in the art (see, for example, Enna, et al, Current Protocols in Pharmacology, J. Wiley & Sons, hie, New York, NY).
In general, current animal models for screening anti-tumour compounds are xenograft models, in which a human tumour has been implanted into an animal. Examples of xenograft models of human cancer include, but are not limited to, human solid tumour xenografts in mice, implanted by sub-cutaneous injection and used in tumour growth assays; human solid tumour isografts in mice, implanted by fat pad injection and used in tumour growth assays; experimental models of lymphoma and leukaemia in mice, used in survival assays, and experimental models of lung metastasis in mice. Non-limiting examples of human cancer cell lines that can be used in these assays are provided in Table 1.
For example, the candidate compounds can be tested in vivo on solid tumours using mice that are subcutaneously grafted bilaterally with 30 to 60 mg of a tumour fragment, or implanted with an appropriate number of cancer cells, on day 0. Subcutaneous xenografts metastasize infrequently and seldom invade adjacent tissue, therefore, rate of tumour growth or delay of significant tumour growth are the endpoints used in this model. The animals bearing tumours are mixed before being subjected to the various treatments and controls. In the case of treatment of advanced tumours, tumours are allowed to develop to the desired size, animals having insufficiently developed tumours being eliminated. The selected animals are distributed at random to undergo the treatments and controls. Suitable controls will be dependent on the actual compound being tested and may include untreated animals. Animals not bearing tumours may also be subjected to the same treatments as the tumour-bearing animals in order to be able to dissociate the toxic effect from the specific effect on the tumour. Experiments to test the efficacy of various compounds can readily be designed by a skilled technician. Chemotherapy generally begins from 1 to 22 days after grafting, depending on the type of tumour, and the animals are observed every day. Alternatively, to evaluate the preventative properties of the compounds, the compounds can be administered prior to tumour implantation, for example, about 7 days prior. The compounds of the present invention can be administered to the ammals, for example, orally, by i.p. injection or bolus infusion. The different animal groups are weighed about 3 or 4 times a week until the maximum weight loss is attained, after which the groups are weighed at least once a week until the end of the trial.
The tumours are measured about 2 or 3 times a week until the tumour reaches a pre-determined size and / or weight, or until the animal dies if this occurs before the tumour reaches the predetermined size / weight. The animals are then sacrificed and the tissue histology, size and / or proliferation of the tumour assessed.
Orthotopic xenograft models are an alternative to subcutaneous models and may more accurately reflect the cancer development process. In this model, tumour cells are implanted at the site of the organ of origin and develop internally. Daily evaluation of the size of the tumours is thus more difficult than in a subcutaneous model. A recently developed technique using green fluorescent protein (GFP) expressing tumours in non-invasive whole-body imaging can help to address this issue (Yang and al, Proc. Nat. Aca. Sci, (2000), pp 1206-1211). This technique utilises human or murine tumours that stably express very high levels of the Aqueora vitoria green fluorescent protein. The GFP expressing tumours can be visualised by means of externally placed video detectors, allowing for monitoring of details of tumour growth, angiogenesis and metastatic spread. The use of this model thus allows for simultaneous monitoring od several features associated with tumour progression and has high preclinical and clinical relevance.
In addition, the ability of the candidate compound to inhibit formation of a solid tumour can also be assessed in the chick chorioallantoic membrane (CAM) assay using published protocols (Brooks et al, in Methods in Molecular Biology, Vol. 129, pp. 257-269 (2000), ed. A.R. Howlett, Humana Press Inc., Totowa, NJ). For the study of the effect of the candidate compounds on leukaemias, the animals are grafted with a particular number of cells, and the anti-tumour activity is detennined by the increase in the survival time of the treated mice relative to the controls. To study the effect of the compositions of the present invention on tumour metastasis, various models of experimental metastasis known in the art can be employed. Typically, this involves the treatment of neoplastic cells with the compound ex vivo and subsequent injection or implantation of the cells into a suitable test animal. Alternatively, the animals are treated before or after injection or implantation of the neoplastic cells into the animal. The spread of the neoplastic cells from the site of injection, for example spread to the lungs and/or lymphoid nodes, is then monitored over a suitable period of time by standard techniques.
An alternative in vivo model of metastasis utilises highly metastatic, chemotherapy-resistant cultured Lewis lung (LLCl) cells. The cells are administered intravenously to normal non- immune-compromised mice thus allowing for immediate dissemination of cancerous cells. Treatment can be initiated several days before injection of the LLCl cells in order to observe a preventive effect or immediately after injection of the cells in order to observe an attenuating effect. After about 14 days, the mice are sacrificed, the lungs removed and fixed and the number and size of lung tumours determined. The intravenous route of administration for the LLCl cells in this model allows for rapid evaluation of treatments.
An alternate model, LLCl cells are injected subcutaneously to allow the growth of a primary tumour, which is then surgically removed once a certain size is obtained. Following removal of the primary tumour, treatment is initiated for about 14 days, after which the animals are sacrificed and tumours counted as in the intravenous model. The primary tumour is removed in this model is recommended as it can be metastasis-suppressing.
In vivo toxic effects of the compounds of Formula I can be evaluated by standard techniques, for example, by measuring their effect on animal body weight during treatment and by performing haematological profiles and liver enzyme analysis after the animal has been sacrificed.
Table 1 : Examples of xenograft models of human cancer
Figure imgf000044_0001
Figure imgf000045_0001
Additional Tests
In addition to the above tests, the compounds of the invention can be submitted to other standard tests, such as cytotoxicity tests, stability tests, bioavailability tests and the like. As will be readily apparent to one skilled in the art, the compounds of the invention will need to meet certain criteria in order to be suitable for human use and to meet regulatory requirements. Thus, once a compound of the invention has been found to be suitable for animal administration, standard in vitro and in vivo tests can be conducted to determine information about the metabolism and pharmacokinetic (PK) of the compound which can be used to design human clinical trials. Clinical Trials
One skilled in the art will appreciate that, following the demonstrated effectiveness of the compounds of the present invention in vitro and in animal models (i.e. pre-clinical efficacy), the safety profile of the compounds can be determined in at least two non-human species and then the compounds will progress into Clinical Trials in order to ftirther evaluate their efficacy in attenuating the metastasis of tumours and to obtain regulatory approval for therapeutic use. As is known in the art, clinical trials progress through phases of testing, which are identified as Phases I, II, III, and IV. In vitro and in vivo information about the metabolism and pharmacokinetic (PK) of the compounds determined from pre-clinical studies facilitates the design of initial Phase I and Phase II clinical studies.
Phase I
Phase I clinical trials are normally performed in healthy human volunteers or in advanced cancer patients.These studies are conducted to investigate the safety, tolerability and PK of the compositions and to help design Phase II studies, for example, in terms of appropriate doses, routes of administration, administration protocols. Phase I studies could incorporate pharmacodynamic assays to evaluate proof of principle in inhibition of target inhumans. An adequate pharmacodynamic endpoint would be to determine the inhibitory activity measured from the plasma of healthy volunteers.
Phase II
Phase I studies allow the selection of safe dose levels for Phase II studies. An important factor in the protocol design of the Phase II studies is the adequate recruitment of the patient population to be studied based on stringent selection criteria defining the demographics (age, race and sex) of the study, the previous medical history of the patient, the type of cancer and stage of its development as well as any previous cancer treatment history. The latter factor can be important when the composition is intended as an adjuvant to first line therapy rather than a treatment to refractory disease. A protocol for Phase II studies typically specifies baseline data that can be used to characterise the population, to evaluate the success of randomization in achieving balance of important prognostic factors, and to allow for consideration of adjusted analyses. Staging of the cancers of interest
Staging of the cancer being investigated can be important and, when possible, patients should be recruited such that the cancer stage is as homogeneous as possible across the population to facilitate statistical analysis and interpretation of the data. As is known in the art, methods and criteria for staging of a cancer vary depending on the particular cancer being investigated.
Clinical biomarkers
Selection of a clinical biomarker for evaluation of efficacy and/or prediction of outcome (including toxicity) is important for Phase II studies, often this clinical biomarker can be used as a selection criteria for inclusion of patient in the Phase II studies. Clinical biomarkers can be defined as follows (Atkinson A et al: Clin. Pharmacol. Ther. 69, 89-95 (2001):
Biological marker (biomarker): a characteristic that is objectively measured and evaluated as an indicator of normal biological process, pathogenic process, or pharmacological response to a therapeutic intervention.
Clinical endpoint: a characteristic or variable that reflects how a patient feels or functions, or how long a patient survives.
Surrogate endpoint: biomarker intended to substitute for a clinical endpoint. A clinical investigator uses epidemiological, therapeutic, pathophysiological, or other scientific evidence to select a surrogate endpoint that is expected to predict benefit, harm or the lack of benefit or harm. The FDA defines a surrogate endpoint, or marker, as a laboratory measurement or physical sign that is used in therapeutic trials as a substitute for a clinically meaningful endpoint that is a direct measure of how a patient feels, functions or survive and is expected to predict the effect of the therapy.
Phase III
Phase III trials focus on determining how the compound compares to the standard, or most widely accepted, treatment. In Phase III trials, patients are randomly assigned to one of two or more "arms". In a trial with two arms, for example, one arm will receive the standard treatment (control group) and the other arm will be treated with the compound (investigational group). Phase TV
Phase IV trials can be used to further evaluate the long-term safety and effectiveness of the compound. Phase IV trials are less common than Phase I, II and III trials and would take place after the compound has been approved for standard use.
Preparation of Compounds of Formula I
hi one embodiment of the present invention, compounds of Formula I wherein R\ and R2 are both H are prepared by the general method provided below.
B-NH2 + O=C
Figure imgf000048_0001
VI VII
An amine derivative of Formula IV wherein B is as defined for compound of formula (I) is reacted with an isocyanate of Formula V in anhydrous ether under nitrogen atmosphere at room temperature for 2 to 16 hours or until the disappearance on thin layer chromatography (TLC) of the starting amine of Formula II. The solid residue obtained is filtered and dried in vacuo, the filtrate is evaporated under reduced pressure, and the solid remaining therein is also dried in vacuo. Both solids are independently recrystallized with suitable organic solvents and the final crystals are pooled after evaluation of their purity by chromatography, IR, 1H NMR and MS. Where the crystallization is difficult, purification by column chromatography on silica gel may be required prior to the final crystallization. The compounds of Formula IV are either commercially available or can be prepared with the standard procedures known to a worker skilled in the relevant art.
In another embodiment of the invention, compounds of formula I, wherein B is a substituted phenyl, R\ and/or R2 are other than H and X is CI, are prepared by the general method provided below.
Figure imgf000049_0001
VIII IX a = Boc2O, DMAP, CH2C12; b = (R) or (S) NH2CR2R3CH2OH, CH2C12; c = PPh3, CC14/ CH2C12.
Formation of the urea moiety is achieved using the 4-dimethylaminopyridine-catalyzed reaction of the relevant 4-alkylaniline (VI) with di-tert-butyldicarbonate in dichloromethane followed by the trapping of the in situ generated isocyanate with the appropriate (R)- or (ιS)-2-aminoalcohol (see Knolker, et al, Synlett (1996) 502-504). This procedure ensures a racemization-free synthesis of urea under mild conditions and circumvents side reactions such as the formation of symmetrical disubstituted urea (see Knolker, et al, Synlett (1997) 925-928). Subsequently, chloration of the chiral 2-hydroxyethylureas (VII) is achieved using triphenylphosphine in a mixture of carbon tetrachloride and dichloromethane at room temperature, affording the final enantiomerically pure (R)- or (jS)-(l-alkyl-2-chloro)ethylurea derivative.
Other methods of preparing compounds of Formula I are known and can be readily employed by one skilled in the art to obtain the compounds of the invention. Certain compounds of Formula I are also available commercially. Further exemplary methods of preparing the compounds are provided by the Examples. These methods are provided by means of example only and are not intended to limit the scope of the invention in any way.
Administration of Therapeutic Compounds and Pharamceutical Compositions
The present invention provides methods of treating diseases characterized by neoplastic cell migration in a mammal comprising administering an effective amount of one or more compounds of Formula I, or non-toxic metabolically-labile esters or amides thereof, or pharmaceutically acceptable salts thereof.
The compounds of the present invention are typically formulated prior to administration. The present invention thus provides pharmaceutical compositions comprising one or more compounds of Formula I and a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical compositions are prepared by known procedures using well-known and readily available ingredients. Pharmaceutical compositions comprising one or more compounds of Formula I in combination with one or more known cancer chemotherapeutics are also contemplated by the present invention.
Compounds of the general Formula I or pharmaceutical compositions comprising the compounds may be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles, hi the usual course of therapy, the active compound is incorporated into an acceptable vehicle to form a composition for topical administration to the affected area, such as hydropohobic or hydrophilic creams or lotions, or into a form suitable for oral, rectal or parenteral administration, such as syrups, elixirs, tablets, troches, lozenges, hard or soft capsules, pills, suppositiories, oily or aqueous suspensions, dispersible powders or granules, emulsions, injectables, or solutions. The term parenteral as used herein includes subcutaneous injections, intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal injection or infusion techniques.
Compositions intended for oral use may be prepared in either solid or fluid unit dosage forms. Fluid unit dosage form can be prepared according to procedures known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. An elixir is prepared by using a hydroalcoholic (e.g., ethanol) vehicle with suitable sweeteners such as sugar and saccharin, together with an aromatic flavoring agent. Suspensions can be prepared with an aqueous vehicle with the aid of a suspending agent such as acacia, tragacanth, methylcellulose and the like.
Solid fonnulations such as tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate: granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc and other conventional ingredients such as dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, methylcellulose, and functionally similar materials. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil.
Aqueous suspensions contain active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gxun tragacanth and gum acacia: dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example hepta-decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or fl-propyl- /j-hydroxy benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin.
Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.
Pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions. The oil phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally- occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or a suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution, hi addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Adjuvants such as local anaesthetics, preservatives and buffering agents can also be included in the injectable solution or suspension. The compound(s) of the general Formula I may be administered, together or separately, in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.
For ophthalmic applications, the compounds can be formulated into solutions, suspensions, and ointments appropriate for use in the eye (see, for example, Mitra (ed.), (1993) Ophthalmic Drug Delivery Systems, Marcel Dekker, Inc., New York, N.Y.; Havener, (1983) Ocular Pharmacology, CN. Mosby Co., St. Louis).
Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philidelphia, PA (2000).
Typically in the treatment of cancer, therapeutic compounds are administered systemically to patients, for example, by bolus injection or continuous infusion into a patient's bloodstream. In one embodiment of the present invention, one or more compounds of Formula I are administered systemically to a patient in need of therapy. When used in conjunction with one or more known chemotherapeutic agents, the compounds can be administered prior to, or after, administration of the chemotherapeutic agents, or they can be administered concomitantly. The one or more chemotherapeutic may be administered systemically, for example, by bolus injection or continuous infusion, or it may be administered orally.
The dosage to be administered is not subject to defined limits, but it will usually be an effective amount. It will usually be the equivalent, on a molar basis of the pharmacologically active free form produced from a dosage formulation upon the metabolic release of the active free drug to achieve its desired pharmacological and physiological effects. The compositions may be formulated in a unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Examples of ranges for the compound(s) in each dosage unit are from about 0.05 to about 100 mg, or more usually, from about 1.0 to about 30 mg.
Daily dosages of the compounds of the present invention will typically fall within the range of about 0.01 to about 100 mg/kg of body weight, in single or divided dose. However, it will be understood that the actual amount of the compound(s) to be administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. The above dosage range is given by way of example only and is not intended to limit the scope of the invention in any way. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing harmful side effects, for example, by first dividing the larger dose into several smaller doses for administration throughout the day.
To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way.
Pharmaceutical Kits
The present invention additionally provides for therapeutic kits containing the compounds of the invention for use in the inhibition of cancer cell migration in a mammal in need of such therapy. Individual components of the kit would be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects , approval by the agency of manufacture, use or sale for human administration. When the components of the kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution, for example a sterile aqueous solution. In this case the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the composition may be administered to a patient or applied to and mixed with the other components of the kit.
The components of the kit may also be provided in dried or lyophilised form and the kit can additionally contain a suitable solvent for reconstitution of the lyophilised components. Irrespective of the number or type of containers, the kits of the invention also may comprise an instrument for assisting with the administration of the composition to a patient. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
EXAMPLES
The following abbreviations are used in the Examples: EtOAc, ethyl acetate; THF, tetrahydrofuran; EtOH, ethanol; TLC, thin layer chromatography; GC, gas chromatography; HPLC, high pressure liquid chromatography; m-CPBA, m-chloroperbenzoic acid; Et2O, diethyl ether; DMSO, dimethyl sulfoxide; DBU, l,8-diazabicyclo-[5.4.0]undec-7-ene, MTBE, methyl t-butyl ether; and FDMS, field desorption mass spectrometry.
EXAMPLE 1: Preparation of l-(4-tert-butylphenyl)-3-(2-chloroethyl)urea [1]
+ OC=N-CH2CH2CI *.
Figure imgf000055_0002
Figure imgf000055_0001
2-Chloroethyl isocyanate (1.15 equiv.) was slowly added dropwise to a magnetically stirred and cooled solution (ice bath) of freshly distilled 4-t-butylaniline (1 equiv.) in dichloromethane (18 mL of solvent/g of aniline). The ice bath was then removed and the reaction mixture was stirred at room temperature for 20 h. After completion of the reaction, the solvent was removed by vacuum distillation to give a white solid, which was purified by recrystallization from dichloromethane/hexane to obtain 88% of l-(4-tert-butylphenyl)-3-(2-chloroethyl)urea. NMR 1H (CDCI3, 300 MHz) δ 7.28 (d, 2H, J = 8.5 Hz), 7,17 (d, 2H, J = 8.5 Hz), 3.52 (m, 4H), 1.27 (s, 9H).
EXAMPLE 2: Preparation of l-(2-chloroethyι)-3-(4-cyclohexylphenyl)urea [2]
Figure imgf000056_0001
This compound was prepared according to the process of Example 1, except that 4-cyclohexyl aniline was used instead of -t-butyl aniline. The final product was recrystallized from THF/hexane to obtain 82% yield.
1H NMR (CDCI3 + MeOD, 300 MHz) δ 7.10 (d, 2H, J - 8.4 Hz), 6.98 (d, 2H, J = 8.4 Hz), 3.48 (m, 2H), 3.38 (m, 2H), 2.31 (m, IH), 1.6-1.7 (m, 5H), 1.1-1.3 (m, 5H).
EXAMPLE 3: Preparation of l-(2-chloroethyl)-3-(4-hepthylphenyl)urea [3]
Figure imgf000056_0002
H H
This compound was prepared according to the process of Example 1, except that 4-heptylaniline was used instead of ¥-t-butyl aniline. The final product was recrystallized from THF/hexane to obtain 93% Yield.
1H NMR (CDC13, 300 MHz) δ 7.15 (d, 2H, J= 8.4 Hz), 7.01 (d, 2H, J= 8.4 Hz), 3.53 (t, 2H, J=
5.2 Hz), 3.45 (t, 2H, J= 5.2 Hz), 2.46 (t, 2H, J= 7.7 Hz), 1.49 (m, 2H), 1.21 (m, 8H), 0.80 (t,
3H, J= 6.7 Hz). EXAMPLE 4
Figure imgf000057_0001
EXAMPLE 5: Preparation of l-(2-chloroethyl)-3-(4-iodophenyl)urea [5]
Figure imgf000057_0002
H H
This compound was prepared according to the process of Example 1, except that 4-iodoaniline was used instead of -t-butyl aniline. The final product was recrystallized from THF/hexane. Yield 60%
1H NMR (DMSO, 300 MHz) δ 8.80 (s, IH), 7.53 (d, 2H, J = 8.7 Hz), 7.24 (d, 2H, J = 8.7 Hz), 6.45 (m, IH), 3.64 (m, 2H), 3.39 (m, 2H).
EXAMPLE 6: Preparation of l-(2-chloroethyl)-3-(4-phenoxyphenyl)urea [6]
Figure imgf000057_0003
This compound was prepared according to the process of Example 1, except that 4- phenoxyaniline was used instead of -t-butyl aniline. 1H NMR (CDC13, 300 MHz) δ 7.1-7.3 (m, 5H), 6.96 (m, 4H), 3.63 (m, 2H), 3.58 (m, 2H).
EXAMPLE 7: Preparation of l-(4-benzyloxyphenyl)-3-(2-chloroethyl)urea [7]
Figure imgf000058_0001
This compound was prepared according to the process of Example 1, except that 4- benzyloxyaniline was used instead of ^-t-butyl aniline.
