SK108897A3 - Novel farnesyl transferase inhibitors, preparation thereof, and pharmaceutical compositions containing same - Google Patents

Abstract

Novel farnesyl transferase inhibitors of general formula (I), preparation thereof, and pharmaceutical compositions containing same. In general formula (I), R1 is Y-S-A1- (where Y is a hydrogen atom, an amino acid residue, a fatty acid residue, an alkyl or alkoxycarbonyl radical, or a radical R4-S-, where R4 is a C1-6 alkyl radical optionally substituted by a phenyl radical, or a radical of general formula (II), wherein A1, X1, Y1, R2, R'2, X2, Y2, X, R3, R'3 and R are as defined below, and A1 is a C1-4 alkylene radical optionally alpha -substituted in the >C(X1)(Y1) grouping by an amino, alkylamino, alkanoylamino or alkoxycarbonylamino radical); X1 and Y1 are each a hydrogen atom or, taken together with the carbon atom to which they are attached, a >C=O grouping; R2 is a straight or branched C1-4 alkyl radical optionally substituted by a cyclohexyl radical; R'2 is hydrogen or alkyl; X2 and Y2 are each a hydrogen atom or, taken together with the carbon atom to which they are attached, a >C=O grouping; R3 is a C1-4 alkyl radical optionally substituted by hydroxy, alkoxy, mercapto, alkylthio, alkylsulphinyl or alkylsulphonyl, with the proviso that, when R3 is an alkyl radical substituted by a hydroxy radical, R3 may form a lactone with the alpha -carboxy radical; R'3 is hydrogen or alkyl; X is an oxygen or sulphur atom; and R is a hydrogen atom or an optionally substituted alkyl radical or an optionally substituted phenyl radical. These novel products have anticancer properties.

Description

Farnesyl transferase inhibitors, process for their preparation and pharmaceutical compositions containing these inhibitors

Technical field>

The present invention relates to farnesyl transferase inhibitors, processes for their preparation and pharmaceutical compositions containing them as active ingredients.

BACKGROUND OF THE INVENTION

Inhibition of farnesyl transferase and hence farnesylation of the Ras protein blocks the ability of the mutated Ras protein to transform normal cells into cancer cells.

The C-terminal sequence of the Ras gene contains the motif CAAX 'or Cys-Aaa _x -Aaa ₂ -Xaa, wherein Aaa represents an aliphatic amino acid and Xaa represents any amino acid.

It is known; that tetrapeptides with the CAAX sequence may inhibit the farnesylation of the Ras protein. For example, EP 0 618 221 discloses 1,2,3,4-tetrahydroisoquinoline peptide derivatives whose side chain is attached at the 3-position of the heterocyclic backbone. In WO 91/16340 PCT and EP Application 0461869 discloses peptide inhibitor of farnesyl transferase, Cys-Aaa _and -Aaa ₂ -Xaa, which are more particularly represented Cys-Val-Leu-Ser, Cys-Val-Ile-Met and Cys-Val-Val-Met, and which exhibit inhibitory activity at concentrations close to 10 ^e or 10 ^-7 M.

SUMMARY OF THE INVENTION

It has now been found novel, and this finding constitutes the essence of the invention that the peptides of formula I

show inhibitory activity (IC _so) even at a concentration of about 10 ^s M

In said general formula, IR ¹ is a group of the formula YSA ¹ - in which Y is a hydrogen atom or an amino acid or fatty acid residue or an alkyl or alkoxycarbonyl group or an R ⁴ -S- group in which R ⁴ is an alkyl group containing 1 to 6 carbon atoms optionally substituted by a phenyl group or a group of formula II wherein A ¹ ,

> 2 I

COOR (II)

X ² , Y ^:

X, R ³ , R ³ 'and R have the following meanings, and represents a straight or branched alkylene group having 1 to 4 carbon atoms, optionally substituted at the alpha position of the> C (X ¹ ) (Y ¹ ) amino, alkylamino group (C 1 -C 4), (C 1 -C 4) alkanoyl, or (C 1 -C 4) alkoxy group containing 1 to 4 carbonylamino;

X ¹ and Y ^x each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O,

^R2 is straight or branched alkyl of 1 to 4 carbon atoms, optionally substituted cyclohexyl,

