CA1067073A - Amide derivatives of vlb, leurosidine, leurocristine and related dimeric alkaloid - Google Patents
Amide derivatives of vlb, leurosidine, leurocristine and related dimeric alkaloidInfo
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- CA1067073A CA1067073A CA242,012A CA242012A CA1067073A CA 1067073 A CA1067073 A CA 1067073A CA 242012 A CA242012 A CA 242012A CA 1067073 A CA1067073 A CA 1067073A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
- C07D519/04—Dimeric indole alkaloids, e.g. vincaleucoblastine
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Abstract
Abstract The invention relates to a process for preparing novel dimeric alkaloid derivatives of vinblastine, vin-cadioline and leurocolombine by reacting vinblastine, vincadioline or leurocolombine with an amine and, if desired, reacting the resulting compound with a nitrosating agent and an amine.
Description
~V6q~73 The invention relates to novel amide derivatives of dimeric alkaloids and to a process for the preparation thereof. The compounds are useful as anti-viral and anti-neoplastic agents or as intermediates in the prepa-ration of such agents.
The invention provides a compound of the formula , ~ R~
~ CH2-CH3 ~ ,~o 14 't--- y 117 ~ 5 ~ CH2-CH3 CH3 o-l~ 3~I_Rl H ' Formula I
wherein R is NH2, NH-NH2, N(CH3)2, pyrrolidinyl, NH-alk-X, N~-(C3-C8)-cyclo-alk, NH-alk-(oH)l 3, or N3;
_ . . . .
20 wherein alk is (Cl-C6) aLkyl, _ __ and X is hydrogen;
_ . . . . . .
Rl is hydrogen, hydroxyl, 0-(Cl-C3)-alkanoyl or O-chloro-(Cl-C3)-alkanoyl;
R2, R3 and R4 are hydrogen or hydroxyl with the provisos that when R2 is hydrogen, R3 and R4 are hydrogen;
when R2 is hydroxyl at least one of R3 or R4 is hydroxyl.
The invention also provides a process for preparing a compound of Formula I by (a) reacting a compound having ~o67~73 a structure of Formula I wherein R is O-CH3, R ls hydrogen, hydroxyl or acetyloxy, and R , R and R are as defined in Formula I with a c~mpound of the formula NH2R5 Formula II
where R5 is hydrogen, methyl or amino and/or (b) when R is NH-NH2 reacting the compound so obtained with a nitrosating agent and with a compound of the formula 0 ~R7 " wherein R6 is hydrogen or methyl and R7 is methyl, -alk-X, (C3-C8)-cycloalk, or -alk-(OH)l 3 wherein alk is (Cl-C6) alkyl and X is as defined in Formula I, and/or (c) acylating the compound obtained in (a) or Ib) above wherein Rl is hydroxyl to provide a compound wherein Rl is other than hydroxyl and, if desired, reacting any of the products obtained above with a non-toxic inorganic acid - or organic acid to provide the pharmaceutically acceptable acid addition salt of the product.
2u Several naturally-occurring alkaloids obtainable from Vinca rosea have been found active in the treatment of experimental malignancies in animals. Among these are leurosine (U. S. Patent No. 3,370,û57), vinblastine (vincaleukoblastine) (U.S. Patent No. 3,097,137), leuro-sidine (vinrosidine) and leurocristine (vincristine) (both in U. S. Patent No. 3,2û5,220). Two of these alkaloids, vinblastine and leurocristine, are now marketed as drugs for the treatment of malignancies, particularly the leukemias and related diseases in humans. Of these marketed compounds, X-3754M -3_ ~.
r leurocristine is a most active and useful agent in the treatment of leukemias but is also the least abundant of the anti-neoplastic alkaloids of Vinca rosea.
Chemical modification of the Vinca alkaloids has been rather limited. In the first place, the molecular structures involved are extremely complex and chemical reactions which affect a specific function of the molecule are difficult to develop. Secondly, alkaloids lacking ~ desirable chemotherapeutic properties have been recovered ; 10 from Vinca rosea fractions, and a determination of their structures has led to the conclusion that these compounds are closely related to the active alkaloids. Thus, anti-neoplastic activity seems to be limited to very specific structures, and the chances of obtaining more active drugs by modification of these structures would seem to be cor-respondingly slight. Among the successful modifications of physiologically-actlve alkaloids has been the preparation of dihydro vinblastine (U. S. Patent No. 3,352,868) and the replacement of the acetyl group at C-4 (carbon no. 4 of vinblastine ring system-see Formula I) with a higher al-kanoyl group or with unrelated acyl groups. (See U. S.
Patent No. 3,392,173.) Several of these derivatives are capable of prolonging the life of mice inoculated with P1534 leukemia. One of the derivatives in which a chloracetyl group replaced the C-4 acetyl group of vinblastine was also a useful intermediate for the preparation of structurally modified vinblastine compounds in which an N,N-dialkylglycl group replaced the C-4 acetyl group of vinblastine (see U. S. Patent No. 3,387,001). An intermediate compound, namely 4-desacetyl vinblastine, was produced during the 1~67073 chemical reactions leading to these latter derivatives.
This intermediate, in which the C-4 acyl group was lacking, leaving an unesterified hydroxy group, has been reported to be a toxic material having little in vivo chemotherapeutic activity against the Pl534 murine leukemia system by Hargrove, Lloydia, 27, 340 (1964).
A preferred group of compounds of Formula I
includes the amides of vincadioline, leurocolombine, 4-desacetoxyvinblastine, 3'-hydroxy-4-desacetoxyvinblastine, deoxyvinblastine and the 4-desacetyl derivative of any of the above dimeric alkaloids having a C-4 acetoxy group, and the pharmaceutically-acceptable salts of the above bases, except those in which R is NH-NH2 and N3, formed with nontoxic acids.
Illustrative of alk-(OH)l 3, alk-Am and alk-X in the above formula are the following: methyl, 2-methyl-pentyl, isohexyl, isopentyl, n-pentyl, n-hexyl, sec-hexyl, ethyl, isopropyl, n-butyl, sec-butyl, cyanomethyl, cyano-ethyl, 2-hydroxy-n-hexyl, 5-cyano-n-pentyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-dimethylaminoethyl, 2-aminoethyl, 2-methylaminoethyl, 2-hydroxypropyl, benzyl, phenethyl, 4-phenylbutyl, 2-aminopropyl, 2-aminohexyl, 2-dimethylamino-propyl, 2,2'-dihydroxyisopropyl, 2,2'-dihydroxy-t-butyl,
The invention provides a compound of the formula , ~ R~
~ CH2-CH3 ~ ,~o 14 't--- y 117 ~ 5 ~ CH2-CH3 CH3 o-l~ 3~I_Rl H ' Formula I
wherein R is NH2, NH-NH2, N(CH3)2, pyrrolidinyl, NH-alk-X, N~-(C3-C8)-cyclo-alk, NH-alk-(oH)l 3, or N3;
_ . . . .
20 wherein alk is (Cl-C6) aLkyl, _ __ and X is hydrogen;
_ . . . . . .
Rl is hydrogen, hydroxyl, 0-(Cl-C3)-alkanoyl or O-chloro-(Cl-C3)-alkanoyl;
R2, R3 and R4 are hydrogen or hydroxyl with the provisos that when R2 is hydrogen, R3 and R4 are hydrogen;
when R2 is hydroxyl at least one of R3 or R4 is hydroxyl.
The invention also provides a process for preparing a compound of Formula I by (a) reacting a compound having ~o67~73 a structure of Formula I wherein R is O-CH3, R ls hydrogen, hydroxyl or acetyloxy, and R , R and R are as defined in Formula I with a c~mpound of the formula NH2R5 Formula II
where R5 is hydrogen, methyl or amino and/or (b) when R is NH-NH2 reacting the compound so obtained with a nitrosating agent and with a compound of the formula 0 ~R7 " wherein R6 is hydrogen or methyl and R7 is methyl, -alk-X, (C3-C8)-cycloalk, or -alk-(OH)l 3 wherein alk is (Cl-C6) alkyl and X is as defined in Formula I, and/or (c) acylating the compound obtained in (a) or Ib) above wherein Rl is hydroxyl to provide a compound wherein Rl is other than hydroxyl and, if desired, reacting any of the products obtained above with a non-toxic inorganic acid - or organic acid to provide the pharmaceutically acceptable acid addition salt of the product.
2u Several naturally-occurring alkaloids obtainable from Vinca rosea have been found active in the treatment of experimental malignancies in animals. Among these are leurosine (U. S. Patent No. 3,370,û57), vinblastine (vincaleukoblastine) (U.S. Patent No. 3,097,137), leuro-sidine (vinrosidine) and leurocristine (vincristine) (both in U. S. Patent No. 3,2û5,220). Two of these alkaloids, vinblastine and leurocristine, are now marketed as drugs for the treatment of malignancies, particularly the leukemias and related diseases in humans. Of these marketed compounds, X-3754M -3_ ~.
r leurocristine is a most active and useful agent in the treatment of leukemias but is also the least abundant of the anti-neoplastic alkaloids of Vinca rosea.
Chemical modification of the Vinca alkaloids has been rather limited. In the first place, the molecular structures involved are extremely complex and chemical reactions which affect a specific function of the molecule are difficult to develop. Secondly, alkaloids lacking ~ desirable chemotherapeutic properties have been recovered ; 10 from Vinca rosea fractions, and a determination of their structures has led to the conclusion that these compounds are closely related to the active alkaloids. Thus, anti-neoplastic activity seems to be limited to very specific structures, and the chances of obtaining more active drugs by modification of these structures would seem to be cor-respondingly slight. Among the successful modifications of physiologically-actlve alkaloids has been the preparation of dihydro vinblastine (U. S. Patent No. 3,352,868) and the replacement of the acetyl group at C-4 (carbon no. 4 of vinblastine ring system-see Formula I) with a higher al-kanoyl group or with unrelated acyl groups. (See U. S.
Patent No. 3,392,173.) Several of these derivatives are capable of prolonging the life of mice inoculated with P1534 leukemia. One of the derivatives in which a chloracetyl group replaced the C-4 acetyl group of vinblastine was also a useful intermediate for the preparation of structurally modified vinblastine compounds in which an N,N-dialkylglycl group replaced the C-4 acetyl group of vinblastine (see U. S. Patent No. 3,387,001). An intermediate compound, namely 4-desacetyl vinblastine, was produced during the 1~67073 chemical reactions leading to these latter derivatives.
This intermediate, in which the C-4 acyl group was lacking, leaving an unesterified hydroxy group, has been reported to be a toxic material having little in vivo chemotherapeutic activity against the Pl534 murine leukemia system by Hargrove, Lloydia, 27, 340 (1964).
A preferred group of compounds of Formula I
includes the amides of vincadioline, leurocolombine, 4-desacetoxyvinblastine, 3'-hydroxy-4-desacetoxyvinblastine, deoxyvinblastine and the 4-desacetyl derivative of any of the above dimeric alkaloids having a C-4 acetoxy group, and the pharmaceutically-acceptable salts of the above bases, except those in which R is NH-NH2 and N3, formed with nontoxic acids.
