IE42385B1 - Novel amide derivatives of dimeric alkaloids - Google Patents

Abstract

Novel amide derivatives of dimeric vinblastin alkaloids of formula wherein the substituents are defined in Claim 1, are prepared. These compounds are obtained by reacting corresponding compounds in which R denotes methoxy and R<1> represents H, OH or acetoxy, with ammonia, methylamine or hydrazine. Resulting compounds in which R denotes an NH-NH2 group can be converted by reaction with a nitrosylating agent into the azide, which can subsequently be converted by means of primary or secondary amines into further amide derivatives. The novel compounds may be used as antiviral or antineoplastic agents.

Description

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 preparation of such agents.

The invention provides a compound of the formula e'x\?S ,- / \ ♦---N 4 A-CHs-CHj Z\8' \ \ZR3 I7 \jo V , \gz (I it , 7 χ Λ i c A ι h ' 1 A,1 Tjf1' •--Q-O-GH:·.

CH; ίο ’T °/\ Γ •g / \- \ Z\ / • is ·-♦ s···*·*CHs-CHs S-0-«3O A .·» 4,·—-R1 ν/θ\’/„/ν ¥ HO:H3 Formula I wherein R is NHg, NH-NHg, NfCHgJgi pyrrolidinyl, NH-alk-X, NH-(Cg-Cg)-cyclo-alkyl, NH-alk-Am, NH-alk-(OH) , or N^; wherein alk is (Cg-Cg) alkylene, Am is Nllg, NHCII^ or NlCII^Jg and X is hydrogen, cyano, optionally substituted phenyl, carboxyl, carbo (Cj-Cg) alkoxy or carbamoyl; is hydrogen, hydroxyl, 0-(Cg-C^)-alkanoyl or O-chloro-(Cg-C^)-alkanoyl; R2, R2 and / are hydrogen or hydroxyl with the provisos that when R2 is hydrogen, R2 and R^ are hydrogen and 2 when R is other than hydrogen and R is hydroxyl at least one 7 1 λ of R' or R is hydroxyl.

The invention also provides a process for preparing a compound of Formula 1 by (a) reacting a compound having -2a structure of Formula I wherein R is O-CII^, R3 is hydrogen or acetoxy, and R2, R3 and R3 are as defined in Formula I with a compound of the formula NH2R5 Formula II where R is hydrogen, methyl or amino and optionally, if desired, (b) when R is reacting the compound so obtained with a nitrosating agent and with a compound of the formula 7 I(i wherein R is hydrogen or methyl and R is methyl, -alk-X, (C ,-C.. )-cyc I oalky I , -alk-Λιιι u r- -alk-(OII) wherein a I k i.s ((' -C J .1 ' ’ - 1-1 — yl 1' alkylene and AM and X are as defined in Formula I or K and R' taken together form an alkylene chain of formula (Cll,, and if desired, acylating the compound obtained in (a) or (b) above wherein R3 is hydroxyl to provide a compound of formula 1 wherein 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 acid addition salt of the product. 2() 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. 3,370,057), vinblastine (vincaleukoblastine) (U.S. Patent No. 3,097,137), leuro25 sidine (vinrosidine) and leurocristine (vincristine) (both in U. S. Patent No. 3,205,220). 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, -342385 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. Xn 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 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, antineoplastic 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 correspondingly slight. Among the successful modifications of physiologically-active 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 alkanoyl 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 -44 2 3 8 5 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 P1534 murine leukemia system by Hargrove, Lloydia, 27, 340 (1964).

A preferred group of compounds of Formula I includes the amides of vincadioline, lourocolombine, 4desacetoxyvinblastine, 3 ' -hydroxy- 4-desaci· Loxy vi nbl as I: i no, deoxyvinblastine and the 4-desaeety! d<-rivnt ive of .my 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 N?, formed with nontoxic acids.