R ² is hydrogen or a straight or branched alkyl of 1 to 6 carbon atoms,

X ² and Y ² each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O,

R ³ is straight or branched alkyl of 1 to 4 carbon atoms, optionally substituted by hydroxy, alkoxy of 1 to 4 carbon atoms, mercapto, an alkylthio radical containing 1 to 4 carbon atoms, alkylsulfinyl of 1 to 4 carbon atoms or alkylsulphonyl a group containing 1 to 4 carbon atoms, provided that when R ³ represents an alkyl group substituted with a hydroxy group, R ³ may form with the carboxyl group in the alpha position a lactone,

R ³ 'represents a hydrogen atom or a straight or branched (C 1 -C 6) alkyl group,

X is oxygen or sulfur; and

R is hydrogen or alkyl optionally substituted with 1 to 4 carbon atoms, 1 to 4 carbon atoms alkylthio, 1 to 4 carbon atoms alkylsulfinyl, 1 to 4 carbon atoms alkylsulfonyl, phenyl, phenoxy , phenylthio, phenylsulfinyl, phenylsulfonyl, C 1 -C 4 alkylamino or a dialkylamino group, each alkyl radical of 1 to 4 carbon atoms, or a phenyl group optionally substituted by one or more of the same or different substituents selected from the group consisting of halogens, C 1 -C 4 alkyl, C 1 -C 4 alkyloxy, C 1 -C 4 alkylthio and C 1 -C 4 alkanoyl.

In particular, in formula (I):

R ¹ is a group of formula YSA ¹ -, wherein Y represents a hydrogen atom or a lysine residue or a fatty acid residue containing up to 20 carbon atoms and ^A1 represents an ethylene or propylene radical optionally substituted by amino,

X ¹ and Y ¹ each represent a hydrogen atom or together with the carbon atom to which they are attached form a> C = O group,

R ² represents isopropyl, 1-methylpropyl, tert-butyl or cyclohexylmethyl radical,

R ² is hydrogen or methyl;

X ² and -Y ² each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O,

R ³ is methyl or ethyl, which is substituted by hydroxyl, methoxy, mercapto, methylthio, methylsulphinyl or methylsulfonylamino onyl,

R ³ 'represents a hydrogen atom or a methyl group,

X represents an oxygen atom and

R represents a hydrogen atom or a (C 1 -C 4) alkyl group optionally substituted by an alkoxy group or a phenyl group.

Especially in the above formula I

R ¹ represents a group of the formula YSA ¹ - in which

Y represents a hydrogen atom and A ¹ represents an ethylene or propylene group which is optionally substituted with an amino group,

X ¹ and Y ¹ each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O,

R ² represents isopropyl, 1-methylpropyl, tert-butyl or cyclohexylmethyl radical,

R ²¹ represents a hydrogen atom,

X ² and Y ² each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O,

R ³ represents a methyl or ethyl group which is substituted by a hydroxy, methoxy, mercapto or methylthio group,

R ³ 'represents a hydrogen atom and

R represents a hydrogen atom or an alkyl group containing 1 to 4 carbon atoms.

Of particular interest are peptides of the invention wherein the peptides of formula (I) are:

R ¹ represents a 2-mercaptoethyl or 1-amino 2-mercaptoethyl radical,

X ¹ and Y ^x each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O,

R ² is isopropyl,

X ² and Y ² each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O,

R ²¹ represents a hydrogen atom,

R ³ is 2-methylthioethyl or 2-metylsulfinyletylovú group,

R ³¹ represents a hydrogen atom and

R represents a hydrogen atom.

The invention also relates to the preparation of stereoisomers of the compounds of formula (I). The amino acid residues R ^x C (X ^x ) (Y ^x ), R ² 'CH (NR ² ') [C (X ² ) (Y ² )] and R ³ CH (NR ³ ') CO-OH have a preference for the natural amino acids.

The invention also relates to mineral or organic salts and esters of the compounds of formula (I).

According to the invention, these novel products of formula (I) can be obtained in solid phase using the synthesis of a 9-fluoromethoxycarbonyl derivative (EMOC). In this case, the thiol groups are protected by trityl or acetamidomethyl groups, the amine functional groups are protected by Boe (t-butoxycarbonyl) groups and the acid functional groups are protected in the form of butyl esters, the alcohol functional groups are protected by t-butyl and amide and imidazole functional groups. trityl.