Illustrative of alk-(OH)l 3, alk-Am and alk-X in the above formula are the following: methyl, 2-methyl-pentyl, isohexyl, isopentyl, n-pentyl, n-hexyl, sec-hexyl, ethyl, isopropyl, n-butyl, sec-butyl, cyanomethyl, cyano-ethyl, 2-hydroxy-n-hexyl, 5-cyano-n-pentyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-dimethylaminoethyl, 2-aminoethyl, 2-methylaminoethyl, 2-hydroxypropyl, benzyl, phenethyl, 4-phenylbutyl, 2-aminopropyl, 2-aminohexyl, 2-dimethylamino-propyl, 2,2'-dihydroxyisopropyl, 2,2'-dihydroxy-t-butyl,
2,2',2''-trihydroxy-t-butyl, and the like.
In Formula I, the terms "(Cl-C3)-alkanoyl" and "chloro-(Cl-C3)-alkanoyl" include groups such as acetyl, chloroacetyl, propionyl, 2-chloropropionyl, 2-chlorobutyryl and butyryl, these terms being represented by the formula (Cl-C3)-alkyl-CO, an alkanoyl group, or by the formula X-3754M -5_ (Cl-C3)-alkyl (Cl)-CO, a chloroalkanoyl group. The term "NH-(C3-CB)-cycloalk" includes the radicals cyclopropyl-amino, cyclobutylamino, cyclopentylamino, cyclohexylamino, cycloheptylamino, and cyclooctylamino. The term "carbo-(Cl-C3~-alkoxy" includes the radicals carbomethoxy, carbo-ethoxy, carboisopropoxy and carbo-n-propoxy.
When "X" in the radical "alk-X" is phenyl, the phenyl group can contain the standard aromatic substituents including lower alkyl, lower alkoxy, hydroxy, halo, nitro and the like and a given phenyl group can contain more than one of the above substituents, either the same or different from the original substituent; examples of such groups are 4-hydroxyphenyl, 2,4-dichlorophenyl, 2-methyl-4-chloro-phenyl, 2,4-dinitrophenyl, 3,5-xylyl, 4-tolyl, 2-tolyl,
In Formula I, the terms "(Cl-C3)-alkanoyl" and "chloro-(Cl-C3)-alkanoyl" include groups such as acetyl, chloroacetyl, propionyl, 2-chloropropionyl, 2-chlorobutyryl and butyryl, these terms being represented by the formula (Cl-C3)-alkyl-CO, an alkanoyl group, or by the formula X-3754M -5_ (Cl-C3)-alkyl (Cl)-CO, a chloroalkanoyl group. The term "NH-(C3-CB)-cycloalk" includes the radicals cyclopropyl-amino, cyclobutylamino, cyclopentylamino, cyclohexylamino, cycloheptylamino, and cyclooctylamino. The term "carbo-(Cl-C3~-alkoxy" includes the radicals carbomethoxy, carbo-ethoxy, carboisopropoxy and carbo-n-propoxy.
When "X" in the radical "alk-X" is phenyl, the phenyl group can contain the standard aromatic substituents including lower alkyl, lower alkoxy, hydroxy, halo, nitro and the like and a given phenyl group can contain more than one of the above substituents, either the same or different from the original substituent; examples of such groups are 4-hydroxyphenyl, 2,4-dichlorophenyl, 2-methyl-4-chloro-phenyl, 2,4-dinitrophenyl, 3,5-xylyl, 4-tolyl, 2-tolyl,
3-ethoxyphenyl and the like.
Non-toxic acids useful for forming pharmaceu-tically-acceptable acid addition salts of the amine bases include salts derived from inorganic acids such as: hydro-chloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous aci~, phosphorous acid and the like, as well as salts of non-toxic organic acids including aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkandioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc.
Such pharmaceutically-acceptable salts thus include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, 1(~67073 isobutyrate, caprate, heptoanate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chloro-benzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonates, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, 2-hydroxybutyrate, glycollate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-l-sulfonate, naphthalene-2-sulfonate and the like salts.
A majority of the compounds of Formula I can be described generally as derivatives of vinblastine. Vin-blastine is a compound having the structure of Formula I
when R is CH30, Rl is acetoxy, R2 is ~-hydroxyl (the C-4'-ethyl group is alpha) and R3 and R4 are hydrogen. Deoxy-vinblastine occurs when in Formula I all of R2, R3 and R4 are hydrogen, R is CH30 and R is acetoxy. There are two isomers of deoxyvinblastine identified as "A" and "B". In the case of the "A" isomer, the R2 hydrogen is Beta(up) and the C-4'ethyl group is Alpha(down). The "B" isomer has a ~-ethyl group and an a-hydrogen at C-4'.
Vincadioline is the 3'-hydroxy derivative (i.e.
R3 is hydroxyl) of vinblastine. Leurocolombine is the 2'-hydroxy derivative (i.e. R is hydroxyl) of vinblastine.
Vincadioline has the following characteristics:
X-3754M _7_ Melting point = 218-220.5C. with decomposition;
X-ray powder diffraction pattern, using filtered chromium radiation; ~ = 2.2896A.
d in A I/Il d in A I/I
9.55 100 -1 3.99 60 8.87 9o)-2 3.71 20 8.63 90) 3.64 15 7.78 05 3.44 10 B
7.57 60 3.19 20 7.21 50 3.05 05 6.00 40 2.85 20 5.88 40 2.78 10 5.58 70 -3 2.61 10 5.22 20 2.44 15 B
5.08 20 2.21 05 B
Non-toxic acids useful for forming pharmaceu-tically-acceptable acid addition salts of the amine bases include salts derived from inorganic acids such as: hydro-chloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous aci~, phosphorous acid and the like, as well as salts of non-toxic organic acids including aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkandioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc.
Such pharmaceutically-acceptable salts thus include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, 1(~67073 isobutyrate, caprate, heptoanate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chloro-benzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonates, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, 2-hydroxybutyrate, glycollate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-l-sulfonate, naphthalene-2-sulfonate and the like salts.
A majority of the compounds of Formula I can be described generally as derivatives of vinblastine. Vin-blastine is a compound having the structure of Formula I
when R is CH30, Rl is acetoxy, R2 is ~-hydroxyl (the C-4'-ethyl group is alpha) and R3 and R4 are hydrogen. Deoxy-vinblastine occurs when in Formula I all of R2, R3 and R4 are hydrogen, R is CH30 and R is acetoxy. There are two isomers of deoxyvinblastine identified as "A" and "B". In the case of the "A" isomer, the R2 hydrogen is Beta(up) and the C-4'ethyl group is Alpha(down). The "B" isomer has a ~-ethyl group and an a-hydrogen at C-4'.
Vincadioline is the 3'-hydroxy derivative (i.e.
R3 is hydroxyl) of vinblastine. Leurocolombine is the 2'-hydroxy derivative (i.e. R is hydroxyl) of vinblastine.
Vincadioline has the following characteristics:
X-3754M _7_ Melting point = 218-220.5C. with decomposition;
X-ray powder diffraction pattern, using filtered chromium radiation; ~ = 2.2896A.
d in A I/Il d in A I/I
9.55 100 -1 3.99 60 8.87 9o)-2 3.71 20 8.63 90) 3.64 15 7.78 05 3.44 10 B
7.57 60 3.19 20 7.21 50 3.05 05 6.00 40 2.85 20 5.88 40 2.78 10 5.58 70 -3 2.61 10 5.22 20 2.44 15 B
5.08 20 2.21 05 B
4.70 50 2.07 05 4.57 40 1.98 15 4.42 05 1.91 05 4.31 05 nmr spectrum, ~ at 7.13, 7.53, 8.04, 3.60, 6.61, 6.09, 3.79, 2.70, 9.77, 5.47, 2.09, 0.80, 5.85, 5.29, 5.63, 3.84, 0.91;
infra-red absorption maxima at 3480, 1745 and 1725 cm molecular weight, 826;
Empirical formula, C46H58N4O]o; and Mass ions, m/e = 826, 170, 371.
Vincadioline is prepared according to the following pro-cedure: Leaves of plants containing crude vinca alkaloids;
ie, Catharanthus roseus (Vinca rosea), are extracted with a water-immiscible solvent such as benzene. The benzene is distilled from the extract in the presence of aqueous tartaric acid. The pH of the resulting aqueous acidic extract is adjusted to pH=6 by the addition of base.
Alternatively, the leaves are contacted with an aqueous acid at pH=3, and the resulting acidic layer extracted with benzene. The benzene layer is separated and discarded, and lQ67073 the pH of the aqueous layer adjusted to pH=6 as before. The dimeric alkaloids are then extracted from the aqueous layer into an organic solvent, customarily benzene. An optional ` gel exclusion filtration step can be carried out on the extracted alkaloids using a cross-linked dextran gel ("Sephadex G-25F"*), the mobile phase being a pH=3.0 ~.Ir ammonium citrate buffer. A pressure of about 15 psi is employed during gel-e~clusion chromatography. In this process, the dimeric alkaloid fraction containing leluro-cristine, vinblastine, des-N-methyl vinblastine, leuro-formine, leurosine and leurosidine is eluted first. The dimeric alkaloids are extracted from the pH = 3 buffer by adjusting the pH to 7.0 with base and then contacting the resulting aqueous solution with a water-immiscible solvent, preferably again benzene. Evaporation of the benzene yields a residue which can be dissolved in ethanol and leurosine crystallized directly therefrom. The leurosine crystals are separated by decantation, and the supernate thus obtained is acidified to pH = 4.2 with 3 percent ethanolic sulfuric acid to convert the remaining dimeric alkaloids to their sulfate salts which precipitate. The precipitated salt~ are collected and are converted to the corresponding free alkaloidal bases by standard procedures as, for example, by dissolving the salts in water, adjusting the pH to 8.0 with ammonium hydroxide and extracting the dimeric alkaloids with a water-immiscible organic solvent, preferably methylene-dichloride. Evaporation of the methylenedichloride yields the mixed dimeric alkaloids which are then chromatographed at high pressure over alumina (Activity III) using an ethyl acetate-methylenedichloride-water ~25:75:0.4) solvent system X-3754M _g_ *Trademark t~.. i _. .
~ 067073 as the eluant.
Operating pressures employed have been in the range 150-350 psi. As will be understood by those skilled in the art of high-pressure chromatography, equipment is available to carry out procedures at 4000-5000 psi and pres-sures in the range 7500-8000 psi appear feasible. Alkaloidal separation is in general more efficient at the higher pres-sures. High-pressure chromatography procedures are carried out in stainless steel equipment equipped with pressure-resistant fittings.
The alkaloids are eluted in the following order in this chromatographic procedure: residual leurosine, vin-blastine, des-N-methyl vinblastine, leurocristine and leurosidine. Identification of the dimeric alkaloid in the eluant fraction is carried out by standard procedures known to the art, as by thin layer chromatography~
After elution of the known alkaloids, there remain on the column several more polar dimeric alkaloids. These are eluted with methanol and rechromatographed until vinca-dioline is obtained as a separate fraction substantially ` free from other dimeric alkaloids present in the polar alkaloid fraction~
Leurocolombine has the following characteristics:
pKa's at 5~05, 6~3;
Infra-red absorption maxima at 2~80, 2~88, 3.35,
infra-red absorption maxima at 3480, 1745 and 1725 cm molecular weight, 826;
Empirical formula, C46H58N4O]o; and Mass ions, m/e = 826, 170, 371.