Illustrative of alk-(Oil) -,, alk-Am and alk-X in the above formula are the following: methyl, 2-methylpentyl, isohexyl, isopentyl, n-pentyl, n-hexyl, sec-hexyl, ethyl, isopropyl, n-butyl, sec-butyl, cyanomethyl, 2-cyanoethyl, 2-hydroxy-n-hexyl, 5-cyano-n-pentyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-dimethylaminoethyl, 2-aminoethyl, 2methylaminoethyl, 2-hydroxypropyl, benzyl, phenethyl , 4pheny Lbuty I , 2-am inop ropy I , 2 - am i noh'-xy ! , 2-d i mol hy I am i nopropy l , 2,2' -di hydr oxyisopropy ( , , '-d j hydroxy-t-butyl , 2,2',2''-trihydroxy-t-butyl .

In Formula I, the terms (C„-C .)-alkanoyl and — 4 chloro-(C„-C,)-alkanoyl'· include groups such as acetyl, chloroacetyl, propionyl, 2-chloropropionyl, 2-chlorobutyryl and butyryl, these terms being represented by the formula (Cj-C-,) -alkyl-CO, an alkanoyl group, or by the formula (C^-C-j)-alkyl (Cl)-CO, a chloroalkanoyl group. The term NH-(C^-Cg)-cycloalkyl includes the radicals cyclopropylamino, cyclobutylamino, cyclopentylamino, cyclohexylamino, cycloheptylamino, and cyclooctylamino. The term carbo5 (C^-Cg)-alkoxy includes the radicals carbomethoxy, carboethoxy, carboisopropoxy and carbo-n-propoxy.

When X in the radical alk-X is optionally substituted phenyl, the phenyl group can contain substituents selected from lower alkyl, lower alkoxy, hydroxy, halo and nitro and a given phenyl; group can contain more than one of the above substituents, either the sami- or tl i fl'erent; examples of sueli groups are 4-hydroxyphenyl, 2,4-dichlorophenyl, 2-methyl-4-chlorophenyl, 2,4-dinitrophenyl, 3,5-xylyl, 4-tolyl, 2-tolyl, and if 3-e thoxypheny 1.

Non-toxic acids useful for forming pharmaceutically-acceptable acid addition salts of the amine bases include salts derived from inorganic acids such as: hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, 2Q hydrobromic acid, hydroiodic acid, nitrous acid, and phosphorus acid, as well as salts Of non-toxic organic acids including aliphatic mono and dicarboxylates, phenylsubstituted alkanoates, hydroxy alkanoates and alkandioates, aromatic acids, aliphatic and aromatic sulfonic acids.

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, -642385 isobutyrate, caprate, heptoanate, propiolate, oxalate, malonate, succinate, suberats, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonat.es, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetatp, phenylpropionate, phenylbutyrate, citrate, lactate, 2-hydroxybutyrate, glycollate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-l-sulfonate, and naphfchaleno-2-stilfonate salts.

A majority of the compounds of Formula I can be described generally as derivatives of vinblastine. Vinblastine is a compound having the structure of Formula I 2 when R is CH^O, R is acetoxy, R is β-hydroxyl (the C-4'3 4 ethyl group is alpha) and R and R are hydrogen. Deoxy2 3 4 vinblastine occurs when in Formula I all of R , R and R are hydrogen, R is CHand i'1 is acetoxy. There .ire two isomers of deoxyvinblasline identified a:; A and ft. In the case of the A isomer, the if hydrogen is Beta(up) and the C-4'ethyl group is Aipha(down). The B isomer has a p-ethyl group and an α-hydrogen at C-4'.

Vincadioline is the i'-hydroxy derivative (i.e.

R is hydroxyl) of vinblastine, beurocolombine is the 2'hydroxy derivative (i.e. R* is hydroxyl) of vinblastine.

Vincadioline has the following characteristics: -742385 Melting point = 218-220.5°C. with decomposition; X-ray powder diffraction pattern, using chromium radiation; λ - 2.2896°A. filtered Ι/Ιχ o d in AIZI1 o d in A 9.55 100 -1 3.99 60 8.87 8.63 90) , 90) Z 3.71 3.64 20 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, 6 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, and Mass ions, m/e = 826, 170, 371.

