DK141511B - Analogous process for the preparation of derivatives of wind blastin, leurosidine or leurocristine. - Google Patents

Description

(11) PUBLICATION NOTICE 1 ^ + 1511 DENMARK ln * · C | S c 07 D 519/04 '(21) Application No 1787/74 (22) Filed on 1 · aPr · 1974 (23) Running Day 1 · Apr · 1974

(44) The application presented and in qQA

the submission letter published on o · apr. I you DIRECTORATE OF W u PATENT AND TRADEMARKET (30) The prlont requested from the

Apr 2 1973, 347275 US

(71) ELI LILLY IND COMPANY, JO7 East McCarty Street, Indianapolis, In = dlaria 46204, US.

(72) Inventor: George Joseph £ ullInan, 7819 Rea Hoad, Indianapolis, In = diana, US: Koert Gerzon, 4921 Hawthorne Terrace, Apt. C., Indlanapo * lis, Indiana, US.

(74) Plenipotentiary in the proceedings:

Hofman-Bang & Boutard Engineering Company.

(64) Analogous process for the preparation of derivatives of vinblastine, leurosidine or leurocristine.

The invention relates to an analogous process for the preparation of novel derivatives of vinblastine, leurosidine or leurocristine of the general formula I set out in the preamble of the claim, which is characterized by the characterizing part of the claim.

Several naturally occurring alkaloids obtainable from Vinca rosea have been found to be active in the treatment of experimental malignancies in animals. Among these alkaloids are leurosine (U.S. Patent No. 3370057), vincaleukoblastin (vinblastine), (U.S. Patent No. 3097137), leurosidine (vinrozidine), and leurocristine (vineristin) (both in U.S. Pat.

2 141511 3205220). Two of these alkaloids, vinblastine and leurocristine, are now marketed as medicines for the treatment of malignant diseases, in particular leukemia and other related diseases in humans. Of these marketed compounds, leurocristine is the most active and useful compound in the treatment of leukemia, but is also the compound found in least amounts among the antineoplastic alkaloids in Vinca rosea.

Chemical modification of Vinca alkaloids has occurred only to a limited extent. First, the molecular structures involved are extremely complex, and chemical reactions affecting a specific function of the molecule are difficult to perform. Second, alkaloids lacking the desired chemotherapeutic properties have been recovered from Vinca rosea fractions, and a determination of their structure has led to the conclusion that these compounds are closely related to the active alkaloids.

Thus, the antineoplastic activity appears to be limited to very specific structures, and changes to obtain more active drugs upon modification of these structures appear to be correspondingly difficult. Among the successful modifications of physiologically active alkaloids have been the preparation of dihydrovinyl blastine (U.S. Patent No. 3352868) and replacement of the acetyl group in C-4 (carbon atom # 4 in the vinblastine system, see Formula I) with a higher alkanoyl group or with other acyl groups (U.S. Patent No. 3392173). Several of these derivatives are capable of extending the life of mice inoculated with P 1534 leukemia. One of the derivatives in which a chloroacetyl group replaced the C-4 acetyl group in vinblastine was also a useful intermediate for the preparation of a structurally modified vinblastine compound in which a Ν, Ν-dialkylglycyl group replaced the C-4 acetyl group in vinblastine (U.S. Pat. 3,387,001). An intermediate, namely 4-deacetyl-vinblastine, was prepared during the chemical reactions leading to these latter derivatives.

This intermediate, in which the C-4 acyl group was missing and which had an unesterified hydroxy group, is reported in the literature to be a toxic substance with little chemotherapeutic effect in vivo against P 1534 leukemia, Hargove, Lloydia, 27, 340 (1964 ).

The compounds of the present invention have useful antiviral and anti-various effects.

3 141511

Illustrative of the alk-X groups of formula I are the following: methyl, 2-methylpentyl, isohexyl, isopentyl, n-pentyl, n-hexyl, sec-hexyl, ethyl, isopropyl, n-butyl, sec-butyl, cyanmethyl, cyanethyl, 5-cyan-n-pentyl, 2-dimethylaminoethyl, 2-aminoethyl, 2-methylaminoethyl, benzyl, phenethyl, 4-phenylbutyl, dimethylamino-methyl, 2-aminopropyl, 2-aminohexyl and 2-dimethylaminopropyl.

Nontoxic acids which may be used to form pharmaceutically acceptable acid addition salts of the amine bases include salts derived from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrogeribromic, hydroiodic, nitric and phosphoric acids, as well as non-salts derived from organic acids comprising aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids. Thus, such pharmaceutically acceptable salts include e.g. sulfate, pyrosulfate, hydrogen sulfate, sulfite, hydrogen sulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, carpoate lat, malonate, succinate} suberate, sebacate, fumarate, maleate, butyn 1,4-dioate, hexyn-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzene sulfone toluene sulfonate, chlorobenzenesulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, 2-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate and naphthalene