Vincadioline is prepared according to the following pro-cedure: Leaves of plants containing crude vinca alkaloids;
ie, Catharanthus roseus (Vinca rosea), are extracted with a water-immiscible solvent such as benzene. The benzene is distilled from the extract in the presence of aqueous tartaric acid. The pH of the resulting aqueous acidic extract is adjusted to pH=6 by the addition of base.
Alternatively, the leaves are contacted with an aqueous acid at pH=3, and the resulting acidic layer extracted with benzene. The benzene layer is separated and discarded, and lQ67073 the pH of the aqueous layer adjusted to pH=6 as before. The dimeric alkaloids are then extracted from the aqueous layer into an organic solvent, customarily benzene. An optional ` gel exclusion filtration step can be carried out on the extracted alkaloids using a cross-linked dextran gel ("Sephadex G-25F"*), the mobile phase being a pH=3.0 ~.Ir ammonium citrate buffer. A pressure of about 15 psi is employed during gel-e~clusion chromatography. In this process, the dimeric alkaloid fraction containing leluro-cristine, vinblastine, des-N-methyl vinblastine, leuro-formine, leurosine and leurosidine is eluted first. The dimeric alkaloids are extracted from the pH = 3 buffer by adjusting the pH to 7.0 with base and then contacting the resulting aqueous solution with a water-immiscible solvent, preferably again benzene. Evaporation of the benzene yields a residue which can be dissolved in ethanol and leurosine crystallized directly therefrom. The leurosine crystals are separated by decantation, and the supernate thus obtained is acidified to pH = 4.2 with 3 percent ethanolic sulfuric acid to convert the remaining dimeric alkaloids to their sulfate salts which precipitate. The precipitated salt~ are collected and are converted to the corresponding free alkaloidal bases by standard procedures as, for example, by dissolving the salts in water, adjusting the pH to 8.0 with ammonium hydroxide and extracting the dimeric alkaloids with a water-immiscible organic solvent, preferably methylene-dichloride. Evaporation of the methylenedichloride yields the mixed dimeric alkaloids which are then chromatographed at high pressure over alumina (Activity III) using an ethyl acetate-methylenedichloride-water ~25:75:0.4) solvent system X-3754M _g_ *Trademark t~.. i _. .
~ 067073 as the eluant.
Operating pressures employed have been in the range 150-350 psi. As will be understood by those skilled in the art of high-pressure chromatography, equipment is available to carry out procedures at 4000-5000 psi and pres-sures in the range 7500-8000 psi appear feasible. Alkaloidal separation is in general more efficient at the higher pres-sures. High-pressure chromatography procedures are carried out in stainless steel equipment equipped with pressure-resistant fittings.
The alkaloids are eluted in the following order in this chromatographic procedure: residual leurosine, vin-blastine, des-N-methyl vinblastine, leurocristine and leurosidine. Identification of the dimeric alkaloid in the eluant fraction is carried out by standard procedures known to the art, as by thin layer chromatography~
After elution of the known alkaloids, there remain on the column several more polar dimeric alkaloids. These are eluted with methanol and rechromatographed until vinca-dioline is obtained as a separate fraction substantially ` free from other dimeric alkaloids present in the polar alkaloid fraction~
Leurocolombine has the following characteristics:
pKa's at 5~05, 6~3;
Infra-red absorption maxima at 2~80, 2~88, 3.35,
5.74, 6.18, 6.65, 6.83, 6.95, 7.25, 7.50, 8.11, 9.60, 9~90 and 10~75 microns;
Ultra-violet absorption maxima at 217 (am=51~091) and 265 (am=15,666) millimicrons;
Molecular weight, 826;
Empirical formula, C46H58N4Olo;
Ion fragments by mass spectroscopy, m/e 795, 767, 749, 667, 649, 282, 170, 156, 154, 152, 144, 143;
proton nmr spectrum, chemical shifts in ppm at 7.51, 7.13, 0.90, 3.60, 3.75, 7.01, 3.84, 6.15, 5.29, 5.85, 5.48, 0.78, 2.68, 3.79, 2.70, 2.10 and 4.16; and forming a sulfate salt with the following x-ray powder diffraction pattern using filtered chromium radiation at 2.2896A.
d in A I/ 2 17.00 30 12.50 100 9.45 50 7.70 10 7.20 60
Ultra-violet absorption maxima at 217 (am=51~091) and 265 (am=15,666) millimicrons;
Molecular weight, 826;
Empirical formula, C46H58N4Olo;
Ion fragments by mass spectroscopy, m/e 795, 767, 749, 667, 649, 282, 170, 156, 154, 152, 144, 143;
proton nmr spectrum, chemical shifts in ppm at 7.51, 7.13, 0.90, 3.60, 3.75, 7.01, 3.84, 6.15, 5.29, 5.85, 5.48, 0.78, 2.68, 3.79, 2.70, 2.10 and 4.16; and forming a sulfate salt with the following x-ray powder diffraction pattern using filtered chromium radiation at 2.2896A.
d in A I/ 2 17.00 30 12.50 100 9.45 50 7.70 10 7.20 60
6.20 20 5.70 30 4.95 05 4.65 20 Leurocolombine is prepared according to the following procedure: Leaves of plants containing crude vinca alkaloids; ie, Catharanthus roseus (Vinca rosea), are extracted with a water-immiscible solvent such as benzene.
The benzene is distilled from the extract in the presence of aqueous tartaric acid. The pH of the resulting aqueous acidic extract is adjusted to pH=6 by the addition of base.
Alternatively, the leaves are contacted with an aqueous acid at ph=3, and the resulting acidic layer extracted with benzene. The benzene layer is separated and discarded, and the pH of the aqueous layer adjusted to pH=6 as before. The dimeric alkaloids are then extracted from the aqueous layer 1067~)'73 into an organic solvent, customarily benzene. An optional gel exclusion filtration step can be carried out on the ex-tracted alkaloids using a cross-linked dextran gel ("Sepha~ex G-25 F"*), the mobile phase being a pH=3.0, O.lM ammonium citrate buffer. A pressure of about 15 psi is employed during gel-exclusion chromatography. In this process, the dimeric alkaloid fraction containing leurocristine, vin-blastine, des-N-methyl vinblastine, leuroformine, leurosine and leurosidine is eluted first. The dimeric alkaloids are extracted from the pH=3 buffer by adjusting the pH to pH=7.0 with base and then extracting the resulting aqueous solution with a water-immiscible solvent, preferably again benzene.
Evaporation of the benzene yields a residue which can be dissolved in ethanol and leurosine crystallized directly therefrom. The leurosine crystals are separated by decan-tation, and the supernatant thus obtained is acidified to pH=4.2 with 3 percent ethanolic sulfuric acid to convert the remaining dimeric alkaloids to their sulfate ~alts which precipitate. The precipitated salts are collected and are converted to the corresponding free alkaloidal bases by standard procedures as, for example, by dissolving the salts in water, adjusting the pH ~ 8.0 with ammonium hydroxide and extracting the dimeric alkaloids with a water-immiscible organic solvent, preferably methylenedichloride. Evapo-ration of the methylenedichloride yields the mixed dimeric alkaloids which are then chromatographed at high pressure over alumina (Activity III-IV) using a ethyl acetate-methylenedichloride-water (25:75:0.4) solvent system as the eluant.
*Trademark ... .
Operating pressures employed have been in the range 150-350 psi. As will be understood by those skilled in the art of high-pressure chromatography, equipment is available to carry out procedures at 4000-5000 psi and pressures in the range 7500-8000 psi appear feasible.
Alkaloidal separation is in general more efficient at the higher pressures. High-pressure chxomatography procedures are carried out in stainless steel equipment equipped with pressure-resistant fittings.
The alkaloids are eluted in the following order in this chromatographic procedure: residual leurosine, vin-blastine, leurocolombine, des-N-methyl vinblastine, leuro~
cristine and leurosidine. Identification of the dimeric alkaloids in the eluant fraction is carried out by standard procedures known to the art, as by thin layer chromatography.
4-DesacetoxyVinblastine has the following physical and chemical characteristics: melting point = 183-190C.
with decomposition after recrystallization from methanol;
[]26= +95,3o (chloroform); molecular ion M+ = 752, corre-sponding to an empirical formula C44H56N4O7.
Analysis Calcd. for: C44H56N4O7 Analysis Calc.: C, 70.19; H, 7.50; N, 7.44;
O, 14.87 Found: C, 69.71; H, 7.47; N, 7.08;
O, 15.00 4-Desacetoxyvinblastine is prepared according to the following procedure: Leaves of plants containing crude vinca alkaloids; i.e., Catharanthus roseus (Vinca rosea), previously moistened with aqueous ammonia, are extracted with a water-immisclble solvent such as benzene. The ~067C~73 benzene is distilled from the extract in the presence of aqueous tartaric acid. The tartaric acid layer is extracted with a water-immiscible organic solvent and is then made basic by the addition of ammonia. The dimeric alkaloids are then extracted from the alkaline layer into an organic solvent, customaril~ benzene. Evaporation of the benzene yields a mixture of amorphous dimeric alkaloids which are dissolved in benzene and chromatographed over alumina (CAMAG - Activity III3.
The alkaloids are eluted in the following order:
leurosine, vinblastine, des-N-methyl vinblastine, leuro-cristine and leurosidine. Identification of the dimeric alkaloids in the eluant fraction is carried out by standard procedures known to the art, as by thin layer chromatography.
Vinblastine is customarily eluted with a benzene-chloroform (1:1) solvent mixture. The procedure for obtaining the V~B
fraction is more fully set forth in U. S. Patent 3,225,030.
Vinblastine fractions thus obtained were shown by thin layer chromatography to contain small quantities of a second alkaloid, identified as 4-desacetoxyvinblastine.
This second alkaloid is isolated as follows: The vinblastine fraction is converted to the corresponding sulfate salts by standard procedure and the sulfates subjected to a gradient pH separation procedure in which the sulfates are dissolved in 2 percent aqueous citric acid, and the citric acid solution extracted twice with benzene. The pH is then raised to pH = 5.5 by the addition of ammonia and two more benzene extractions are carried out. The second fraction is chromatographed over alumina (activity III). The chroma-togram is developed with benzene. Fractions shown by thin .. . .
.. _ _ . . _ .. . . .
:1067073 layer chromatography to contain a secGnd alkaloid inaddition to vinblastine are combined and rechromatographed over alumina and the chromatogram again developed with benzene. This procedure is repeated. Fractions containing substantially only the second alkaloid, 4-desacetoxy vin-blastine with only minor amounts of vinblastine are combined and recrystallized from methanol. 4-Desacetoxy vinblastine thus recrystallized is then further purified by preparative thin layer chromatography over silica using as eluant a 3:2;4 diethyla~ine-chloroform-benzene solvent mixture.
3'-Hydroxy-4-desacetoxyvinblastine has the following physical characteristics:
proton nmr spectrum peaks at ~4.075~5), 5.85(15), 5.46-5.78 (broad m~ltiplet) mass spectrum: ions at m/e 768, 411, 371, 224, 170, 102.
3'-Hydroxy-4-desacetoxyvinblastine is prepared according to the following procedure: Defatted leaves of plants containing crude vinca alkaloids; i.e., Catharanthus roseus (Vinca rosea), previously moistened with aqueous ammonia, are extracted with a water-immiscible solvent such as benzene. The benzene is distilled from the extract in the presence of aqueous tartaric acid. The tartaric acid layer is then made basic by the addition of ammonia. The dimeric alkaloids are extracted from the alkaline layer into an organic solvent, customarily benzene. Evaporation of the solvent yields a mixture of amorphous dimeric alkaloids.