EXAMPLE 8: Preparation of l-(biphenyl-4-yl)-3-(2-chloroethyl)urea [8]
Figure imgf000058_0002
This compound was prepared according to the process of Example 1, except that 4- biphenylamine was used instead of ^-t-butyl aniline. The final product was recrystallized from methanol/water. Yield 79%. 1H NMR (CDC13, 300 MHz) δ 7.44 (m, 4H), 7.2-7.35 (m, 5H), 3.55 (m, 2H), 3.46 (m, 2H).
EXAMPLE 9: Preparation of l-(2-chloroethyl)-3-(4-hydroxyphenyl)urea [9]
Figure imgf000058_0003
H H
This compound was prepared according to the process of Example 1, except that 4- hydroxyaniline was used instead of ^-t-butyl aniline. The final product was recrystallized from
THF/hexane. Yield 30%
1H NMR (CDC13, 300 MHz) δ 7.04 (d, 2H, J= 8.7 Hz), 6.69 (d, 2H, J= 8.7 Hz), 3.53 (m, 2H),
3.44 (t, 2H, J= 5.5 Hz). EXAMPLE 10: Preparation of N-{3-[3-(2-chloroethyl)ureido]phenyl}acetamide [10]
Figure imgf000059_0001
This compound was prepared according to the process of Example 1, except that 3'- aminoacetanilide was used instead of ^-t-butyl aniline. The final product was recrystallized from ethyl acetate/methanol/hexane. Yield 46%.
1H NMR (DMSO, 300 MHz) δ 9.86 (br s, IH), 8.67 (br s, IH), 7.66 (br s, IH), 7.12 (s, 4H), 6.35 (t, IH, J= 5.7 Hz), 3.65 (t, 2H, J= 6.1 Hz), 3.41 (t, 2H, J= 6.1 Hz), 2.02 (s, 3H).
EXAMPLE 11: Preparation of N-Butyl-3-[3-(2- chloroethyl)ureido]benzenesulfonamide [11]
Figure imgf000059_0002
This compound was prepared according to the process of Example 1, except that 3-amino-N- butylbenzenesulfonmide was used instead of ^-t-butyl aniline. The final product was recrystallized from from ethanol/water. Yield 50%.
1H NMR (DMSO, 300 MHz) δ 9.02 (s, IH), 7.98 (s, IH), 7.50 (m, 2H), 7.43 (t, IH, J= 7.9 Hz), 7.30 (d, IH, J= 7.9 Hz), 6.48 (t, IH, J= 5.7 Hz), 3.67 (t, 2H, J= 6.0 Hz), 3.43 (q, 2H, J= 6.0 Hz), 2.72 (q, 2H, J= 6.5 Hz), 1.34 (m, 2H), 1.24 (m, 2H), 0.79 (t, 3H, J= 7.1 Hz).
EXAMPLE 12: Preparation of l-(2-chloroethyl)-3-[3-(l-hydroxyethyl)phenyl]urea [12]
Figure imgf000059_0003
This compound was prepared according to the process of Example 1, except that 3-(l- hydroxyethyl) aniline was used instead of ^-t-butyl aniline. The final product was purified by flash chromatography on silica gel (3/2 ethyl acetate/chloroform). Yield 63% 1H NMR (CDC13, 300 MHz) δ 7.1-7.3 (m, 4H), 4.85 (q, IH, J = 6.6 Hz), 3.63 (m, 2H), 3.58 (m, 2H), 1.46 (d, 3H, J = 6.6 Hz).
EXAMPLE 13: Preparation of l-(2-chloroethyl)3-[4-(2-methoxyethyI)phenyl)urea [13]
Figure imgf000060_0001
To a cold (ice bath) suspension of NaH 60% (483 mg, 12.1 mmol) in dry THF (20 mL) was added a solution of 2-(4-nitrophenyl)ethanol (1.04 g, 6.25 mmol) in dry THF (5 mL). The mixture was stirred at 0 °C for 10 minutes, then methyl iodide (0.50 mL, 8.0 mmol) was added. The ice bath was removed and the solution was stirred at room temperature for 24 h. Excess of NaH was quenched carefully with water and the solution was poured into brine. The aqueous phase was extracted with ether (3 times). The organic portions were washed with brine, dried over MgSO4 and concentrated to give l-(2-methoxyethyl)-4-nitrobenzene which was purified by flash chromatography on silica gel (ether/petroleum ether 1/1).
l-(2-Methoxyethyl)-4-nitrobenzene was dissolved in a mixture of ethanol (5 mL) and water (0.5 mL) and cone. HCl (0.25 mL). Iron powder was added (140 mg) and the mixture was refluxed for 2 hours. The solid was removed by filtration on Celite. The solution was neutralized with NaOH IM (to pH 8) and extracted with ethyl acetate (3 times). The organic portions were reunited, washed with brine, dried over K2CO3 and concentrated to yield 4-(2- methoxyethyl)aniline which was purified by flash chromatography (CH2C12). 4-(2-methoxyethyl)aniline was then reacted with 2-Chloroethyl isocyanate as described in Example 1 to prepare compound 13. The final product was purified by flash chromatography on silica gel (ether/petroleum ether 13/7). Yield 84%.
1H NMR (CDCI3, 300 MHz) δ 7.16 (d, 2H, J = 8.5 Hz), 7.11 (d, 2H, J = 8.5 Hz), 3.5-3.6 (m, 6H), 3.34 (s, 3H), 2.81 (t, 2H, J= 6.9 Hz).
EXAMPLE 14: Preparation of l-(2-chloroethyl)-3-[4-(4-ethoxybutyl)phenyl)urea [14]
Figure imgf000061_0001
To a suspension of powdered potassium hydroxide (2.42 g, 43.1 mmol) in DMSO (20 mL) was added a solution of 4-(4-nitrophenyl)butanol (2.09 mg, 10.7 mmol) and ethyl iodide (3.4 mL, 42 mmol) in DMSO (10 mL) over a 1 h period while the temperature was maintained below 25 °C. The resulting mixture was stirred for an additional hour, poured into water (150 mL), and extracted with methylene chloride (3 times). The combined organic extracts were washed with 10% sodium bisulfite (twice), water and brine, dried over MgSO4, and concentrated in vacuo to yield l-(4-ethoxybutyl)-4-nitrobenzene (838 mg, 35%).
A mixture of l-(4-ethoxybutyl)-4-nitrobenzene (838 mg, 3.75 mmol), iron powder (1.58 g), and coned HCl (0.05 mL) in of a mixture of EtOH, CH3COOH and H2O (2:2:1, 25 mL) was refluxed for 4h. The solution was filtered, diluted with H20 (100 mL), and extracted with CH C12 (3 x 50 mL). The combined organic layers were washed with a saturated aqueous solution of NaHCO , water, and dried over K2CO . Evaporation of the solvent afforded 4-(4-ethoxybutyl)aniline which was used without further purification to prepare compound 14 by reacting with 2- Chloroethyl isocyanate as described for compound 1 to 13. The final product was purified by flash chromatography on silica gel (dichloromethane/ether 9/1). Yield 82%.
1H NMR (CDCI3, 300 MHz) δ 7.14 (d, 2H, J= 8.4 Hz), 7.03 (d, 2H, J = 8.4 Hz), 3.3-3.5 (m, 8H), 2.53 (t, 2H, J= 6.8 Hz), 1.60 (m, 4H), 1.18 (t, 3H, J= 6.9 Hz).
EXAMPLE 15: Preparation of l-(2-chloroethyl)-3-[4-(4-fluorobutyl)phenyl]urea [15]
Figure imgf000062_0001
To a cold solution (-12 °C) of 4-(4-nitrophenyl) butanol (788 mg, 4.04 mmol) in dry CH2C12 (8 mL) was slowly added, under nitrogen, bis(2-methoxyethyl)aminosulfur trifluoride (0.8 mL, 4.3 mmol). The reaction was kept at -12 °C for 15 min and warmed up to room temperature. The mixture was stirred for 24 h. The solution was poured into saturated aqueous NaHCO3 (25 mL), and after CO evolution ceased it was extracted three times with dichloromethane. The organic portions were reunited and washed with brine, dried over Na2SO4, filtered, and evaporated in vacuo. Flash chromatography on silica gel (ether/hexane 1/9) afforded l-(4-fluorobutyl)-4- nitrobenzene (490 mg, 62%).
To a solution of l-(4-fluorobutyl)-4-nitrobenzene (70 mg, 0.36 mmol) in ethanol (2 mL) was added SnC|2'2 H2O (409 mg, 1.81 mmol) and the mixture was refluxed for 2 hours. The solution was cooled and poured into chilled water. The aqueous phase was alkalized by adding a solution of NaHCO3 and extracted with ethyl acetate (3 times). The organic portions were reunited, washed with brine, dried over K2CO3 and evaporated in vacuo to afford 4-(4-fluorobutyl)aniline which was purified by flash chromatography (CHCI3) (53 mg, 88%).
4-(4-fluorobutyl)aniline was then reacted with 2-Chloroethyl isocyanate as described in examples 1-12 to obtain compound 15. Purified by flash chromatography on silica gel (dichloromethane/ether 85/15). Yield 74%.
1H NMR (CDCI3, 300 MHz) δ 7.16 (d, 2H, J= 8.3 Hz), 7.07 (d, 2H, J= 8.3 Hz), 4.43 (dt, 2H, J = 47.4, 5.5 Hz), 3.53 (m, 4H), 2.58 (t, 2H, J= 7.0 Hz), 1.65-1.75 (m, 4H). EXAMPLE 16: Preparation of l-(2-chloroethyl)-3-[3-(5-hydroxypent-l-ynyl)phenyl)urea [16]
Figure imgf000063_0001
3-Iodoaniline (5.05 g, 23.0 mmol), K2CO3 (7.97 g, 57.7 mmol), Cul (183 mg, 0.96 mmol), PPh3 (499 mg, 1.90 mmol), and 10% Pd/C (492 mg, 0.46 mmol Pd) were mixed in 1,2- dimethoxyethane (30 mL) and water (30 mL) at 25 °C under a nitrogen atmosphere. The reaction mixture was stirred for 30 minutes and 4-pentyn-l-ol (4.8 mL, 51.6 mmol) was added. The mixture was heated at 80 °C for 16 hours, cooled to room temperature and filtered through Celite. The organic solvents were removed in vacuo, and the aqueous residue acidified with IM HCl. This solution was extracted with ethyl acetate, and the aqueous phase basified with KOH. The water layer was then extracted with ethyl acetate (3X). The organic portions were reunited, washed with brine, dried over Na2SO4 and concentrated in vacuo. 3-(5-hydroxypent-l- ynyl)aniline was used for the next reaction without any purification. (5.62 g), which was then reacted with 2-Chloroethyl isocyanate as described in examples 1-12 to obtain compound 16.
Purified by flash chromatography on silica gel (chloroform/ethyl acetate 1/1). Yield 72%. 1H NMR (CDC13, 300 MHz) δ 7.26 (m, 2H), 7.10 (t, IH, J= 7.9 Hz), 6.94 (d, IH, J= 7.5 Hz), 3.68 (t, 2H, J = 6.3 Hz), 3.55 (m, 2H), 3.47 (m, 2H), 3.31 (br s, 3H), 2.42 (t, 2H, J- 6.9 Hz), 1.75 (quint, 2H, J= 6.6 Hz).
EXAMPLE 17: Preparation of l-(2-chloroethyl)-3-[3-(5-hydroxypentyl)phenyl)urea [17]
Figure imgf000063_0002
The crude 3-(5-hydroxypent-l-ynyl) aniline (5.62 g, 32.0 mmol) (as prepared in example 16) was dissolved in ethanol (40 mL) and placed in a hydrogenation bottle with 10% Pd/C (604 mg). The bottle was filled with 40 psi of hydrogen and shaken for 4 h. The product was filtered through Celite, and the solvent was removed in vacuo. The aniline was purified by vacuum distillation to give 3-(5-hydroxypentyl)aniline as a clear oil (3.49 g, 84% for two steps, from 3- iodoaniline). 3-(5-hydroxypentyi)aniline was then reacted with 2-Chloroethyl isocyanate as described in examples 1-12 to obtain compound 17.
Purified by flash chromatography on silica gel (ethyl acetate/hexane 45/55. Yield 36%.
1H NMR (CDC13, 300 MHz) δ 7.0-7.15 (m, 3H), 6.77 (d, IH, J= 7.2 Hz), 3.45-3.6 (m, 6H), 3.31
(br s, 3H), 2.50 (t, 2H, J= 7.6 Hz), 1.45-1.6 (m, 4H), 1.30 (m, 2H).
EXAMPLE 18: Preparation of Acetic acid 5-{4-[3-(2-chloroethyl)ureido]phenyl}pentyl ester [18]
Figure imgf000064_0001
To a cold (0 °C) solution of 3-(5-hydroxypentyl)aniline (708 mg, 3.95 mmol) (as prepared in example 17) in dry dichloromethane (20 mL) was added, under nitrogen, (Boc)2θ (1.00 g, 4.60 mmol). The solution was allowed to warm to room temperature and stirred for 2 days (the reaction was monitored by TLC). The solution was diluted with ethyl acetate. The organic phase was washed with a 5% solution of citric acid (twice), brine, dried over Na2SO4 and concentrated under vacuum. [3-(5-Hydroxypentyl)phenyl]carbamic acid tert-butyl ester was used without any ftirther purification.
To a cold (0 °C) solution of [3-(5-Hydroxypentyl)phenyl]carbamic acid tert-butyl ester (112 mg, 0.40 mmol) in dry dichloromethane (3 mL) was added triethylamine (0.06 mL, 0.43 mmol) and acetyl chloride (32 μL, 0.45 mmol) under nitrogen. The solution was allowed to warm to room temperature and stirred for 5 hours. The solution was diluted with ethyl acetate. The organic phase was washed successively with a 5% solution of citric acid (3 times), a saturated solution of NaHCO3 (twice), brine, dried over Na2SO4 and evaporated to give acetic acid 5-(3-tert- butoxycarbonylaminophenyl)pentyl ester which was purified by flash chromatography on silica gel (ethyl acetate/hexane 15/85) (88 mg, 68 %).
Acetic acid 5-(3-tert-butoxycarbonylamino-phenyl)-pentyl ester (88 mg, 0.27 mmol) was dissolved in a mixture of trifluoroacetic acid (4.5 mL) and water (0.5 mL) and the solution was stirred for 10 min at room temperature. The solvents were evaporated and the residue was taken up in ethyl acetate. The organic phase was washed with a saturated solution of NaHCO (twice), brine, dried over Na2SO and concentrated in vacuo to give acetic acid 5-(3-aminophenyl)pentyl ester (36 mg, 60%).
Acetic acid 5-(3-aminophenyl)pentyl ester was then reacted with 2-Chloroethyl isocyanate as described in examples 1-12 to obtain compound 18.
Purified by flash cliromatography on silica gel (ethyl acetate/hexane 3/7). Yield 86%
1H NMR (CDC13, 300 MHz) δ 7.19 (d, 2H, J= 8.3 Hz), 7.10 (d, 2H, J= 8.3 Hz), 4.04 (t, 2H, J=
6.7 Hz), 3.60 (m, 2H), 3.55 (m, 2H), 2.56 (t, 2H, J= 7.6 Hz), 2.04 (s, 3H), 1.63 (m, 4H), 1.38 (m,
2H).
EXAMPLE 19: Preparation of 6-{3-[3-(2-chloroethyl)ureido]phenoxy}hexanoic acid ethyl ester [19]
Figure imgf000065_0001
To a solution of 3-nitrophenol (1.72 g, 12.3 mmol) and K2CO3 (3.40 g, 24.6 mmol) in acetone (30 mL) was added dropwise 6-bromohexanoic acid ethyl ester (2.3 mL, 15 mmol). The mixture was refluxed under nitrogen for 48 hours. The solution was diluted with ethyl acetate. The organic phase was successively washed with water, a saturated solution of NaHCO3 (3 times), brine, dried over MgSO4 and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel (hexane/ethyl acetate 9/1) to yield 6-(3- nitrophenoxy)hexanoic acid ethyl ester (3.34 g, 48%).
To a solution of 6-(3-nitrophenoxy)hexanoic acid ethyl ester (442 mg, 1.57 mmol) in ethanol (20 mL) was added SnC|2 • 2 H2O (1.78 g, 7.90 mmol) and the mixture was refluxed for 4 hours. The solution was cooled and poured into chilled water. The aqueous phase was alkalized by adding a solution of IM NaOH and extracted with ethyl acetate (3 times). The organic portions were reunited, washed with brine (twice), dried over Na2SO4 and evaporated under reduced pressure to yield 6-(3-aminophenoxy)hexanoic acid ethyl ester which was used without any further purification (371 mg, 94%).
6-(3-aminophenoxy)hexanoic acid ethyl ester was then reacted with 2-Chloroethyl isocyanate as described in examples 1-12 to obtain compound 19. Purified by flash chromatography (hexane/ethyl acetate 3/2). Yield 85%.
1H NMR (CDCI3, 300 MHz) δ 7.11 (t, IH, J = 8.1 Hz), 6.96 (t, IH, J = 2.1 Hz), 6.77 (dd, IH, J = 8.1, 1.1 Hz), 6.54 (dd, IH, J = 8.1, 2.1 Hz), 4.11 (q, 2H, J = 7.1 Hz), 3.86 (t, 2H, J = 6.4 Hz), 3.54 (m, 4H), 2.30 (t, 2H, J = 7.5 Hz), 1.6-1.8 (m, 4H), 1.4-1.5 (m, 2H), 1.24 (t, 3H, J = 7.1 Hz).
EXAMPLE 20: Preparation of l-(2-chloroethyl)-2-(2-heptylphenyl)urea [20]
Figure imgf000066_0001
2-Iodoaniline (1.31 g, 5.97 mmol), K2CO3 (2.07 g, 15.0 mmol), Cul (47 mg, 0.25 mmol), PPh3 (134 mg, 0.51 mmol), and 10% Pd/C (125 mg, 0.12 mmol Pd) were mixed in 1,2- dimethoxyethane (15 mL) and water (15 mL) at 25 °C under a nitrogen atmosphere. This was stirred for 30 minutes and hept-1-yne (2.0 mL, 15 mmol) was added. The mixture was heated at 80 °C for 16 hours, cooled to room temperature and filtered through Celite. The organic solvents were removed in vacuo, and the aqueous residue acidified with IM HCl. This solution was extracted with ethyl acetate, and the aqueous phase basified with KOH. The water layer was then extracted with ethyl acetate (3X). The organic portions were reunited, washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (THF/hexane 5/95) to yield 2-(hept-l-ynyl)aniline (909 mg, 81%).
2-(hept-l-ynyl)aniline (628 mg, 3.35 mmol) was dissolved in ethanol (20 mL) and placed in a hydrogenation bottle with 10% Pd/C (88 mg). The bottle was filled with 40 psi of hydrogen and shaken for 4 h. The product was filtered through Celite, and the solvent was removed in vacuo to obtain 2-heptanylaniline. The aniline was used without any further purification (602 mg, %)
2-heptanylaniline was then reacted with 2-Chloroethyl isocyanate as described in examples 1-12 to obtain compound 20. Purified by flash chromatography (ethyl acetate/hexane 3/7). Yield 83%.
1H NMR (CDC13, 300 MHz) δ 7.37 (m, IH), 7.1-7.25 (m, 3H), 6.63 (br s, IH), 5.44 (t, IH, J = 5.5 Hz), 3.58 (m, 2H), 3.49 (m, 2H), 2.57 (t, 2H, J = 7.7 Hz), 1.52 (m, 2H), 1.25-1.55 (m, 8H), 0.86 (t, 3H, J = 6.7 Hz).
EXAMPLE 21: Preparation of l-(2-Chloroacetyl)-3-(4-iodophenyl)urea [21]
Figure imgf000067_0001
2-Chloroacetyl isocyanate (1.15 mL, 13.5 mmol) was slowly added dropwise to a magnetically stirred and cooled solution (ice bath) of freshly recrystallized 4-iodoaniline (2.60 g, 11.8 mmol) in dichloromethane (70 mL). The ice bath was then removed and the reaction mixture was stirred at room temperature for 20 h. After completion of the reaction, the solvent was removed under reduced pressure to give a yellow solid, which was purified by recrystallization (THF/hexane) to give 21 (3.23 g, 81%).
1H NMR (DMSO, 300 MHz) δ 10.93 (br s, IH), 10.15 (br s, IH), 7.66 (d, 2H, J= 8.6 Hz), 7.37 (d, 2H, J= 8.6 Hz), 4.38 (s, 2H).
