CA1042428A - Amide derivatives of vlb and leurocristine - Google Patents

Abstract

AMIDE DERIVATIVES OF VLB
AND LEUROCRISTINE

ABSTRACT OF THE DISCLOSURE

The invention relates to process for preparing derivatives of vinblastine, leurosidine, and leurocristine, pharma-ceutical preparations containing the derivatives as the active ingredient and methods for treating neoplastic disease by administering the derivatives to a mammal.

Description

The invention relates to derivatives o~ vinblastine, leurosidine, and leurocristine, pharmaceutical preparations containing the derivatives as the active inyredient and methods for treating neoplastic disease ~y administering the derivatives to a mammal.
The invention provides a process for preparing a compound of the formula 5' 7' 6 ~ C;5 I a t'~'.CH2-C~
CH3- o I~6 e ~ $~-0-R
~o/1~ W ~ ~ / Formula I

. R~ C-R
ll ;

wherein R is NH2~ NH-NH2,-N(CH3)2, NH-alk-X, NH-alk-Am, ~:
NH-alk(OH)1_3, or N3 wherein alk is (Cl-C6)-alkyl, Am is NH2, NHCH3 or N(CH3)2 and X is hydrogen, cyano, phenyl; R' is (Cl-C3)-alkanoyl or chloro-(Cl-C3)-alkanoyl; R'' is hydrogen, (Cl-C3)-alkyl, formyl or (Cl-C3) alkanoyl; and one of R' ' ' and R' ' ' ' is hydroxyl and the other is ethyl; and its pharmaceutically acceptable salts; which comprises reacting a compound of Formula I wherein R is O-CH3, R' is hydrogen .B.~ ' .:
,. ~ - - . . .
,' . . ' ~, . .
~ , .

i!L~4~Z~
or acetyl and R'', R'l' and R'''' are as defined above with ammonia, methylamine, ethylamine or hydrazine to Obtain a compound of Formula I wherein R is NH2, NH NH2, NH-C~3 or NH-C2H5 and, if desired, when R is NH-NH2 (a) reacting said compound with a nitrosating agent to form an azide in which R is N3 and then reacting said azide with ammonia, an amine of the formula HN(CH3)2, NH2-alk-X, NH2-alk- ~ or NH2-alk-(OH)l 3 wherein alk is (Cl-C6)-alkyl and Am and X are as defined above, to yield a compound of formula I wherein R' is X ~r aoetyl;
or (b) hydrogenolyzing said compound with Raney nickel in an inert solvent to yield a compound of formuia I wherein R is N~2 and R' is hydrogen or acetyl; and (c) acylating the compound wherein r' R' is hydrogen with an acid chloride of the formula (Cl-C3)-alkyl-COCl or chloro-(Cl-C3)-alkyl-COCl or an acid anhydride of the formula [(Cl-C3)-alkyl-CO]2O or [chloro-(Cl-C3)-alkyl-CO]2O to form a compound fo~m~a I in which R' is (~C31-alkanoyl or chloro-(Cl-C3)-alkanoyl, and R'', R''' and R'''' are as defined above; and optionally, converting said compound to a pharmaceutically acceptable salt.
Several naturally-occurring alkaloids obtainable from Vinca rosea have been found active in the treatment of experimental malignancies in an.imals. Among these are leurosine (U.S. Patent No. 3,370,057), vincaleukoblastine (vinblastine) (U.S. Patent No. 3,097,137), leurosidine (vinrosidine) and leurocristine (vincristine) (both in ' .

~.,~' , , ` ".
.....

~4~;28 U. S. Patent No. 3,205,220)~ Two of these alkaloids, vin-blastine and leurocris-tine, are now marketed as drugs for the treatment of malignancies, par1:icularly the leukemias and related diseases in humans. of these marketed compounds, 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 chemo-therapeutic 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, anti-neoplastic activity seems to-be limited to very specific structures, and the chances of obtaining more active drugs by modification o~ 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. 3,352,368) and the replacement o~ the acetyl group at C-4 (carbon no. 4 of the - vinblastine ring system, see Formula I~ with a highex 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 Pl534 leukemia. One of the derivatives in which a chloracetyl group replaced the C-4 acetyl group of vinblastine was also ~0~2~28 a useful intermediate for the preparation o~ structurally modified vinbla~ti~e compound~ in which a~ N,N-dlalkylglycyl 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 chemical reactions leading to these latter derivatives.
This intermediate, in which the C-4 acyl yroup was lacking, leaving an unesterified hydroxy group, has been reported to be a toxic material having little ln vlvo chemotherapeutic activity against the Pl534 murine leukemia system by Hargrove, Lloyd , 27, 340 (1964).
Illustrative of alk-(OH)l 3, _lk-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, s_ -butyl, cyanomethyl, cyanoethyl, 2-hydroxy-n-hexyl, 5-cyano-n-pentyl, 2-hydroxyethyl, 3-hydroxy-propyl, 2 dimethylaminoethyl, 2-aminoethyl, 2-methylamino-ethyl, 2-hydroxypxopyl, benzyl, phenethyl, 4-phenylbutyl, di-methylaminomethyl, 2 aminopropyl, 2-aminohexyl, 2-dimethyl-aminopropyl, 2,2-dihydroxyisopropyl, 2,2-dihydroxy-t-butyl,

2,2,2-trihydroxy-t-butyl, and the like.
In the above formula, 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 (CI-C3)-alkyl (Cl)-CO, a chloroalkanoyl group. The term "NH-(C3-C8)-cycloalkyl" includes the radicals cyclopropyl-amino, cyclobutylamino, cyclopentylaminoj cyclohexylamino, cycloheptylamino, and cyclooctylamino. The texm "carbo-I

,'.. ' '' ' . ' ' ;. ~ ' .: . ' '' , .