The dimeric alkaloid fraction is dissolved in ethanol and the corresponding sulfate salts formed by the addition of ethanolic sulfuric acid. The crystalline mixed sulfate salts are collected and then converted to the corresponding X-3754M -lS-, _ -free bases by solution in water, basifying the aqueous solu-tion and extracting the alkaloids into a water-immiscible organic solvent, customarily methylene dichloride. Evapora-tion of the solvent yields a mixture of amorphous dimeric alkaloids which are redissolved in methylene dichloride and chromatographed over alumina (CAMAG - Activity III-IV).
The alkaloids are eluted in the following order:
leurosine, vinblastine, des-N-methyl vinblastine, leuro-cristine and leurosidine. Identification of the dimeric alkaloids in the eluant fraction is carried out by standard procedures known to the art, as by thin layer chromatography.
Chromatography was carried out in a stainless steel column, 5 cm. by 730 cm., at a pressure of 200-400 psi. The alumina-to-charge ratio was approximately 300 to 1. The eluate was monitored at 280 m~, and fractions were separated based upon the peaks observed in the ultraviolet profile.
Fractions were identified containing predominatntly leuro-sine, vinblastine, des-N-methylvinblastine, and leuro-cristine by thin layer chromatography. Post-des-N-methyl-vinblastine, pre-leurocristine fractions were accumulated, i.e., fractions containing more than one dimeric alkaloid occurring after the peak des-N-methylvinblastine fraction and prior to the peak leurocristine fraction, and were converted to the corresponding sulfate salts by treatment with an excess of 1 percent ethanolic sulfuric acid. The sulfate salts were subjected to a gradient pH separation procedure in which a solution of the sulfate salts in citric acid buffer at pH = 3.4 was extracted with benzene. The pH
of the citric acid solution was raised in increments of one-half pH unit, and the resulting aqueous layer extracted with benzene. 4-Desacetoxy-3'-hyaroxyvinblastine was found to be present by thin layer chromatography in extracts at pH
= 5.4 and 5.9. Sulfates (VLB and leurocristine were shown by TLC to be the chief dimeric alkaloid impurities present), recovered from the pH = 5.4 extract, were dissolved in 5 ml.
of water and the acidity of the aqueous solution adjusted to pH = 9 by the addition of ammonium acetate. The precip-itated alkaloidal free bases were separated by centri-fugation, dissolved in 3 ml. of methylenechloride and chromatographed at high pressure in a stainless steel 5/16"
by 6 meter column packed with neutral alumina [Woelm N-18 (18-30 ~)~ using a linear gradient of 0-5 percent ethanol in methylene chloride. The column was operated at about 1100 psi with a consequent flow rate of 180 ml/hr. Fractions were collected every 3 minutes after material began to appear in the column effluent as determined by ultra-violet profile. Fractions 30-32 contained 4-desacetoxy-3'-hydroxylvinblastine, as shown by TLC on silica gel using an etherdiethylamine-toluenemethanol (100:5:5:5) solvent system.
Illustrative compounds include:
3'-hydroxy-4-desacetoxyvinblastine C-3 N-methyl carboxamide 3'-hydroxy-4-desacetoxyvinblastine C-3 N-cyclo-propylmethyl carboxamide 2'-hydroxy-4-desacetoxyvinblastine C-3 N-cyano-ethylamide 2'-hydroxy-4-desacetoxyvinblastine C-3 N-(2-hydroxypropyl~amide deoxyvinblastine "A" C-3 carboxazide deoxyvinblastine "A" C-3 N-~2-dimethylamino-ethyl) carboxamide deoxyvinblastine "B" C-3 N-(2,3-dihydroxypentyl) carboxamide 4-desacetoxyvinblastine C-3 N-(3-hydroxypropyl) carboxamide 4-desacetoxyvinblastine C-3 N-(2-aminoethylamino) carboxamide 2'-hydroxyvinblastine C-3 N-(2-acetoxyethyl) carboxamide 2'-hydroxyvinblastine C-3 N-(2-phenylethyl) carboxamide 2'-hydroxyvinblastine C-3 N-(3-phenylpropyl) carboxamide 3'-hydroxyvinblastine C-3 carboxhydrazide and the like.
The present compounds are derivatives in which the carbomethoxyl group at C-3 of certain known indole-dihydroindole alkaloids is transformed to a carboxhydrazidegroup, a carboxazide group, a carboxamide group or a derivative thereof. Not all of these derivatives are ordinarily prepared by one process. The compounds in which R in formula I above is NH2, NH-NH2 or NH-CH3 are prepared as follows: Treatment of leurocolumbine, vin-cadioline, their respective 4-desacetyl compounds or deoxyvinblastine with either ammonia, methylamine or hydrazine yields the corresponding amide, N-methylamide or hydrazide. The product of this reaction with starting materials having an intact 4-acetyl group is usually a mixture of compounds in which the carbomethoxy group at C-3 is transformed to a carboxamide, N-methylcarboxamide or carboxhydrazide group, but also in which the acetyl group at C-4 is completely or partially removed. For purification, the C-4 desacetyl derivatives thus prepared are separated by chromatography.
The compounds in which R is N(CH3~2, NH-alk-X
wherein X is H, CN or phenyl, NH-(C3-C8)-cycloalk, NH-alk-Am or NH-alk-(OH)l 3 and alk and Am are as previously defined are prepared by the following procedure: a hydrazide, (Formula I wherein R is NH-NH2) prepared by reaction of the C-3 carbomethoxyl compound with anhydrous hydrazine, is transformed into the corresponding azide by treatment with nitrous acid, nitrosyl chloride, nitrogen tetroxide, amyl nitrite or a similar reagent according to conventional procedures. The C-3 azide thus prepared is then reacted with the primary of secondary amines HN (CH3)2, NH2-alk-X, NH2-(C3-C8)-cycloalk, NH2-alk-(OH)1 3 or NH2-alk-Am, to yield the desired C-3 amide. This C-3 azide-amine reaction does not affect the C-4 acyl group which if present remains intact during the reaction and workup. The above azide-amine transformation follows the procedure originated by Stoll and Huffman, Helv. Chim. Acta., 26, 944 (1943) -- see also U. S. Patents 2,090,429 and 2,090,430.
Compounds in which there is an acetyl group at C-4 can be prepared, as has been stated above, by reaction of vincadioline, leurocolumbine, or deoxyvinblastine - directly with ammonia, methylamine or hydrazine followed by separation of the 4-acetyl derivative from the 4-desacetyl derivative, and, in the case of the hydrazide, conversion to ~067073 the azide followed by reaction of the azide with an amine to yield the amides. More generally, however, because of the lability of the 4-acetyl group under basic reaction conditions, the hydrazine-azide-amide reaction sequence is carried out with a 4-desacetyl derivative. In general, the 4-desacetyl amides according to Formula I above can be acylated with an aliphatic anhydride or acid chloride to yield the corresponding C-4 acetate, propionate or butyrate or a chloro derivative thereof. An acid chloride (Cl-C3)-alkyl-COCl or chloro-(Cl-C3)-alkyl-CQ-Cl or an acid anhydride l(Cl-C3)-alkyl-CO]=O or [chloro-tCl-C3)-alkyl-CO]2=O can be used in the acylation reaction. The preferred acylation procedure is that described in U. S. Patent 3,392,173 for vinblastine or leurocristine in which a diacyl derivative is the first product of the reaction, and this derivative is selectively hydrolysed to yield a 4-acyl compound. Other procedures involving selective acylating or multiple acylation followed by selective hydrolysis can be employed to prepare the 4-acyl derivatives of this invention.
There are, however, certain provisos which must be kept in mind when an acylation procedure is contemplated.
If the C-3 carboxamide group contains an acylable group;
i.e., hydroxy or amino, the C-4 acylation procedure must be carried out prior to the azide-amine reaction which yields the ultimate C-3 carboxamide group. The preferred procedure here is to acylate, by the above procedures, the C-3 carboxhydrazide, first protecting the hydrazide group itself, which would otherwise also be acylated. The pre-ferred hydrazide protecting group is the propylidene group formed by reaction of the NH2 portion of the hydrazide ~067073 moiety with acetone. This group can be readily removed by treatment with acid or, preferably, the propylidene deri-vative itself can be reacted directly with nitrite to form an azide group (see U. S. Patent 3,470,210, Example VII).
Other procedures involving selective acylation or multiple acylation followed by selective hydrolysis or selective protection of an acylable function followed by acylation and subsequent removal of the protecting group will be apparent to those skilled in the art.
Compounds according to Formula I above in which R
is NH-alk-X and X is carboxyl, carboxamido or carbo-(Cl-C3)-alkoxy are prepared by reacting an amino acid, amino amide preferably an amino ester of the structure NH2-C-COQ
wherein Z
Q is OH, NH2 or O-alk and Z is H or a Cl-C5 alkyl group, with the chosen dimeric indole-dihydroindole azide. Amino acids useful for this purpose, and coming within the scope of the above formula, include leucine, isoleucine, valine, glycine, alanine, norleucine and the like. As will be apparent to those skilled in the art, other amino acids and polypeptides can also be used to react with, for example, 4-desacetyl vinblastine C-3 carboxazide, to yield substi-tuted C-3 carboxamides having anti-tumor properties.
An alternative method of preparing the primary amide (R is NH2) from the hydrazide (R is NH-NH2) involves the use of a procedure based on that of Ainsworth, U. S.
Patent 2,756,235, in which the hydrazide is hydrogenolyzed with Raney nickel.
. _ _, . _ _ . , _ _ . . . . . . . . _ .... _ 1067~73 The novel derivatives will be named with referenceonly to the new group formed at a given carbon atom. For example, the compound produced by replacing the methyl ester function in vinblastine at C-3 with an amide function will be called simply vinblastine C-3 carboxamide, and not vin-blastine C-3 descarbomethoxy C-3 carboxamide.
The compounds in the form of their free bases, including both carboxamides, carboxazides and carboxhydra-zides are white or tan-colored amorphous solids. It is preferable, however, where possible to isolate and crys-tallize the amides in the form of their anionic salts formed with non-toxic acids. Such salts are high-melting, white, crystalline or amorphous, water-soluble solids.
The preparation of the compounds is more fully illustrated in the following specific examples:
! Example 1 4-Desacetyl deoxyvinblastine "B" C-3 carboxhydrazide Deoxyvinblastine "B" in the amount of 2.55 grams was reacted with 30 ml. of anhydrous hydrazine in anhydrous methanol in a sealed reaction vessel at about 60C for about 18 hours. The reaction vessel was cooled, and opened, the contents removed, and the volatile constituents evaporated therefrom in vacuo. The resulting residue, comprising 4-desacetyl deoxyvinblastine "s" C-3 carboxhydrazide, was taken up in methylenechloride, the methylenechloride solution washed with water, separated and dried, and the methylenechloride removed in vacuo to yield an amorphous powder with the following characteristics: infrared maxima;
3440 cm 1 (N-H), 1735 cm 1 (COO), 1675 cm (CON); molecular ion spectrum; m/e = 752 (cc,nsistent with C43H56N6O6); nmr 1~67073 spectrum; ~3.78 (ArOCH3), ~3.58 (C18COOC_3), ~2.77 (N-CH3), ~4.15 (C4-H).