EXAMPLE 22: Preparation of l-(2-chloroacetyl)-3-[3-(5-hydroxypentyl)phenyl]urea [22]
Figure imgf000068_0001
Prepared as described for compound 21 from 3-(5-hydroxypentyl)aniline (see previous example for its preparation).
Purified by flash chromatography (chloroform/ethyl acetate 1/1). Yield 51%.
!H NMR (CDC13, 300 MHz) δ 7.34 (s, IH), 7.15 (m, 2H), 6.87 (m, IH), 4.43 (s, 2H), 4.11 (s,
2H), 4.08 (m, 2H), 2.55 (t, 2H, J= 7.2 Hz), 1.5-1.65 (m, 4H), 1.36 (m, 2H).
EXAMPLE 23: Preparation of (R)-l-(2-chloro-l-methylethyl)-3-(4-iodophenyl)urea [(E)- 23]
Figure imgf000068_0002
To a stirred solution of phenyl chloroformate (4.04 g, 25.8 mmol) in dry THF (60 mL) at 0 °C and under nitrogen was added 4-iodoaniline (5.59 g, 25.5 mmol) over 5 minutes. After the addition was complete, triethylamine (4.0 mL, 28.7 mmol) was added. The ice bath was removed and the mixture was stirred at room temperature for 4 h. The mixture was diluted with ethyl acetate and the organic solution was washed successively with 1 M HCl (2X), 1 M NaOH (3X), brine (IX), dried over MgSO4 and evaporated down to afford a white solid (6.99 g, 81%) which was used directly.
h a round bottom flask equipped with a drying tube filled with CaC ., (R)-alaninol (698 mg, 9.17 mmol) was dissolved in acetonitrile (35 mL). To the solution was added the carbamate (2.575 g, 7.59 mmol) and the mixture was stirred at room temperature for 3 days. The solution was then diluted with ethyl acetate (heating might be necessary to dissolve completely the urea). The organic portion was washed successively with IM HCl (2X), IM NaOH (3X), brine (IX), dried over MgSO4 and the solvent was evaporated under reduce pressure. The pure product was obtained after crystallization from CHCl3/THF/hexanes (1.542 g, 63%).
To a suspension of the alcohol (1.79 g, 5.61 mmol) in dry THF (30 mL) and under nitrogen was added SOCI2 (0.6 mL, 8.2 mmol). The mixture was then heated for 30 min. The cooled solution was poured into brine and the product was extracted three times with ethyl acetate. The organic portions were reunited, washed successively with IM HCl (2X), brine, dried over MgSO4 and the solvent s were removed under reduced pressure. The residue was purified twice by crystallization from THF/hexane (1.42 g, 75%).
1H NMR (CDC13 + MeOD, 300 MHz) δ 7.44 (d, 2H, J = 8.8 Hz), 7.07 (d, 2H, J = 8.8 Hz), 4.09 (m, IH), 3.59 (dd, IH, J = 11.0, 4.6 Hz), 3.50 (dd, IH, J = 11.0, 3.6 Hz), 1.17 (d, 3H, J = 6.8 Hz).
EXAMPLE 24: Preparation of (S)-l-(2-chloro-l-methylethyl)-3-(4~iodophenyl)urea [(S)-23]
Figure imgf000069_0001
Prepared as described for (R)-23 using (ιS)-alaninol instead of (R)-alaninol. 1H NMR (CDC13 + MeOD, 300 MHz) δ 7.44 (d, 2H, J = 8.8 Hz), 7.07 (d, 2H, J = 8.8 Hz), 4.09 (m, IH), 3.59 (dd, IH, J = 11.0, 4.6 Hz), 3.50 (dd, IH, J = 11.0, 3.6 Hz), 1.17 (d, 3H, J = 6.8 Hz).
EXAMPLE 25: Preparation of (R)-l-(4-tert-Butylphenyl)-3-(2-chloro-l-methylethyl)urea K -24]
Figure imgf000070_0001
Prepared as described for (R)-23 starting with 4-tert-butylaniline.
EXAMPLE 26: Preparation of (S)-l-(4-tert-Butylphenyl)-3-(2-chloro-l-methylethyl)urea [(S)-24]
Figure imgf000070_0002
Prepared as described for (R)-23 starting with 4-tert-butylaniline and using (S)-alaninol instead of (R)-alaninol.
EXAMPLE 27: Preparation of l-(4-tert-Butylphenyl)-3-(2-chloro-l,l-dimethylethyl)urea [25]
Figure imgf000070_0003
Prepared as described for (R)-23 starting with 4-tert-butylaniline and using 2-amino-2-mefhyl-l- propanol instead of (R)-alaninol.
EXAMPLE 28: Preparation of l-(2-Bromoethyl)-3-(3-iodophenyl)urea [26]
Figure imgf000071_0001
2-Bromoethyl isocyanate (0.21 mL, 2.32 mmol) was slowly added dropwise to a magnetically stirred and cooled solution (ice bath) of freshly recrystallized 3-iodoaniline (465 mg, 2.12 mmol) in dichloromethane (10 mL). The ice bath was then removed and the reaction mixture was stirred at room temperature for 20 h. After completion of the reaction, the solvent was removed under reduced pressure to give a solid, which was purified by recrystallization (THF/hexane) to give 26 (650 mg, 83%)
1H NMR (CDC13, 300 MHz) δ 7.72 (d, IH, J= 2.0 Hz), 7.25 (m, 2H), 6.89 (t, IH, J= 8.0 Hz), 3.53 (M, 2H), 3.41 (M, 2H).
EXAMPLE 29: Preparation of 3-[3-(2-bromoethyI)ureido]benzoic acid ethyl ester [27]
Figure imgf000071_0002
Prepared as described for compound 26 using 3-aminobenzoic acid ethyl ester. Purified by flash chromatography on silica gel (ethyl acetate/hexane 2/3). Yield 87%.
1H NMR (CDC13, 300 MHz) δ 7.87 (s, IH), 7.72 (d, 2H, J= 8.0 Hz), 7.36 (t, IH, J = 8.0 Hz), 4.36 (q, 2H, J= 7.1 Hz), 3.67 (m, 2H), 3.51 (m, 2H), 1.38 (t, 3H, J= 7.1 Hz). EXAMPLE 30: Preparation of 4-tert-Butylphenyl(4,5-dihydrooxazoI-2-yl) amine [27]
Figure imgf000072_0001
4-(tert-Butylphenyl)-3-(2-chloroethyl)urea (102 mg, 0.40 mmol) and KF supported on silica gel 40% (220 mg) were suspended in acetonitrile (5 mL). The mixture was stirred at room temperature for 3 days. The solvent was removed under reduced pressure and the solid was purified by flash chromatography on silica gel (methanol/dichloromethane 5/95) to yield the oxazoline (41 mg, 47%).
1H NMR (CDC13, 300 MHz) δ 7.28 (d, 2H, J = 8.7 Hz), 7.18 (d, 2H, J = 8.7 Hz), 4.37 (t, 2H, J = 8.4 Hz), 3.80 (t, 2H, J = 8.4 Hz), 1.27 (s, 9H).
EXAMPLE 31: Preparation of l-(2-Chloro-ethyl)-3-(3-iodo-phenyl)-urea [28]
Figure imgf000072_0002
2-Chloroethyl isocyanate (1,2 Eq, 1.640 mmol, 0.173 g) was slowly added dropwise to a magnetically stirred and cooled solution (ice bath) of freshly distilled 3-iodobenzenamine (1.0 Eq, 1.370 mmol, 0.300 g) in dichloromethane (15 mL of solvent/g of aniline). The ice bath was then removed and the reaction mixture was kept at room temperature for 20 h. After completion of the reaction, the solvent was removed by vacuum distillation to give a white solid, which was purified by flash chromatography on silica gel: dichloromethane/ MeOH (98 / 2). Yield = 100% NMR 1H (Acetone) δ: 8.18 (s, NHCONH, IH), 8.10 (s, Ar, IH), 7.37 (d, Ar, IH J = 8,01), 7.30 (d, Ar, IH, J = 7,86), 7,02 (t, Ar, IH, J = 8,01), 6.17 (s, NHCONH, IH), 3.68 (m, 2H), 3.53 (q, 2H, J = 5,85) NMR 13C (Acetone) : 158.0, 143.0, 131.1, 127.4, 125.0, 118.0, 102.8, 44.7, 42.4.
EXAMPLE 32: Preparation of l-(2-Chloro-ethyl)-3-[3-(7-hydroxy-heptyl)-phenyl]- urea (29)
Figure imgf000073_0001
3-iodonitrobenzene (1.0 Eq, 3.655 mmol, 0.910 g), Pd/C 10% (0.02 Eq, 0.073 mmol, 0.078 g), PPh3 (0.08 Eq, 0.292 mmol, 0.077 g), Cul (0.04 Eq, 0.146 mmol, 0.028 g), K2CO3 (2.52 Eq, 9.212 mmol, 1.273 g), 1,2-DME (5 mL), H2O (5 mL) were mixed at 25 °C under a nitrogen atmosphere. The reaction mixture was stirred for 30 minutes and 6-Heptyn-l-ol (1.0 Eq, 3.655 mmol, 0.410 g) was added. The mixture was heated at 80 °C for 16 hours, cooled to room temperature. The organic solvents were removed in vacuo, and the product was purified by flash crhomatographie on silica gel: Hexane / AcOEt (75/25).
7-(3-nitrophenyl)hept-6-yn-l-ol (1.0 Eq, 0.276 mmol, 0.064 g) was dissolved in ethanol (10 mL) and placed in a hydrogenation bottle with 10% Pd/C (0.03 Eq, 0.009 mmol, 0.010 g). The bottle was filled with 38 psi of hydrogen and shaken for 4 h. The product was filtered through Celite, and the solvent was removed in vacuo. The resulting aniline was purified by flash crhomatography on silica gel: Hexane / AcOEt (70 / 30).
In a round bottom flask equipped with a dry tube filled with CaCl , 7-(3-aminophenyl)heptan-l- ol (1.0 Eq, 0.060 mmol, 0.012 g) was dissolved in dichloromethane (10 mL). 2-chloroethyl isocyanate (1.1 Eq, 0.067 mmol, 0.007 g) was added and the mixture was stirred at room temperature overnight. The solvent was removed and the residue was purified by flash chromatography on silica gel: Dichloromethane / MeOH (98 / 2). NMR 1H (Acetone) δ: 8.01 (s, NHCONH, IH), 7.29 (m, Ar, 3H), 6.78 (m, Ar, IH), 6.09 (s, NHCONH, IH), 3.67 (m, 2H), 3.52 (m, 4H), 2.87 (m, 4H), 2.54 (m, 2H), 1.35 (m, 6H).
EXAMPLE 33: Preparation of l-(2-Chloro-ethyl)-3-[3-(5-hydroxy-pentyl)-phenyl]-urea (30)
Figure imgf000074_0001
3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g), 4-Pentyn-l-ol (2.67 Eq, 21.400 mmol, 1.800 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K2CO3 (2.52 Eq, 20.200 mmol, 2.790 g), 1.2-DME (10 mL), H2O (10 mL) were mixed as described in Example 32. The procedure was similar. The purification technique was flash chromatography on silica gel : Hexane / AcOEt (70 / 30). 5-(3-nitrophenyl)pent-4-yn-l-ol (1.0 Eq, 0.970 mmol, 0.200 g) in Pd / C 10% (0.05 Eq, 0.047 mmol, 0.050 g), H2 (38 PSI), ETOH (10 mL) was hydrogenated as described in Example 32 to yield 5-(3-aminophenyl)pentan-l-ol. The product was purified by flash chromatography on silica gel : Hexane / AcOEt (70 / 30).
2-Chloroethyl isocyanate (1.2 Eq, 0.600 mmol, 0.063 g) and 5-(3-aminophenyl)pentan-l-ol (1.0 Eq, 0.500 mmol, 0.090 g) were mixed in dichloromethane (10 mL) as described in Example 31. The product was purified by flash cliromatography on silica gel: dichloromethane/ MeOH (98 / 2). Yield = 20%
1H NMR (CDC13 and MD3OD) δ: 7.03 (m, Ar, 3H), 6.70 (t, Ar, IH, J = 6.54) 3.89 (s, 3H), 4.03 (t, 2H, J = 6.54), 3.45 (m, 6H), 2.45 (t, 2H, J = 7.56), 1.45 (m, 4H), 1.23 (m, 2H). 13C NMR (CDC13 and MD3OD) δ: 156.3, 143.4, 138.9, 128.6, 122.8, 119.3, 116.6, 62.1, 44.2, 41.6, 35.7, 32.2, 30.8, 25.2. EXAMPLE 34: Preparation of 1-(2-Chloro-ethyl)-3-[3-(6-hydroxy-hexyl)-phenyl]-urea (31)
Figure imgf000075_0001
3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 5-Hexyn-l-ol (2.58 Eq, 20.670 mmol, 2.030 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K2CO3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H2O (10 mL) as described in Example 32.
6-(3-nitrophenyl)hex-5-yn-l-ol (1.0 Eq, 0.958 mmol, 0.210 g) was hydrogenated with Pd / C 10% (0.05 Eq, 0.047 mmol, 0.050 g), H2 (38 PSI), ETOH (10 mL) as described in Example 32.
6-(3-aminophenyl)hexan-l-ol (1.0 Eq, 0.671 mmol, 0.147 g) was mixed with 2-chloroethyl isocyanate (1.1 Eq, 0.739 mmol, 0.078 g) in dichloromethane (10 mL) as described in Example 31. The product was purified by flash chromatography on silica gel: Dichloromethane/ MeOH (97 / 3). Yield = 42%
1H NMR (Acetone) δ: 8.17 (s, NHCONH, IH), 7.20 (m, Ar, 3H), 6.79 (d, Ar, IH J = 6.42), 6.25 (s, NHCONH, IH), 3.60 (m, 6H), 2.51 (t, 2H, J = 6,87), 1.48 (m, 8H). 13C NMR (Acetone) δ: 156.2, 144.1, 141.0, 129.2, 122.7, 119.2, 116.6, 62.4, 44.8, 42.5, 36.5, 33.6, 32.1, 29.7, 26.4.
EXAMPLE 35: Preparation of l-(2-Chloro-ethyl)-3-[3-(4-hydroxy-butyl)-phenyl]-urea (32)
Figure imgf000075_0002
3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 3-Butyn-l-ol (2.58 Eq, 20.670 mmol, 1.449 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K2CO3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H2O (10 mL) as described in Example 32.
4-(3-nitrophenyl)but-3-yn-l-ol (1.0 Eq, 1.046 mmol, 0.200 g) was hydrogenated with Pd/C 10% (0.05 Eq, 0.047 mmol, 0.050 g), H2 (38 PSI), ETOH (10 mL) as described in Example 32.
4-(3-aminophenyl)butan-l-ol (1.0 Eq, 0.726 mmol, 0.120 g) was mixed with 2-chloroethyl isocyanate (1.2 Eq, 0.870 mmol, 0.091 g) in dichloromethane (10 mL) as described in Example 31. The product was purified by flash chromatography on silica gel: Hexane / AcOEt (65 / 35). Yield = 18%
1H NMR (Acetone) δ: 8.17 (s, NHCONH, IH), 7.20 (m, Ar, 3H), 6.79 (d, Ar, IH J = 6.42), 6.25 (s, NHCONH, IH), 3.60 (m, 6H), 2.51 (t, 2H, J = 6,87), 1.48 (m, 8H). 13C NMR (Acetone) δ: 156.2, 144.1, 141.0, 129.2, 122.7, 119.2, 116.6, 62.4, 44.8, 42.5, 36.5, 33.6, 32.1, 29.7, 26.4.
EXAMPLE 36: Preparation of l-(2-Chloro-ethyl)-3-[3-(3-hydroxy-propyl)-phenyl]-urea
(33)
Figure imgf000076_0001
To a mixture of 3-iodonitrobenzene (7 g, 28.1 mmoles), K2CO3 (11.63 g, 84.3 mmoles) in 80 mL 1,2-DME/water (1:1) were added successively Cul (229.50 mg, 1.21 mmoles), PPh3 (591.20 mg, 2.25 mmoles), Pd/C 10% (598.0 mg, 0.562 mmoles). The mixture was stirred at room temperature for 1 hour. 4-butyn-l-ol (5.90 g, 84.30 mmoles) was added, then the mixture was heated to reflux overnight. After cooling, the mixture was filtered on Celite and the organic layer was evaporated under reduced pressure. Tha aqueous layer was acidified with concentrated Chlorhydric acid and extracted with AcOEt. The organic layer were washed with brine, dried, filtered and evaporated. Purified by flash chrmatography on silica gel AcOEt/Hexanes (35 :65). Yield : 81%
1H NMR (CDCI3, 300 MHz) δ: 8.29 (s, Ar, IH), 8.17 (m, Ar, IH), 7.74 (d, Ar, IH, J = 8Hz), 7.51 (t, Ar, IH, J = 8Hz), 4.53 (d, 2H, J = 6Hz).
A mixture of 3-(3-nitrophenyl)-prop-2-yn-l-ol (100 mg, 0.564 mmoles), Pd/C 10% (10 mg, 0.094 mmoles) was hydrogenated under 38 psi overnight. The mixture was filtered on Celite and the filtrate was evaporated to dryness. Purified by flash chrmatography on silica gel AcOEt/Hexanes (25:75). Yield : 99%
1H NMR (CDCI3, 300 MHz) δ: 7.08 (t, Ar, IH, J = 8.0Hz), 6.61 (d, Ar, IH, J = 7.5Hz), 6.53 (m, Ar, 2H), 3.67 (t, 2H, J = 6.5Hz), 2.84 (s, 3H), 2.62 (t, 2H, J = 8.0Hz), 1.87 (m, 2H).
3-(3-aminophenyl)propan-l-ol was then reacted with 2-chloroethylisocyanate as described in examples 1-12 to obtain desired product. Purified by flash chromatography on silica gel EtOH/CH2Cl2 (2:98). Yield: 28%
1H NMR (Acetone) δ: 8.00 (s, NHCONH, IH), 7.36 (s, Ar, IH), 7.29 (d, Ar, IH J = 8.01), 7.13 (t, Ar, IH J = 7.83), 6.80 (d, Ar, IH J = 7.32), 6.09 (s, NHCONH, IH), 3.68 (m, 2H), 3.55 (m, 4H), 2.63 (t, 2H, J = 7.62), 1.79 (m, 2H). 13C NMR (Acetone) δ: 155.9, 143.8, 141.2, 129.2, 122.6, 119.1, 116.5, 61.7, 42.6, 42.4, 35.3, 32.8.
EXAMPLE 37: Preparation of l-(3-Bromo-phenyl)-3-(2-chloro-ethyι)-urea (34)
Figure imgf000077_0001
3-bromobenzenamine (1.0 Eq, 1.740 mmol, 0.300 g) was mixed with 2-chlroethyl isocyanate (1,2 Eq, 2.090 mmol, 0.220 g) in dichloromethane (10 mL) as described in Example 31. The solvent was removed and the product was purified by flash chromatography on silica gel: dichloromethane / MeOH (98 / 2). Yield = 100%
1H NMR (Acetone) δ: 8.26 (s, NHCONH, IH), 7.93 (m, Ar, IH), 7.31 (m, Ar, IH), 7.15 (m, Ar, 2H), 6.20 (s, NHCONH, IH), 3.67 (m, 2H), 3.52 (q, 2H, J = 2,11). 13C NMR (Acetone) δ: 155.6, 142.9, 131.0, 125.0, 122.7, 121.3, 117.4, 44.7, 42.5.
EXAMPLE 38: Preparation of l-(2-Chloro-ethyl)-3-(3-chloro-phenyl)-urea (35)
Figure imgf000078_0001
3-chlorobenzenamine (1.0 Eq, 2.350 mmol, 0.300 g) was mixed with 2-chlroethyl isocyanate (1,2 Eq, 2.820 mmol, 0.298 g) in dichloromethane (15 mL) as described in Example 31. The solvent was removed and the product was purified by flash chromatography on silica gel: dichloromethane / MeOH (98 / 2). Yield = 99%.
1H NMR (Acetone) δ: 8.28 (s, NHCONH, IH), 7.76 (m, Ar, IH), 7.24 (m, Ar, 2H), 6.95 (m, Ar,
IIHH)),, 66..2211 ((ss,, NNHHCCOONNHH,, IIHH)),, 33..6688 ((mm,, 22HH)),, 33..5544 ((qq,, 2H, J = 2,13). 13C NMR (Acetone) δ: 155.6, 142.7, 134.6, 130.7, 122.0, 118.6, 117.0, 44.6, 42.4.