104~
(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 substituent5 including lower alkyl, lower alkoxy, hydroxy, halo, nitro and the like and a yiven phenyl group can contain more than one of the above substituents, either the same or different from the ori~inal substituent; examples of such groups are 4-hydroxyphenyl, 2,4-dichlorophenyl, 2-methyl-4-chlorophenyl, 2,4-dinitrophenyl, 3,5-xylyl, 4-tolyl, 2-tolyl, 3-ethoxy-phenyl and the like.
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, hydrobromic acid, h~droiodic acid, nitrous acid, phosphorous acid and the like, as well as salts of non-toxic organic acids including - ;
aliphatic mono and dicarboxylates, phenyl-substituted alkano-ates, hydroxy alkanoates and alkandioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Such pharma-ceutically-acceptable salts thus include the sulfate, pyro-sulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptoanate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, tolunesul~onate, chloro-~Z~28 ben~enesulfonate, xylenesulfonate, phenylacetate, phenyl-propionate, phenylbutyrate, citrate, lactate, 2-hydroxy-butyrate, glycollate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-l-sulfonate, naphthalene-2-sulfonate and the like salts.
Compounds of the above f~ormula can be described generically as either derivatives of vinblastine where R' is acetyl, R" is methyl, R " ' is hydroxyl and R" '' i9 ethyl, or of desacetyl vinblastine where R", R''' and R'''' remain the same but R' is hydrogen or of leurocristine where R' is acetyl, R" is formyl, R''' is hydroxyl and R " '' is ethyl, or of desacetyl leurocristine where R", R''' and R" '' remain the same but R' is hydrogen, or as derivatives of desmethyl vinblastine (also known as desformylleurocristine) where R' is acetyl, R" is hydrogen, and R " ' and R " '' are hydroxyl and ethyl, respectively or of desacetyl desmethyl vinblastine (or desacetyl desformyl leurocristine) where both R' and R"
are hydrogen and R''' and R " '' are hydroxyl and ethyl, re-spectively, or of leurosidine where R' is acetyl, R" is methyl, R " ' is ethyl and R'l'' is hydroxyl, or of desmethyl leuro-sidine where R', R " l and R'' " remain the same but R" is hydrogen, or of desacetyl desmethyl leurosidine where R' and R" are hydrogen, R''' is ethyl and R " '' iq hydroxyl or of desacetyl leurosidine where R' is hydrogen, R" is methyl and R " ' and R'''' are ethyl and hydroxyl, respectively. In each instance, the term "desacetyl" refers to the lack of an acetyl group on the hydroxyl at C-4 of the complex indole-dihydro-indole ring system.
The derivatives exemplified by Formula I are those in which the carbomethoxyl group at C-3 of certain known .: . . : . .
":', ' ' . . ' :

~ Z428 indol~-dihydroindole alkaloids is trans~ormed to a carbox-hydrazide group, a carboxazide group, a carboxamide group or a derivative thereof. Not all o~ 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 vinblastine, leurocristine, leurosidine, desmethyl vinblastine, desmethyl leurosidine, or their respective 4-desacetyl compounds with either ammonia, methylamine or hydrazine yields the corresponding amide, N-methylamide or hydrazide. The product of this reaction withstarting materials having an intact 4-acetyl group is usually a mixture of compounds in which the carbomethoxy group at C-3 is transfoxmed 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 of this invention in which R is N(CH3)2, NH-alk-X wherein X is H, CN or phenyl, NH-~C3-C8)-~0 cycloalk, N~-alk-Am or NH-alk-(OH)l 3 and alk and Am are as previously defined are prepared by the following procedure:
a hydrazide, of Formula I wherein R is NH-NH2, prepared by reaction of the C-3 carbomethoxyl compound with anhydrous hydrazine, is transfor,med into the corresponding azide by treat-ment with a nitrosating agent such as nitrous acid, nitro~yl chloride, nitrogen tetroxide, amyl nitrite or a similar reagent according to conventional proceduresc The C-3 azide thus prepared is'then reacted with the primary or secondary amines HN(CH3)2, NH2-alk-X, NH2-(C3-C8)-cycloalk, NH2~alk-(OH)l 3 or NH2-alk-Am, to yield the desired C-3 amide coming X-37~4 -8-: ' ~4Z~Z8 within the scope of Formula I above. This C-3 azide-amine reaction does not affect the C-4 acyl group which if present remains intact during the reaction and work-up. 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 ls an acetyl group at C-4 can be prepared, as has been stated above, by reaction of vinblastine, leurocristine or leurosidine directly with am- `
monia, methylamine or hydrazine followed by separation of the ~-acetyl derivative from the 4-desacetyl derivative, and, in -~
the case of the hydrazide, conversion *o the azide followed by reaction of the azide with an amine to yield the amides of Formula I. More generally, however, because o~ the liability 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 ox butyrate or a chloro derivative there-of. An acid chloride (Cl-C3)-alkyl-COCl or chloro-(Cl-C3)-alkyl-CO-Cl or an acid anhyaride l(Cl-C3)~alkyl-CO]2 O or Ichloro-(Cl-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 fDr vinbl~t ~ ~r leunx~istk~ in w~ch 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 acylation or multiple acylation followed by selective hydrolysis can be employed to prepare the 4-acyl derivatives.
X-3754 _9_ .~ , .

; ~

~04;~42b~

There are, however, certain provi~os which must be kept in mind when an acylation procedure is contemplated.
In the first place, as will be ob~ious to those skill~d in the art, acylation of a l-desformyl leurocristine ~l-desmethyl vinblastine) or of a l~desmethyleurosidine will result in acylation also at N-l. Therefore, a l-desformyl or l-desmethyl compound must be formylated, acylated or alkylated to give the desired substituent at N-l prior to the C-4 acylation pro-cedure. In addition if the C-3 carboxamide group contains an acylable group; i.e., hydroxy or amino, the C-4 acylation pro-cedure must be carried out prior to the azide--amine reaction which yields the ultimate C-3 carboxamide group. The pre-ferred 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 NH2 portion of the hydrazide moiety with ace-tone. 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 a~ide 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 ~hose 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 e9ter or , .

~4Z~2~3 H
amino amide of the structure NH2-C-COQ wherein 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 ~cope o~ Formula I, in-clude leucine, isoleucine, valine, glycine, alanine, nor-leucine and the like. As will be apparent to those skilled in the art, other amino acids and polypeptides can al50 be used to react with, for example, 4-desacetyl vinblastine C-3 carboxazide, to yield substituted C-3 carboxamides havinq 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. - -The derivatives of Formula I 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 be called simply vinblastine C-3 carboxamide, and not vinblastine C-3 descarbomethoxy C-3 carboxamide.
The compounds of Formula I, in the orm 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 cry-stallize 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 of Formula I is more fully illustrated in the following specific examples:

,: . ,. . :. .
, . ~- ............................................... ..
::, 104~4ZB

Example 1 Vinblastine C-3 N-methyl carboxamide and 4-desacetyl vin-blastine C-3 N-methylcarboxamide A solution containing 21 g. of methylamine was prepared in 100 ml o~ anhydrous ~ethanol at -78C. About

3.5 g. of vinblastine were added ~o this solution. The reaction vessel was sealed and placed in a constant temperature bath at 50 C. for eight days. The reaction vessel was then opened, and the volatile contents removed by evaporation in vacuo. The nmr and infrared spectra of the residue in-dicated that it was a mixture of 4-desacetyl vinblastine C-3 N-methylcarboxamide and of 4-desacetyl vinblastine. The residue was dissolved in 100 ml. of pyridine, and 20 ml. of acetic anhydride were added. The resulting solution was al-lowed to stand at ambient temperature for about 18 hours. The volatile constituents were removed by evaporation in vacuo, and the resulting residue chromatographed over alumina using an ethyl acetate-chloroform (1:1) solvent mixture as the eluant. Fractions containing vinblastine C-3 N-methylcarbox-amide as indicated by thin layer chromatography were combined,and the solvent evaporated therefrom in vacuo.
The above acetylation procedure not only re-acetylates the C~4 hydroxyl but also acetylates the C-3 hydroxyl. The acetyl group at C-3 was removed by the procedure of Hargrove, Lloydia, 27, 340 (1964), in which procedure the product containing C~3 acetylated material is treated with silica gel in aqueous methanol at room temperature for a period varying from 6 hours to several days to yield a product lacking tha C-3 acetyl function. In this particular instance, the residue from the combined ., ~ 04~28 chromatographic fractions was dis~olved in 50 ml o~ methanol.
Twenty ml of water and 2 g. Q~ sili.ca gel were added. The resulting mixture was filtered, the filtrate evaporated to dryness in vacuo, and the resulting residue chromatographed over silica gel. A benzene-chloroform-triethylamine (100:50:7.5) solvent mixture was u$ed as the eluant. Fractions containing ~he desired vinblastine C-3 N-methylcarboxamide were collected, and the solvent removed there~rom by evapora-tion in vacuo. The residue was dis~olved in aqueous methanol, and the pH adjusted to 2.9 with 1 percent sulfuric acia. Evap-oration of the resulting mixture in vacuo yielded vinblastine C-3 N-methylcarboxamide sulfate which crystallized from an-hydrous ethanol to yield material melting with decomposition at 272-5 C.
An infrared spectrum of vinblastine C-3 N-methyl-carboxamide base obtained as indicated above showed a band at 1672 cm 1 (absent in the spectrum of vinblastine) indicative of the presence of an amide group. -The nmr spectrum was in complete agreement with the proposed structure of vinblastine C-3 N-methylcarboxamide including the newly introduced N-methylcarbGxamide function which is represented by the sharp methyl resonance at 2.81 ppm.