The synthesis can be carried out on a resin which is enclosed in 3 cm ³ solid-density extraction syringes of high density polyethylene. The syringes are equipped with a Teflon filter and a two-way Teflon cap, closed with a single-use high density polyethylene wing plug. The swinging movement of the syringe is ensured by the rotary device for hemolysis tubes. Washing and filtration are performed at the solid phase extraction station.

The synthesis is carried out using 50 μιηοΐ resin. Amino acids are bound within one hour. 250 mmol amino acid suitably protected in the presence of 250 mmol of 2- (lH-benzotriazol-1 -) - 1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 250 ° F ^m l of N-hydroxybenzotriazole and 750 1 of diisopropylethylamine μταοί , 2 ml of Ν-2-methylpyrrolidone (NMP) / dimethylformamide (1/1 v / v). Removal of the EMOC protecting group is accomplished by three successive resin treatments over two minutes, followed by 2 ml piperidine in a 2% (v / v) solution in Ν-2-methylpyrrolidone over twenty minutes.

The following example illustrates the invention in more detail without limiting its scope in any way.

DETAILED DESCRIPTION OF THE INVENTION

Cys- (NMe) Val - [(R, S) -1,2,3,4-tetrahydroisoquinoline-1-carbonyl] -Met can be prepared as follows:

M ^mo1 Fmoc-Met resin [Wang resin, Wang et al., J.

Amer. Chem. Soc., 95, 1328, (1973)] is subjected to the following procedure:

- removal of the FMOC protecting group

- wash 5 times with 2 ml NMP

- acid bond

FMOC- (R, S) -1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid i

- wash 5 times with 2 ml NMP

- removal of the FMOC protecting group

- wash 5 times with 2 ml NMP

- FMOC-N-methylvaline binding

- wash 5 times with 2 ml NMP

- removal of the FMOC protecting group

- wash 5 times with 2 ml NMP

- FMOC-cysteine (S-trityl) bond

- wash 5 times with 2 ml NMP

- removal of the FMOC protecting group

- wash 5 times with 2 ml NMP.

The synthesis ends with the isolation of the products. The products are isolated by treating the resin with 10 ml of a mixture of trifluoroacetic acid j-phenol-ethanedithiol-thioanisole-water in a ratio of 40: 3: 1: 2: 2 by volume for one hour and 30 minutes. The resin is then separated by filtration. The filtrate was concentrated under reduced pressure on a rotary evaporator (RC10-10 Jouan) equipped with a vane pump and a pantograph (-90 ° C) for 1.5 hours while maintaining the evaporation chamber temperature at 50 ° C. The final volume of the concentrate is approximately 1 ml. The product is then precipitated by the addition of 15 ml of a mixture of methyl tert-butyl ether and petroleum ether (2: 1 by volume), then centrifuged. The pellet is further dissolved in 1 ml of trifluoroacetic acid and precipitated again by addition of 15 ml of methyl tert-butyl ether and washed with a further 15 ml of methyl tert-butyl ether. The product was then dried under reduced pressure (3.5 kPa) and purified by high pressure liquid chromatography (HPLC) on a C18 100 Å column (250 x 10 mm, BioRad), eluting with an acetonitrile gradient containing 0.07% trifluoroacetic acid (v / v). .) in water containing 0.07 trifluoroacetic acid (v / v) at a flow rate of 6 ml / min and then lyophilized. The product obtained is characterized by its mass spectrum.

1,2,3,4-tetrahydro-1-isoquinolinecarboxylic acid, in racemic form, can be prepared by hydrogenation of 1-isoquinolinecarboxylic acid under the conditions described by R. T. Schuman et al., J. Med. Chem., 36, 314 (1993).

Introduction of the FMOC. the amino acid is performed by treating the amino acid with 9-fluoromethylchloroformate (FMOC-chloride) in a basic environment.

The farnesyltransferase inhibitory activity and the farnesylation of Protein Ras are described in more detail in the following assay:

Farnesyltransferase activity is determined by the amount of ( ³ H) farnesyl transferred from ( ³ H) farnesyl pyrophosphate ( ³ H) FPP) to the p21 protein. The standard reaction mixture consists, for a final volume of 60 ml, of 50 mM Tris-HCl, 5 mM MgCL ₂ , 5 mM dithiothreitol, 0.2% octyl-BD-glucopyranoside, 200 picomolar p21, 4.5 picomolar (3H) FPP (61000 dpm / picomolar).