EXAMPLE 39: Preparation of (36)
Figure imgf000078_0002
H H
3-aminophenol (1.0 Eq, 2.740 mmol, 0.300 g) was mixed with 2-chloroethyl isocyanate (1.2 Eq, 3.300 mmol, 0.348 g) in THF (4 mL) and dichloromethane (10 mL) as described in Example 31. The solvent was removed and the product was purified by flash chromatography on silica gel: dichloromethane / MeOH (99 / 1). Yield = 96%.
11 1H NMR (Acetone)δ: 8.57 (s, Ph, IH), 8.23 (s, NHCONH, IH), 7.25 (s, NHCONH, IH), 7.06 (m, Ar, IH), 6.80 (d, Ar, IH, J = 7.71), 6.50 (m, Ar, IH), 6.35 (m, Ar, IH), 3.64 (m, 2H), 3.55 (q, 2H, J = 5.73). 13C NMR (Acetone) δ: 158.7, 156.8, 141.7, 130.4, 110.9, 110.3, 107.0, 44.8, 42.6.
EXAMPLE 40: Preparation of Acetic acid 3-[3-(2-chloro-ethyl)-ureido]-phenyl ester (37)
Figure imgf000079_0001
l-(2-chloroethyl)-3-(3-hydroxyphenyl)urea (1MO-365) was obtained as described in example 39. l-(2-chloroethyl)-3-(3-hydroxyphenyl)urea (1.0 Eq, 0.326 mmol, 0.070 g) was mixed with triethylamine (3.0 Eq, 0.978 mmol, 0.099 g), acetic anhydride (3.0 Eq, 0.978 mmol, 0.100 g) and 4-pyπolidinopyridine (0.02 Eq, 0.007 mmol, 0.001 g) at room temperature. The solvent was removed and the residue was purified by flash chromatography on silica gel, Dichloromethane / MeOH (98 / 2). Yield = 27%
1H NMR (Acetone) δ: 8.32 (s, NHCONH, IH), 7.48 (s, Ar, IH), 7.20 (m, Ar, 2H), 6.68 (m, Ar, IH), 6.22 (s, NHCONH, IH), 3.67 (m, 2H), 3.55 (q, 2H, J = 5.82). 13C NMR (Acetone) δ: 169.5, 155.4, 152.2, 142.4, 129.8, 115.8, 115.5, 112.3, 44.7, 42.4, 20.8.
EXAMPLE 41: Preparation of l~(2-Chloro-ethyl)-3-(3-hydroxymethyl-phenyl)-urea (38)
Figure imgf000079_0002
3-nitrobenzylalcohol (1.0 Eq, 1.959 mmol, 0.300 g) was reduced on SnCl2.2H20 (6.0 Eq, 11.750 mmol, 2.650 g) and EtOH (20 mL). (3-aminophenyl)methanol (1.0 Eq, 1.620 mmol, 0.200 g) was mixed with 2-chlroethyl isocyanate (1.2 Eq, 1.940 mmol, 0.206 g)in THF (20 mL) as described in Example 31. The solvent was removed and the product was purified by flash chromatography on silica gel: dichloromethane / MeOH (98 / 2). Yield = 56%
1H NMR (Acetone) δ: 8.09 (s, NHCONH, IH), 7.46 (s, Ar, IH), 7.37 (m, Ar, IH), 7.18 (t, Ar, IH, J = 7.86), 6.95 (d, Ar, IH, J = 7,35), 6.15 (s, NHCONH, IH), 4.57 (s, 2H), 4.23 (s, OH, IH), 3.67 (m, 2H), 3.53 (q, 2H, J = 6,00). 13C NMR (Acetone) δ: 156.0, 143.9, 141.0, 129.2, 120.7, 117.6, 117.3, 64.6, 44.8, 42.4.
EXAMPLE 42: Preparation of Acetic acid 5-{3-[3-(2-chloro-ethyl)-ureido]-phenyl}-pentyl ester (39)
Figure imgf000080_0001
3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 4-pentyn-l-ol (2.67 Eq, 21.400 mmol, 1.800 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K2CO3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H2O (10 mL) as described in Example 32. The solvent was removed and the residue was purified by flash chromatography on silica gel: hexane / AcOEt (70 / 30).
5-(3-nitrophenyl)pent-4-yn-l-ol (1.0 Eq, 1.460 mmol, 0.300 g) was mixed with triethylamine (3.0 Eq, 4.380 mmol, 0.443 g), acetic anhydride (3.0 Eq, 4.380 mmol, 0.447 g) and 4- pyπolidinopyridine (0.02 Eq, 0.029 mmol, 0.004 g) at room temperature. The solvent was removed and the residue was purified by flash chromatography on silica gel: hexane / AcOEt (75
/ 25).
5-(3-nitrophenyl)pent-4-ynyl acetate (1.0 Eq, 0.978 mmol, 0.242 g) in Pd / C 10% (0.05 Eq, 0.047 mmol, 0.050 g), H2 (38 PSI) and EtOH (10 mL) was hydrogenated and purified as described in Example 32. The solvent was removed and the product was purified by flash chromatography on silica gel: hexane / AcOEt (75 / 25).
5-(3-aminophenyl)pentyl acetate (1.0 Eq, 0.704 mmol, 0.156 g) was mixed with 2-chlroethyl isocyanate (1.2 Eq, 0.808 mmol, 0.085 g) in dichloromethane (15 mL) as described in Example 31. The solvent was removed and the product was purified by flash cliromatography on silica gel: dichloromethane / AcOEt (70 / 30). Yield = 54%
1H NMR (CDC13) δ: 7.81 (s, NHCONH, IH), 7.09 (m, Ar, 3H), 6.80 (d, Ar, IH, J = 7.32), 6.07 (s, NHCONH, IH), 3.48 (m, 4H), 2.48 (t, 2H, J= 7.68), 2.02 (s, Ac, 3H), 1.56 (m, 4H), 1.31 (m, 2H). 13C NMR (CDCl3) δ: 171.5, 156.4, 143.5, 138.8, 128.9, 123.3, 120.1, 117.5, 64.5, 44.3, 41.9, 35.7, 30.9, 28.4, 25.5, 21.0.
EXAMPLE 43: Preparation of Acetic acid 4-{3-[3-(2-chloro-ethyl)-ureido]-phenyl}-butyl ester (40)
Figure imgf000081_0001
3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 3-Butyn-l-ol (2.58 Eq, 20.670 mmol, 1.449 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K2CO3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H2O (10 mL) as described in Example 32. The solvent was removed and the residue was purified by flash chromatography on silica gel: Hexane / AcOEt (65 / 35).
4-(3-nitrophenyl)but-3-yn-l-ol (1.0 Eq, 1.046 mmol, 0.200 g) was mixed with triethylamine (3.0 Eq, 3.1380 mmol, 0.318 g), acetic anhydride (3.0 Eq, 3.138 mmol, 0.320 g), and 4- pyrrolidinopyridine (0.02 Eq, 0.021 mmol, 0.003 g) at room temperature. The solvent was removed. 4-(3-nitrophenyl)but-3-ynyl acetate (1.0 Eq, 0.988 mmol, 0.231 g) in Pd / C 10% (0.05 Eq, 0.047 mmol, 0.050 g), H2 (38 PSI), ETOH (10 mL) was hydrogenated and purified as described in Example 32. The solvent was removed and the product was purified by flash chromatography on silica gel: hexane / AcOEt (75 / 25).
4-(3-aminophenyl)butyl acetate (1.0 Eq, 0.471 mmol, 0.098 g) was mixed with 2-chlroethyl isocyanate (1.2 Eq, 0.539 mmol, 0.057 g) in dichloromethane (15 mL) as described in Example 31. The solvent was removed and the product was purified by flash chromatography on silica gel: hexane / AcOEt (65 / 35). Yield = 48%
1H NMR (CDC13) δ: 7.69 (s, NHCONH, IH), 7.11 (m, Ar, 3H), 6.83 (d, Ar, IH, J = 7.32), 6.07 (s, NHCONH, IH), 4.03 (t, 2H, J = 6.57), 3.53 (m, 4H), 2.52 (t, 2H, J= 7.89), 2.03 (s, Ac, 3H), 1.60 (m, 4H). 13C NMR (CDC13) δ: 171.5, 156.3, 143.2, 138.7, 129.0, 123.5, 120.3, 117.8, 64.4, 44.4, 41.9, 35.4, 28.1, 27.5, 21.0.
EXAMPLE 44: Preparation of Acetic acid 3-[3-(2-chloro-ethyl)-ureido]-benzyl ester (41)
Figure imgf000082_0001
3-nitrobenzylalcohol (1.0 Eq, 1.959 mmol, 0.300 g) was mixed with triethylamine (3.0 Eq, 5.880 mmol, 0.595 g), acetic anhydride (3.0 Eq, 5.880 mmol, 0.600 g) and 4-pyπolidinopyridine (0.02 Eq, 0.039 mmol, 0.006 g) at room temperature. The solvent was removed and the product was purified by flash chromatography on silica gel: hexane / AcOEt (75 / 25).
3-nitrobenzyl acetate (1.0 Eq, 0.649 mmol, 0.127 g) was reduced on SnCl2.2H20 (6.0 Eq, 3.890 mmol, 0.879 g) and EtOH (20 mL). The solvent was removed and the product was purified by flash chromatography on silica gel: dichloromethane / MeOH (95 / 5). 2-(3-aminophenyl)ethyl acetate (1.0 Eq, 0.352 mmol, 0.058 g) was mixed with 2-chlroethyl isocyanate (1,2 Eq, 0.422 mmol, 0.045 g) in dichloromethane (10 mL) as described in Example 31. The solvent was removed and the product was purified by flash chromatography on silica gel: dichloromethane / MeOH (95 / 5). Yield = 31%
1H NMR (CDC13) δ: 7.79 (s, NHCONH, IH), 7.23 (m, Ar, 3H), 6.98 (m, Ar, IH), 6.08 (s, NHCONH, IH), 4.98 (s, 2H), 3.53 (m, 4H), 2.05 (s, Ac, 3H). 13C NMR (CDCI3) 171.2, 156.2, 139.0, 136.9, 129.3, 122.9, 119.9, 119.8, 66.1, 44.4, 41.9, 21.0.
EXAMPLE 45: Preparation of Acetic acid 3-{3-[3-(2-chloro-ethyl)-ureido]-phenyl}-propyl ester (42)
Figure imgf000083_0001
To a mixture of 3-iodonitrobenzene (1 g, 4.56 mmoles), K2CO3 (1.57 g, 11.4 mmoles) in 30 mL 1.2-DME/water (1:1) were added successively Cul (34.78 mg, 0.18 mmoles), PPh3 (95.80 mg, 0.36 mmoles), Pd/C 10% (97.05 mg, 0.09 mmoles). The mixture was stirred at room temperature for 1 hour. Propargyl alcohol (807 mg, 14.40 mmoles) was added, then the mixture was heated to reflux overnight. After cooling, the mixture was filtered on Celite and the organic layer was evaporated under reduced pressure. Tha aqueous layer was acidified with concentrated Chlorhydric acid and extracted with AcOEt. The combined organic layers were washed with brine, dried, filtered and evaporated. Purified by flash chromatography on silica gel CH2Cl2/EtOH (95: 5). Yield : 81%
1H NMR (CDCI3, 300 MHz) δ: 8.29 (s, Ar, IH), 8.17 (m, Ar, IH), 7.74 (d, Ar, IH, J = 8Hz), 7.51 (t, Ar, IH, J = 8Hz), 4.53 (d, 2H, J = 6Hz).
To an ice-cold 3-(3-nitrophenyl)-prop-2-yn-l-ol (150 mg, 0.85 mmoles) in diethylether (10 mL) were added acetic anhydride (254.23 mg, 2.54 mmoles), triethylamine (256.54 mg, 2.54 mmoles), 4-pyπolidinopyridine (2.52 mg, 0.017 mmoles) and the mixture was stirred at room temperature for 12 hours. The reaction was quenched by saturated solution of Na2CO3 and the mixture was extracted with AcOEt. The extracts were washed with brine, dried and evaporated. Purified by flash chromatography on silica gel AcOEt Hexanes (8 :2). Yield : 99%
!H NMR (CDC13, 300 MHz) δ: 8.24 (s, Ar, IH), 8.14 (d, Ar, IH, J = 8.5Hz), 7.71 (d, Ar, IH, J = 7.5Hz), 7.48 (t, Ar, IH, J= 8Hz), 4.88 (s, 2H), 2.12 (s, 3H).
A mixture of acetic acid 3-(3-nitrophenyl)-prop-2-ynyl ester (100 mg, 0.48 mmoles), Pd/C 10% (10 mg, 0.094 mmoles) in 30 mL of dry ethanol was hydrogenated under 38 psi overnight. The mixture was filtered on Celite and the filtrate was evaporated to dryness. Purified by flash chromatography on silica gel AcOEt/Hexanes (25:75). Yield : 81%
1H NMR (CDCI3, 300 MHz) δ: 7.08 (m, Ar, IH), 6.59 (d, Ar, IH, J = 7.5Hz), 6.53 (m, Ar, 2H), 4.09 (t, 2H, J = 6.5Hz), 3.66 (s, 2H), 2.60 (t, 2H, J = 8Hz), 2.06 (s, 3H), 1.93 (m, 2H).
Acetic acid 3-(3-aminophenyl)-prop-2-ynyl ester was then reacted with 2-chloroethylisocyanate as described in examples 1-12 to obtain desired product. Purified by flash cliromatography on silica gel AcOEt/CH2Cl2 (2:8). Yield : 93%
1H NMR (CDCI3) δ: 7.51 (s, NHCONH, IH), 7.11 (m, Ar, 3H), 6.98 (m, Ar, IH), 6.84 (d, Ar, IH, J = 7.38) 5.94 (s, NHCONH, IH), 4.03 (t, 2H, J = 6.54), 3.55 (m, 4H), 2.59 (t, 2H, J = 7.92), 2.04 (s, Ac, 3H), 1.88 (m, 2H) NMR. 13C (CDCI3) δ: 171.5, 156.2, 142.4, 138.7, 129.2, 123.6, 120.5, 118.1, 63.9, 44.5, 41.9, 32.1, 30.0, 21.0.
EXAMPLE 46: Preparation of l-(2-Chloro-ethyl)-3-[3-(2-hydroxy-ethyl)-phenyl]-urea (43)
Figure imgf000084_0001
2-(3-nitrophenyl)ethan-l-ol (1.0 Eq, 1.200 mmol, 0.200 g) was reduced on SnCl2.2H20 (6.0 Eq, 7.200 mmol, 1.625 g) and EtOH (20 mL). The solvent was removed and the product was purified by flash chromatography on silica gel: dichloromethane / MeOH (95 / 5).
2-(3-aminophenyl)ethan-l-ol (1.0 Eq, 0.437mmol, 0.060 g) was mixed with 2-chloroethyl isocyanate (1.2 Eq, 0.524 mmol, 0.055 g), in dichloromethane (15 mL) as described in Example 31. The solvent was removed and the product was purified by flash chromatography on silica gel: Ether / Hexane (90 / 10). Yield = 20%
!H NMR (Acetone-d6) δ: 8.08 (brs, NH, IH), 7.33 (m, Ar, 2H), 7.13 (m, Ar, IH), 6.82 (d, Ar, IH, J = 7.44), 6.15 (brs, NH, IH), 3.69 (m, 4H), 3.53 (m, 2H), 2.93 (s, OH, IH), 2.74 (t, 2H, J = 7.02). 13C NMR (Acetone-d6) δ: 177.3, 155.9, 140.9, 129.2, 123.1, 119.5, 116.7, 63.7, 44.8, 42.4, 40.3.
EXAMPLE 47: Preparation of Acetic acid 2-{3-[3-(2-chloro-ethyl)-ureido]-phenyl}-ethyl ester (44)
Figure imgf000085_0001
2-(3-nitrophenyl) ethan-1-ol (1.0 Eq, 1.495 mmol, 0.250 g) was mixed with triethylamine (3.0 Eq, 4.485 mmol, 0.454 g), acetic anhydride (3.0 Eq, 4.485 mmol, 0.457 g) and 4- pyπolidinopyridine (0.02 Eq, 0.030 mmol, 0.004 g) ) at room temperature. The solvent was removed and the product was purified by flash chromatography on silica gel: Hexane / AcOEt (75 / 25).
3-nitrophenethyl acetate (1.0 Eq, 0.871 mmol, 0.182 g) in Pd / C 10% (0.05 Eq, 0.047 mmol, 0.050 g), H2 (38 PSI), and ETOH (10 mL) was hydrogenated and purified as described in Example 32. The solvent was removed and the product was purified by flash chromatography on silica gel: hexane / AcOEt (60 / 40). 2-(3-aminophenyl)ethyl acetate (1.0 Eq, 0.726 mmol, 0.130 g) was mixed with 2-chloroethyl isocyanate (1,2 Eq, 0.871 mmol, 0.092 g), in dichloromethane (15 mL) as described in Example 31. The solvent was removed and the product was purified by flash chromatography on silica gel: dichloromethane / MeOH (98 / 2). Yield = 87%
1H NMR (Acetone) δ: 7.90 (s, NHCONH, IH), 7.11 (m, Ar, 3H), 6.83 (d, Ar, IH, J = 6.63), 6.21 (s, NHCONH, IH), 4.18 (t, 2H, J = 6.96), 3.50 (m, 4H), 2.79 (t, 2H, J = 6.99), 1.99 (s, Ac, 3H) NMR 13C (Acetone) δ: 171.4, 156.5, 138.9, 138.8, 129.1, 123.8, 120.5, 118.4, 64.8, 44.3, 41.9, 34.9, 21.0.
EXAMPLE 48: Preparation of Acetic acid 6-{3-[3-(2-chloro-ethyl)-ureido]-phenyl}-hexyl ester (45)
Figure imgf000086_0001
3-iodonitrobenzene (1.0 Eq, 8.010 mmol, 1.995 g) was mixed with 5-Hexyn-l-ol (2.58 Eq, 20.670 mmol, 2.030 g), Pd/C 10% (0.02 Eq, 0.160 mmol, 0.170 g), PPh3 (0.08 Eq, 0.630 mmol, 0.166 g), Cul (0.04 Eq, 0.320 mmol, 0.061 g), K2CO3 (2.52 Eq, 20.200 mmol, 2.790 g), 1,2- DME (10 mL), H2O (10 mL) under a nitrogen atmosphere. The reaction was carried out as described in Example 32. The organic portion was purified by flash chromatography on silica gel : Hexane / AcOEt (75 / 25).
6-(3-nitroρhenyl)hex-5-yn-l-ol (1.0 Eq, 1.140 mmol, 0.255 g) was mixed with triethylamine (3.0 Eq, 3.420 mmol, 0.346 g), acetic anhydride (3.0 Eq, 3.420 mmol, 0.349 g) and 4- pyrrolidmopyridine (0.02 Eq, 0.023 mmol, 0.003 g) ) at room temperature. The solvent was removed and the product was purified by flash chromatography on silica gel: Hexane / AcOEt (75 / 25). 6-(3-aminophenyl)hex-5-ynyl acetate (1.0 Eq, 0.643 mmol, 0.168 g) in Pd / C 10% (0.07 Eq, 0.047 mmol, 0.050 g), H2 (38 PSI), and ETOH (10 mL) was hydrogenated and purified as described in Example 32. The solvent was removed and the product was purified by flash cliromatography on silica gel: dichloromethane / MeOH (99 / 1).
6-(3-aminophenyl)hexyl acetate (1.0 Eq, 0.348 mmol, 0.082 g) was mixed with 2-chloroethyl isocyanate (1.2 Eq, 0.418 mmol, 0.0446 g), in dichloromethane (15 mL) as described in Example 31. The solvent was removed and the product was purified by flash chromatography on silica gel: dichloromethane / MeOH (98 / 2). Yield = 64%
1H NMR (Acetone) δ: 7.80 (s, NHCONH, IH), 7.12 (m, Ar, 3H), 6.84 (m, Ar, IH), 6.15 (s, NHCONH, IH), 4.05 (m, 2H), 3.51 (m, 4H), 2.42 (m, 2H), 2.08 (s, Ac, 3H) 1.56 (m, 4H), 1.32 (m, 4H). 13C NMR (Acetone) δ: 171.7, 156.4, 143.8, 138.8, 128.9, 123.4, 120.2, 117.6, 64.7, 44.4, 41.9, 35.8, 31.2, 28.9, 28.8, 25.7, 21.0.