4-Desacetyl vinblastine C-3 N-methylcarboxamide was isolated from the mixture with 4-desacetyl vinblastine ob-tained in the amidation step by the following procedure. The residue obtained by evaporation of the original ~prior to reacetylation) reaction mixture to dryness in vacuo was chromatographed over silica gel using as eluant the benzene-chloroformtriethylamine solvent system referred to above.
Fractions shown by thin layer chromatography to contain 4-, :

1~)42~28 mixture sealed ancl maintained at abou-t 100 C for 60 hours.
The reaction vessel was opened, and the contents removed and evaporated to dryness in vacuo. The resulting residue, was rechromatographed and fractions containing 4-desacetyl vin-blastine C-3 carboxamide, as shown by thin layer chroma-tography, were combined and the solvent evaporated therefrom in vacuo, yielding as a residue purified 4-desacetyl vin-blastine C-3 carboxamide free base. The nmr and ir spectra of the solid free base confirmed the structure indicated. The free base showed a band in the infrared at 1687 cm l, charac-teristic of the amide function. The molecular weight of the free base determined by mass spectroscopy was 753 which is in agreement with the theoretical value calculated for C43H~5N~07.
600 mg of the above residue were converted to the sulfate salt in accordance with the procedure of Example l.
- Evaporation of the reaction mixture to dryness yielded 4-desacetyl vinblastine C-3 carboxamide sulfate which crystal-lized from an ethanol-isopropyl solvent mixture and melted above 250 C. with decomposition. The salt was freely soluble in water.
4-Desacetyl vinblastine C-3 carboxamide was con-verted to vinblastine C-3 carboxamide by the procedures of Example l as follows: 2.8 g of 4-desacetyl vinblastine C-3 carboxamide free bases were acetylated with a mixture of an-hydrous pyridine and acetic anhydride. The reaction mixture was maintained for three days at room temperature. The volatile constituents were removed by evaporation in vacuo, and the resulting residue dissolved in methylenechloride. The methylenechloride solution was washed with water, dried, and evaporated to dryness in vacuo leaving as a residue vin-, ~04~42~
desacetyl vinbla~tine C-3 N-methylcarboxamide were combined and the solvent evaporated therefrom in vacuo to yield the N-methyl carboxamide as an amorphous solid. An in~rared spectrum of the compound showed a band at 1672 cm 1 character-istic of an amide group. The absence of the 4-acetyl group was evidenced by the lack of a resonance for ~his function at 2,1 ppm in the nmr (present in the nmr spectrum of vin-blastine), The molecular weight determined by mass spectro-scopy was 767 which is in agreement with the theoretical value calculated for C4~H57N507.
The sulfate salt o~ 4-desacetyl vinblastine C-3 N-methylcarboxamlde was prepared by the procedure described for the preparation of vinblastine C-3 N-methylcarboxamide sulfate. The resulting product was an amorphous, water soluble solid.
Following the above procedure, 2 g of 4-desacetyl vinblastine were dissolved in- a mixture of 75 ml of anhydrous methanol and 20 g. of ethyl amine. The reaction mixture was sealed and heated at about 60C for about 8 days. 4-Desacetyl vinblastine C-3 N-ethylcarboxamide thus obtained was separated by thin layer chromatography. The resulting solid showed a band at 1670 cm 1 in the infrared spectrum characteristic of the substituted carboxamide function.
Example 2 Preparation of 4-desacetyl vinblastine C-3 carboxamide About 10 g of vinblastine sulfate were converted by standard procedures to vinblastine free base. The free base, obtained as a residue after evaporation of the dried ethereal solvent, was dissolved in about 200 ml of anhydrous methanol.
Anhydrous liquid ammonia (300 ml) was added, and the reaction . : , . . . .
,:: ', . , ' . ' , : - ' ~)4~42~3 blastine C-3 carboxamide. The amide was puri~ied by chroma-tography over silica gel using an ethyl acetate-ethanol ~
solvent mixture as the eluant. Fractions shown to contain the vinblastine C-3 carboxamide by thin layer chromatography were combined, and the solvent removed therefrom by evaporation in vacuo, yielding as a residue vinbLastine C-3 carboxamide free base. The free base had the characteristic amide band ln the infrared occurring at about 1700 cm . Molecular weight by mass spPctroscopy was 795 which is in agreement with the theoretical value calculated for C45H57N5O8. Vinblastine C-3 carboxamide sulfate was prepared by the method of Example l and crystallized from ethanol; m.p.= above 250 C.
Example 3 4-Desacetyl vinblastine C-3 carboxhydrazide Following the procedure of Example l, 4-desacetyl vinblastine was heated in anhydrous ethanol wikh an excess of anhydrous hydrazine in a sealed reaction vessel at ab~ut 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, com-prising 4-desacetyl vinblastine C-3 carboxhydrazide, was taken up in methylenechloride, the methylenechloride solution washed with water, separated and dried, and the methylenechloride re-moved by evaporation in vacuo. The resulting residue was dis-solved in a l:l chloroform:benzene solvent mixture and chroma-tographed over silica gel. The benzene-chloro~orm-triethyl-amine eluant of Example 1 was employed to develop the chroma-togram. The initial chromatographic fractions contained un-reacted 4-desacetyl vinblastine. Further fractions were found to contain 4-desacetyl 18-descarbomethoxy vinblastine C-3 ' ''' ' ' ' "~'' ' ~ . . ' " " ' ., ~ L~4242~
carboxhydrazide previously described by Neu~s et al, Tetrahedron Lek-ters, 19~8, 783. The next ~ractions, found to contain 4~desacetyl vinblastine C-3 carboxhydrazide by thin layer chromatography~ were combined, and the solvents evap-orated therefrom in vacuo. The resulting solid melted at about 210-220 C. with decomposition. 4--Desacetyl vinblastine C-3 carboxhydrazide thus prepared had a carbomethoxy absorption band in the IR at 1725-1735 cm 1 thereby differentiating it from the 18-descarbomethoxy compound of Neuss et al supra, and a 1690 cm 1 band in the IR attributable to the hydrazide function. Molecular weight by mass spectrography was 768 in agreement with the theoretical value calculated for C43~56N6O7.
The nmr spectrum contained the prominent resonance at 3.6 ppm representing the methyl group of the C-18 carbomethoxy function.
Following the above procedure, 4-desacetyl leuro-cristine available from Hargrove U. S. Patent 3,392,173 was reacted with anhydrous hydrazine in anhydrous methanol to yield 4-desacetyl l-desformyl leurocristine C-3 carboxhydra-zide, isolated as an amorphou~ powder. Infrared spectrum;
absorption maxima at 1730 cm 1 (ester), 1670 cm 1 (hydrazide);
molecular ion spectrum: m/e=754 (consistent for C~2H54N6O7);
nmr ~3.60 (C18 methyl) ~3.74 (C16 methyl) ~4.05 (C4-hydrogen) ~6.34 (amide hydrogen).
Example 4 - 4-Desacetyl leurocristine C-3 N-methylcarboxamide A solution of 900 mg of leurocristine and 473 ml of anhydrous methanol was saturated with gaseous hydrogen chloride at about 0C. The reaction flask was then fitted with a drying tube and warmed to room temperature. After X-375~ -17~