Vincadioline 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 -842385 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 (Sepbadex G-25F), (Sephadcx is a registered Trade Mark), the mobile phase being a pII=3-0 0.1M ammonium citrate buffer. A pressure of about 15 psi is employed during gel-exclusion chromatography. In this process, the dimeric alkaloid fraction containing leurocristine, vinblastine, 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 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 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 to 8.0 with ammonium hydroxide and extracting the dimeric alkaloids with a water-immiscible organic solvent, preferably methylenedichloride. 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 -9as 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 pressures in the range 7500-8000 psi appear feasible. Alkaloidal separation is in general more efficient at the higher pressures. Iligh-pressure chromatoqraphy procedures are carried out in stainless steel equipment equipped with pressure10 resistant fittings.

The alkaloids are eluted in the following order in this chromatographic procedure: residual leurosine, vinblastine, 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 vincaί 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, .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, C46H5gN4°^o? 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, .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 o pattern using filtered chromium radiation at 2.2896A. 0 d in A I/I 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 -114 2385 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-25 F), the mobile phase being a pH=3.0, 0.1M ammonium citrate buffer. A pressure of about 15 psi is employed during gel-exclusion chromatography. In this process, the dimeric alkaloid fraction containing leurocristine, vinblastine, 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 decantation, 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 salts which precipitate. Tho 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. Evaporation of the methylenedichloride yields the mixed dimeric alkaloids which are then chromatographed at high pressure over alumina (Activity III-IV) using a ethyl acetatemethylenedichloride-water (25:75:0.4) solvent system as the eluant. -124 2 3 8« 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 chromatography procedures are carried out in stainless steel equipment equipped with pressure-resistant fitt mgs.

The alkaloids are eluted in the following order in this chromatographic procedure: residual leurosine, vinblastine, leurocolombine, des-N-methyl vinblastine, leurocristine 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 = 183-190°C. with decomposition after recrystallization from methanol; (a]p = +95.3° (chloroform); molecular ion M+ = 752, corresponding to an empirical formula Ο^Η,-^Ν^Ο?.

Analysis Analysis Calcd. Calc.: for: C44H56N4O7 c, 70.19; H, 7.50; N, 7.44; 14.87 Found: c. 69.71; H, 7.47; N, 7.08; 0, 15.00 4-Desacetoxyvinblastine is prepared according to the following procedure: Leaves of plants containing crude vinca alkaloids; i.e., Citharanthus roseus (Vinca rosea), previously moistened with aqueous ammonia, arc extracted with a water-immiscible solvent such as benzene. The -1342385 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, customarily benzene. Evaporation of the benzene yields a mixture of amorphous dimeric alkaloids which are dissolved in benzene and chromatographed over alumina (CAMAG - Activity III).

The alkaloids are eluted in the following order: leurosine, vinblastine, des-N-methyl vinblastine, leurocrlstine 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 VLB 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 2$ 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 chroma30 togram is developed with benzene. Fractions shown by thin -14layer chromatography to contain a second alkaloid in addition 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 vinblastine with only minor amounts of vinblastine are combined and recrystallized from inethanol. 4-Desacetoxy vinblastine thus recrystallized is then further purified by preparative thin layer chromatography over silica using as eluant a 3:2:4 diethylamine-ehloroform-benzene solvent mixture. 31-Hydroxy-4-desacetoxyvinblastino has the following physical characteristics: proton nmr spectrum peaks at ^4.075^5), 5.85(15), 5.46-5.78 (broad multiplet) mass spectrum: ions at m/e 768, 411, 371, 224, 170, 102.

*-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. Tlie 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, oust' ·. ..iri Ly benzene. Evaporation of the solvent yields a mixture of amorphous dimeric alkaloids.