The reaction is initiated by the addition of about 5 ng of human farnesyltransferase purified from THP1 cell culture. After 20 min incubation at 37 ° C in a 1 ml 96-well microtiter plate (Titer Plate ^R , Beckman), the reaction is terminated by the addition of 0.4 ml 0.1% SDS in methanol at 0 ° C. Add the mixture to 0.4 ml of 30% trichloroacetic acid ^R (TCA) in methanol. The plates are kept in the freezer for one hour. The precipitate content is then retained on a glass fiber membrane ¹¹ (Filtermat ^R , Pharmacia) with a filter unit (Combi Cell Harvester ¹¹ , Skarton) and rinsed with 6% trichloroacetic acid in distilled water. The membranes are dried in a microwave, then saturated with warm air Meltilex ¹¹ (Pharmacia). Inhibitory activity is calculated in cpm on a B-Plate ^R computer (LKB). Each experiment is repeated three times.

The unit of activity is defined by one picomolar (3H) FPP, which is transferred to p21 in 20 minutes.

Percent inhibition was obtained by comparison of the tests with and without inhibitor after deduction of blanks, _the IC was measured for the inhibitions obtained with 9 different concentrations using known under the name Enzfitter ¹ * or graphite ^11th

The results obtained are summarized in Table I.

Table I

Product inhibitory activity IC 50 Cys- (N-Me) Val - [(R, S) -tetrahydro-l-1,2,3,4izochinolín-carbonyl] Met 2.6 X 10 ' ^and M

The novel peptides (I) may exist in the form of pharmaceutically acceptable non-toxic salts. These non-toxic salts include salts of mineral acids (such as hydrochloric, sulfuric, hydrobromic, phosphoric, nitric) or salts of organic acids (acetic, propionic, succinic, maleic, hydroxymaleic, benzoic, fumaric, methanesulfonic, oxalic) of mineral bases (sodium hydroxide) , potassium hydroxide, lithium hydroxide, calcium hydroxide) or organic bases (tertiary amines such as triethylamine, piperidine, benzylamine) according to the nature of the amino acids that make up peptide (I).

The peptides of the present invention inhibit farnesyltransferase and farnesylation of the Ras protein. They are significantly anticancer substances, acting at the level of solid and liquid tumors.

The present invention also relates to pharmaceutical compositions comprising at least one peptide of formula (I) in combination with one or more diluents or acceptable pharmaceutical excipients which are inert or physiologically active.

These pharmaceutical compositions may be administered orally, parenterally or rectally.

Formulations for oral administration include tablets, pills, powders, or granules. In these compositions, the active ingredient of the invention is mixed with one or more inert fillers such as sucrose, lactose or starch. These compositions may contain other substances such as fillers, for example lubricants such as magnesium stearate.

As liquid compositions for oral administration, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, alcohol-containing syrups containing inert fillers such as water or paraffin oil may be used. These compositions may also contain other excipients, for example wetting, sweetening, or flavoring agents.

The compositions of the patent for parenteral administration may be in the form of sterile water or non-aqueous phase solutions as a suspension or emulsion. As the solvent or vehicle, propylene glycol, polyethylene glycol, vegetable oils, in particular olive oil or injectable organic esters, e.g. ethyl oleate.

These compositions may also contain adjuvants, in particular wetting, emulsifying or dispersing agents. Sterilization can be carried out in a number of ways, for example by means of bacteriological filters, by introducing the sterilizing agent into the preparation or by heating. The agents may also be prepared in the form of sterile solids, which may be dissolved in sterile water or other sterile injectable formulations immediately prior to use.

Compositions for rectal administration are presented as suppositories, which may contain other active agents, a vehicle such as e.g. cocoa butter.

The compositions of the invention are particularly useful in human therapy in the treatment of various cancers.

In human therapy, the dose depends on the desired effect, the time of use and the individual requirements of the patient for whom the substance is intended.

Generally, the human dose is between 0.1 and 20 mg / kg daily (intraperitoneal administration).