~LV4;2~28 being maintained at that temperature for about 24 hours, the volatile constituents were removed by evaporation in vacuo, and the resulting residue dissolved in water. The aqueous solution so formed was made basic with 14 N ~nmonium hydroxide and the alkali insoluble bases present extracted with methylenechloride. The m~th~lenechloride extracts were combined, dried, and evaporated to dryness in vacuo.
The resulting residue, comprising 4-desacetyl l-desformyl leurocristine, was a light brown amorphous solid. Molecular weight by mass spectrography was 754 in agreement with that calculated for C43H54N408. In the infrared, an amide band at 1670 cm , characteristic of the l-formyl group, was lacking, but there was a strong band at 173Q ~m 1 char-acteristic of ester absorption. Likewise the nmr spectrum showed pPaks at 3.61 ppm and 3.85 ppm characteristic of the two carbomethoxy groups present; there was an additional peak at 4.11 ppm, characteristic of the proton at C-4.
A solution was prepared containing 500 mg of 4- -desacetyl l-desformyl leurocristine prepared as above in 75 ml of anhydrous methanol previously saturated with methylamine at -78 C. (The quantity of methylamine present was about 20 g~) The reaction vessel was sealed and heated at 60 C. for about one week. The volatile constituents were then removed by evaporation in vacuo to yield 4-desacetyl 1-desformyl leurocristine C-3 N-methylcarboxamide, a light brown amorphous solid. Infrared spe~trography of the above com-pound showed a strong amide band at 1668 cm in addition to an ester band at 1730 cm 1.
4-Desacetyi l-desformyl leurocristine C-3 N-methyl-carboxamide thus prepared was formylated with a mixture of .r~

. . ~ .-- - . . . ~

4~8 24 ml of 97 perc~nt formic acid and 2 ml of acetic anhydride.
The reaction mixture ~as allowed to stand at room temperature for about 24 hours, after which time the volatile constituents were removed in vacuo. The residue thus obtained was dis-solved in water, and the aqueous solution made basic with 14 N ammonium hydroxide. The nitrogenous bases insoluble in the alkaline solution were extracted into methylenechloride.
The methylenechloride extracts were combined, dried, and the methylenechloride removed therefrom by evaporation in vacuo 1~ to yield 4-desacetyl leurocristine C-3 N-methylcarboxamide, a light brown amorphous solid having the following char- -acteristics:
Molecular weight by mass spectro~raphy was 781 in agreement with the theoretical value calculated for 4 desacetyl leurocristine C-3 N-methylcarboxamide, C44H55N5O8.
The nmr spectrum had peaks at 3.65 ppm, character-istic of the carbomethoxy group, and at 2.79 ppm, character~
istic of the N-methylcarboxamide group.
Infrared spectrum contained a strong amide band at 1678 cm 1, indicating the presence of more than one amide function in the molecule, and a characteristic ester band at 1728 cm 1.
Following the procedure of Example 1, 4-desacetyl leurocristine C-3 N-methylcarboxamide free base was converted to the corresponding sulfate salt with one percent sulfuric acid. The sulfate was a white amorphous powder.
In the above procedure, other amide derivatives of 4-desacetyl l-desformyl leurocristine C-3 N-methylcarboxamide can be formed by substituting acetic or propionic anhydride for the formylation reagent -specified above to form the ~:, , . :
, . ' ~ ' ',, ' , ~ . ' ' . . .

~)4~ 8 corresponding 4-desacetyl l-desformyl l~acetyl ~or l-propion~l) leurocri~tine C-~ carboxamide derivative. Su~prisingly, re-acylation of a 4-desacPtyl leurocr:istine does not affect the l-formyl group which remains intact during the synthetic pro-cedure.
Example 5 4-Desacetyl vinblastine C-3 carhoxaæide A solution of 678 mg. of 4-desacetyl vinblastine C-3 carboxhydrazide (from Example 3) was prepared in 15 ml. of anhydrous methanol. About 50 ml. of lN aqueous hydrochloric acid were added, and the resulting solution cooled to about 0C. Approximately 140 mg. of sodium nitrite were then added, and the resulting reaction mixture stirred for 10 minutes while maintaining the temperature at about 0C. The solution turned dark red-brown upon the addition of the 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. 4-Desacetyl vinblastine C-3 carboxazide formed in the above reaction passed into the methylene dichloride.
While ordinarily the methylene dichloride solution of 4-desacetyl vinblastine C-3 carboxazide is used without further purification, an aliquot was treated as follows in order to characterize the azide: Evaporation of the methylene dichloride left the azide in an amorphous state. The azide residue was washed with ether, and the resulting suspension filtered, The residual tan powder had the following dis-tinguishing physical characteristics: ultraviolet spectrum lambda maX=269 mu. (epsilon = 16,700); shoulder at about 290 mu; 309 mu. (epsilon = 7,100); infrared absorption max-...

'!