The dimeric alkaloid frac ion 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 -1542385 free bases by solution in water, basifying the aqueous solution and extracting the alkaloids into a water-immiscible organic solvent, customarily methylene dichloride. Evaporation of the solvent yields a mixture of amorphous dimeric alkaloids which are redissolved in methylene dichloride and chromatographed over alumina (CAMAfi - Activity ITI-IV).

The alkaloids are eluted in the following order: leurosine, vinblastine, des-N-methyl vinblastine, leurocristine 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, 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 mp, and fractions were separated based upon the peaks observed in the ultraviolet profile. Fractions were identified containing predominatntly leurosine, vinblastine, des-N-methylvinblastine, and leurocristine by thin layer chromatography. Post-des-N-methylvinblastine, 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 -16with benzene. 4-Desacetoxy-3'-hydroxyvinblastine 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 oi the aqueous solution adjusted to pH - 9 by the addition of ammonium acetate. The precipitated alkaloidal free bases were separated by centrifugation, 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 μ)] 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: 31-hydroxy-4-desacetoxyvinblastine C-3 N-methyl carboxamidej 2'-hydroxy-4-desacetoxyvinblastine C-3 N-(2-cyanoethyl.) 25 carboxamide, 2'-hydroxy-4-desacetoxyvinblastine C-3 N-(2hydroxypropyl)carboxami de, -1742385 deoxyvinblastine Λ C-3 carboxazide, deoxyvinblastine Λ” C-3 N-(2-dimethylaminoethyl ) carboxamide, deoxyvinblastine ”B C-3 N-(2,3-diiiydroxypentyl) carboxamide, 2’-hydroxyvinblastine C-3 N-(2-phenylethyi) carboxamide, *-hydroxyvinbiastine C-3 N-(3-phenylpropyl) carboxamide and 3’-hydroxyvinbiastine C-3 carboxhydrazide.

The present compounds arc derivatives in which the carbomethoxy group at C-3 of certain known indo 1 edi.hydro! ndote alkaloids is transformed to a carboxhydrazide group,a carboxazide group, a carboxamide group or a derivative thereof. Not all of these derivatives are ordinarly prepared by one process. The compounds in which R in formula I above is NH2, NII-NI(2 or NH-CHg are prepared as follows: Treatment of leurocolumbine, vincadioline or deoxyvinblastine, or their respective 4desacetoxy compounds with either ammonia, methylamine or hydrazine yields the corresponding amide, N-methylamide or hydrazide. The products of this reaction with starting materials having a 4-acetoxy group are usually IS 42385 < ι mi pi 11111 < I;in which the cnrhoiiicl.lioxy group al. C-3 is transformed t.o a carboxamide, N-mothylcarboxnmidu or carboxhydrazi.de group, but also in which the acetyl croup 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 NtCH^)2, HH-alk-X wherein X is as del'ined above;, Nn-(Cg-Cs)-cycloalk, NUalk-Am or NH-alk-(OH)-, and alk and Am are as previously defined are prepared by the following procedure: a hydrazide, (Formula I wherein R is NH-NH^) prepared by reaction of the C-3 carbornothoxyi. 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 (CH^)^, NIb,-alk-X, NIl,-(C.j-C8)-eyo|oalk, NH-alk-(OH) , or NH?alk-Am, to yield the desired C-Ί amide. This C-3 azide20 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, leurocolumbino, or deoxyvinblastine directly with ammonia, methylamine or hydrazine followed by separation of the 4-acetyi derivative from the 4-dosac»ty 1. derivative, ami, in the ease of the hydrazide, conversion to 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 (CgCg) -alky1-COC1 or chloro-(Cg-Cg)-alkyl-CO-Cl or an acid anhydride [(Cg-Cg)-alkyl-CO]=O or [chloro-(Cg-Cg)-alkylC0)2=0 can be used in the acylation reaction. The preferred acylation procedure is that described in u. S. Patent 3,392,173 for vinblastine or leurocristinc 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 acylatable 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 preferred hydrazide protecting group is the propylidene group formed by reaction of the NH,. portion of the hydrazide £-20moiety with acetone. This group can be readily removed by treatment with acid or, preferably, the propylidene derivative itself can be reacted directly with nitrite to form an azide group (see U. S. Patent 3,470,210, Example VII) . g 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, carbamoyl or carbo-(C^-Cg)alkoxy may be prepared, by reacting an appropriate amino acid, amino amide oi· amino ester, preferably having the structure II I NH,-C-COO where ί ιι X Q is OH, NH, or O-alk and 7. is I! or a C(-f· alkyl i|t.>up, with the chosen dimeric i tide I e-d i hydro) nd>' Ie azide. Λπΐϊπο acids useful, for this purpose, and coming within the scope of the above formula, include leucine, isoleucine, valine, glycine, alanine, and nor·leucine. Λ.Κ will be apparent to Lhose skilled in the art, cither amino acids can also be used to react with, for example, 4-desacetyl vinblastine C-3 earboxazide, to yield substituted C-3 carboxamides having anti-tumor properties.