EXAMPLE 49: Preparation of l-(2-Chloro-ethyl)-3-[3-(3-methoxy-propyl)-phenyl]-urea
(46)
Figure imgf000087_0001
NaH (60%) (97.40 mg, 4.06 mmoles) was suspended in dry THF (8 mL) and 3-(3-nitrophenyl)- pent-4-yn-l-ol (see synthesis of O-371) (200 mg, 0.98 mmoles) in dry THF (3 Ml) was added dropwide at 0°C. The mixture was stirred for 15 minutes at 0°C. The Mel (332.5 mg, 2.34 mmoles) was added dropwise and the mixture was stured at room temperature for 3 hours. Saturated solution of NaHCO3 (10 mL) and MeOH (10 mL) were added. The mixture was extracted with AcOEt, dried, filtered and evaporated to dryness. Purified by flash chromatography on silica gel AcOEt. Yield : 58% 1H NMR (CDCI3, 300 MHz) δ: 8.17 (s, IH), 8.07 (d, IH, J = 8Hz), 7.64 (d, IH, J = 7.5Hz), 7.42 (m, IH), 3.49 (t, 2H, J = 6Hz), 3.34 (s, 3H), 2.49 (t, 2H, J = 6Hz), 1.86 (t, 2H, J = 6Hz), 1.22 (m, 2H).
A mixture of l-(5-methoxypent-l-ynyl)-3-nitrobenzene (100 mg, 0.425 mmoles), Pd/C 10% (10 mg, 0.094 mmoles), dry ehtanol was hydrogenated under 38 psi overnight. The mixture was filtered on Celite and the filtrate was evaporated to dryness. Purified by flash chrmatography on silica gel AcOEt. Yield : 85%
1H NMR (DMSO-d6, 300MHz) δ: 7.17 (m, IH), 6.55 (m, 3H), 3.37 (m, 5H), 2.51 (m, 2H), 1.61 (m, 2H).
3-(5-methoxypentyl) phenylamine was then reacted with 2-chloroethylisocyanate as described in examples 1-12 to obtain desired product. Purified by flash chromatography on silica gel AcOEt. Yield : 87%
1H NMR (CDCI3, 300 MHz) δ: 8,18 (brs, NH, IH), 7,14 (m, 3H), 6,86 (d, Ar, IH, J = 7,2Hz), 3,57 (m, 4H), 3,33 (m, 4H), 2,55 (t, 2H, J = 7Hz), 1,59 (m, 4H).
EXAMPLE 50: Preparation of (47) 1-(3-pentyl-phenyϊ)-3-(2-chloro-ethyl)-urea (47)
Figure imgf000088_0001
To a mixture of 3-iodonitrobenzene (1 g, 4.01 mmoles), K2CO3 (1.38 g, 10.0 mmoles) in 20 mL 1,2-DME/water (1 :1) were added successively Cul (30.54 mg, 0.16 mmoles), PPh3 (84.14 mg, 0.32 mmoles), Pd/C 10% (85.35 mg, 0.080 mmoles). The mixture was stirred at room temperature for 1 hour. 1-pentyne (725.40 mg, 84.30 mmoles) was added, then the mixture was heated to reflux overnight. After cooling, the mixture was filtered on Celite and the organic layer was evaporated under reduced pressure. Tha aqueous layer was acidified with concentrated Chlorhydric acid and extracted with AcOEt. The organic layer were washed with brine, dried, filtered and evaporated. Purified by flash chrmatography on silica gel Hexanes/ AcOEt (60 :40). Yield : 58%
1H NMR (CDCI3, 300 MHz) δ: 8.17 (s, IH), 8.06 (d, IH, J = 8Hz), 7.63 (d, IH, J = 7.5Hz), 7.41 (m, IH), 2.37 (m, 2H), 1.02 (m, 2H).
A mixture of l-nitro-3-pentynylbenzene (100 mg, 0.523 mmoles), Pd/C 10% (10 mg, 0.094 mmoles) in 30 mL of dry ethanol was hydrogenated under 38 psi overnight. The mixture was filtered on Celite and the filtrate was evaporated to dryness. Purified by flash chrmatography on silica gel CH2Cl2/EtOH (95 :5). Yield : 97%
1H NMR (DMSO-d6, 300MHz) δ: 7.09 (m, IH), 6.63 (d, IH, J = 7.5Hz), 6.53 (m, 2H), 3.55 (brs, 2H), 2.54 (m, 2H), 1.63 (m, 2H), 1.37 (m, 4H), 0.93 (m, 2H).
3-pentylphenylamine was then reacted with 2-chloroethylisocyanate as described in examples 1- 12 to obtain desired product. Purified by flash chrmatography on silica gel CH2O2/E1OH (95 :5). Yield : 91%
1H NMR (CDCI3, 300 MHz) δ: 7,94 (brs, NH, IH), 7,01 (m, Ar, IH), 6,77 (brs, Nh, IH), 3,47 (m, CH2, 8H), 2,45 (t, CH2, 2H, J = 7), 1,21 (m, CH2, 4H).
EXAMPLE 51: Preparation of 5-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-pentanoic acid (48)
Figure imgf000089_0001
To a mixture of 3-iodonitrobenzene (5 g, 20.3 mmoles), K2CO3 (7.03 g, 50.9 mmoles) in 80 mL 1,2-DME/water (1 :1) were added successively Cul (166.0 mg, 0.87 mmoles), PPh3 (427.11 mg, 1.82 mmoles), Pd/C 10% (432.06 mg, 0.406 mmoles). The mixture was stkred at room temperature for 1 hour. 5-pentanoic acid (5.90 g, 84.30 mmoles) was added, then the mixture was heated to reflux overnight. After cooling, the mixture was filtered on Celite and the organic layer was evaporated under reduced pressure. Tha aqueous layer was acidified with concentrated Chlorhydric acid and extracted with AcOEt. The organic layer were washed with brine, dried, filtered and evaporated.
Purified by flash chromatography on silica gel CH2Cl2/EtOH (95 :5). Yield : 45%
1H NMR (DMSO-d6, 300 MHz) δ: 12.39 (brs, IH), 8.19 (d, IH, J = 8Hz), 8.13 (s, IH), 7.82 (d, IH, J = 7.5Hz), 7.66 (t, IH, J = 8Hz), 2.63 (m, 4H).
A mixture of 5-(3-nitrophenyl)-pent-4-ynoic acid (100 mg, 0.456 mmoles), Pd/C 10% (10 mg, 0.094 mmoles) was hydrogenated under 38 psi overnight. The mixture was filtered on Celite and the filtrate was evaporated to dryness.
Purified by flash chromatography on silica gel EtOH/CH2Cl2 (2 :98). Yield : 57%
1H NMR (DMSO-d6, 300 MHz) δ: 7.08 (t, IH, J = 8Hz), 6.61 (d, IH, J = 7Hz), 6.54 (m, 2H), 6.17 (m, 2H), 2.56 (m, 2H), 2.36 (m, 2H), 1.67 (m, 4H).
5-(3-aminophenyl) pent-4-ynoic acid was then reacted with 2-chloroethylisocyanate as described in examples 1-12 to obtain desired product.
Purified by flash chromatography on silica gel EtOH/CH2Cl2 (5 :95). Yield : 37% 1H NMR (Acetone-d6, 300MHz) δ: 7.57 (brs, NH, IH), 7.36 (m, Ar, 2H), 7.07 (m, Ar, 2H), 6.87 (d, Ar, IH, J = ,0), 6.74 (brs, NH, IH), 3.66 (m, CH2, 4H), 3.46 (m, CH2, 4H), 2.59 (m, CH2, 2H), 1.63 (m, CH2, 2H).
EXAMPLE 52: Preparation of 5-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-pentanoic acid ethyl ester (49)
Figure imgf000091_0001
To a mixture of 3-iodonitrobenzene (5 g, 20.3 mmoles), K2CO3 (7.03 g, 50.9 mmoles) in 80 mL 1,2-DME/water (1 :1) were added successively Cul (166.0 mg, 0.87 mmoles), PPh3 (427.11 mg, 1.82 mmoles), Pd/C 10% (432.06 mg, 0.406 mmoles). The mixture was stirred at room temperature for 1 hour. 5-pentanoic acid (5.90 g, 84.30 mmoles) was added, then the mixture was heated to reflux overnight. After cooling, the mixture was filtered on Celite and the organic layer was evaporated under reduced pressure. Tha aqueous layer was acidified with concentrated Chlorhydric acid and extracted with AcOEt. The organic layer were washed with brine, dried, filtered and evaporated.
Purified by flash chromatography on silica gel CH2Cl2/EtOH (95 :5). Yield : 45%
1H NMR (DMSO-d6, 300 MHz) δ: 12.39 (brs, IH), 8.19 (d, IH, J = 8Hz), 8.13 (s, IH), 7.82 (d, IH, J = 7.5Hz), 7.66 (t, IH, J = 8Hz), 2.63 (m, 4H).
A mixture of 5-(3-nitrophenyl)-pent-4-ynoic acid (250 mg, 1.14 mmoles), 4 mL of dry EtOH, 8.5 mL of dry CH2C12 and 8 mg of APTS was stirred at reflux for 12 hours. After cooling, the mixture was evaporated under reduce pressure. The residue was dissolved in saturated solution Na2CO3 and extracted with CH2C12. The organic layer was dried, filtered and evaporated to dryness. Purified by flash chromatography on silica gel CH2Cl2/EtOH (98 :2). Yield : 87% 1H NMR (CDCI3, 300 MHz) δ: 8.14 (s, IH), 8.05 (d, IH, J = 8Hz), 7.61 (d, IH, J = 8Hz), 7.41 (m, IH), 4.15 (q, 2H, J = 7Hz), 2.71 (m, 2H), 2.59 (m, 2H), 1.23 (q, 3H, J = 7Hz).
A mixture of 5-ethyl-(3-nitrophenyl)-pent-4-ynoic acid ester (100 mg, 0.404 mmoles), Pd/C 10% (10 mg, 0.094 mmoles) was hydrogenated under 38 psi overnight. The mixture was filtered on Celite and the filtrate was evaporated to dryness. Purified by flash chromatography on silica gel EtOH/CH2Cl2 (2 :98). Yield : 57%
1H NMR (CDCI3, 300 MHz) δ: 7.06 ( , IH), 6.58 (d, IH, J = 7.5Hz), 6.50 (m, 2H), 4.11 (q, 2H, J = 7Hz), 2.54 (m, 2H), 2.32 (m, 2H), 1.64 (m, 4H), 1.26 (t, 3H, J = 7Hz).
5-ethyl-(3-aminophenyl)pent-4-ynoic acid ester was then reacted with 2-chloroethylisocyanate as described in examples 1-12 to obtain desired product. Purified by flash chromatography on silica gel EtOH/CH2Cl2 (5 :95). Yield : 77%
1H NMR (CDCI3, 300 MHz) δ: 7.23 (m, Ar, 3H), 6.89 (m, Ar, 2H), 5.63 (brs, NH, IH), 4.31 (q, CH2, 2H, J = 7,0), 2.56 (m, CH2, 2H), 2.39 (m, CH2, 2H), 1.81 (m, CH2, 4H), 1.23 (t, CH3, 3H, J = 7.0).
EXAMPLE 53: Preparation of l-(2-Chloro-ethyl)-3-(3-cyanomethyl-phenyl)-urea (50)
Figure imgf000092_0001
A mixture of nitrobenzene acetic acid (1 g, 5.52 mmoles), 1.66 mL of SOCI2, 10 mL dry CHCI3 was refluxed for 14 hours. CHCI3 and excess thionyl chlorid were removed in vacuo, and the residue was evaporated twice with 25 mL of toluene to remove traces of thionyl chlorid. The residue was taken into 10 mL of toluene and 30 mL of cold concentrated ammonium hydroxyde were added. The white solid formed was collected and dried with etanol in vacuo. Yield : 79 %. 1H NMR (DMSO-d6, 300MHz) δ: 8.14 (m, 2H), 7.71 (m, 2H), 7.06 (brs, 2H), 3.58 (s, 2H).
2-(3-nitrophenyl) acetamide was added with 10 mL of POCl3 and the mixture was heated at reflux for 2 hours. After cooling, the mixture was poured into ice, basified with Na2CO3 and extracted with CH2CI2. The organic layer was dried over Na2SO4, filtered and evaporated. Purified by flash chromatography on silica gel CH2C12. Yield : 41%)
1H NMR (CDCI3, 300 MHz) δ: 8.14 (m, 2H), 7.69 (d, IH, J = 8Hz), 7.56 (m, IH), 3.88 (s, 2H).
A solution of 15 mL HBr 48% was cooled to 0°C. (430 mg, 2.65 mmoles) of 3-nitrobenzonitrile, (574 mg, 4.85 mmoles) of Sn were added successively. The mixture was stirred at room temperature for 3 hours, then poured into ice. The solution was basified with Na CO3, extracted with CH2C12, dried, filtered and evaporated. Purified by flash chromatography on silica gel EtOH/CH2Cl2 (2 :98). Yield : 32%
1H NMR (DMSO-d6, 300MHz) δ: 7.37 (m, 2H), 6.81 (m, 2H), 3.86 (s, 2H).
3-aminobenzonitrile was then reacted with 2-chloroethylisocyanate as described in examples 1- 12 to obtain desired product. Purified by flash chrmatography on silica gel EtOH/CH2Cl2 (5 :95). Yield : 78%
1H NMR (CDCI3, 300 MHz) δ: 8.12 (brs, NH, IH), 7.26 (m, Ar, 4H), 6.97 (brs, NH, IH), 3.63 (m, CH2, 6H).
EXAMPLE 54: Preparation of 2-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-acetamide (51)
Figure imgf000093_0001
A mixture of 2-(3-nitrophenyl) acetamide (510 mg, 2.83 mmoles), Pd/C 10% (29 mg, 0.272 mmoles), dry ehtanol was hydrogenated under 38 psi overnight. The mixture was filtered on Celite and the filtrate was evaporated to dryness. The crude product was then reacted with 2- chloroethylisocyanate as described in examples 1-12 to obtain desired product. Purified by flash chromatography on silica gel EtOH/CH2Cl2 (10 :90). bYield : 37%.
1H NMR (DMSO-d6, 300MHz) δ: 8.17 (brs, NH, IH), 8.12 (d, Ar, IH, J = 8.2Hz), 7.65 (m, Ar, 3H), 7.03 (brs, NH, IH), 3.68 (s, CH2, 2H), 3.45 (m, CH2, 4H).
The following exemplary compounds were also prepared.
Example 55: l-(2-Chloro-ethyl)-3-»t-tolyl-urea (52):
Figure imgf000094_0001
1H NMR (CDC13) δ: 7.20-7,12 (m, Ar, 3H), 6.90 (d, Ar, IH, J = 7), 3.57 (m, Ch2, 4H), 2.29 (s, CH3, 3H). 13C NMR δ: 156.1, 139.4, 138.0, 129.2, 125.1, 122.1, 118.4, 44.5, 42.1, 21,4.
Example 56: l-(2-Chloro-ethyl)-3-(3-ethyl-phenyl)-urea (53);
Figure imgf000094_0002
1H NMR (Acetone-d6) δ: 8.09 (brs, NH, IH), 7.29 (m, Ar, 2H), 7.13 (t, Ar, IH, J = 7.8), 6.80 (d, Ar, J = 7.8), 6.20 ( brs, NH, IH), 2.54 (q, CH2, 2H, J = 7.1), 3.57 (m, CH2, 4H), 1.19 (t, CH3,
3H, J = 7). 113JC NMR δ: 156.2, 145.5, 141.0, 129.3, 122.1, 118.7, 116.7, 44.8, 42.5, 29.0, 14.9. Example 57: l-(2-ChIoro-ethyl)-3-(3-methoxy-phenyϊ)-urea (54);
Figure imgf000095_0001
1H NMR (CDC13) δ: 8.12 (brs, NH, IH), 7.21 (m, Ar, 3H), 6.87 (d, Ar, IH, J = 7), 3.97 (s, CH3, 3H), 3.57 (m, CH2, 4H). 13C NMR δ: 156.1, 139.4, 138.0, 129.2, 125.1, 122.1, 118.4, 57.8, 44.5, 42.1.
Example 58: l-(2-Chloro-ethyl)-3-[4-(4-hydroxy-butyl)-phenyl]-urea (55);
Figure imgf000095_0002
1H NMR (Acetone-d6) δ: 8.00 (brs, NH, IH), 7.39 (d, CH2, 2H, J = 8.2), 7.07 (d, CH2, 2H, J = 8.2), 6.50 ((brs, NH, IH), 3.51 (m, 8H), 2.57 (t, 2H, J = 7.5), 1.58 (m, 2H). 13C NMR δ: 155.9, 139.0, 136.3, 130.0, 119.2, 64.8, 42.8, 42.5, 42.4, 35.2.
Example 59: l-(2-Chloro-ethyl)-3-[4-(3-hydroxy-propyl)-phenyl]-urea (56);
Figure imgf000095_0003
1H NMR: 8.23 (brs, NH, IH), 7.30 (d, Ar, IH, J = 7.9), 7.06 (d, Ar, IH, J = 8.2), 6.36 (brs, Nh, IH), 3.58 (m, 4H), 3.42 (m, 6H). 1 3C NMR δ: 155.2, 138.2, 134.0, 128.5, 118.0, 63.3, 44.5, 43.1, 35.7, 30.4. Example 60: l-(2-Chloro-ethyl)-3-[4-(5-hydroxy-pentyl)-phenyl]-urea (57);
Figure imgf000096_0001
1H NMR: 8.63 (brs, NH, IH), 7.36 (d, 2H, J = 7.9), 7.13 (d, 2H, J = 7.9), 6.43 (brs, NH, IH), 3.96 (t, 2H, J = 6.5), 3.57 (m, 6H), 2.57 (t, 2H, J = 7.5), 1.54 (m, 2H), 1.42 (m, 2H). 13C NMR δ: 155.2, 129.5, 128.4, 123.2, 117.9, 60.6, 43.4, 40.9, 34.4, 32.7, 25.0.
Example 61: l-(2-Chloro-ethyl)-3-[3-(5-hydroxy-pent-l-ynyl)-phenyl]-urea (58);
Figure imgf000096_0002
1H NMR (acetone-d6) δ: 8.12 (brs, Nh, IH), 7.03 (m, IH), 6.79 (d, Ar, IH, J = 7), 6.69 (brs, Ar, IH), 6.57 (d, Ar, IH, j= 8.0), 3.74 (t, 2H, j = 7), 3.49 (m, 4H), 2.47 (t, 2H, J = 7), 1.81 (t, 2H, J = 7), 1.23 (t, 2H, j=7). "C MR δ: 156.1, 146.3, 129.2, 125.3, 124.4, 122.0, 118.0, 114.9, 88.9, 81.3, 61.6, 45.1, 42.8, 31.4, 16.0.
Example 62: 3-[3-(2-Chloro-ethyl)-ureido]-benzoic acid ethyl ester (59);
Figure imgf000096_0003
1H NMR (CDC13) δ: 8.22 (brs, Nh, IH), 7.93 (s, Ar, IH), 7.63 (d, Ar, IH, J = 7,8), 7.57 (d, Ar, IH, J = 7), 7.30 (m, IH), 6.35 (brs, Nh, IH), 4.31 (q, CH2, 2H, J = 7), 3.57 (m, 4H), 1.34 (t, CH3, 3H, J = 7). 13C NMR δ: 166.7, 156.0, 139.1, 131.0, 129.1, 124.3, 124.1, 120.5, 61.2, 44.5, 42.0, 14.2.
Example 63: l-(2-Chloro-ethyl)-3-[3-(5-methoxy-pentyl)-phenyl]-urea (60);
Figure imgf000097_0001
1H NMR (CDC13) δ: 8.18 (brs, Nh, IH), 7.14 (m, 3H), 6.86 (d, Ar, IH, J = 7,2), 3.57 (m, 4H), 3.33 (m, 4H), 2.55 (t, 2h, J = 7), 1.59 (m, 4H). 13C NMR δ: 156.0, 144.0, 138.4, 129.1, 123.9, 120.8, 118.2, 58.5, 44.6, 42.0, 35.9, 31.2, 29.5, 25.9.
Example 64: {3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-acetic acid (61);
Figure imgf000097_0002
!H NMR (DMSO-d6) δ: 8.95 (brs, OH), 7.89 (s, IH, Ar), 7.61 (d, IH, J = 8.0), 7.41 (d, IH, J = 7.9), 7.29m, IH, Ar), 6.61 (brs, NH, IH), 3.68 (m, 4H), 3.34 (s, 2H). 13C NMR δ: 168.8, 155.7, 148.6, 135.2, 128.5, 116.5, 114.7, 113.2, 47.3, 44.8, 38.2.