~09~Z~8 imum at 1690 cm. 1 (carboxhydrazicle) was absent, while the maximum at 1730 cm 1 was not affected. Furthermore, a sharply ~efined maximum at 2135 cm 1 was noted characteristic of the carboxazide function. The mass spectrogram revealed a molecu-lar ion m/e = 708 showing a loss of 71 mass units (~, CON3) from the molecular weight calculated for C43H53N707 = 77g.
Example 6 4-Desacetyl vinblastine C-3 N-ethylcarboxamide A solution of 4-desacetyl vinblastine C-3 carbox~
azide was prepared in methylene dichloride solution according to the above procedure from 900 mg. of 4-desacetyl vinblastine C-3 carboxhydrazide. The methylene dichloride solution was dried, and the volume reduced to about 20 ml.
The solution of the azide in methylene dichloride was then placed in a flask fitted with a drying tube and stirrer. 50 ml. of anhydrous ethylamine were added thereto, and the reaction mixture was stirred at room temperature for about two hours. Evaporation of the volatile constituents ~n vacuo yielded a tan amorphous powder which was chroma-,~ - .
t~ographed over silica gel. The chromatogram was developed ~th an ethyl acetate-anhydrous ethanol (3:1) solvent mixture.
F~-ractions containing 4-desacetyl vinblastine C-3 N-ethyl-carboxamide as determined by thin-layer chromatography were combined, and the solvent was removed from the combined fractions ln vacuo. 450 mg. of a tan amorphous powder was obtained with the following distinctive physical character-istics: molecular ion spectrum, m/e = 781 (corresponding to C45H59N507)~ infrared spectrum; absorption maxima at 1730 cm 1 (ester), 1670 cm (amide), 3420 cm 1 (N-H amide), nmr.
~1.18 (triplet-~-methyl of ethyl amide group), ~3.28 :

104~42~
(~uartet-~-methylene of ethyl ami~e group), ~3.59 ~singlet-methyl ester), 4-desacetyl vinblastine C-3 N-ethylcarboxamide sul-fate was prepared by dissolving the above amorphous powder in anhydrous ethanol and adjusting the p~ to about 4.0 with 2 per-cent sulfuric acid in anhydrous ethanol. Evaporation of the solvent in vacuo yielded a water-soluble tan powder comprising 4-desacetyl vinblastine C-3 N-ethylcarboxamide sulfate.
Following the above procedure, 4-desacetyl vin-blastine C-3 N-isopropylcarboxamide was prepared. The com-pound had the following distinguishing characteristics:
molecular ion spectrum, m/e = 795 (corresponding to C46H51N507); infrared spectrum: maxima at 1730 cm 1 (ester);
1660 cm 1 (amide); nmr; ~1.16, ~1.22 (doublet-for isopropyl methyl groups); 4-desacetyl vinblastine C-3 N-isopropylcarbox-amide sulfate was prepared by the above procedure and was a tan water-soluble amorphous powder.
Following the above procedure, 4-desacetyl vin-blastine C-3 N,N-dimethylcarboxamide was prepared having the following distinctive physical characteristics: molecular ion spectrum; m/e = 781 (consistent with C45H59N507; infrared spectrum: absorption maxima at 1730 cm 1 (ester), 1620 cm 1 (amide) nmr ~2.96 (singlet-N-methyl) ~3.46 (singlet-N-methyl).
The sulfate salt of 4-desacetyl vinblastine C-3 N,N-dimethyl-carboxamide was pxepared by the above procedure and was a water-soluble, tan amorphous powder.
Following the above procedure, 4-desacetyl vin-blastine C-3 N-~2 (N,N-dimethylaminoethyl)]carboxamide was prepared from the carboxazide and N,N-dimethyl ethylamine, it had the following distinguishing physical characteristics:

~ 042~8 Infrared spectrum; absorption maxima at 3410 cm 1 (N-H amide), 1740 cm 1 (ester), 1670 cm 1 (amide); nmr ~2.44, ~3.41 (methylenes from ethylidene function ~2.23 (methyls of di-methylamino group); molecular ion spectrum; m/e = 824 (con-C47H64N6O7); pKa = 4,,85, 7.0, 8 5 The corresponding sulfate salt prepared by the above procedure was a tan, water-soluble amorphous powder, 4-Desacetyl vinblastine C-3 N-benzylcarboxamide was prepared using benzylamine by the above procedure and had tha following distinctive physical characteristics: Infrared spectrum; maxima a~ 3420 cm 1 (N-H amide), 1735 cm 1 (ester), 1675 cm 1 (amide); nmr; ~7.32 (aromatic protons) ~3.69 (methyl-ene group of benzyl amide); molecular ion spectrum, m~e = 843 50H61N5O7~, The corresponding sul~ate salt was made by the above procedure and was a water-soluble, tan amorphous powder, Following the above procedure, 4-desacetyl vln-blastine C-3 N-cyanomethylcarboxamide was prepared by reacting 4-desacetyl vinblastine C-3 carboxazide with cyanomethylamine, The novel amide had the following distinctive physical charac-teristics: Infrared absorption maxima at 1690 cm 1 ~amide~t 3420 cm~l (amide NH); nmr; ~4,17 (C4-H) ~2,80 ~N-CH3) ~3.77 (aromatic H) ~3,69 (methylester H) ~4,48 (J = 6Hz) ~3.92 (J = 17Hz); both cyanomethylene, Molecular ion spectrum m/e ~ 792 (consistent with C44H56N6O7), Following the above procedure, l-desformyl 4-des-acetyl leurocristine C-3 carboxhydrazide, furnished by the procedure of Example 3, was reacted with sodium nitrite in dilute hydrochloric acid to form l-desformyl 4-desacetyl leurocristine C-3 carboxazide which was obtained as a yellow 10424Z~
amorphous powder. A methylene dichloride solution of 1-desformyl 4-desacetyl leurocristine C-3 carboxazide was then reacted with ethylamine to yield l-desformyl 4-desacetyl leurocristine C-3 N-ethylcarboxamide. The compound was purified by chromatography over silica gel using an ethyl acetate-anhydrous ethanol (1:1) solvent mixture as the eluant.
Fractions, determined by thin-layer chromatography to contain the N-ethyl amide, were combined, and the combined fractions evaporated to dryness. l-Desformyl 4-desacetyl leurocristine C-3 N-ethylcarboxamide was obtained as a yellow amorphous powder having the following distinctive physical character-istics: Infrared spectrum; absorption maxima at 1665 cm 1 (amide), 1745 cm 1 and 1730 cm 1 (ester bands, 1 hydrogen bonded), 3430 cm 1 (N-H amide); molecular ion spectrum;
m/e = 767 (consistent with C44H57N507).
Example 7 4-Desacetyl leurosidine C-3 carboxhydrazide Following the method of Example 3, 1500 mg. of leurosidine were reacted with 25 ml. of anhydrous hydrazine in anhydrous methanol solution. The reaction mixture was sealed in a reaction flask, and the flask heated to 50C. for 12 days. Evaporation of the volatile constituents in vacuo yielded 4-desacety~ leurosidine C-3 carboxhydrazide having the following distinctive physical characteristics: Infra-red maxima 1735 cm 1 (ester), 1660 cm 1 (hydrazide);
molecular ion spectrum; m/e = 768 (consistent with 43 56N6O7) 4-Desacetyl leurosidine C-3 carboxhydrazide was con-verted to the corresponding azide by treatment with sodium nitrite, and the azide reacted with methanol-saturated with ~ ~ .
: - . , . . - ~ :