An alternative method of preparing the primary amide (R is Nil.,) from the hydrazide (P is NH-NIIj) involves the use of a procedure based on that of Ainsworth, 0. s. Patent 2,756,235, in which the hydrazide is hydrogenolyzed with Raney nickel. -214 2385 The novel derivatives will be named with reference only 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 t be called simply vinblastine C-3 carboxamide, and not vinblastine C-3 descarbomethoxy C-3 carboxamide.

The compounds in the form of their free bases, including both carboxamides, carboxazides and carboxhydrazides are white or tan-colored amorphous solids. It is preferable, however, where possible to isolate and crystallize 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 60°C 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 B“ 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 3 (N-H), 1735 cm 3 (COO), 1675 cm 3 (CON); molecular ion spectrum; m/e = 752 (consistent with ^.jHggNgOg); nmr -2242385 spectrum; <53.78 (ArOCH3), 63.58 (ClgCOOCH3), 62.77 (N-CH3), 64.15 (C4-H).

Example 2 4-Desacetoxyvinblastine C-3 N-(2-IIydroxyethyl) Carboxamide Following the procedure of Example? 1, 4-desacetoxyvinblastine was reacted with anhydrous hydrazine in methanol solution in a sealed tube at 46°C. 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 characteristics: pK = 5.61 and 7.38; ultraviolet spectrum; a Amax = 215 and 267 nm; infrared spectrum, peaks at 3450 cm-1 (N-H), 1725 cm'^ (COO), 1680 cm-1 (CON); molecular spectrum m/e = 252, 411, 355, 244, 154. Molecular ion M+ 752 consistent with empirical formula C^HggNgOg. The compound was a tan amorphous powder.

The 4-desacot.oxyv inblastine C-3 carboxhydrazide was converted Lo the corresponding carboxazide in hydrochloric acid solution at ()°C. 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 ethanolamine wcr<· added to a .solution containing about 1.2 g. of 4-desacetoxyvinblastino C-i 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 -2342385 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-Desacetoxyvinblastine C-3 (N-(2-hydroxyethyl) carboxamide thus prepared was a tan amorphous material with the following physical characteristics: molecular ion; M+ = 781 con15 sistent with empirical formula infrared spectrum peaks at 3420 cm-1 (NH), 1735 cm-1 (COO), 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 hy the procedure of Example 8 and the azide was reacted with methylamine according to the procedure of the same example. The product of this reaction, 4-desacetyl vincadioline C-3 N-methylamide, had the following physical characteristics: infrared spectrum peaks -244238 at 2.95 my, 5.75 mu and 5.97 mp; nmr spectrum consistent with postulated structure with added doublet at 63.82 (amidemethyl hydrogens); molecular spectrum, molecular ion M+ ~ 783 consistent with C,.Hr_N_O„. 57 5 8 'i Following the above procedure 4-desacetyl]curocoloinbine C-3 N-methylamide is prepared.