The compounds of formula I are described as derivatives either of vinblastine, wherein R 'is acetyl, R "is methyl, R, M is hydroxyl and R" is ethyl, or of desacetylvinblastine, wherein R n, R same, but R * is hydrogen or as derivatives of leu- rocristine, where R * is acetyl, Rw is formyl, Rn * is hydroxyl, and R "'1 is ethyl, or of desacetyl-leurocristine where R", Rn * and R ”is the same meaning, but R 'is hydrogen, or as derivatives of desmethyl-vinblastine (also known as desformyl-leurocristine), wherein R' is acetyl, R" is hydrogen, and R "and Rπ, are hydroxyl and ethyl, or of desacetyl-desmethyl-vinblastine (desacetyl-des-formyl-leurocristine), wherein R 1 and R 4 are hydrogen and R 2 and R 2 are hydroxy and ethyl respectively, or which derivatives of leurosidine, wherein R 'is acetyl, R "is methyl, R, M is ethyl and R" M is hydroxy, or of desmethylleurosidine where R 1, R "and R" "are the same, but R" is hydrogen , or as derivatives of desacetyl-desmethyl-leurosidine, wherein R 'o g R "is hydrogen, R" * is ethyl and R "11 is hydroxy, or of desacetyl-leurosidine wherein R 'is hydrogen, R" is methyl and R, n and R, rM are ethyl and hydroxyl, respectively. In each case, the term desacetyl refers to a lack of an acetyl group on the hydroxy group at C-4 in the indole-dihydroindole ring system.

In the process of the invention, the compounds of formula I wherein R is ML · NH-NHg »Mi-CH2 or NH-CgH2 are prepared by the treatment of vinblastine, leurocristine, leurosidine, desmethyl-vinblastine, desmethyl-leurosidine or their respectively 4-desacetyl compounds with ammonia, methylamine, ethylamine or hydrazine to give the corresponding amide, N-methylamide, N-ethylamide or hydrazide. The product of this reaction with starting materials having an intact 4-acetyl group is usually a mixture of compounds in which, on the one hand, the methoxycarbonyl group at C-3 is converted to a carboxamide, N-methylcarboxamide,. N-ethylcarboxamide or carboxhydrazide group, but on the other hand also the acetyl group at C-4 is completely or partially removed. These C-4 desacetyl derivatives are separated by chromatographic methods.

The compounds of formula I wherein R is N 1, N (CH 2) 2 or Mi-alk-X, wherein alk and X are as defined herein, are prepared by reaction of a hydrazide of formula I wherein R is NH Prepared as described above, with a nitrosating agent such as nitric acid, nitrochloride, nitrogen tetroxide, amyl nitrite or a similar agent, according to known methods to form the corresponding azide. The azide thus prepared is reacted, if desired, then with the primary or secondary amine, HN (CH2) 2 or Mi2-alk-X, to give the desired C-3 amide. This C-3 azide-amine reaction does not affect the C-4 acetyl group, which, if present, remains intact during the reaction and work-up. The above azide U1511 amine transformation occurs according to methods described in Helv. Chim, Acta., 26, 944 (1943) and U.S. Patent Nos. 2090429 and 2090430.

Compounds of formula I, wherein there is an acetyl group at carbon 4, can be prepared, as stated above, by reacting vinblastine, leurocristine or leurosidine directly with ammonia, methylamine, ethylamine or hydrazine, followed by separation of 4 -acetyl derivative from the 4-desacetyl derivative, and optionally conversion of hydrazides to azide followed by reaction between the azide and an amine to give the other amides of formula I. In general, however, the hydrazine-azide amide reaction is carried out with a 4-desacetyl derivative the reliability of the acetyl group under basic reaction conditions. Generally, 4-desacetylamides of formula I can be acetylated with acetic anhydride or acetic acid chloride to give the corresponding C-4 acetate.

The preferred acylation method is that described in US Patent No. 3392173 for vinblastine or leurocristine, wherein the diacyl derivative is the first reaction product and this derivative is selectively hydrolyzed to give a 4-acyl compound, Other methods comprising selective acylation or multiple acyl ring followed by selective hydrolysis can be used to prepare 4-acetyl derivatives of formula I.

However, there are certain precautions that must be observed when performing an acylation reaction. First, it will be apparent to those skilled in the art that an acylation of a 1-desformyl-leurocristine (1-desmethyl-vinblastine) or of a 1-desmethyl-leurosidine will result in acylation also at N-1. Therefore, the 1-desformyl or the 1-desmethyl compound must be formylated, acylated or alkylated to obtain a desired substituent at N-1 prior to the C-4 acylation reaction. Furthermore, if the C-3 carboxamide group contains an acylatable amino group, the C-4 acylation reaction must be carried out prior to the azide-amine reaction giving the final C-3 carboxamide group.

The preferred method is to acylate the C-3 carboxhydrazide by the above methods, protecting the hydrazide group first, otherwise it would be acylated. The preferred hydrazide protecting group is the propylidene group formed by reaction of the NH 2 moiety 6 µm of the hydrazide moiety with acetone. This group can be easily removed by treatment with acid, or preferably the propylene derivative itself can be reacted directly with nitrite to form an azide group (U.S. Patent No. 3470210, Example VII).

Other methods which include 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 are well known to those skilled in the art.