Claims (5)

PATENT CLAIMS Peptides of the general formula I N ^ COOR R ^J (I) wherein R ¹ is a group of the formula YSA ¹ - in which Y is a hydrogen atom or an amino acid or a fatty acid residue or an alkyl or alkoxycarbonyl group or an R ⁴ -S- group in which R ⁴ is an alkyl group having 1 to 6 carbon atoms, which is optionally substituted by a phenyl group or a group of formula II R N COOR Ŕ ³ '(II) in which A ¹ , X ¹ , Y ¹ , R ² , R ² ', X ² , Y ² , X, R ³ , R ³ 'and R have the following meanings and means straight or branched (C 1 -C 4) alkylene optionally substituted at the alpha position of> C (X ¹ ) (Y ¹ ) amino, C 1 -C 4 alkylamino, C 1 -C 4 alkanoylamino or alkoxycarbonylamino whose alkyl radical contains 1 to 4 carbon atoms, X ¹ and Y ^x each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O, ^R2 is straight or branched alkyl of 1 to 4 carbon atoms, optionally substituted cyclohexyl, R ² is hydrogen or a straight or branched alkyl of 1 to 6 carbon atoms, X ² and Y ² each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O, R ³ represents a straight or branched (C 1 -C 4) alkyl group optionally substituted by hydroxy, (C 1 -C 4) alkoxy, mercapto, (C 1 -C 4) alkylthio, (C 1 -C 4) alkylsulfinyl 1 to 4 carbon atoms or alkylsulphonyl of 1 to 4 carbon atoms, it being understood that when R ³ is alkyl substituted by hydroxy, then R ³ can form a carboxyl group at the alpha lactone, R ³ 'represents a hydrogen atom or a straight or branched (C 1 -C 6) alkyl group, X is oxygen or sulfur; and R represents a hydrogen atom or an alkyl group optionally substituted by a (1-4C) alkoxy group, (1-4C) alkylthio, (1-4C) alkylsulfinyl, (1-4C) alkylsulfonyl, phenyl, phenoxy , phenylthio, phenylsulfinyl, phenylsulfonyl, C 1 -C 4 alkylamino or a dialkylamino group in which each alkyl radical contains 1 to 4 carbon atoms, or a phenyl group optionally substituted by one or more of the same or different substituents selected 2 groups comprising halogen atoms, a C 1 -C 4 alkyl group, a C 1 -C 4 alkyloxy group, a C 1 -C 4 alkylthio group, and a C 1 -C 4 alkanoyl group. Peptides according to claim 1 of the general formula I, in which R ¹ is a group of formula YSA ¹ -, wherein Y represents a hydrogen atom or a lysine residue or a fatty acid residue containing up to 20 carbon atoms and ^A1 represents an ethylene or propylene radical optionally substituted by amino, X ¹ and Y ¹ each represent a hydrogen atom or together with the carbon atom to which they are attached form a> C = O group, R ² represents isopropyl, 1-methylpropyl, tert-butyl or cyclohexylmethyl radical, R ² is hydrogen or methyl; X ² and Y ² each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O, R ³ is methyl or ethyl, which is substituted by hydroxyl, methoxy, mercapto, methylthio, methylsulphinyl or methylsulphonyl, R ³ 'represents a hydrogen atom or a methyl group, X represents an oxygen atom and R represents a hydrogen atom or a (C 1 -C 4) alkyl group optionally substituted by an alkoxy group or a phenyl group. Peptides according to claim 1 of the formula I, in which R ¹ represents a group of the formula YSA ¹ -, in which Y represents a hydrogen atom and A ¹ represents an ethylene or propylene group which is optionally substituted with an amino group, X ¹ and Y ¹ each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O, R ² represents isopropyl, 1-methylpropyl, tert-butyl or cyclohexylmethyl radical, R ² is hydrogen, X ² and Y ² each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O, R ³ represents a methyl or ethyl group which is substituted by a hydroxy, methoxy, mercapto or methylthio group, ³ · R is H and R represents a hydrogen atom or an alkyl group containing 1 to 4 carbon atoms. Peptides according to claim 1, in which R ¹ represents a 2-mercaptoethyl group or 1 amino-2-mercaptoethyl group, X ^x and Y ^x each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O, R ² is isopropyl, X ² and Y ² each represent a hydrogen atom or together with the carbon atom to which they are attached form> C = O, R ² is hydrogen, R ³ is 2-methylthioethyl or 2-metylsulfinyletylovú group, R ³ 'represents a hydrogen atom and R represents a hydrogen atom. A pharmaceutical composition comprising a sufficient amount of a peptide of formula (I) according to any one of claims 1 to 4 in combination with one or more pharmaceutically acceptable diluents or one or more pharmaceutically acceptable excipients which are inert or physiologically active.