Example 4 Preparation of salts Other salts, including salts with inorganic anions such as chloride, bromide, phosphate, and nitrate, as well as salts with organic anions such as acetate, chloroacetate, trichloroacetate, benzoate, alkyl or aryl sulfonates, arc 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, transesterification 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, 2' 66-72 (1961). -2542385 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 inhibition of tumor growth. (ROS is an abbreviation for Ridgeway osteogenic sarcoma; GLS for Gardner lymphosarcoma; and CA 755 is an adenocarcinoma).

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 vin25 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. -26c o 4-> -H C 4-> Q) -rt ϋ J3 Jh ·η H O O pH j M* ft m* in (fl Dose Tumor mg,/? vo o in ft . ft X I in in γ** o o CG co O 1-4 « c I CN I uo OJ tJ c4 § ra c « .4 o 27' 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 NH-NH. or N„, formed with a non-toxic acid is mixed with starch or other excipient and the mixture placed in telescoping 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 7.5-50 mg. The tablets may be scored if lower or divided dosages arc 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-NHj or N^ 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 NII-NHg or N^), are also useful as intermediates 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 hydrogenolysis. The azido can in turn be made to react with primary or secondary amines to yield the amides.

Claims (9)

1. Λ compound of the formula —z , z w. «'·. · ν ίΐ'- ι '·:( vy .v Vy«\4·· A CHa-CHo R M 10 .--Z \ 7 14 I l H ;/\i.V\ /\ Z 6 /, x-. a,,-0,,3 CH.-.-0-idc A A : 1*-- R 17 I., : 3I,J C-R Formula I and non-toxic acid addition salts thereof, wherein R is NH 2 , NH-NH^, N(CHg) 2 , pyrroiidinyl, NH-alk-X, NH-fC^-Cg)-cyclo-alkyl, Nil-alk-Am, Μ-alk-(OHor Nj wherein alk is (C 2 ~Cg) alkylene, Am is NH 2 , NHCII^ or N(CII^) 2 and X is hydrogen, cyano, optionally substituted phenyl, carboxyl, carbo (Cj-C^) alkoxy or carboxamoyl; R 3 is hydrogen, hydroxyl, 0-(C 2 ~C 4 )-alkanoyl or 0-chloro- (.C 2 ~C^) -alkanoyl ; 2. 3 4 R , R and R are hydrogen or hydroxyliwith the 2 3 4 provisos that when R is hydrogen, R and R are hydrogen; and

1 . 2 . when R is other than hydrogen and R is hydroxyl at least one 1 5 of R 3 or R 3 Is hydroxyl.

2. 4-uesacetyl deoxyvinblastine B C-3 carboxhydrazide.

3.

4. -Desacetoxyvinblastine C-3 N-(2-hydroxyethyl) carboxamide. 2o 4. 4-Desacetylvincadioline C-3 N-methylamide.

5. 4-Desacetylleurocolombine C-3 N-methylamide. 8S

6. A process for preparing a compound of Formula I of claim 1 which comprises (a) reacting a compound having a structure of Formula I wherein R is O-CH 3 , R^ is hydrogen 2 3 4 or acetoxy and R , R and R are as defined in Claim 1 with a compound of the formula NH 2 r5 Formula II wherein R is hydrogen, methyl or amino and optionally, if desired, (b) when R is NH-NH^ reacting the compound so obtained with a nitrosating agent and with a compound of the formula 6 7 wherein R is hydrogen or methyl and R is methyl, -alk-X, (C^-Cg) -cycloalkyl', -alk-Am or -alk-(OHwherein alk is (C^-Cfi) alkylene and Am and X are as defined in Formula I or R (1 and R taken together form an alkylene chain of formula (CH„)., and if desired, acylating the compound obtained in (a) or (b)^ above wherein R 3 is hydroxyl to provide a compound of formula 1 wherein R^ 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 acid addition salt of the product.

7. The compound of Formula J substantially as herein described.

8. The process for preparing a compound of Formula I substantially as herein described with reference to any one of Examples 1 to 4·

9. A compound of formula 1 whenever prepared by the process claimed in claim 6 or claim 8.