Example 2 4-Desacetoxyvinblastine C-3 N-(2-Hydroxyethyl) Carboxamide Following the procedure of Example 1, 4-des-acetoxyvinblastine was reacted with anhydrous hydrazine in methanol solution in a sealed tube at 46C. for three days.
4-Desacetoxyvinblastine C-3 carboxhydrazide thus prepared was isolated and purified by the procedure of the same example. The compound had the following physical char-acteristics: PKa = 5.61 and 7.38; ultraviolet spectrum;
~max = 215 and 267 nm; infrared spectrum, peaks at 3450 cm 1 (N-H), 1725 cm 1 (COO), 1680 cm 1 (CON); molecular spectrum m/e = 252, 411, 355, 244, 154. Molecular ion M+ =
752 consistent with empirical formula C43H56N6O6. The compound was a tan amorphous powder.
The 4-desacetoxyvinblastine C-3 carboxhydrazide was converted to the corresponding carboxazide in hydro-chloric acid solution at 0C. with sodium nitrite. The reaction mixture was next made basic by the addition of an excess of cold 5 percent aqueous sodium bicarbonate. The aqueous solution was extracted three times with methylene dichloride.
Five ml. of ethanol amine were added to a solution containing about 1.2 g. of 4-desacetoxyvinblastine C-3 carboxazide. The reaction mixture was sealed and protected from light. After being allowed to stand for one day at room temperature, the reaction vessel was opened and the volatile constituents removed from the reaction mixture by ~-3754M -23-evaporation in vacuo. The resulting residue containing 4-desacetoxyvinblastine C-3 N-(2-hydroxyethyl) carboxamide formed in the above reaction was dissolved in methylene dichloride and the methylene dichloride layer washed several times with water. The methylene dichloride layer was separated and dried and the solvent removed by evaporation in vacuo. The resulting residue was chromatographed over silica gel using a 3:1 ethyl acetate-ethanol solvent mixture as the eluant. Fractions shown to contain the desired product as determined by thin layer chromatography were combined and evaporated to dryness in vacuo. 4-Desacetoxy-vinblastine C-3 (N-(2-hydroxyethyl) carboxamide thus prepared was a tan amorphous material with the following physical characteristics: molecular ion; M+ = 781 con-sistent with empirical formula C45H59N507; infrared spectrum peaks at 3420 cm 1 (NH), 1735 cm 1 (C00), 1665 cm 1 (CON~.
The sulfate salt was prepared using ethanolic sulfuric acid and adjusting the pH in the range 3.8-4.2. The sulfate salt was recovered by evaporation of the volatile constituents in vacuo.
Example 3 4-Desacetylvincadioline C-3 N-Methylamide Following the procedure of Example 1, vincadioline was reacted with hydrazine to form the corresponding C-3 carboxhydrazide. The hydrazide was in turn converted to the corresponding carboxazide by the procedure of Example 8 and the azide was reacted with methylamine according to the procedure of the same example. The product of this re-action, 4-desacetyl vincadioline C-3 N-methylamide, had the following physical characteristics: infrared spectrum peaks 1067~73 at 2.95 m~, 5.75 m~ and 5.97 m~; nmr spectrum consistent with postulated structure with added doublet at ~3.82 (amidemethyl hydrogens); molecular spectrum, molecular ion M+ = 783 consistent with C44H57N508 Following the above procedure 4-desacetylleuro-colombine C-3 N-methylamide is prepared.
Example 4 Preparation of salts Other salts, including salts with inorganic anions such as chloride, bromide, phosphate, nitrate and the like, as well as salts with organic anions such as acetate, chloroacetate, trichloroacetate, benzoate, alkyl or aryl sulfonates and the like, are prepared from the amide bases of this invention by a procedure analogous to that set forth in Example 1 above for the preparation of the sulfate salt by substituting the appropriate acid in a suitable diluent in place of the 1 percent aqueous sulfuric acid of that example.
As will be apparent to those skilled in the art, the presence of other ester and/or amide groups in the indole-dihydroindole components requires extra care in the preparation of salts so as to avoid hydrolysis, trans-esterification and other reactions which take place at high - temperatures, extremely acid pH's, etc.
The compounds have shown antiviral activity in vitro against herpes virus employing a tissue culture system in a plaque suppression test similar to that described by Siminoff, Applied Microbiology~ 9, 66-72 (1961).
,~
10671:~73 In addition, the compounds have been shown to be active against transplanted mouse tumors in vivo. of particular interest, however, is the activity of the compounds against Ridgeway osteogenic sarcoma (ROS) and Gardner lymphosarcoma (GLS). In demonstrating activity of the drugs against these tumors, a protocol was used which involved the administration of the drug, usually by the intraperitoneal route, at a given dose level for 7-10 days after inoculation with the tumor.
The following table - Table 1 - gives the results of several experiments in which mice bearing transplanted tumors were treated successfully with a compound of this invention. In the table, column 1 gives the name of the compound; column 2, the transplanted tumor; column 3, the dose level or dose level range and the number of days the dosage was administered; and column 4, the percent inhi-bition of tumor growth. (ROS is an abbreviation for Ridgeway osteogenic sarcoma; GLS for Gardner lymphosarcoma;
and CA 755 is an adenocarcinoma).
~o The compounds like leurocristine and vinblastine become toxic to mice at doses above those at which they produce 100 percent inhibition of the transplanted tumor.
In addition, for reasons that are not well understood, all drugs in a given test including control drugs like vin-blastine may show toxicity at dose levels where they ordinarily give tumor inhibition without toxicity. Thus, the results set forth in Table 1 are of typical experiments where the control drugs give expected results and are not an average of all runs.
1~67073 o o ~ ~ o a) ~1 C) Q
~1 ~
H
U~
X X
~ O Ln O ~1 a ~ ,, .Y X
10 ~s In r~
O O
E~ ~
~ o~
.. E~ ~; ~
~ .~
o C) ~ ^
, X
~a ~ o o s~
~ d o I
o I ~:~
~ ~r ~ m The compounds are also active against other transplanted tumors. For example, with Mecca lymphosarcoma, parenteral injection of 0.25 mg./kg. for 9 days of vin-blastine C-3 N-methylcarboxamide sulfate gave a 54 percent inhibition of growth and of vinblastine C-3 amide, a 28 percent inhibition. At the same dose levels, vinblastine itself was completely inactive.
In addition, in studies against CA 755 adeno-carcinoma, 4-desacetyl vinblastine C-3 carboxamide sulfate gave a 67 percent inhibition of tumor growth, 4-desacetyl vinblastine C-3 N-methylcarboxamide sulfate 61 percent inhibition and vinblastine C-3 carboxamide sulfate 49 per-cent inhibition at a dose level of 0.25 mg./kg. for eight days and 72 percent inhibition at 0.3 mg./kg. In a similar experiment, vinblastine gave a 31 percent inhibition while leurocristine at the somewhat lower dose level of 0.2 mg./kg.
gave 79 percent inhibition with an excellent effectiveness rating. Against L5178Y lymphocytic leukemia, vinblastine C-3 carboxamide sulfate at a dose level of 0.25 mg./kg. for ten days in an experiment using five mice gave three indefinite survivors; the life span of the two diseased mice in this experiment was prolonged by 26 percent over that of the control mice. In the same experiment, vinblastine gave a 36 percent prolongation but with no indefinite survivors and rated only a minimal effectiveness rating.
In utilizing the novel amides and hydrazides as anti-neoplastic agents, either the parenteral or oral route of administration may be employed. For oral dosage, a suitable quantity of a pharmaceutically-acceptable salt of a base according to Formula I except those in which R is ~067073 NH-NH2 or N3, formed with a non-toxic acid is mixed with starch or other excipient and the mixture placed in tele-scoping gelatin capsules each containing from 7.5-50 mg. of active ingredients. Similarly, the anti-neoplastic salt can be mixed with starch, a binder, and a lubricant and the mixture compressed into tablets each containing from the
The benzene is distilled from the extract in the presence of aqueous tartaric acid. The pH of the resulting aqueous acidic extract is adjusted to pH=6 by the addition of base.
Alternatively, the leaves are contacted with an aqueous acid at ph=3, and the resulting acidic layer extracted with benzene. The benzene layer is separated and discarded, and the pH of the aqueous layer adjusted to pH=6 as before. The dimeric alkaloids are then extracted from the aqueous layer 1067~)'73 into an organic solvent, customarily benzene. An optional gel exclusion filtration step can be carried out on the ex-tracted alkaloids using a cross-linked dextran gel ("Sepha~ex G-25 F"*), the mobile phase being a pH=3.0, O.lM ammonium citrate buffer. A pressure of about 15 psi is employed during gel-exclusion chromatography. In this process, the dimeric alkaloid fraction containing leurocristine, vin-blastine, des-N-methyl vinblastine, leuroformine, leurosine and leurosidine is eluted first. The dimeric alkaloids are extracted from the pH=3 buffer by adjusting the pH to pH=7.0 with base and then extracting the resulting aqueous solution with a water-immiscible solvent, preferably again benzene.
Evaporation of the benzene yields a residue which can be dissolved in ethanol and leurosine crystallized directly therefrom. The leurosine crystals are separated by decan-tation, and the supernatant thus obtained is acidified to pH=4.2 with 3 percent ethanolic sulfuric acid to convert the remaining dimeric alkaloids to their sulfate ~alts which precipitate. The precipitated salts are collected and are converted to the corresponding free alkaloidal bases by standard procedures as, for example, by dissolving the salts in water, adjusting the pH ~ 8.0 with ammonium hydroxide and extracting the dimeric alkaloids with a water-immiscible organic solvent, preferably methylenedichloride. Evapo-ration of the methylenedichloride yields the mixed dimeric alkaloids which are then chromatographed at high pressure over alumina (Activity III-IV) using a ethyl acetate-methylenedichloride-water (25:75:0.4) solvent system as the eluant.
*Trademark ... .
Operating pressures employed have been in the range 150-350 psi. As will be understood by those skilled in the art of high-pressure chromatography, equipment is available to carry out procedures at 4000-5000 psi and pressures in the range 7500-8000 psi appear feasible.
Alkaloidal separation is in general more efficient at the higher pressures. High-pressure chxomatography procedures are carried out in stainless steel equipment equipped with pressure-resistant fittings.
The alkaloids are eluted in the following order in this chromatographic procedure: residual leurosine, vin-blastine, leurocolombine, des-N-methyl vinblastine, leuro~
cristine and leurosidine. Identification of the dimeric alkaloids in the eluant fraction is carried out by standard procedures known to the art, as by thin layer chromatography.
4-DesacetoxyVinblastine has the following physical and chemical characteristics: melting point = 183-190C.
with decomposition after recrystallization from methanol;
[]26= +95,3o (chloroform); molecular ion M+ = 752, corre-sponding to an empirical formula C44H56N4O7.