Example 65: 3-[3-(2-Chloro-ethyl)-ureido]-phenyl]-carboxamide (62);
O
H2N XI
H H 1H NMR (DMSO-d6) δ: 8.92 (brs, NH2, 2H), 7.89 (brs, IH, NH), 7.83 (s, IH, Ar), 7.40 (d, IH, J = 7.9), 7.32 (m, 2H), 6.57 (brs, NH, IH), 3.69 (m, 4H). 13C NMRδ: 168.1, 155.1, 140.4, 135.1, 128.5, 120.5, 120.1, 117.3, 44.4, 41.3.
Example 66: l-(2-Chloro-ethyl)-3-(3-heptyl-phenyl)-urea (63);
Figure imgf000098_0001
1H NMR: 7.83 (brs, NH, IH), 7.08 (m, 3H), 6.79 (d, Ar, IH, J = 7), 6.23 (brs, NH, IH), 3.50 (m, 6H), 2.48 (t, Ar, 2H, J = 7), 1.21 (m, 8H). 13C NMR δ: 156.0, 144.0, 138.9, 128.8, 123.3, 120.0, 117.2, 61.1, 44.7, 43.9, 42.7, 41.9, 36.0, 31.7, 31.4, 29.3.
Example 67: 5-{3-[3-(2-Chloro-ethyι)-ureido]-phenyl}-pentanoic acid amide (64);
Figure imgf000098_0002
lB NMR (DMSO-d6) δ: 8.03(brs, NH, IH, 7.11 (m, Ar, 3H), 6.69 (d, Ar, IH, J = 7), 6.21 (brs, NH, IH), 3.47 (m, 4H), 3.34 (m, 2H), 2.67 (t, CH2, 2H, J = 7), 2.53 (m, 2H), 2.39 (t, CH2, 2H, J = 7). 13C NMR δ: 172.3, 155.8, 147.9, 137.5, 130.3, 125.6, 124.7, 122.9, 58.3, 44.9, 41.2, 33.9, 29.3, 15.0.
Example 68: Pentanedioic acid mono-{3-[3-(2-chloro-ethyl)-ureido]-phenyl} ester (65);
Figure imgf000098_0003
1H NMR (Acetone-d6) δ: 8.23 (brs, NH, IH), 7.23 (m, Ar, 3H), 6.69 (m, Ar, IH), 6.20 (brs, NH, IH), 3.67 (m, 2H), 3.52 (m, 2H), 2.66 (m, 2H), 2.44 (m, 2H), 2.04 (m, 2H). 13C NMR (Acetone- d6) δ: 174.1, 171.9, 155.7, 152.2, 142.3, 129.9, 115.8, 115.6, 112.4, 44.7, 42.4, 33.6, 33.0, 20.8.
Example 69: l-(2-Chloro-ethyl)-3-(3-cyano-phenyl)-urea (66);
Figure imgf000099_0001
1H NMR (DMSO-d6) δ: 8.92 (brs, NH, IH), 7.76 ( s, Ar, IH), 7.40 (d, Ar, IH, j = 6.7), 7.18 ( d, Ar, IH, J = 7.2), 6.43 (brs, Nh, IH), 3.51 (t, CH2, 2H, J = 7.0), 3.47 (t, CH2, 2H, J = 7.0). 13C NMR δ: 154.8, 141.2, 130.0, 123.9, 122.6, 120.8, 118.9, 111.4, 44.8, 41.4.
Example 70: 3-[3-(2-Chloro-ethyl)-ureido]-benzoic acid (67);
Figure imgf000099_0002
1H NMR (DMSO-d6) δ: 12.87 (brs, OH, IH), 8.91 (brs, NH, IH), 8.08 ( s, Ar, IH), 7.68 (d, Ar, IH, J = 7.3), 7.61 (d, Ar, IH, J = 7.5), 7.36 (m, Ar, IH), 6.48 (brs, Nh, IH), 3.68 (m, CH2, 2H), 3.45 m, CH2, 2H. 13C NMR δ: 167.4, 155.0, 140.6, 131.3, 128.9, 122.1, 121.9, 118.5, 44.3, 41.3.
Example 71: 5-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-pentanoic acid methyl ester (68);
Figure imgf000099_0003
1H NMR (CDC13) δ: 8.21 (brs, NH, IH), 7.07 (m, Ar, IH), 6.57 (m, Ar, 3H), 6.17 (brs, NH, IH), 3.66, (s, CH3, 3H), 3.51 (m, CH2, 4H), 2.58 (m, CH2, 2H), 2.33 (t, CH2, 2H, J = 7.0), 1.65 (m, CH2, 2H), 1.25 (m, CH2, 2H). 13C NMR δ: 174.1, 155.3, 146.3, 1434, 129.2, 118.8, 115.3, 112.8, 51.3, 44.7, 41.8, 35.5, 34.2, 30.4, 24.6.
Example 72: l-(2-Chloro-ethyl)-3-(3-hexyl-phenyl)-urea (69);
Figure imgf000100_0001
1H NMR (CDC13) δ: 7.58 (brs, NH, IH), 7.11 (m, Ar, 3H), 6.79 (d, Ar, IH, J = 69), 3.53 (m, CH2, 4H), 3.46 (m, CH2, 4H), 2.48 (m, CH2, 2H), 1.20 (m, CH2, 8H). 13C NMR δ: 156.6, 144.0, 138.8, 128.8, 123.3, 120.0, 117.2, 61.1, 44.7, 43.9, 42.7, 41.9, 36.0, 31.8, 31.4, 29.3, 29.1, 22.6.
Example 73: 6-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-hexanoic acid amide (70);
Figure imgf000100_0002
1H NMR (DMSO-d6) δ: 8.21 (brs, NH, IH), 7.38 (m, Ar, 2H), 6.55 (m, Ar, 2H), 6.38 (brs, NH, IH), 3.45 (m, CH2, 6H), 2.56 (m, CH2, 2H), 2.23 (m, CH2, 4H). 13C NMR δ: 172.3, 154.8, 137.4, 129.2, 123.7, 121.1, 117.2, 60.1, 44.7, 42.1, 35.9, 32.4, 30.9, 23.8.
Example 74: 6-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-hexanoic acid ethyl ester (71);
Figure imgf000100_0003
1H NMR (CDC13) δ: 7.14 (m, Ar, NH, 4H), 6.86 (d, Ar, IH, J = 7,2), 5.66 (brs, NH, IH), 4.10 (q, CH2, 2H, J = 7.0), 3.59 (m, CH2, 4H), 2.54 (m, CH2, 2H), 2.27 (m, CH2, 2H), 1.62 (m, CH2, 4H), 135 (m, CH2, CH3, 5H). 13C NMR δ: 174.9, 155.9, 143.9, 138.4, 129.1, 124.0, 120.9, 118.3, 60.3, 44.8, 42.0, 35.6, 34.3, 30.9, 29.7, 28.7, 24.8, 14.2.
Example 75: 6-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-hexanoic acid (72);
Figure imgf000101_0001
1H NMR (Acetone-d6) δ: 8.17 (brs, NH, IH), 7.31 (m, Ar, IH), 7.12 (m, Ar, 2H), 6.79 (d, Ar, IH, J = 7.4), 6.23 (brs, Nh, IH), 3.64 (m, CH2, 2H), 3.54 (m, CH2, 2H), 2.55 (m, CH2, 2H), 2.20 (m, CH2, 2H), 1.71 (m, CH2, 4H), 1.22 (m, CH2, 2H). 13C NMR δ: 174.1, 156.1, 143.9, 141.1, 129.2, 122.6, 119.2, 116.8, 44.9, 44.8, 42.5, 36.3, 34.1, 31.8, 30.6, 30.2, 25.4.
Example 76: 6-{3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-hexanoic acid methyl ester (73);
Figure imgf000101_0002
1H NMR (CDC13) δ: 8.11 (brs, Nh, IH), 7.48 (m, Ar, 2H), 6.51 (m, Ar, 2H), 6.38, (brs, Nh, IH), 3.57 (m, CH2, 6H), 3.81 (s, CH3, 3H), 2.51 (m, CH2, 2H), 2.17 (m, CH2, 4H). 13C NMR δ: 174.1, 155.3, 138.3, 131.7, 124.2, 121.1, 116.8, 51.4, 44.6, 41.9, 35.9, 32.6, 30.8, 23.6.
Example 77: 3-[3-(2-Chloro-ethyl)-ureido]-benzoic acid methyl ester (74);
Figure imgf000101_0003
1H NMR (CDC13) δ: 8.21 (brs, NH, IH), 7.87 (s, Ar, IH), 7.61, Ar, IH, J = 7.0), 7.58 (d, Ar, IH, J = 7.), 7.21 brs, Nh, IH), 3.76 (s, CH3, 3H), 3.57 (m, CH2, 4H). 13C NMR δ: 167.1, 156.2, 139.3, 131.1, 129.0, 125.0, 123.9, 120.4, 52.2, 46.4, 42.0.
Example 78: l-(2-Chloro-ethyl)-3-[3-(4-hydroxy-but-l-ynyl)-phenyl]-urea (75);
Figure imgf000102_0001
1H NMR (Acetone-d6) δ: 8.21 (brs, NH, IH), 7.03 (m, Ar, IH), 6.79 (d, Ar, IH, J = 7.0), 6.71 (s, Ar, IH), 6.57 (d, Ar, IH, J = 7.9), 6.23 (brs, NH, IH), 3.71 (m, CH2, 6H), 3.45 (m, CH2, 2H) 2.601 (t, CH2, 2H, J = 7.0). 13C NMR δ: 155.8, 146.3, 129.3, 124.1, 122.1, 118.2, 115.2, 86.2, 82.5, 61.1, 44.7, 42.3, 23.7.
Example 79: l-(2-Chloro-ethyl)-3-[3-(3-hydroxy-prop-l-ynyl)-phenyl]-urea (76);
Figure imgf000102_0002
1H NMR (Acetone-d6) δ: 8.06 (brs, NH, IH), 7.08 (m, Ar, IH), 6.83 (d, Ar, IH, J = 7.5), 6.75 (s, Ar, IH), 6.63 (d, Ar, IH J = 7.9), 3.57 (m, CH2, 4H), 2.04 (s, CH2, 2H). 13C NMR δ: 155.2, 129.8, 123.3, 123.1, 118.0, 115.6, 86.7, 85.9, 51.6, 44.8, 42.3.
Example 80: 3-[3-(2-Chloro-ethyl)-ureido]-phenyl}-acetic acid ethyl ester (77);
Figure imgf000102_0003
1H NMR (CDC13) δ: 7.89 (brs, NH, IH), 7.17 (s, Ar, IH), 7.08 (m, Ar, 2H), 6.81 (d, Ar, IH, J = 7.9), 4.09 (q, CH2, 2H, J = 7,0), 3.46 (m, CH2, 6H), 1.21 (t, CH3, 3H, J = 7.0). 13C NMR δ: 172.1, 156.2, 139.2, 134.9, 129.7, 123.8, 120.8, 118.6, 61.1, 44.4, 42.0, 41.8, 14.1.
Example 81: Acetic acid 3-{3-[3-(2-chloro-ethyl)-ureido]-phenyl}-propyl ester (78).
Figure imgf000103_0001
1H NMR (CDC13) δ: 7.57 (brs, NH, IH), 7.21 (m, Ar, 3H), 6.79 (m, Ar, IH), 5.91 (m, Ar, IH), 3.57 (m, CH2, CH3, 9H). 13C NMR δ: 172.5, 156.0, 139.1, 134.7, 129.2, 124.0, 120.9, 118.8, 52.1, 44.4, 40.9, 40.3.
EXAMPLE 82: Cell Proliferation Assays
The compounds can be assayed initially for their ability to inhibit cell growth (i.e. their cytotoxicity) in vitro using standard techniques, hi general, cells of a specific test cell line are grown to an appropriate density (e.g. approximately 1 x 104) and the candidate compound is added. After an appropriate incubation time (for example 48 to 74 hours), cell survival is assessed, for example, by using the resazurin reduction test (see Fields & Lancaster (1993) Am. Biotechnol. Lab. 11:48-50; O'Brien et al, (2000) Eur. J. Biochem. 267:5421-5426 and U.S. Patent No. 5,501,959), the sulforhodamine assay (Rubinstein et al, (1990) J. Natl. Cancer Inst. 82:113-118) or the neutral red dye test (Kitano et α/., (1991) Euro. J. Clin. Investg. 21:53-58; West et al, (1992) J Investigative Derm. 99:95-100). Inhibition of cell growth is determined by comparison of the cell survival in the treated culture with the cell survival in one or more control cultures, for example, cultures not pre-treated with the candidate compound and/or those pre- treated with a control compoimd (typically a known therapeutic).
The candidate compounds can also be tested in vitro for their ability to inhibit anchorage- independent growth of tumour cells. Anchorage-independent growth is known in the art to be a good indicator of tumourigenicity. In general, anchorage-independent growth is assessed by plating cells from an appropriate cancer cell-line onto soft agar and determining the number of colonies formed after an appropriate incubation period. Growth of cells treated with the compound can then be compared with that of cells treated with an appropriate control (as described above).
EXAMPLE 83: Cytotoxicity of Compounds of Formula I in Various Cancer Cell Lines
Cells were grown in an appropriate medium. Cell growth inhibition was assessed using a modified Alamar Blue assay as described by Lancaster et al. (U.S. Patent No. 5,501,959). For the proliferating state, cells were seeded in 96-well plates and preincubated for 24h (or 72 hours for the quiescent state). After addition fresh medium containing increasing concentrations of the candidate compound, cells were incubated at 37°C for 48 hours. The culture medium was removed, cells were washed and contacted with a resazurin solution. Cell survival was calculated from fluorescence (excitation, 485 mn; emission, 590 nm) measured with a FL 600 Reader (BioTek Instruments). Cell growth inhibition was expressed as the dose of drug required to inhibit cell growth by 50%) (GI50). Values are the means of at least three independent detenninations.
Table 2: hihibition of Cell Growth by Various Compounds of Formula I
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0002
1 GI50 is the dose required to inhibit cell growth by 50%. Different values obtained from different synthetic batches of compound.
Figure imgf000107_0001
EXAMPLE 84: MDA-MB-231 Cell Growth Inhibition by Compounds of the Invention The human tumour cell line MDA-MB-231 was grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. Cells were inoculated into 96 well microtiter plates in 100 μL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of the cell line. After cell inoculation, the microtiter plates was incubated at 37° C, 5 % CO2, and 100 % relative humidity for 24 h prior to addition of experimental drugs. After 24 h, two plates of each cell line were fixed in situ with trichloro acetic acid (TCA), to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drugs were solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration. An aliquot of concentrate was diluted to twice the desired final maximum test concentration with complete medium. Additional seven Y serial dilutions were made to provide a total of eight drug concentrations plus control. Aliquots of 100 μL of these different drug dilutions were added to the appropriate microtiter wells already containing 100 μL of medium, resulting in the required final drug concentrations. Following drug addition, the plates were incubated for an additional 48 h at 37°C, 5 % CO2, and 100 %> relative humidity. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 μl of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded, and the plates were washed five times with tap water. Sulforhodamine B (SRB) solution (100 μL) at 0,1% (w/v) in 1 % acetic acid (v/v) was added to each well, and plates were incubated for 10 minutes at room temperature. After staining, unbound dye was removed by washing five times with 1 %> acetic acid (v/v) and the plates were air dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was read on plate reader at a wavelength of 575 mn. Using the absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], growth inhibition of 50 % (GI50) was calculated from [(Ti- Tz)/(C-Tz)] x 100 = 50. GI50 was the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation.
Table 3: MDA-MB-231 Cell Growth Inhibition by Compounds of the Invention
Figure imgf000109_0001
1 GI50 is the dose required to inhibit cell growth by 50%.
EXAMPLE 85: Inhibition of tumour cell growth by the compounds of the instant invention in a dose- and time- dependent manner.
M21 and HT1080 tumour cell lines were inoculated into 96 well tissue culture plates in 100 μL containing 2 X 103 cells and were incubated at 37 °C. After 24 h, freshly solubilized drugs in DMSO were diluted in fresh medium. Aliquots of 100 μl containing escalating concentration of drugs (0.3 μM to 100 μM; A) compound 1, B) compound 2, C) tBEU and D) cDDP) were added to the appropriate microtiter wells already containing 100 μl of culture medium. The cells were incubated for different period of time ranging from 3 h to 48 h. The supernatant was removed, the cells were washed and incubated with fresh medium to complete the total incubation time to 48 h for each condition. Assays were stopped by addition of cold TCA to the wells (final concentration, 10 %), followed by their incubation for 60 min at 4 °C. The supernatant was discarded and the plates were washed five times with tap water and air-dried. Sulforhodamine B solution (50 μl) at 0.1 % (w/v) in 1 % acetic acid was added to each well, and plates were incubated for 15 min at room temperature. After staining, unbound dye was removed by washing five times with 1 % acetic acid and the plates were air-dried. Bound stain was solubilized with 10 mM trizma base, and the absorbance was read using a μQuant Universal Microplate Specfrophotometer (Biotek, Winooski, VT) at 585 nm. The results were compared with those of a control reference plate fixed on the freatment day and the growth inhibition percentage was calculated for each drug contact period. The growth inhibition percentage is expressed as the mean of triplicates for each drug contact period, compared with those of a control reference plate fixed on the day of the treatment.
The cytotoxicity of the test compounds was compared with that of a classical and strong alkylating agent, namely cisplatin (cDDP). The antimicrotubule agents colchicine, vinblastin and paclitaxel were also tested in this assay but was found not cytotoxic until they reach 48 h of exposure (data not shown). When they were in contact for less than 6 h with either cell lines, virtually none of the tested compounds showed inhibition of tumour cell growth and proliferation. However, as the time of contact between the test compounds and tumour cells was increased from 6 to 48 h, the comparable GI50 of compound 1 and 5 markedly shifted to the left hand-side of the graph (Figure 1 A and B) and this was shown in the low micromolar range for all tumour cell lines tested. This was strikingly different from the cDDP effect, which displayed cytotoxicity after only 3 h of treatment, with a GI50 that was essentially in the same range for all time of contact tested (Figure ID). Interestingly, at all concentrations tested, the non-alkylating compound 1 showed no apparent cytotoxicity (Figure IC), suggesting that the chemical alkylating property of the test compounds of the instant invention was essential for their cytotoxicity. Overall, soft alkylating haloethyl urea compounds were as cytotoxic as cDDP. However they require a longer time of contact to display their proliferative inhibitory activity, which is compatible to the incubation time of other anti-antimicrotubule agents tested, such as colchicine and paclitaxel (data not shown). EXAMPLE 86: Microtubule depolymerization and cytoskeleton disruption induced by compounds of the instant invention
M21 cells were seeded at lxlO5 cells in in 35-mm Petri dishes and incubated for 16 h at 37 °C. Cells were treated for 24h with either 100 μM of compounds 1 and 5 or tBEU or classical antimicrotubule agents (50μM of cisplatin (cDDP), 25 μM of colchicine (COL), 5 μM of vinblastine (VINB) or 50 μM of paclitaxel (TAX)). Following the treatment, the cells were washed twice with phosphate-buffered saline (PBS, pH 7.4) and then fixed with 3.7 % formaldehyde in PBS for 20 min. After two washes, the cells were penneabilized and blocked with 0.1 % saponin and 3 % (w/v) BSA in PBS, during 1 h at 37 °C. The cells were then further incubated during 1 h at 37 °C with anti-tubulin (clone TUB2.1, that is specific to β-tubulin and does not cross-react with other-tubulin isoforms; Sigma-Aldrich; St-Louis, MO) (1: 200) in 0.1 % saponin and 3 % BSA in PBS. The cells were washed three times with PBS containing 0.05 % of Tween 20 and incubated 1 h at 37 °C in blocking buffer containing anti-mouse IgG Alexa-488 (1 : 1000), DAPI (2.5 μg/ml in PBS) to stain nuclei and Rhodamine-labeled phalloidin (1 : 600) to stain the actin cytoskeleton. The observations were made using a Nikon Eclipse E800 microscope (Tokyo, Japan) equipped with a 40X objective. Images were captured as 16 bit TIFF files with a Hamamatsu ORCA ER cooled (-20°C) digital camera (Photonics Management Management Corp., Bridgewater, N.J.) driven by SimplePCI AIC software (Compix Inc. C Imaging systems, Pennsylvania). Representative fields are shown from three separate experiments.