~424Z8 ammonia at -7~C. to yield 4-desacetyl leurosidine C-3 amide which was purified by chromatography as before. The compound had the following physical characteristics: Infrared maxima 3400 cm 1 (N-H amide) 1740 cm 1 (lester), 1690 cm 1 (amide);
molecular ion spectrum, m~e = 753 (consistent with C43H55N507);
~3.58 (C18 methyl ester), ~3.78 (C16, methoxyl), ~2.88 (C
methyl), ~5.78 (C-3 amide hydrogens), ~4.18 (C4 hydrogen).
The corresponding sulfate salt was prepared by a procedure involving neutralization of an ethanol solution of the base with ethanolic sulfuric acid to yi~ld a tan amorphous powderO
Other amides of leurosidine can be prepared in similar fashion.
Each of the desacetyl l-desformyl l-alkanoyl deriva-tives, as well as _he 4-desacetyl vinblastine, leurocristine, or leurosidine C-3 carboxamides with exception of the carbox-hydrazides and those amide moieties containing a reactive function as discussed above, can be reacted with cther an-hydrides such as chloroacetic acid anhydride, butyric an-hydride, 2-chloropropionic anhydride and the like to yield using, for example, chloroacetic anhydride, a mixture of the 3,4-bis-chloroacetyl and the 4-chloroacetyl derivatives whlch can be converted to the pure 4-chloroacetyl derivative by treatment with wet silica gal. Other 4-acyl derivatives are prepared in similar fashion.
Example 8 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, chloro-acetate, trichloroacetate, benzoate, alkyl or aryl sulfonates - : .

l.C~42428 and the like, are prepared from the amide bases by a procedure analogous to that set forth in Example 1 above for the prep-aration of the sulfate salt by substituting the appropriate acid in a suitable diluent in place of the 1 percent aqueous suluric 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 prep-aration of salts so as to avoid hydrolysis, transesterification and other reactions which take place at high temperatures, ex-tremely acid pH's etc.
The compounds have shown antiviral activity in vitro against erpes virus employing a tissue culture system in a plaque suppression test similar to that described by Siminoff, Applied Microbiology, 9, 66-72 (1961). For example, vin-blastine C-3 carboxamide sulfate gave a 20 mm inhibition zone with a 3 rating and without toxicity at a dose level of 125 ~g/ml. The same compound was shown to be able to induce meta-phase arrest in cultured Chinese hamster ovary cells at doses ranging from 2 x 10 ~g/ml to 2 x 10 ~g/ml. A most active inducer of such arrest was shown to be 4-desacetyl leuro-cristine C-3 N-methylcarboxamide sulfate which was effective at doses on the order of 10 6 ~g/ml.
In addition, the compounds of this invention have been shown to be active against transplanted mouse tumors in vivo. For example, 4-desacetyl vinblastine C-3 carboxamide sulfate, 4-desacetyl vinblastine C-3 N-methylcarboxamide sul-fate, vinblastine C-3 carboxamide sulfate, vinblastine C-3 N-methylcarboxamide sulfate, 4-desacetyl vinblastine C-3 N-(2-3C hydroxyethyl) carboxamide sulfate, 4-desacetyl vinblastine iO4~428 C-3 N,N-dimethylcarboxamide sulfate, 4-desacetyl leurosidine C-3 amide sulfate, 4-desacetyl vinblastine C-3 N-benzyl carboxamide sulfate and 4-desacetyl vinblastine C-3 carbox-hydrazide, as well as other compounds coming within the scope of the above formula, demonstrated such activity. Of par-ticular interest, however, is the activity of the compounds of this invention, especially the 4-desacetyl vinblastine C-3 carboxamide sulfate, and its N-alkyl and N-hydroxyalkyl deriva-tive against Ridgeway osteogenic sarcoma (ROS) and Gardner lymphosarcoma (GLS). In demonstrating activity of the drugs of this invention against these tumors, 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 innoculation with the tumor.
The following table - Table 1 - gives the resultq of several experiments in which mice bearing transplanted tumors were treated successfully with a compound of Formula I.
In the table, column 1 gives the name of the compound; column 2, the transplanted tumor; column 3, the dose level or doce 2~ level range and the number of days the dosage was admin-istered; 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).

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~V42~;28 The compounds of Formula I, like leurocristine and vinblastine, are 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 inhihition 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.
The compounds of Formula I are also active against other transplanted tumors. For example, with Mecca lympho-sarcoma, parenteral injection of 0.25 mg/kg for 9 days of vin-blastine C-3 N-methylcarboxamide sulfate gave a 54 percent in-hibition 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 adenocarcinoma, 4-desacetyl vinblastine C-3 carboxamide sulfate gave a 67 per-cent inhibition of tumor growth, 4-desacetyl vinblastine C-3 N-methylcarboxamide sulfate 61 percent inhibition and vin blastine C-3 carboxamide sulfate 49 percent inhibition at a dose level of 0.25 mg/kg for eight days and 72 percent in-hibition at 0.3 mg/kg. In a similar experiment, vinblastine gave a 31 percent inhibition while leurocristine at the some-what 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 3Q the two diseased mice in this experiment was prolonged by 26 104Z42~
percent over that of the control mice. In the same experi-ment, vinblastine gave a 36 percent prolongation but with no indefinite survivors and rated only a minimal effectiveness rating.
As would be expected, the amides and hydrazides differ in their anti-tumor spectrum from vinblastine leuro-cristine and leurosine, as well as from the C-4 N,N-dialkyl-glycyl esters of vinblastine in the same way that the anti-tumor spectra of those compounds differ among themselves, some being more effective against certain tumors or classes of tumors and less effective against others.
In utilizing the 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 i5 NH-NH2 or N3, formed with a non-toxic acid is mixed with starch or other excipient and the mixture placed in telescoping gelatin cap-sules 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 mgs. 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 mammalian body weight once a week, depending on both the activity and the toxicity of the drug. Free bases of compounds according ~-3754 -31-' ' :

1~42428 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 are useful as anti-neoplastic or antiviral drugs, two types of derivatives, the hydrazides and azides (compounds o~ Formula I wherein R is NH-NH2 or N3), are also useful as intermediates as has been set forth above, in that the hydrazide can be transformed to the azide by reaction with a nitrosating agent, or to the simple amide by hydrogenolysis. The azide can in turn be made to react with primary or secondary amines to yield the amides of this invention.

. ~ - ': .

Claims (36)

The embodiments of the invention for which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a compound of the formula Formula I

wherein R is NH2, NH-NH2, N(CH3)2, NH-alk-X, NH-alk-Am, NH-alk(OH)1-3, or N3 wherein alk is (C1-C6)-alkyl, Am is NH2, NHCH3 or N(CH3)2 and X is hydrogen, cyano, phenyl; R' is (C1-C3)-alkanoyl or chloro-(C1-C3)-alkanoyl; R'' is hydrogen, (C1-C3)-alkyl, formyl or (C1-C3) alkanoyl; and one of R''' and R'''' is hydroxyl and the other is ethyl;
and its pharmaceutically acceptable salts; which comprises reacting a compound of Formula I wherein R is O-CH3, R' is hydrogen or acetyl and R'', R''' and R'''' are as defined above, with ammonia, methylamine, ethylamine or hydrazine to obtain a compound of Formula I wherein R is NH2, NH-NH2, NH-CH3 or NH-C2H5 and, if desired, when R is NH-NH2 (a) reacting said compound with a nitrosating agent to form an azide in which R is N3 and then reacting said azide with ammonia, an amine of the formula HN(CH3)2, NH2-alk-X, NH2-alk-Am or NH2-alk-(OH)1-3 wherein alk is (C1-C6)-alkyl and Am and X are as defined above, to yield a compound of formula I
wherein R' is H or acetyl,or (b) hydrogenolyzing said compound with Raney nickel in an inert solvent to yield a compound of formula I wherein R is NH2 and R' is hydrogen or acetyl, and (c) acylating the compound wherein R' is hydrogen with an acid chloride of the formula (C1-C3)-alkyl-COC1 or chloro-(C1-C3)-alkyl-COCl or an acid anhydride of the formula [(C1-C3)-alkyl-CO]2O or [chloro-(C1-C3)-alkyl-CO]2O to form a compound of formula I in which R' is (C1-C3)-alkanoyl or chloro-(Cl-C3)-alkanoyl, and R " , R''' and R'''' are as defined above;and optionally,converting said compound to a phar-maceutically acceptable salt.