Analysis Calcd. for: C44H56N4O7 Analysis Calc.: C, 70.19; H, 7.50; N, 7.44;
O, 14.87 Found: C, 69.71; H, 7.47; N, 7.08;
O, 15.00 4-Desacetoxyvinblastine is prepared according to the following procedure: Leaves of plants containing crude vinca alkaloids; i.e., Catharanthus roseus (Vinca rosea), previously moistened with aqueous ammonia, are extracted with a water-immisclble solvent such as benzene. The ~067C~73 benzene is distilled from the extract in the presence of aqueous tartaric acid. The tartaric acid layer is extracted with a water-immiscible organic solvent and is then made basic by the addition of ammonia. The dimeric alkaloids are then extracted from the alkaline layer into an organic solvent, customaril~ benzene. Evaporation of the benzene yields a mixture of amorphous dimeric alkaloids which are dissolved in benzene and chromatographed over alumina (CAMAG - Activity III3.
The alkaloids are eluted in the following order:
leurosine, vinblastine, des-N-methyl vinblastine, leuro-cristine and leurosidine. Identification of the dimeric alkaloids in the eluant fraction is carried out by standard procedures known to the art, as by thin layer chromatography.
Vinblastine is customarily eluted with a benzene-chloroform (1:1) solvent mixture. The procedure for obtaining the V~B
fraction is more fully set forth in U. S. Patent 3,225,030.
Vinblastine fractions thus obtained were shown by thin layer chromatography to contain small quantities of a second alkaloid, identified as 4-desacetoxyvinblastine.
This second alkaloid is isolated as follows: The vinblastine fraction is converted to the corresponding sulfate salts by standard procedure and the sulfates subjected to a gradient pH separation procedure in which the sulfates are dissolved in 2 percent aqueous citric acid, and the citric acid solution extracted twice with benzene. The pH is then raised to pH = 5.5 by the addition of ammonia and two more benzene extractions are carried out. The second fraction is chromatographed over alumina (activity III). The chroma-togram is developed with benzene. Fractions shown by thin .. . .
.. _ _ . . _ .. . . .
:1067073 layer chromatography to contain a secGnd alkaloid inaddition to vinblastine are combined and rechromatographed over alumina and the chromatogram again developed with benzene. This procedure is repeated. Fractions containing substantially only the second alkaloid, 4-desacetoxy vin-blastine with only minor amounts of vinblastine are combined and recrystallized from methanol. 4-Desacetoxy vinblastine thus recrystallized is then further purified by preparative thin layer chromatography over silica using as eluant a 3:2;4 diethyla~ine-chloroform-benzene solvent mixture.
3'-Hydroxy-4-desacetoxyvinblastine has the following physical characteristics:
proton nmr spectrum peaks at ~4.075~5), 5.85(15), 5.46-5.78 (broad m~ltiplet) mass spectrum: ions at m/e 768, 411, 371, 224, 170, 102.
3'-Hydroxy-4-desacetoxyvinblastine is prepared according to the following procedure: Defatted leaves of plants containing crude vinca alkaloids; i.e., Catharanthus roseus (Vinca rosea), previously moistened with aqueous ammonia, are extracted with a water-immiscible solvent such as benzene. The benzene is distilled from the extract in the presence of aqueous tartaric acid. The tartaric acid layer is then made basic by the addition of ammonia. The dimeric alkaloids are extracted from the alkaline layer into an organic solvent, customarily benzene. Evaporation of the solvent yields a mixture of amorphous dimeric alkaloids.
The dimeric alkaloid fraction is dissolved in ethanol and the corresponding sulfate salts formed by the addition of ethanolic sulfuric acid. The crystalline mixed sulfate salts are collected and then converted to the corresponding X-3754M -lS-, _ -free bases by solution in water, basifying the aqueous solu-tion and extracting the alkaloids into a water-immiscible organic solvent, customarily methylene dichloride. Evapora-tion of the solvent yields a mixture of amorphous dimeric alkaloids which are redissolved in methylene dichloride and chromatographed over alumina (CAMAG - Activity III-IV).
The alkaloids are eluted in the following order:
leurosine, vinblastine, des-N-methyl vinblastine, leuro-cristine and leurosidine. Identification of the dimeric alkaloids in the eluant fraction is carried out by standard procedures known to the art, as by thin layer chromatography.
Chromatography was carried out in a stainless steel column, 5 cm. by 730 cm., at a pressure of 200-400 psi. The alumina-to-charge ratio was approximately 300 to 1. The eluate was monitored at 280 m~, and fractions were separated based upon the peaks observed in the ultraviolet profile.
Fractions were identified containing predominatntly leuro-sine, vinblastine, des-N-methylvinblastine, and leuro-cristine by thin layer chromatography. Post-des-N-methyl-vinblastine, pre-leurocristine fractions were accumulated, i.e., fractions containing more than one dimeric alkaloid occurring after the peak des-N-methylvinblastine fraction and prior to the peak leurocristine fraction, and were converted to the corresponding sulfate salts by treatment with an excess of 1 percent ethanolic sulfuric acid. The sulfate salts were subjected to a gradient pH separation procedure in which a solution of the sulfate salts in citric acid buffer at pH = 3.4 was extracted with benzene. The pH
of the citric acid solution was raised in increments of one-half pH unit, and the resulting aqueous layer extracted with benzene. 4-Desacetoxy-3'-hyaroxyvinblastine was found to be present by thin layer chromatography in extracts at pH
= 5.4 and 5.9. Sulfates (VLB and leurocristine were shown by TLC to be the chief dimeric alkaloid impurities present), recovered from the pH = 5.4 extract, were dissolved in 5 ml.
of water and the acidity of the aqueous solution adjusted to pH = 9 by the addition of ammonium acetate. The precip-itated alkaloidal free bases were separated by centri-fugation, dissolved in 3 ml. of methylenechloride and chromatographed at high pressure in a stainless steel 5/16"
by 6 meter column packed with neutral alumina [Woelm N-18 (18-30 ~)~ using a linear gradient of 0-5 percent ethanol in methylene chloride. The column was operated at about 1100 psi with a consequent flow rate of 180 ml/hr. Fractions were collected every 3 minutes after material began to appear in the column effluent as determined by ultra-violet profile. Fractions 30-32 contained 4-desacetoxy-3'-hydroxylvinblastine, as shown by TLC on silica gel using an etherdiethylamine-toluenemethanol (100:5:5:5) solvent system.
Illustrative compounds include:
3'-hydroxy-4-desacetoxyvinblastine C-3 N-methyl carboxamide 3'-hydroxy-4-desacetoxyvinblastine C-3 N-cyclo-propylmethyl carboxamide 2'-hydroxy-4-desacetoxyvinblastine C-3 N-cyano-ethylamide 2'-hydroxy-4-desacetoxyvinblastine C-3 N-(2-hydroxypropyl~amide deoxyvinblastine "A" C-3 carboxazide deoxyvinblastine "A" C-3 N-~2-dimethylamino-ethyl) carboxamide deoxyvinblastine "B" C-3 N-(2,3-dihydroxypentyl) carboxamide 4-desacetoxyvinblastine C-3 N-(3-hydroxypropyl) carboxamide 4-desacetoxyvinblastine C-3 N-(2-aminoethylamino) carboxamide 2'-hydroxyvinblastine C-3 N-(2-acetoxyethyl) carboxamide 2'-hydroxyvinblastine C-3 N-(2-phenylethyl) carboxamide 2'-hydroxyvinblastine C-3 N-(3-phenylpropyl) carboxamide 3'-hydroxyvinblastine C-3 carboxhydrazide and the like.
The present compounds are derivatives in which the carbomethoxyl group at C-3 of certain known indole-dihydroindole alkaloids is transformed to a carboxhydrazidegroup, a carboxazide group, a carboxamide group or a derivative thereof. Not all of these derivatives are ordinarily prepared by one process. The compounds in which R in formula I above is NH2, NH-NH2 or NH-CH3 are prepared as follows: Treatment of leurocolumbine, vin-cadioline, their respective 4-desacetyl compounds or deoxyvinblastine with either ammonia, methylamine or hydrazine yields the corresponding amide, N-methylamide or hydrazide. The product of this reaction with starting materials having an intact 4-acetyl group is usually a mixture of compounds in which the carbomethoxy group at C-3 is transformed to a carboxamide, N-methylcarboxamide or carboxhydrazide group, but also in which the acetyl group at C-4 is completely or partially removed. For purification, the C-4 desacetyl derivatives thus prepared are separated by chromatography.
The compounds in which R is N(CH3~2, NH-alk-X
wherein X is H, CN or phenyl, NH-(C3-C8)-cycloalk, NH-alk-Am or NH-alk-(OH)l 3 and alk and Am are as previously defined are prepared by the following procedure: a hydrazide, (Formula I wherein R is NH-NH2) prepared by reaction of the C-3 carbomethoxyl compound with anhydrous hydrazine, is transformed into the corresponding azide by treatment with nitrous acid, nitrosyl chloride, nitrogen tetroxide, amyl nitrite or a similar reagent according to conventional procedures. The C-3 azide thus prepared is then reacted with the primary of secondary amines HN (CH3)2, NH2-alk-X, NH2-(C3-C8)-cycloalk, NH2-alk-(OH)1 3 or NH2-alk-Am, to yield the desired C-3 amide. This C-3 azide-amine reaction does not affect the C-4 acyl group which if present remains intact during the reaction and workup. The above azide-amine transformation follows the procedure originated by Stoll and Huffman, Helv. Chim. Acta., 26, 944 (1943) -- see also U. S. Patents 2,090,429 and 2,090,430.
Compounds in which there is an acetyl group at C-4 can be prepared, as has been stated above, by reaction of vincadioline, leurocolumbine, or deoxyvinblastine - directly with ammonia, methylamine or hydrazine followed by separation of the 4-acetyl derivative from the 4-desacetyl derivative, and, in the case of the hydrazide, conversion to ~067073 the azide followed by reaction of the azide with an amine to yield the amides. More generally, however, because of the lability of the 4-acetyl group under basic reaction conditions, the hydrazine-azide-amide reaction sequence is carried out with a 4-desacetyl derivative. In general, the 4-desacetyl amides according to Formula I above can be acylated with an aliphatic anhydride or acid chloride to yield the corresponding C-4 acetate, propionate or butyrate or a chloro derivative thereof. An acid chloride (Cl-C3)-alkyl-COCl or chloro-(Cl-C3)-alkyl-CQ-Cl or an acid anhydride l(Cl-C3)-alkyl-CO]=O or [chloro-tCl-C3)-alkyl-CO]2=O can be used in the acylation reaction. The preferred acylation procedure is that described in U. S. Patent 3,392,173 for vinblastine or leurocristine in which a diacyl derivative is the first product of the reaction, and this derivative is selectively hydrolysed to yield a 4-acyl compound. Other procedures involving selective acylating or multiple acylation followed by selective hydrolysis can be employed to prepare the 4-acyl derivatives of this invention.
There are, however, certain provisos which must be kept in mind when an acylation procedure is contemplated.
If the C-3 carboxamide group contains an acylable group;
i.e., hydroxy or amino, the C-4 acylation procedure must be carried out prior to the azide-amine reaction which yields the ultimate C-3 carboxamide group. The preferred procedure here is to acylate, by the above procedures, the C-3 carboxhydrazide, first protecting the hydrazide group itself, which would otherwise also be acylated. The pre-ferred hydrazide protecting group is the propylidene group formed by reaction of the NH2 portion of the hydrazide ~067073 moiety with acetone. This group can be readily removed by treatment with acid or, preferably, the propylidene deri-vative itself can be reacted directly with nitrite to form an azide group (see U. S. Patent 3,470,210, Example VII).