In comparison with untreated cells, 100 μM of compound 1 during 24 h considerably affected the β-tubulin fibers (Figure 2) showing a punctated β-tubulin staining that lead to a microtubule depolymerization phenotype. This was established by a comparison with a 24 h cells exposure to paclitaxel (50 μM), colchicine (25 μM), or to vinblastine (5 μM), classical antimicrotubule agents having opposite effect on microtubule network by their non-covalent binding to β-tubulin. Paclitaxel stabilizes the microtubules, thus inhibiting their depolymerization, whereas the others rather blocking their polymerization, inducing therefore a depolymerization phenotype (Figure 2). In fact, the compound 1 effect on β-tubulin was indeed drastically different from what was observed after paclitaxel treatment, not as severe as vinblastine, but rather similar to that observed after colchicine cell exposure. As expected, the bioisosteric derivative compound 5 showed a similar microtubule dissolution activity as compound 1. On the contrary, tBEU did not exhibit any effect on the microtubule network nor did affected the filamentous structure of actin. However, compounds 1 and 5, colchicine and vinblastine considerably decreased the amounts of filamentous actin in both cell lines, presumably as an indirect consequence of β-tubulin depolymerization, showing a more punctate actin distribution. This collapse of the actin structure was not observed in response to paclitaxel. Interestingly, the toxic effect of cDDP on β- tubulin and on actin filaments seemed rather associated to its pro-apoptotic mechanism, actin and microtubules being dissolved only in cells showing a typical apoptotic nuclear fragmentation phenotype (Deschesnes et al., Mol. Biol. Cell. 12:1569-1582 (1996); Desbiens et al., Biochem. J. 372:631-641 (2003). Stress fibers were rather observed in cDDP-treated cells that are still non- apoptotic. This was still contrasting with CEUs that were rather inducing a non-classical nuclear condensation phenotype consequently or in parallel to their microtubules disruption effect.
EXAMPLE 87: Generation of an alkylated form of β-tubulin by compound 1 and compound 5.
To confirm that compound 5 was as active as compound 1 to bind to β-tubulin, western blot analysis of β-tubulin was carried out, as reported by Legault et al. (Cancer Res. 60:985-992 (2000)) procedure. Briefly, M21 or MDA-MB-231 cells were seeded at 5 X 105 cells in 6-well plates and incubated for 16 h. The cells were treated with 50 μM of compound 1, compound 5 or tBEU and 50 μM of cDDP, 25 μM of COL, 5 μM of VINB or 50 μM of TAX for 6, 12, 24, 30 or 48 h. After the treatments with tested drugs, floating and adherent M21 cells or MDA-MB-231 cells were washed in ice-cold PBS, pooled and then solubilized in buffer containing 62.5 mM Tris, pH 6.8, 2 % SDS, 6 M urea, 10 % glycerol, 0.00125 % bromophenol blue, and 720 mM β- mercaptoethanol. The cell extracts were boiled for 5 min, separated on 10 % SDS-PAGE electrophoresis gel and transfeπed onto nitrocellulose membrane. The membranes were blocked for 1 h at 37 °C with 5 % (w/v) milk in Tris buffered saline (TBS) containing 0.1 % Tween 20 (TBST) and then incubated, 1 h at 37 °C, with the appropriate antibody diluted in 5 % milk in TBST. The apparition of an additional immunoreactive band of β-tubulin was evaluated with the monoclonal anti-tubulin antibody (1: 500). Membranes were incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1: 2500) (Amersham Canada, Oakville, Canada) diluted in 5 % milk in TBST, 1 h at room temperature, followed by chemiluminescent detection, using an enhanced chemoluminescence (ECL) detection kit (Amersham Pharmacia Biotech).
Legault et al. (Cancer Res. 60:985-992 (2000)) initially reported that the cell incubation with [urea-14C]- compound 1 revealed the apparition of a radioactive protein on SDS-PAGE, that exactly coincided with a second immunoreactive band of β-tubulin monomer, detected by western blotting, demonstrating the covalent binding of 14C- compound 1 to β-tubulin. Here, the time-dependent detection of this additional immunoreactive band in M21 (Figure 3) and MDA- MB-231 cells (data not shown) was confirmed, h line with this previous study compound 1 and compound 5 was shown to generate a band that is presumed to be the result of β-tubulin alkylation (Figure 3). This second band was however not observed after cell exposure to tBEU and cDDP, nor after treatment with paclitaxel, colchicine or vinblastine, presumably due to their non-covalent binding to β-tubulin. Hence, our results confirmed that compound 1 and its bioisosteric derivative, compound 1, are both potent anti-antimicrotubule and soft alkylating agents, that covalently bind to β-tubulin, inducing a microtubule depolymerization phenotype.
EXAMPLE 88: Generation of an alkylated form of β-tubulin by exemplary compounds 1, (R)24; S(24); 28; 5; (R)23; 81, 82, 83 and 84 of the invention
Figure imgf000114_0001
81 82
Figure imgf000114_0002
83 84
Cell Culture. Human breast carcinoma cell line, MDA-MB-231 was grown in RPMI 1640 medium supplemented with 10% bovine calf serum iron supplemented (Hyclone, Road Logan, Utah) in a humidified atmosphere at 37°C in 5% CO2. After trypsinization with 1 mL trypsin- EDTA, cells were seeded in 12 well-plates. Each well received around 220 000 cells. After an incubation of 24 hours, the culture medium was removed and drugs, diluted in fresh medium, added.
Drugs. Each of the ten drugs was dissblved in dimethyl sulfoxide to yield a 40mM stock solution. An aliquot of stock solution is mixed with the culture to a final volume concentration of 30 μM. Eight incubation times for each drug was performed, i.e. 0, 1, 2, 4, 8, 12, 24 and 48 hours. After each incubation time, the medium of each well is collected. 1 mL of cold PBS is added to each well. Remaining cells are scratched from the well surface, centrifugated and washed anew in 500 μL of cold PBS. Both cell aliquots are mixed.
Cell suspension. 100 μL from the cell suspension was pipetted and used to determine cell concentration with NaOH/DO280. The remaining cell solution was centrifugated to obtain a pellet. The pellet is brought to a concentration of 60 000 cells/35 μL in a solution Laemmli IX + 5 % Beta-mercaptoethanol, resuspended, sonicated 5 seconds, boiled 5 minutes and then centrifugated. SDS-PAGE Analysis and Immunoblotting of /3-tubulin. Samples (60 000 cells) were analyzed by 10% SDS-PAGE. Gels were transferred to a nitrocellulose membrane. Membranes were then incubated with PBSMT [PBS (pH 7.4), 5% fat-free dry milk, and 0.1% Tween-20]. After Ponceau staining, the membrane is immersed with a solution of 1:500 monoclonal anti-/3-tubulin (clone TUB 2.1, Sigma) for 1 h at room temperature. Membranes were washed with PBSMT and incubated with 1:2500 peroxidase-conjugated antimouse imnrunoglobulin (Amersham Canada, Oakville, Canada) in PBSMT for 30 min. Detection of the immunoblot was carried out with the ECL Western blotting detection reagent kit (Amersham Canada, Oakville, Canada).
The results are presented in Figure 4A.
EXAMPLE 89: Generation of an alkylated form of β-tubulin by exemplary compounds 1; (R)24; (S)24; (R) 85; (S) 85, 5; (R)23; (S)23; (R) 86, (S) 86 of the invention (II)
Figure imgf000115_0001
(R) 85 (S) 85
Figure imgf000115_0002
(R) 86 (R) 86
Cell Culture. Human breast carcinoma cell line, MDA-MB-231 was grown in RPMI 1640 medium supplemented with 10%> bovine calf serum iron supplemented (Hyclone, Road Logan, Utah) in a humidified atmosphere at 37°C in 5% CO2. After trypsinization with 1 mL trypsin- EDTA, cells were seeded in 12 well-plates. Each well received around 220 000 cells. After an incubation of 24 hours, the culture medium was removed and drugs, diluted in fresh medium, added. Drugs. Each of the ten drugs was dissolved in dimethyl sulfoxide to yield a 40mM stock solution. An aliquot of stock solution is mixed with the culture to a final volume concentration of 30 μM. Eight incubation times for each drug was performed, i.e. 0, 8, 12, 16, 24, 32, 40 and 48 hours. After each incubation time, the medium of each well is collected. 1 mL of cold PBS is added to each well. Remaining cells are scratched from the well surface, centrifugated and washed anew in 500 μL of cold PBS. Both cell aliquots are mixed.
Cell suspension. 100 μL from the cell suspension was pipetted and used to determine cell concenfration with NaOH/DO280. The remaining cell solution was centrifugated to obtain a pellet. The pellet is brought to a concentration of 60 000 cells/35 μL in a solution Laemmli IX + 5 % Beta-mercaptoethanol, resuspended, sonicated 5 seconds, boiled 5 minutes and then centrifugated.
SDS-PAGE Analysis and Immunoblotting of -tubulin. Samples (60 000 cells) were analyzed by 10% SDS-PAGE. Gels were transfeπed to a nitrocellulose membrane. Membranes were then incubated with PBSMT [PBS (pH 7.4), 5% fat-free dry milk, and 0.1% Tween-20]. After Ponceau staining, the membrane is immersed with a solution of 1:500 monoclonal anti- 3-tubulin (clone TUB 2.1, Sigma) for 1 h at room temperature. Membranes were washed with PBSMT and incubated with 1:2500 peroxidase-conjugated antimouse immunoglobulin (Amersham Canada, Oakville, Canada) in PBSMT for 30 min. Detection of the immunoblot was carried out with the ECL Western blotting detection reagent kit (Amersham Canada, Oakville, Canada).
The results are presented in Figure 4B.
EXAMPLE 90: Generation of an alkylated form of β-tubulin by exemplary compounds 53; 57; 30; 63, 31, 28, 66, 47, 49, 68, 69 and 87 of the invention
Figure imgf000117_0001
87 Cell Culture. Human breast carcinoma cell line, MDA-MB-231 was grown in DMEM 1640 medium supplemented with 10% bovine calf serum iron supplemented (Hyclone, Road Logan, Utah) in a humidified atmosphere at 37°C in 5%> CO2. After trypsinization with 1 mL trypsin- EDTA, cells were seeded in 6 well-plates. Each well received around 220 000 cells. After an incubation of 24 hours, the culture medium was removed and drugs, diluted in fresh medium, added.
Drugs. Each of the ten drugs was dissolved in dimethyl sulfoxide to yield a 40mM stock solution. An aliquot of stock solution is mixed with the culture to a final volume concentration of 5 μM. Eight incubation times for each drug was performed, i.e. 2, 7, 17, 25 and 48 hours. After each incubation time, the medium of each well is collected. 1 mL of cold PBS is added to each well. Remaining cells are scratched from the well surface, centrifugated and washed anew in 500 μL of cold PBS. Both cell aliquots are mixed.
Cell suspension. 100 μL from the cell suspension was pipetted and used to determine cell concentration with NaOH/DO280. The remaining cell solution was centrifugated to obtain a pellet. The pellet is brought to a concentration of 100 000 cells/25 μL in a solution Laemmli IX + 5 % Beta-mercaptoethanol, resuspended, sonicated 5 seconds, boiled 5 minutes and then centrifugated.
SDS-PAGE Analysis and Immunoblotting of -tubulin. Samples (100 000 cells) were analyzed by 10% SDS-PAGE. Gels were transferred to a nitrocellulose membrane. Membranes were then incubated with PBSMT [PBS (pH 7.4), 5% fat-free dry milk, and 0.1% Tween-20]. After Ponceau staining, the membrane is immersed with a solution of 1:500 monoclonal anti- 3- tubulin (clone TUB 2.1, Sigma) for 1 h at room temperature. Membranes were washed with PBSMT and incubated with 1:2500 peroxidase-conjugated antimouse immunoglobulin (Amersham Canada, Oakville, Canada) in PBSMT for 30 min. Detection of the immunoblot was carried out with the ECL Western blotting detection reagent kit (Amersham Canada, Oakville, Canada).
The results are presented in Figure 4C.
Example 91: Inhibition of MDA_MB-231 Cancer Cell Migration in the Wound Assay by Compounds of the Invention
MDA-MB-231 cells were grown in DMEM plus 5% FBS (Hyclone, Road Logan, Utah) and L-glutamine without antibiotics. Cells were seeded at 3xl05 cells in 6 well plate and grown to confluence. 16 hours prior the experiments, the different drugs were diluted in fresh medium and added to the wells. For a series of measurements, the concentrations selected were 0,5, 3 and 5 μM. For the other series of measurements, the concentrations selected were 0,25, 0,5 and 1 μM. The following day, the tip of a cell scraper was used to scratch the bottom of each well, producing a "wound" of roughly 1000 μm. The medium was washed to remove the detached cells, and two pictures per well were taken at 0, 3, 6, 8 and 24 hours. By superimposing a hemacytometer grid over the picture, the distance between the two fronts was measured at numerous positions. Migration velocity was determined by a graph of the "wound" front distance over time. The assay was repeated at least twice for each drug, at each concentration. For IMO- 236, the concentration at 3 and 5 μM were too toxic for the cells.
Results are presented in Figure 5. Of note, two compounds 75 and 76 which did not show a GI50 (see table 3 in Example 84) smaller than lOμm significantly reduced cell mobility.
EXAMPLE 92: Inhibition of HT1080 tumour cell migration by compounds 1 and 5.
The chemotaxis motility of HT1080 was assessed using Boyden chambers. Briefly, the underside of Transwell™ migration chamber membranes (8.0- mm pore size) were coated with collagen IV as described previously (filardo et al., J. Cell Biol. 130:441-450 (1995); Kle ke et al. J. Cell Biol. 127:859-866 (1994) and as modified by Petitclerc et al. (Petitclerc et al. J. Biol. Chem. 275:8051-8061 (2000)). The cells were pre-incubated or not for 16 h with escalating concentrations of different compounds of the invention, then they were added to the top of a collagen IN coated Transwell™ membrane (8.0 mm pore size), separating the lower and upper part of a Boyden chamber in the presence of same drugs. Soluble fibronectin (25 μg/ml) was added to the lower chamber to induce chemotaxis. The cells were allowed to migrate for 4-6 h at 37 °C, fixed and stained for quatification. The number of migrating cells per well were counted. The results expressed the mean ± s.e. of triplicates.
In contrast to the effect of tBEU on HT1080 cells (Figure 6), 50 μM of ompound 1 or compound 5 (Figure 6) induced a strong and reproducible inhibition of cell migration with an overall efficiency of 70 % for either drugs. Because the antiproliferative effect of the compounds of the instant invention depends on the time of contact, the migration assays were conducted with either 0 or 16 hours pretreatments. The inhibitory effect of the test compounds was essentially the same for both conditions for all drugs, indicating that the antimigrational action of the compounds of the instant invention is an early mechanism of action.
EXAMPLE 93: The compounds of the instant invention impede the growth of two unrelated tumour cell lines in the chick chorioallantoic membrane (CAM) assay.
Human HT1080 fibrosarcoma and hamster CS1 melanoma cell lines were used to assess the antitumoural activity of CEUs in the chick chorioallantoic assay (Petitclerc et al. J. Biol. Chem. 275:8051-9061 (2000); Kim et al. Cell 94:353-362 (1998); Lyu et al. Int. J. Cancer 77:257-263 (1998)). In brief, day-0 fertilized chicken eggs were purchased from Couvoirs Victoriaville (Victoriaville, QC, Canada). The eggs were incubated for 10 days in a Pro-FI egg incubator fitted with an automatic egg turner, before being transferred to a RoU-X static incubator for the rest of the incubation time (incubators were purchased from Lyon Electric, Chula Vista, San Diego). The eggs were kept at 37 °C in a 60 % humidity atmosphere for the whole incubation period. Using a hobby drill (Dremel; Racine, WI), a hole was drilled on the side of the egg, and a negative pressure was applied to create a new air sac. A window was opened on this new air sac and was covered with transparent adhesive tape to prevent contamination. A freshly prepared cell suspension (40 μl) of either HT1080 (3.5 x 105 cells/egg) or CS1 (5 x 106 cells/egg) cells was applied directly on the freshly exposed CAM tissue through the window. On day 11, the tested drugs were injected intravenously in 10-12 eggs in a small volume (100 μl). The eggs were incubated until day, at which time the embryos were euthanized at 4 °C, followed by decapitation. Tumours were collected, pictures were taken to illustrate the different groups and the tumour-wet weights were recorded. In all experiments, the number of dead embryos from the different groups was monitored for any sign of toxicity.
Panel A and B of Figure 7 show that incubation of CS1 -derived tumours on the CAM with compound 1 or compound 5 resulted in a significant dose-dependent reduction of the tumours size, as observed also with cDDP (Figure 7D). Moreover, both test compounds also inhibited the formation of HT1080 tumour mass in the same concentration range (data not shown). It is noteworthy that the non-alkylating homologue tBEU failed to influence the growth of CS1 tumours (Figure 7C), supporting the assumption that the antitumoural effect of compound 1 and compound 5 was dependent on their alkylating activity. In the same experimental settings, only 10 μg/egg of cDDP was sufficient to inhibit tumour cell growth. However, a high level of chick embryo toxicity was observed at higher concentration, since 95 to 100 % of embryos died at a cDDP concentration reaching 50 μg/egg (Figure 7). By contrast, the antitumoural effect of compound 1 and compound 5 was shown at doses that were well tolerated by the chick embryo, as we monitored by chick necropsy (up to 150 μg/egg, data not shown).
Cumulative results are shown from three independent experiments. ANOVA test showed a significant difference between doses (p< 0.01). Dunnett test was performed (* p < 0.05, ** p< 0.01). * indicates 95-100 % chick embryos mortality in the group when using 25 μg/ml cDDP. hi panel A), the black circle corresponds to the injection of the solvent used to solubilize the drugs.
EXAMPLE 94: Compounds 1, 2, 5 and 30 impede the growth of CS1 tumour cell line in the chick chorioallantoic membrane (CAM) assay.
The ability of compounds 1, 2, 5 and 33 to impede the growth of CS1 tumour cell line was determined using the chick chorioallantoic membrane (CAM) assay as described in Example #. The results are present in figure 8. EXAMPLE 95: Clonogenic survival of M21 cells on extracellular matrices (ECMs).
It was recently showed that the tumour microenvironment could modulate the tumour cells' ability to resist to chemotoxic agents such as cDDP, triggering a pro-survival signal through integrins (Hazlehurst et al. Oncogene 19:4319-4327 (2000); Rintoul et al. Clin. Sci. (Lond) 102:417-424 (2002): Damiano et al. Blood 93:1658-1667 (1999). To evaluate if this cell adhesion mediated-drug resistance (CAM-DR) mechanism may impede the cytotoxic effect of the compunds of the instant invention, the effect of purified ECMs on the outcome of CEU- treated tumour cells in clonogenic survival assay was determined and compared to the cytotoxic effect of cDDP.
Briefly, M21 skin melanoma cells were plated (1 X 10 cells in 100-nιm Petri dishes) on different ECMs and challenged with test compounds of the instant invention or the strong alkylating agent cDDP, as previously described (Deschesnes et al. Mol. Biol. Cell. 12:1569-1582 (2001)). Briefly, native and heat-denatured type IN collagen (50 μg/ml), fibronectin (25 μg/ml) and fibrin (50 μg/ml) were used to coat non-tissue culture plates (Νunc). After washes with serum-free DMEM, cells were plated in serum-containing or serum-free media on the different matrices for 16 h. Cells were then challenged with cDDP (50 μM) for 3 h or with test compounds (20 μM) for 24 h. Forthwith, the cells were washed, trypsinized and plated at appropriate dilutions ranging from 102 to 105 cells per well. The experiments were conducted in triplicate to have approximately 50-200 viable cells per dish Huot et al. Cancer Res. 56:273-279 (1996)). Relative survival was calculated from the number of single cells that formed colonies of 50 or more cells within 12 days. The survival data were coπected for the plating efficiency of the appropriate controls. One hundred percent is define relative to the number of colonies obtained with the same cells untreated. Representative results from two independent experiments performed in quadruplicates are shown.