2. A compound of the formula Formula I

wherein R is NH2, NH-NH2, N(CH3)2, NH-alk-X, NH-alk-Am, NH-alk(OH)1-3, or N3 wherein alk is (C1-C6)-alkyl, Am is NH2, NHCH3 or N(CH3)2 and X is hydrogen, cyano, phenyl;
R' is (C1-C3)-alkanoyl or chloro-(C1-C3)-alkanoyl; R'' is hydrogen, (C1-C3)-alkyl, formyl or (C1-C3) alkanoyl; and one of R''' and R'''' is hydroxyl and the other is ethyl;
and its pharmaceutically acceptable salts,when prepared by the process of claim 1 or an obvious chemical equivalent thereof.

3. A process for preparing a pharmaceutically acceptable inorganic salt of a compound of the formula Formula I

wherein R is NH2, NH-NH2, N(CH3)2, NH-alk-X, NH-alk-Am, NH-alk(OH)1-3, or N3 wherein alk is (C1-C6)-alkyl, Am is NH2, NHCH3 or N(CH3)2 and X is hydrogen, cyano, phenyl; R' is (C1-C3)-alkanoyl or chloro-(C1-C3)-alkanoyl; R'' is hydrogen, (C1-C3)-alkyl, formyl or (C1-C3) alkanoyl; and one of R''' and R' ' ' ' is hydroxyl and the other is ethyl;
wherein said inorganic salt is a salt of hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, which comprises reacting a compound of Formula I wherein R is O-CH3, R' is hydrogen or acetyl and R'', R''' and R'''' are as defined above with ammonia, methylamine, ethylamine or hydrazine to obtain a compound of Formula I wherein R is NH2, NH-NH2, NH-CH3 or NH-C2H5 and, if desired, when R is NH-NH2 (a) reacting said compound with a nitrosating agent to form an azide in which R is N3 and then reacting said azide with ammonia, an amine of the formula HN(CH3)2, NH2-alk-X, NH2-alk-Am or NH2-alk-(OH)1-3 wherein alk is (C1-C6)-alkyl and Am and X are as defined above, to yield a compound of formula I
wherein R' is H or acetyl; or (b) hydrogenolyzing said compound with Raney nickel in an inert solvent to yield a compound of formula I wherein R is NH2 and R' is hydrogen or acetyl; and (c) acylating the compound wherein R' is hydrogen with an acid chloride of the formula (C1-C3)-alkyl-COC1 or chloro-(C1-C3)-alkyl-COCl or an acid anhydride of the formula [(C1-C3)-alkyl-CO]2O or [chloro-(C1-C3)-alkyl-CO]2O,to form-a compound of formula I in which R' is (C1-C3)-alkanoyl or chloro-(C1-C3)-alkanoyl, and R'', R''' and R'''' are as defined above; and (d) converting said compound to a pharmaceu-tically acceptable salt of an acid as named above.

4. A pharmaceutically acceptable inorganic salt of a compound of the formula Formula I

wherein R is NH2, NH-NH2, N(CH3)2, NH-alk-X, NH-alk-Am, NH-alk(OH)1-3, or N3 wherein alk is (C1-C6)-alkyl, Am is NH2, NHCH3 or N(CH3)2 and X is hydrogen, cyano, phenyl, R' is (C1-C3)-alkanoyl or chloro-(C1-C3)-alkanoyl; R'' is hydrogen, (C1-C3)-alkyl, formyl or (C1-C3) alkanoyl; and one of R''' and R'''' is hydroxyl and the other is ethyl, wherein said inorganic salt is a salt of hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, when prepared by the process of claim 3 or an obvious chemical equivalent thereof.

5. A process for preparing a pharmaceutically acceptable organic salt of a compound of the formula Formula I
wherein R is NH2, NH-NH2, N(CH3)2, NH-alk-X, NH-alk-Am, NH-alk(OH)1-3, or N3 wherein alk is (C1-C6)-alkyl, Am is NH2, NHCH3 or N(CH3)2 and X is hydrogen, cyano, phenyl; R' is (C1-C3)-alkanoyl or chloro-(C1-C3)-alkanoyl; R'' is hydrogen, (C1-C3)-alkyl, formyl or (C1-C3) alkanoyl; and one of R''' and R'''' is hydroxyl and the other is ethyl;
wherein said organic salt is a salt of aliphatic mono- or dicarboxylic acid, phenyl-substituted alkanoic acid, hydroxy alkanoic acid, hydroxy alkandioic acid, aromatic acid, aliphatic sulfonic acid or aromatic sulfonic acid, which comprises reacting a compound of Formula I wherein R is O-CH3, R' is hydrogen or acetyl and R'', R''' and R'''' are as defined above with ammonia, methylamine, ethylamine or hydrazine to obtain a compound of Formula I wherein R is NH2, NH-NH2, NH-CH3 or NH-C2H5 and, if desired, when R is NH-NH2 (a) reacting said compound with a nitrosating agent to form an azide in which R is N3 and then reacting said azide with ammonia, an amine of the formula HN(CH3)2, NH2-alk-X, NH2-alk-Am or NH2-alk-(OH)1-3 wherein alk is (C1-C6)-alkyl and Am and X are as defined above, to yield a compound of formula I
wherein R' is H or acetyl; or (b) hydrogenolyzing said compound with Raney nickel in an inert solvent to yield a compound of formula I wherein R is NH2 and R' is hydrogen or acetyl; and (c) acylating the compound wherein R' is hydrogen with an acid chloride of the formula (C1-C3)-alkyl-COCl or chloro-(C1-C3)-alkyl-COCl or an acid anhydride of the formula [(C1-C3)-alkyl-CO]2O or [chloro-(C1-C3)-alkyl-CO]2O to form a compound of formula I in which R' is (C1-C3)-alkanoyl or chloro-(C1-C3)-alkanoyl, and R'', R''' and R'''' are as defined above; and converting said compound to a pharmaceutically acceptable organic salt of an acid as named above.