Other procedures involving selective acylation or multiple acylation followed by selective hydrolysis or selective protection of an acylable function followed by acylation and subsequent removal of the protecting group will be apparent to those skilled in the art.
Compounds according to Formula I above in which R
is NH-alk-X and X is carboxyl, carboxamido or carbo-(Cl-C3)-alkoxy are prepared by reacting an amino acid, amino amide preferably an amino ester of the structure NH2-C-COQ
wherein Z
Q is OH, NH2 or O-alk and Z is H or a Cl-C5 alkyl group, with the chosen dimeric indole-dihydroindole azide. Amino acids useful for this purpose, and coming within the scope of the above formula, include leucine, isoleucine, valine, glycine, alanine, norleucine and the like. As will be apparent to those skilled in the art, other amino acids and polypeptides can also be used to react with, for example, 4-desacetyl vinblastine C-3 carboxazide, to yield substi-tuted C-3 carboxamides having anti-tumor properties.
An alternative method of preparing the primary amide (R is NH2) from the hydrazide (R is NH-NH2) involves the use of a procedure based on that of Ainsworth, U. S.
Patent 2,756,235, in which the hydrazide is hydrogenolyzed with Raney nickel.
. _ _, . _ _ . , _ _ . . . . . . . . _ .... _ 1067~73 The novel derivatives will be named with referenceonly to the new group formed at a given carbon atom. For example, the compound produced by replacing the methyl ester function in vinblastine at C-3 with an amide function will be called simply vinblastine C-3 carboxamide, and not vin-blastine C-3 descarbomethoxy C-3 carboxamide.
The compounds in the form of their free bases, including both carboxamides, carboxazides and carboxhydra-zides are white or tan-colored amorphous solids. It is preferable, however, where possible to isolate and crys-tallize the amides in the form of their anionic salts formed with non-toxic acids. Such salts are high-melting, white, crystalline or amorphous, water-soluble solids.
The preparation of the compounds is more fully illustrated in the following specific examples:
! Example 1 4-Desacetyl deoxyvinblastine "B" C-3 carboxhydrazide Deoxyvinblastine "B" in the amount of 2.55 grams was reacted with 30 ml. of anhydrous hydrazine in anhydrous methanol in a sealed reaction vessel at about 60C for about 18 hours. The reaction vessel was cooled, and opened, the contents removed, and the volatile constituents evaporated therefrom in vacuo. The resulting residue, comprising 4-desacetyl deoxyvinblastine "s" C-3 carboxhydrazide, was taken up in methylenechloride, the methylenechloride solution washed with water, separated and dried, and the methylenechloride removed in vacuo to yield an amorphous powder with the following characteristics: infrared maxima;
3440 cm 1 (N-H), 1735 cm 1 (COO), 1675 cm (CON); molecular ion spectrum; m/e = 752 (cc,nsistent with C43H56N6O6); nmr 1~67073 spectrum; ~3.78 (ArOCH3), ~3.58 (C18COOC_3), ~2.77 (N-CH3), ~4.15 (C4-H).
Example 2 4-Desacetoxyvinblastine C-3 N-(2-Hydroxyethyl) Carboxamide Following the procedure of Example 1, 4-des-acetoxyvinblastine was reacted with anhydrous hydrazine in methanol solution in a sealed tube at 46C. for three days.
4-Desacetoxyvinblastine C-3 carboxhydrazide thus prepared was isolated and purified by the procedure of the same example. The compound had the following physical char-acteristics: PKa = 5.61 and 7.38; ultraviolet spectrum;
~max = 215 and 267 nm; infrared spectrum, peaks at 3450 cm 1 (N-H), 1725 cm 1 (COO), 1680 cm 1 (CON); molecular spectrum m/e = 252, 411, 355, 244, 154. Molecular ion M+ =
752 consistent with empirical formula C43H56N6O6. The compound was a tan amorphous powder.
The 4-desacetoxyvinblastine C-3 carboxhydrazide was converted to the corresponding carboxazide in hydro-chloric acid solution at 0C. with sodium nitrite. The reaction mixture was next made basic by the addition of an excess of cold 5 percent aqueous sodium bicarbonate. The aqueous solution was extracted three times with methylene dichloride.
Five ml. of ethanol amine were added to a solution containing about 1.2 g. of 4-desacetoxyvinblastine C-3 carboxazide. The reaction mixture was sealed and protected from light. After being allowed to stand for one day at room temperature, the reaction vessel was opened and the volatile constituents removed from the reaction mixture by ~-3754M -23-evaporation in vacuo. The resulting residue containing 4-desacetoxyvinblastine C-3 N-(2-hydroxyethyl) carboxamide formed in the above reaction was dissolved in methylene dichloride and the methylene dichloride layer washed several times with water. The methylene dichloride layer was separated and dried and the solvent removed by evaporation in vacuo. The resulting residue was chromatographed over silica gel using a 3:1 ethyl acetate-ethanol solvent mixture as the eluant. Fractions shown to contain the desired product as determined by thin layer chromatography were combined and evaporated to dryness in vacuo. 4-Desacetoxy-vinblastine C-3 (N-(2-hydroxyethyl) carboxamide thus prepared was a tan amorphous material with the following physical characteristics: molecular ion; M+ = 781 con-sistent with empirical formula C45H59N507; infrared spectrum peaks at 3420 cm 1 (NH), 1735 cm 1 (C00), 1665 cm 1 (CON~.
The sulfate salt was prepared using ethanolic sulfuric acid and adjusting the pH in the range 3.8-4.2. The sulfate salt was recovered by evaporation of the volatile constituents in vacuo.
Example 3 4-Desacetylvincadioline C-3 N-Methylamide Following the procedure of Example 1, vincadioline was reacted with hydrazine to form the corresponding C-3 carboxhydrazide. The hydrazide was in turn converted to the corresponding carboxazide by the procedure of Example 8 and the azide was reacted with methylamine according to the procedure of the same example. The product of this re-action, 4-desacetyl vincadioline C-3 N-methylamide, had the following physical characteristics: infrared spectrum peaks 1067~73 at 2.95 m~, 5.75 m~ and 5.97 m~; nmr spectrum consistent with postulated structure with added doublet at ~3.82 (amidemethyl hydrogens); molecular spectrum, molecular ion M+ = 783 consistent with C44H57N508 Following the above procedure 4-desacetylleuro-colombine C-3 N-methylamide is prepared.
Example 4 Preparation of salts Other salts, including salts with inorganic anions such as chloride, bromide, phosphate, nitrate and the like, as well as salts with organic anions such as acetate, chloroacetate, trichloroacetate, benzoate, alkyl or aryl sulfonates and the like, are prepared from the amide bases of this invention by a procedure analogous to that set forth in Example 1 above for the preparation of the sulfate salt by substituting the appropriate acid in a suitable diluent in place of the 1 percent aqueous sulfuric acid of that example.
As will be apparent to those skilled in the art, the presence of other ester and/or amide groups in the indole-dihydroindole components requires extra care in the preparation of salts so as to avoid hydrolysis, trans-esterification and other reactions which take place at high - temperatures, extremely acid pH's, etc.
The compounds have shown antiviral activity in vitro against herpes virus employing a tissue culture system in a plaque suppression test similar to that described by Siminoff, Applied Microbiology~ 9, 66-72 (1961).
,~
10671:~73 In addition, the compounds have been shown to be active against transplanted mouse tumors in vivo. of particular interest, however, is the activity of the compounds against Ridgeway osteogenic sarcoma (ROS) and Gardner lymphosarcoma (GLS). In demonstrating activity of the drugs against these tumors, a protocol was used which involved the administration of the drug, usually by the intraperitoneal route, at a given dose level for 7-10 days after inoculation with the tumor.
The following table - Table 1 - gives the results of several experiments in which mice bearing transplanted tumors were treated successfully with a compound of this invention. In the table, column 1 gives the name of the compound; column 2, the transplanted tumor; column 3, the dose level or dose level range and the number of days the dosage was administered; and column 4, the percent inhi-bition of tumor growth. (ROS is an abbreviation for Ridgeway osteogenic sarcoma; GLS for Gardner lymphosarcoma;
and CA 755 is an adenocarcinoma).
~o The compounds like leurocristine and vinblastine become toxic to mice at doses above those at which they produce 100 percent inhibition of the transplanted tumor.
In addition, for reasons that are not well understood, all drugs in a given test including control drugs like vin-blastine may show toxicity at dose levels where they ordinarily give tumor inhibition without toxicity. Thus, the results set forth in Table 1 are of typical experiments where the control drugs give expected results and are not an average of all runs.
1~67073 o o ~ ~ o a) ~1 C) Q
~1 ~
H
U~
X X
~ O Ln O ~1 a ~ ,, .Y X
10 ~s In r~
O O
E~ ~
~ o~
.. E~ ~; ~
~ .~
o C) ~ ^
, X
~a ~ o o s~
~ d o I
o I ~:~
~ ~r ~ m The compounds are also active against other transplanted tumors. For example, with Mecca lymphosarcoma, parenteral injection of 0.25 mg./kg. for 9 days of vin-blastine C-3 N-methylcarboxamide sulfate gave a 54 percent inhibition of growth and of vinblastine C-3 amide, a 28 percent inhibition. At the same dose levels, vinblastine itself was completely inactive.
In addition, in studies against CA 755 adeno-carcinoma, 4-desacetyl vinblastine C-3 carboxamide sulfate gave a 67 percent inhibition of tumor growth, 4-desacetyl vinblastine C-3 N-methylcarboxamide sulfate 61 percent inhibition and vinblastine C-3 carboxamide sulfate 49 per-cent inhibition at a dose level of 0.25 mg./kg. for eight days and 72 percent inhibition at 0.3 mg./kg. In a similar experiment, vinblastine gave a 31 percent inhibition while leurocristine at the somewhat lower dose level of 0.2 mg./kg.
gave 79 percent inhibition with an excellent effectiveness rating. Against L5178Y lymphocytic leukemia, vinblastine C-3 carboxamide sulfate at a dose level of 0.25 mg./kg. for ten days in an experiment using five mice gave three indefinite survivors; the life span of the two diseased mice in this experiment was prolonged by 26 percent over that of the control mice. In the same experiment, vinblastine gave a 36 percent prolongation but with no indefinite survivors and rated only a minimal effectiveness rating.
In utilizing the novel amides and hydrazides as anti-neoplastic agents, either the parenteral or oral route of administration may be employed. For oral dosage, a suitable quantity of a pharmaceutically-acceptable salt of a base according to Formula I except those in which R is ~067073 NH-NH2 or N3, formed with a non-toxic acid is mixed with starch or other excipient and the mixture placed in tele-scoping gelatin capsules each containing from 7.5-50 mg. of active ingredients. Similarly, the anti-neoplastic salt can be mixed with starch, a binder, and a lubricant and the mixture compressed into tablets each containing from the
7.5-50 mg. The tablets may be scored if lower or divided dosages are to be used. With parenteral administration, the intravenous route is preferred. For this purpose, isotonic solutions are employed containing 1-10 mg./ml. of a salt of an indole-dihydroindole amide of formula I except for the hydrazides and azides. The compounds are administered at the rate of from 0.1 to 1 mg./kg. of mammalian body weight once a week, depending on both the activity and the toxicity of the drug. Free bases of compounds according to formula I
in which R is NH-NH2 or N3 are compounded into suitable dosage forms and administered in similar fashion at similar dose levels.