Despite its in vitro and in vivo cytotoxic effect, the cytotoxicity of cDDP was markedly decreased by the presence of fibronectin matrix protein, as shown by the relative survival increased, estimated from the number of viable colonies that had resisted to treatments (Figure 10, left panel). Unexpectedly, fibronectin confeπed no protective effect against compound 1 and compound 5 treated-cells. Moreover, it was rather a sensitizing factor, thus enhancing the toxicity of the test compounds of the instant invention on tumour cells (Figure 10, center and right panel). Furthermore, similar results were obtained with a number of ECMs proteins, namely fibrinogen, fibrin and heat denatured type IV collagen (data not shown). Therefore, the compounds of theinstant invention show no sensitivity to CAM-DR that may rather sensitize tumour cells to the effects of these alkylating agents.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A compound having structural formula (I):
Figure imgf000123_0001
or a pharmaceutically acceptable salt thereof, wherein:
X is F, CL Br or l;
RI and R2 are each independently selected from the group of H, -R, -halo, -OR, - SR, -NRR, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NRR, -C(S)NRR, -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, or RI and R2 when taken together form =O, =S or a C3-C6 spiro group;
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
B is substituted with one or more substituents selected from the group of (d- e) alkyl, (C2-C16) alkenyl, (C2-C16) alkynyl, aryl, -O-(d-C16) alkyl, -O-(C2-C16) alkenyl, -O- (C2-Cι6) alkynyl, -O-aryl, -O-CH2-aryl, -S-(C1-Cι6)alkyl, -S-(C2-C16) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2-aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -halo, -NRR, -ONRR -NO2, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, - C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R,
-SC(S)R, -OC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: each R is independently selected from -H, (CrC16) alkyl, substituted (Cι.-C16) alkyl, (C -C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2- C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cycloalkyl, aryl or substituted aryl; the alkyl, alkenyl, alkynyl and aryl are optionally substituted with one or more substituents independently selected from the group of -halo, trihalomethyl, -R, -OR, - SR, -NR'R, -NO2, -CN, -OC(O)R, -OC(S)R, -SC(O)R, -SC(S)R -C(O)R, -C(S)R, - C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NR,R', -C(S)NR'R, -NR'C(O)R' and - NRC(O)OR; the cycloalkyl is optionally substituted with one or more substituents independently selected from the group of R', -halo, OR', -SR', -NRR', -ONRR', -NO2, - CN, -C(O)R', -C(S)R', -OC(O)R', -SC(O)R', -SC(S)R', -OC(S)R', -C(O)OR', -C(S)OR', - C(O)SR', -C(S)SR', -C(O)NRR', -C(S)NRR', -C(O)NR'(OR'), -C(S)NR' -(OR'), - C(O)NR'(SR'), -C(S)NR'(SR , -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, - CH[C(O)OR]2, -CH[C(S)OR']2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NR'C(O)R', - NR'C(O)OR', -S(O)-R', -S(O)OR', -S(O)2OR', -S(O)NRR', -S(O)ONRR', and each R' is independently selected from the group of -H, ( -C^) alkyl, substituted (Ci-C16) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C -C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl; for use as an therapeutic agent for attenuating, inhibiting or preventing cancer cell migration in a mammal in need of such therapy.
A compound having structural formula (I):
Figure imgf000124_0001
or a pharmaceutically acceptable salt thereof, wherein: X is F, CI, Br or I; RI and R2 are each independently selected from the group of H, -R, -halo, -OR, - SR, -NRR, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NRR, -C(S)NRR, -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, or RI and R2 when taken together form =O, =S or a C3-C6 spiro group;
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
B is substituted with one or more substituents selected from the group of (Cι-C16) alkyl, (C2-C16) alkenyl, (C2-C16) alkynyl, aryl, -O-(C1-Cι6) alkyl, -O-(C2-C16) alkenyl, -O- (C2-C16) alkynyl, -O-aryl, -O-CH2-aryl, -S-(d-C16)alkyL -S-(C2-C16) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2-aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -halo, -NRR, -ONRR -NO2, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, - C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R,
-SC(S)R, -OC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: each R is independently selected from -H, (d-C16) alkyl, substituted ( - β) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2- C16) alkynyl, (C -C8) cycloalkyl, substituted (C3-C8) cycloalkyl, aryl or substituted aryl; the alkyl, alkenyl, alkynyl and aryl are optionally substituted with one or more substituents independently selected from the group of -halo, trihalomethyl, -R, -OR, - SR, -NR'R, -NO2, -CN, -OC(O)R, -OC(S)R, -SC(O)R, -SC(S)R -C(O)R, -C(S)R, - C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NR'R, -C(S)NR'R', -NRC(O)R* and - NR'C(O)OR'; the cycloalkyl is optionally substituted with one or more substituents independently selected from the group of R', -halo, OR', -SR', -NRR', -ONRR', -NO2, - CN, -C(O)R', -C(S)R', -OC(O)R', -SC(O)R', -SC(S)R', -OC(S)R', -C(O)OR', -C(S)OR', - C(O)SR', -C(S)SR', -C(O)NRR', -C(S)NRR', -C(O)NR'(OR'), -C(S)NR' -(OR - C(O)NR'(SR -C(S)NR'(SR'), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, - CH[C(O)OR]2, -CH[C(S)OR 2, -CH[C(O)SR]2, -CH[C(S)SR 2, -NR'C(O)R', - NR'C(O)OR', -S(O)-R', -S(O)OR', -S(O)2OR', -S(O)NRR', -S(O)ONRR', and each R' is independently selected from the group of -H, (d-C16) alkyl, substituted (d-dβ) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl; for use as an therapeutic agent for attenuating, inhibiting or preventing cancer cell proliferation in a mammal in need of such therapy.
3. The compound according to claim 1 or 2, wherein said cancer cell is selected from leukaemia, carcinomas, adenocarcinomas, melanomas and sarcomas.
4. The compound according to claim 1 or 2, wherein said leukaemia is selected from acute nonlymphocytic leukaemia, chronic lymphocytic leukaemia, acute granulocytic leukaemia, chronic granulocytic leukaemia, acute promyelocytic leukaemia, adult T-cell leukaemia, aleukaemic leukaemia, aleukocythemic leukaemia, basophylic leukaemia, blast cell leukaemia, bovine leukaemia, chronic myelocytic leukaemia, leukaemia cutis, embryonal leukaemia, eosinophilic leukaemia, Gross' leukaemia, hairy-cell leukaemia, hemoblastic leukaemia, hemocytoblastic leukaemia, histiocytic leukaemia, stem cell leukaemia, acute monocytic leukaemia, leukopenic leukaemia, lymphatic leukaemia, lymphoblastic leukaemia, lymphocytic leukaemia, lymphogenous leukaemia, lymphoid leukaemia, lymphosarcoma cell leukaemia, mast cell leukaemia, megakaryocytic leukaemia, micromyeloblastic leukaemia, monocytic leukaemia, myeloblastic leukaemia, myelocytic leukaemia, myeloid granulocytic leukaemia, myelomonocytic leukaemia, Naegeli leukaemia, plasma cell leukaemia, plasmacytic leukaemia, promyelocytic leukaemia, Rieder cell leukaemia, Schilling's leukaemia, stem cell leukaemia, subleukaemic leukaemia, and undifferentiated cell leukaemia. The compound according to claim 1 or 2, wherein said carcinoma is acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colorectal carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, haematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, non-small cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scrrrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum. The compound according to claim lor 2, wherein said adenocarcinoma is of the breast, lung, pancreas and prostate.
The compound according to claim 1 or 2, wherein said melanoma is acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.
The compound according to claim 1 or 2, wherein said sarcoma is a soft tissue sarcomas, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumour sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented haemoπhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.
The compound according to claim 1 or 2, wherein said cancer cell is selected from Hodgkin's Disease, Non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumours, primary brain tumours, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, gliomas, testicular cancer, thyroid cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, mesothelioma and medulloblastoma.
10. The compound according to claim 1 or 2, wherein the compound is for use in combination with known therapeutics.
11. Use of compound having structural formula (I) :
Figure imgf000129_0001
or a pharmaceutically acceptable salt thereof in the preparation of a medicament. for attenuating, inhibiting or preventing cancer cell migration or cancer cell proliferation in a mammal, wherein:
X is F, CI, Br or I;
RI and R2 are each independently selected from the group of H, -R, -halo, -OR, - SR, -NRR, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NRR, -C(S)NRR, -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, or RI and R2 when taken together form =O, =S or a C3-C6 spiro group;
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
B is substituted with one or more substituents selected from the group of (d-C16) alkyl, (C2-Cι6) alkenyl, (C2-Cι6) alkynyl, aryl, -O-(d-C16) alkyl, -O-(C2-C16) allcenyl, -O- (C2-Cι6) alkynyl, -O-aryl, -O-CH2-aryl, -S-(d-C16)alkyl, -S-(C2-C16) alkenyl, -S-(C2-Cι6) alkynyl, -S-aryl, -S-CH2-aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloaUcyl, -S-(C3-C8) cycloalkyl, -halo, -NRR, -ONRR -NO2, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, - C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R,
-SC(S)R, -OC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: each R is independently selected from -H, (d-C16) alkyl, substituted (d-C16) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2- C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cycloalkyl, aryl or substituted aryl; the alkyl, alkenyl, alkynyl and aryl are optionally substituted with one or more substituents independently selected from the group of -halo, trihalomethyl, -R, -OR, - SR', -NR'R, -NO2, -CN, -OC(O)R', -OC(S)R', -SC(O)R, -SC(S)R -C(O)R', -C(S)R', - C(O)OR, -C(S)OR, -C(O)SR', -C(S)SR, -C(O)NR'R', -C(S)NR'R', -NR'C(O)R' and - NR'C(O)OR'; the cycloalkyl is optionally substituted with one or more substituents independently selected from the group of R', -halo, OR', -SR', -NRR', -ONRR', -NO2, - CN, -C(O)R', -C(S)R', -OC(O)R', -SC(O)R', -SC(S)R', -OC(S)R', -C(O)OR', -C(S)OR', - C(O)SR', -C(S)SR', -C(O)NRR', -C(S)NRR', -C(O)NR'(OR'), -C(S)NR' -(OR'), - C(O)NR'(SR'), -C(S)NR'(SR'), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, - CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NR'C(O)R', - NR'C(O)OR', -S(O)-R', -S(O)OR', -S(O)2OR', -S(O)NRR', -S(O)ONRR', and each R' is independently selected from the group of -H, (d-C16) alkyl, substituted (d-dβ) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl.
12. A method of attenuating, inhibiting, or preventing cancer cell migration contacting cancer cells with an effective amount of a compound having structural formula (I):
Figure imgf000130_0001
or a pharmaceutically acceptable salt thereof, wherein: X is F, CI, Br or I;
RI and R2 are each independently selected from the group of H, -R, -halo, -OR, - SR, -NRR, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NRR, -C(S)NRR, -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, or RI and R2 when taken together form =O, =S or a C3-C6 spiro group;
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
B is substituted with one or more substituents selected from the group of (d-C16) alkyl, (C2-C16) alkenyl, (C2-Cι6) alkynyl, aryl, -O-(Cι-Cι6) alkyl, -O-(C2-C16) alkenyl, -O- (C2-Cj6) alkynyl, -O-aryl, -O-CH2-aryl, -S-(d-Cι6)alkyl, -S-(C2-d6) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2-aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -halo, -NRR, -ONRR -NO2, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, - C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R,
-SC(S)R, -OC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: each R is independently selected from -H, (d-C16) alkyl, substituted (d-C16) alkyl, (C -C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2- C16) alkynyl, (C3-C8) cycloalkyl, substituted (C -C8) cycloalkyl, aryl or substituted aryl; the allcyl, alkenyl, alkynyl and aryl are optionally substituted with one or more substituents independently selected from the group of -halo, trihalomethyl, -R', -OR', - SR, -NR'R, -NO2, -CN, -OC(O)R, -OC(S)R, -SC(O)R, -SC(S)R -C(O)R, -C(S)R, - C(O)OR, -C(S)OR', -C(O)SR, -C(S)SR, -C(O)NR'R, -C(S)NR'R', -NR'C(O)R and - NR'C(O)OR'; the cycloalkyl is optionally substituted with one or more substituents independently selected from the group of R', -halo, OR', -SR', -NRR', -ONRR', -NO2, - CN, -C(O)R', -C(S)R', -OC(O)R', -SC(O)R', -SC(S)R', -OC(S)R', -C(O)OR', -C(S)OR', C(O)SR', -C(S)SR', -C(O)NRR', -C(S)NRR', -C(O)NR'(OR -C(S)NR' -(OR^, C(O)NR'(SR'), -C(S)NR'(SR'), -CH(CN)2, -CHCC^R^, -CH[C(S)R']2, CH[C(O)OR]2, -CH[C(S)OR']2, -CH[C(O)SR]2, -CH[C(S)SR']2, -NR'C(O)R', NR'C(O)OR', -S(O)-R', -S(O)OR', -S(O)2OR', -S(O)NRR', -S(O)ONRR', and each R' is independently selected from the group of -H, (d-C16) alkyl, substituted (d- C16) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl.
13. A method of attenuating, inhibiting, or preventing cancer cell proliferation contacting cancer cells with an effective amount of a compound having structural formula (I):
Figure imgf000132_0001
or a pharmaceutically acceptable salt thereof, wherein:
X is F, CI, Br or I;
RI and R2 are each independently selected from the group of H, -R, -halo, -OR, - SR, -NRR, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NRR, -C(S)NRR, -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, or RI and R2 when taken together form =O, =S or a C3-C6 spiro group;
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; wherein:
B is substituted with one or more substituents selected from the group of (d-C16) alkyl, (C2-Cι6) alkenyl, (C2-C16) alkynyl, aryl, -O-(d-C16) alkyl, -O-(C2-d6) alkenyl, -O- (C2-C16) alkynyl, -O-aryl, -O-CH2-aryl, -S-(Cι-C16)alkyl, -S-(C2-C16) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2-aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -halo, -NRR, -ONRR -NO2, -CN, -C(O)R, -C(S)R, -C(O)OR, -C(S)OR, - C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R,
-SC(S)R, -OC(S)R, -C(O)NRR, -C(S)NRR, -C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: each R is independently selected from -H, (d-C16) alkyl, substituted (d-C16) alkyl, (C2-C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2- C16) alkynyl, (C -C8) cycloalkyl, substituted (C3-C8) cycloalkyl, aryl or substituted aryl; the alkyl, alkenyl, alkynyl and aryl are optionally substituted with one or more substituents independently selected from the group of -halo, trihalomethyl, -R, -OR', - SR, -NR'R, -NO2, -CN, -OC(O)R, -OC(S)R, -SC(O)R, -SC(S)R -C(O)R, -C(S)R, - C(O)OR, -C(S)OR, -C(O)SR, -C(S)SR', -C(O)NR'R, -C(S)NR'R', -NR'C(O)R* and - NR'C(O)OR; the cycloalkyl is optionally substituted with one or more substituents independently selected from the group of R', -halo, OR', -SR', -NRR', -ONRR', -NO2, - CN, -C(O)R', -C(S)R', -OC(O)R', -SC(O)R', -SC(S)R', -OC(S)R', -C(O)OR', -C(S)OR', - C(O)SR', -C(S)SR', -C(O)NRR', -C(S)NRR', -C^NR'O R , -C(S)NR' -(OR , -
Figure imgf000133_0001
-C(S)NR'(SR'), -CH(CN)2, -CH[C(O)R]2, -CH[C(S)R]2, - CH[C(O)OR]2, -CH[C(S)OR']2, -CH[C(O)SR]2, -CH[C(S)SR']2, -NR'C(O)R', - NR'C(O)OR', -S(O)-R', -S(O)OR', -S(O)2OR', -S(O)NRR', -S(O)ONRR', and each R' is independently selected from the group of -H, (d-C16) alkyl, substituted (d- C16) alkyl, (C -C16) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C -C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl.
14. A compound having the structural formula (I):
Figure imgf000133_0002
or a phannaceutically acceptable salt thereof, wherein:
X is F, C Br or l;
RI and R2 are as defined above, and
B is an aryl group selected from phenyl, indane, fluorene, indazole, indole, and pyridine; and substituted with at least one substituent selected from, (d-C16) allcyl, (C2- C16) alkenyl, (C2-C16) alkynyl, -O-(Cι-C16) alkyl, -O-(C2-Cι6) alkenyl, -O-(C2-C16) alkynyl, aryl, substituted aryl, -O-aryl, -O-CH2-aryl, -S-(d-C16) alkyl, -S-(C2-C16) alkenyl, -S-(C2-C16) alkynyl, -S-aryl, -S-CH2-aryl, (C3-C8) cycloalkyl, -O-(C3-C8) cycloalkyl, -S-(C3-C8) cycloalkyl, -ONRR, -C(O)R, -C(S)R -C(O)OR, -C(S)OR, - C(O)SR, -C(S)SR, -OC(O)R, -SC(O)R, -SC(S)R, -OC(S)R, -C(O)NRR, -C(S)NRR, - C(O)NR(OR), -C(S)NR(OR), -C(O)NR(SR), -C(S)NR(SR), -CH(CN)2, -CH[C(O)R]2, - CH[C(S)R]2, -CH[C(O)OR]2, -CH[C(S)OR]2, -CH[C(O)SR]2, -CH[C(S)SR]2, - NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR; wherein: said alkyl is substituted with at least one substituent selected from the group of halo, -CN, -NO2, -NR'R', -O-alkyl, -O-alkenyl, -O-alkynyl, -O-aryl, -OC(O)R, -OC(S)R, -C(O)R, - C(S)R, -C(O)NR'R and -C(S)NR'R; said alkenyl, alkylnyl, -O-alkyl, -S-alkyl, are each independently substituted with at least one group selected from halo, -CN, -NO2, -NR'R', -OH, -OR, -O-aryl, -OC(O)R, - OC(S)R, -C(O)R, -C(S)R, -C(O)OR', -C(O)NR*R and -C(S)NR'R; said -O-alkenyl, -O-alkynyl, -S-alkenyl, -S-alkynyl, cycloalkyl, -O-cycloalkyl are are each optionally and independently substituted with at least one group selected from halo, -CN, -NO2, -NR'R, -OH, -OR', -O-aryl, -OC(O)R, -OC(S)R, -C(O)R, -C(S)R, - C(O)OR, -C(O)NR'R and -C(S)NR'R', and wherein R, R is each independently selected from the group of -H, (d-Ciβ) alkyl, substituted (d-C16) alkyl, (C2-Cι6) alkenyl, substituted (C2-C16) alkenyl, (C2-C16) alkynyl, substituted (C2-C16) alkynyl, (C3-C8) cycloalkyl, substituted (C3-C8) cyloalkyl, aryl or substituted aryl
15. The compound according to claim 14, wherein B is phenyl.
16. The compound according to claim 14, wherein: X is F, CL Br or l;
RI and R2 are as defined above, and
B is phenyl substituted with at least one group selected from, (d-C16) alkyl, (C2- Ciβ) alkenyl, (C2-C16) alkynyl, -O-(d-C16) alkyl, -O-(C2-C16) alkenyl, -O-(C2-C16) alkynyl, -aryl, -O-aryl, -O-CH2-aryl, -OC(O)R, -C(O)R, -C(O)OR, -C(O)NR'R', - NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)NRR, -S(O)ONRR; wherein: said alkyl is substituted with at least one group selected from halo, -CN, -O-alkyl, -O-alkenyl, -O-alkynyl, -O-aryl, -OC(O)R, -C(O)R, and -C(O)NR'R: said alkenyl, alkynyl, -O-alkyl, -O-alkenyl and -O-alkynyl are each independently substituted with at least one group selected from halo, -CN, -OH, -OR', -O-aryl, - OC(O)R, -C(O)R', -C(O)OR* and -C(O)NR'R, and R and R' are as defined above.
17. The compound according to claim 16, wherein:
B is substituted with at least one substituent selected from the group of (d-C16) alkyl,
(d-C16) alkynyl or -O-alkyl; wherein: said allcyl is substituted with at least one substituent selected from the group of -CN, -O- alkyl, -OC(O)R, -C(O)R, , -C(O)NR'R or halo; said alkyny and -O-alkyl are are substituted with at least one substituent selected from -
CN, -OH, -O-alkyl, -OC(O)R, -C(O)R, -C(O)OR, -C(O)NR'R' or halo; and
R is as defined above.
18. The compound according to claim 16, wherein:
B is substituted with at least one group selected from -NRC(O)R, -NRC(O)OR, -S(O)-R, -S(O)OR, -S(O)2OR, -S(O)NRR, -S(O)ONRR, -C(O)R, -C(O)OR, -OC(O)R, -C(O)NRR; wherein R is as defined above.
19. The compound according to claim 16, wherein:
B is substituted with at least one group selected from aryl, -O-aryl, -O-CH2-aryl and halo.
20. A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 14 to 19 and a carrier, diluent or excipient.
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