6. A pharmaceutically acceptable organic salt of a compound of the formula Formula I

wherein R is NH2, NH-NH2, N(CH3)2, NH-alk-X, NH-alk-Am, NH-alk(OH)1-3, or N3 wherein alk is. (C1-C6)-alkyl, Am is NH2, NHCH3 or N(CH3)2 and X is hydrogen, cyano, phenyl; R' is (C1-C3)-alkanoyl or chloro-(C1-C3)-alkanoyl; R'' is hydrogen, (C1-C3)-alkyl, formyl or (C1-C3) alkanoyl; and one of R''' and R'''' is hydroxyl and the other is ethyl;
wherein said organic salt is a salt of an aliphatic mono- or dicarboxylic acid, phenyl-substituted alkanoic acid, hydroxy alkanoic acid, hydroxy alkandioic acid, aromatic acid, aliphatic sulfonic acid or aromatic sulfonic acid, when prepared by the process of claim 5 or an obvious chemical equivalent thereof.

7. A process for the preparation of 4-desacetyl vinblastine C-3 N-methylcarboxamide which comprises reacting vinblastine with methylamine to obtain a mixture of 4-desacetyl vinblastine C-3 N-methylcarboxamide and 4-desacetyl vinblastine, and recovering 4-desacetyl vinblastine C-3 N-methylcarboxamide.

8. 4-Desacetyl vinblastine C-3 N-methylcarbox-amide,when prepared by the process of claim 7 or an obvious chemical equivalent thereof.

9. A process as in claim 7 which comprises the additional step of reacting 4-desacetyl vinblastine C-3 N-methylcarboxamide with sulfuric acid to obtain 4-desacetyl vinblastine C-3 N-methylcarboxamide sulfate.

10. 4-Desacetyl vinblastine C-3 N-methylcarbox-amide sulfate, when prepared by the process of claim 9 or an obvious chemical equivalent thereof.

11. A process for preparing 4-desacetyl vinblastine C-3 carboxamide which comprises reacting vinblastine with ammonia at an elevated temperature.

12. 4-Desacetyl vinblastine C-3 carboxamide,when prepared by the process of claim 11 or an obvious chemical equivalent thereof.

13. A process for preparing 4-desacetyl vinblastine C-3 carboxhydrazide which comprises reacting 4-desacetyl vinblastine with anhydrous hydrazine at an elevated tem-perature.

14. 4-Desacetyl vinblastine C-3 carboxhydrazide, when prepared by the process of claim 13 or an obvious chemical equivalent thereof.

15. A process for preparing 4-desacetyl 1-desformyl leurocristine C-3 carboxhydrazide which comprises reacting 4-desacetyl leurocristine with anhydrous hydrazine at an elevated temperature.

16. 4-Desacetyl 1-desformyl leurocristine C-3 carboxhydrazide, when prepared by the process of claim 15 or an obvious chemical equivalent thereof.

17. A process for preparing 4-desacetyl leuro-cristine C-3 N-methylcarboxamide which comprises reacting 4-desacetyl 1-desformyl leurocristine with methylamine at an elevated temperature, and reacting the resulting 4-desacetyl 1-desformyl leurocristine C-3 N-methylcarboxamide with formic acid and acetic anhydride.

18. 4-Desacetyl leurocristine C-3 N-methylcarbox-amide,when prepared by the process of claim 17 or an obvious chemical equivalent thereof.

19. A process for preparing 4-desacetyl vinblastine C-3 carboxazide which comprises reacting 4-desacetyl vin-blastine with anhydrous hydrazine to form 4-desacetyl vin-blastine C-3 carboxhydrazide, and carrying out the additional step of reacting 4-desacetyl vinblastine C-3 carboxhydrazide with sodium nitrite.

20. 4-Desacetyl vinblastine C-3 carboxazide,when prepared by the process of claim 19 or an obvious chemical equivalent thereof.

21. A process as in claim 19 for preparing 4-desacetyl vinblastine C-3 N-ethylcarboxamide which comprises the additional step of reacting the product 4-desacetyl vinblastine C-3 carboxazide with anhydrous ethylamine.

22. 4-Desacetylvinblastine C-3 N-ethylcarboxamide, when prepared by the process of claim 21 or an obvious chemical equivalent thereof.

23. A process as in claim 19 for preparing 4-desacetyl vinblastine C-3 N-isopropylcarboxamide which com-prises the additional step of reacting the product 4-desacetyl vin-blastine C-3 carboxazide with anhydrous isopropylamine.

24. 4-Desacetylvinblastine C-3 N-isopropylcarbox-amide, when prepared by the process of claim 23 or an obvious chemical equivalent thereof.

25. A process as in claim 19 for preparing 4-desacetyl vinblastine C-3 N,N-dimethylcarboxamide which comprises the additional step of reacting the product 4-desacetyl vinblastine C-3 carboxazide with anhydrous N,N-dimethyl-amine.

26. 4-Desacetyl vinblastine C-3 N,N-dimethyl-carboxamide, when prepared by the process of claim 25 or an obvious chemical equivalent thereof.

27. A process as in claim 19 for preparing 4-desacetyl vinblastine C-3 N[2-(N,N-dimethylaminoethyl)]-carboxamide which comprises the additional step of reacting the product 4-desacetyl vinblastine C-3 carboxazide with N,N-dimethyl-ethylamine.

28. 4-Desacetyl vinblastine C-3 N[2-(N,N-dimethyl-aminoethyl)]carboxamide, when prepared by the process of claim 27 or an obvious chemical equivalent thereof.

29. A process as in claim 19 for preparing 4-desacetyl vinblastine C-3 N-benzylcarboxamide which comprises the additional step of reacting the product 4-desacetyl vinblastine C-3 carboxazide with benzylamine.

30. 4-Desacetyl vinblastine C-3 N-benzylcarbox-amide,when prepared by the process of claim 29 or an obvious chemical equivalent thereof.

31. A process as in claim 19 for preparing 4-desacetyl vinblastine C-3 N-cyanomethylcarboxamide which comprises the additional step of reacting the product 4-desacetyl vinblastine C-3 carboxazide with cyanomethylamine.

32. 4-Desacetyl vinblastine C-3 N-cyanomethyl-carboxamide,when prepared by the process of claim 31 or an obvious chemical equivalent thereof.

33. A process as in claim 15 for preparing 1-desformyl 4-desacetyl leurocristine C-3 N-ethylcarboxamide which comprises the additional steps of reacting 1-desformyl 4-desacetyl leurocristine C-3 carboxhydrazide with sodium nitrite to form 1-desformyl 4-desacetyl leurocristine C-3 carboxazide and reacting said carboxazide with ethylamine.

34. 1-Desformyl 4-desacetyl leurocristine C-3 N-ethylcarboxamide,when prepared by the process of claim 33 or an obvious chemical equivalent thereof.

35. A process for preparing 4-desacetyl leuro-sidine C-3 amide which comprises reacting leurosidine with anhydrous-hydrazine at an elevated temperature, reacting the resulting 4-desacetyl leurosidine C-3 carboxhydrazide with sodium nitrite, and reacting the resulting azide with ammonia.

36. 4-Desacetyl leurosidine C-3 amide,when pre-pared by the process of claim 35 or an obvious chemical equivalent thereof.