While most of the compounds of this invention are useful as antineoplastic or antiviral drugs, two types of derivatives, the hydrazides and azides (compounds of formula I wherein R is NH-NH2 or N3), are also useful as inter-mediates as has been set forth above, in that the hydrazide can be transformed to the azide by nitrosation, as by nitrous acid treatment, or to the simple amide by hydro-genolysis. The azide can in turn be made to react with primary or secondary amines to yield the amides.
in which R is NH-NH2 or N3 are compounded into suitable dosage forms and administered in similar fashion at similar dose levels.
While most of the compounds of this invention are useful as antineoplastic or antiviral drugs, two types of derivatives, the hydrazides and azides (compounds of formula I wherein R is NH-NH2 or N3), are also useful as inter-mediates as has been set forth above, in that the hydrazide can be transformed to the azide by nitrosation, as by nitrous acid treatment, or to the simple amide by hydro-genolysis. The azide can in turn be made to react with primary or secondary amines to yield the amides.
Claims (8)
1. A process for preparing a compound of the formula Formula I
wherein R is NH2, NH-NH2, N(CH3)2, pyrrolidinyl, NH-alk-X, NH-(C3-C8)-cyclo-alk, NH-alk-(OH)1-3, or N3;
wherein alk is (C1-C6) alkyl, and X is hydrogen, R1 is hydrogen, hydroxyl, O-(C1-C3)-alkanoyl or O-chloro-(C1-C3)-alkanoyl;
R2, R3 and R4 are hydrogen or hydroxyl with the provisos that when R2 is hydrogen, R3 and R4 are hydrogen;
when R2 is hydroxyl at least one of R3 or R4 is hydroxyl, which comprises (a) reacting a compound having a structure of Formula I wherein R is O-CH3, R1 is hydrogen, hydroxyl, or acetyloxy, and R2, R3 and R4 are as defined above with a compound of the formula NH2R5 Formula II
wherein R5 is hydrogen, methyl or amino and/or (b) when R is NH-NH2 reacting the compound so obtained with a nitrosating agent and with a compound of the formula wherein R6 is hydrogen or methyl and R7 is methyl, -alk-X, (C3-C8)-cycloalk, or -alk-(OH)1-3 wherein alk is (C1-C6) alkyl and X is as defined in Formula I, and/or (c) acylating the compound obtained in (a) or (b) above wherein R1 is hydroxyl to provide a compound wherein R1 is other than hydroxyl and, if desired, reacting any of the products obtained above with a non toxic inor-ganic acid or organic acid to provide the pharmaceutically acceptable acid addition salt of the product.
wherein R is NH2, NH-NH2, N(CH3)2, pyrrolidinyl, NH-alk-X, NH-(C3-C8)-cyclo-alk, NH-alk-(OH)1-3, or N3;
wherein alk is (C1-C6) alkyl, and X is hydrogen, R1 is hydrogen, hydroxyl, O-(C1-C3)-alkanoyl or O-chloro-(C1-C3)-alkanoyl;
R2, R3 and R4 are hydrogen or hydroxyl with the provisos that when R2 is hydrogen, R3 and R4 are hydrogen;
when R2 is hydroxyl at least one of R3 or R4 is hydroxyl, which comprises (a) reacting a compound having a structure of Formula I wherein R is O-CH3, R1 is hydrogen, hydroxyl, or acetyloxy, and R2, R3 and R4 are as defined above with a compound of the formula NH2R5 Formula II
wherein R5 is hydrogen, methyl or amino and/or (b) when R is NH-NH2 reacting the compound so obtained with a nitrosating agent and with a compound of the formula wherein R6 is hydrogen or methyl and R7 is methyl, -alk-X, (C3-C8)-cycloalk, or -alk-(OH)1-3 wherein alk is (C1-C6) alkyl and X is as defined in Formula I, and/or (c) acylating the compound obtained in (a) or (b) above wherein R1 is hydroxyl to provide a compound wherein R1 is other than hydroxyl and, if desired, reacting any of the products obtained above with a non toxic inor-ganic acid or organic acid to provide the pharmaceutically acceptable acid addition salt of the product.
2. A process as in Claim 1 for preparing 4-desacetyl deoxyvinblastine "B" C-3 carboxhydrazide which comprises reacting deoxyvinblastine "B" with anhydrous hydrazine in anhydrous methanol.
3. A compound of the formula Formula I
wherein R is NH2, NH-NH2, N(CH3)2, pyrrolidinyl, NH-alk-X, NH-(C3-C8)-cyclo-alk, NH-alk-(OH)1-3, or N3;
wherein alk is (C1-C6) alkyl, and X is hydrogen, R1 is hydrogen, hydroxyl, O-(C1-C3)-alkanoyl or O-chloro-(C1-C3)-alkanoyl;
R2, R3 and R4 are hydrogen or hydroxyl with the provisos that when R2 is hydrogen, R3 and R4 are hydrogen;
when R2 is hydroxyl at east one of R3 or R4 is hydroxyl, or a pharmaceutically acceptable acid addition salt thereof, when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.
wherein R is NH2, NH-NH2, N(CH3)2, pyrrolidinyl, NH-alk-X, NH-(C3-C8)-cyclo-alk, NH-alk-(OH)1-3, or N3;
wherein alk is (C1-C6) alkyl, and X is hydrogen, R1 is hydrogen, hydroxyl, O-(C1-C3)-alkanoyl or O-chloro-(C1-C3)-alkanoyl;
R2, R3 and R4 are hydrogen or hydroxyl with the provisos that when R2 is hydrogen, R3 and R4 are hydrogen;
when R2 is hydroxyl at east one of R3 or R4 is hydroxyl, or a pharmaceutically acceptable acid addition salt thereof, when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.
4. 4-Desacetyl deoxyvinblastine "B" C-3 car-boxhydrazide when prepared by the process of Claim 2 or an obvious chemical equivalent thereof.
5. A process as in Claim 1 for preparing 4-desacetylvincadioline C-3 N-methylamide which comprises reacting vincadioline with anhydrous hydrazine in anhydrous methanol to obtain vincadioline C-3 carboxhydrazide and reacting the carboxhydrazide with methylamine.
6. 4-Desacetylvincadioline C-3 N-methylamide when prepared by the process of Claim 5 or an obvious chemical equivalent thereof.
7. A process as in Claim 1 for preparing 4-desacetylleurocolombine C-3 N-methylamide which comprises reacting leurocolombine with anhydrous hydrazine in anhydrous methanol to obtain leurocolombine C-3- carboxhydrazide and reacting the carboxhydrazide with methylamine.
8. 4-Desacetylleurocolombine C-3 N-methylamide when prepared by the process of Claim 7 or an obvious chemical equivalent thereof.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US53968175A | 1975-01-09 | 1975-01-09 |
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| CA242,012A Expired CA1067073A (en) | 1975-01-09 | 1975-12-18 | Amide derivatives of vlb, leurosidine, leurocristine and related dimeric alkaloid |
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|---|---|
| JP (1) | JPS5195100A (en) |
| BE (1) | BE837390A (en) |
| CA (1) | CA1067073A (en) |
| CH (1) | CH624962A5 (en) |
| CS (1) | CS199615B2 (en) |
| DE (1) | DE2558027A1 (en) |
| FR (1) | FR2297043A1 (en) |
| GB (1) | GB1538921A (en) |
| HU (1) | HU176226B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GR69783B (en) * | 1976-09-08 | 1982-07-07 | Lilly Co Eli | |
| USRE30561E (en) | 1976-12-06 | 1981-03-31 | Eli Lilly And Company | Vinca alkaloid intermediates |
| PT67535B (en) * | 1977-01-19 | 1979-06-15 | Lilly Co Eli | A process for the preparation of 1-formyl dimeric indole-dihydroindole |
| US4191688A (en) | 1977-08-08 | 1980-03-04 | Eli Lilly And Company | Amides of leurosine, leuroformine, desacetylleurosine and desacetylleuroformine |
| US4195022A (en) | 1978-03-27 | 1980-03-25 | Eli Lilly And Company | 4-Desacetoxy-4α-hydroxyvinblastine and related compounds |
| US4166810A (en) | 1978-04-20 | 1979-09-04 | Eli Lilly And Company | Derivatives of 4-desacetyl VLB C-3 carboxyhydrazide |
| US4199504A (en) * | 1978-05-15 | 1980-04-22 | Eli Lilly And Company | Bridged cathranthus alkaloid dimers |
| AR225153A1 (en) * | 1978-10-10 | 1982-02-26 | Lilly Co Eli | A PROCEDURE FOR THE PREPARATION OF VINDESINE SULPHATE |
| US4357334A (en) | 1980-03-20 | 1982-11-02 | Eli Lilly And Company | Use of VLB 3-(2-chloroethyl) carboxamide in treating neoplasms |
| LU83822A1 (en) * | 1981-12-08 | 1983-09-01 | Omnichem Sa | N- (VINBLASTINOYL-23) DERIVATIVES OF AMINO ACIDS, THEIR PREPARATION AND THEIR THERAPEUTIC APPLICATION |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AR204004A1 (en) * | 1973-04-02 | 1975-11-12 | Lilly Co Eli | PROCEDURES FOR PREPARING VINBLASTIN LEUROSIDINE AND LEUROCRISTINE DERIVATIVES |
-
1975
- 1975-12-17 IL IL48685A patent/IL48685A/en unknown
- 1975-12-18 CA CA242,012A patent/CA1067073A/en not_active Expired
- 1975-12-19 IE IE2775/75A patent/IE42385B1/en unknown
- 1975-12-19 PH PH17891A patent/PH17563A/en unknown
- 1975-12-22 DE DE19752558027 patent/DE2558027A1/en not_active Ceased
- 1975-12-29 CH CH1684075A patent/CH624962A5/en not_active IP Right Cessation
- 1975-12-31 NL NL7515253A patent/NL7515253A/en not_active Application Discontinuation
-
1976
- 1976-01-06 GB GB281/76A patent/GB1538921A/en not_active Expired
- 1976-01-07 CS CS7699A patent/CS199615B2/en unknown
- 1976-01-08 HU HU76EI664A patent/HU176226B/en not_active IP Right Cessation
- 1976-01-08 BE BE1007122A patent/BE837390A/en not_active IP Right Cessation
- 1976-01-09 JP JP51002380A patent/JPS5195100A/ja active Pending
- 1976-01-09 FR FR7600519A patent/FR2297043A1/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| HU176226B (en) | 1981-01-28 |
| CH624962A5 (en) | 1981-08-31 |
| NL7515253A (en) | 1976-07-13 |
| CS199615B2 (en) | 1980-07-31 |
| FR2297043A1 (en) | 1976-08-06 |
| PH17563A (en) | 1984-10-01 |
| IL48685A0 (en) | 1976-02-29 |
| IL48685A (en) | 1980-03-31 |
| IE42385B1 (en) | 1980-07-30 |
| GB1538921A (en) | 1979-01-24 |
| BE837390A (en) | 1976-07-08 |
| IE42385L (en) | 1976-07-09 |
| FR2297043B1 (en) | 1978-08-11 |
| DE2558027A1 (en) | 1976-07-15 |
| JPS5195100A (en) | 1976-08-20 |
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