The derivatives of formula I will be referred to by reference only to the new group formed at a given carbon atom. For example, the compound produced by substituting the methyl ester function of vinblastine at carbon atom # 3 is called vinblastine C-3 carboxamide and not vinblastine C-3 descarbomethoxy C-3 carboxamide.

Compounds of formula I in the form of their free bases comprising both carboxamides, carboxazides and carboxhydrazides are white or light amorphous solids. However, it is preferred when it is possible to isolate and crystallize the amides in the form of their anionic salts formed with non-toxic acids. Such salts are highly melting, white, crystalline or amorphous water-soluble solids.

The process according to the invention is illustrated in more detail by the following examples.

Example 1

Vinblastine C-3 H-methyl carboxamide and 4-desacetyl-vinblastine C-3 N-methylcarboxamide _____

A solution containing 21 g of methylamine is prepared in 100 ml of anhydrous methanol at -78 c. 3.3 g of vinblastine was added to this solution. The reaction vessel was sealed and placed in a constant temperature bath at 50 ° C for 8 days. The reaction vessel was opened and the volatiles removed by evaporation in vacuo. Nmr and Ir spectra of the residue indicated that it was a mixture of 4-desacetyl-vinblastine C-3 N-methyl-carboxamide and 4-desacetyl-vinblastine. The residue was dissolved in 100 ml of pyridine and 20 ml of acetic anhydride was added. The resulting solution was left at room temperature for 18 hours.

Volatile compounds were then removed in vacuo and the resulting residue was chromatographed over alumina using ethyl acetate chloroform (1: 1) solvent mixture as eluent. Fractions containing vinblastine 0-3 H-methylcarboxamide as indicated by thin layer chromatography were combined and the solvent was evaporated in vacuo.

The above acylation method not only reacylated the 0-4 hydroxyl group but also acetylated the C-3 hydroxyl group. The acetyl group at C-3 was removed by methods described in Lloydia, 27, 340 (1964), in which method the product containing the C-3 acylated material was treated with silica gel in aqueous methanol at room temperature for a period ranging from 6 hours to several days to obtain a product lacking C-3 acetyl function. In this particular case, the residue from the combined chromatographic fractions was dissolved in 50 ml of methanol. 20 ml of water and 2 g of silica gel were added. The resulting mixture was filtered and the filtrate was evaporated to dryness in vacuo and the resulting residue was chromatographed over silica gel. A benzene-chloroform-triethylamine (100: 50: 7.5) solvent mixture was used as the eluent. The fractions containing the desired vinblastine C-3 N-methylcarboxamide were collected and the solvent removed by evaporation in vacuo. The residue was dissolved in aqueous methanol and the pH was adjusted to 2.9 with 1 sulfuric acid. Evaporation of the resulting mixture in vacuo yielded vinblastine C-3 methyl-methylcarboxamide sulfate, which crystallized from anhydrous ethanol to give a material which melted during decomposition at 272 - 275 ° C.

An infrared spectrum of vinblastine C-3 methylcarboxamide base obtained as indicated above showed a band obtained at 1672 cm 8 1415ti (which is not in the spectrum of vinblastine) indicating the presence of an amide group. The Nmr spectrum was fully consistent with the stated structure of vinblastine C-3 N-methylcarboxamide comprising the recently introduced N-methyl-carboxamide function represented by a sharp methyl resonance at 2.81 ppm.

4-desacetyl vinblastine C-3 N-methylcarboxamide was isolated from the mixture with 4-desacetyl vinblastine obtained in the amidation step by the following method. The residue obtained by evaporation of the original (before reacylation) reaction mixture to dryness in vacuo was chromatographed over silica gel using benzene chloroform triethylamine as eluent. The fractions which by thin layer chromatography were found to contain 4-desacetyl-vinblastine C-3 N-methylcarboxamide were mixed and the solvent was evaporated in vacuo to give N-methylcarboxamide as an amorphous solid. The infrared spectrum of the compound showed a band at 1672 cm 2 which is characteristic of an amide group. Absence of the 4-acetyl group was evidenced by the absence of a resonance for this function at 2.1 ppm in the Nmr spectrum. The molecular weight was determined by mass spectroscope! to 767, which is in accordance with the theoretical value calculated for C44H57N507.

The sulfate salt of 4-desacetyl-vinblastine C-3 N-methylcarboxamide was prepared by the method described for the preparation of vinblastine C-3 N-methylcarboxamide sulfate. The resulting product was an amorphous, water-soluble solid.

Following the above method, 2 g of 4-desacetyl-vinblastine was dissolved in a mixture of 75 ml of anhydrous methanol and 20 g of ethylamine. The reaction mixture was sealed and heated at 60 ° C for 8 days. 4-desacetyl-vinblastine C-3 N-ethylcarboxamide, thereby obtained, was separated by thin layer chromatography. The resulting solid showed a band at 1670 cm in the infrared spectrum, which is characteristic of a substituted carboxamide function.

9 U15T1

Example 2

Preparation of 4-desacetyl-vinblastine C-3 carboxamide

Ca. 10 g of vinblastine sulfate was converted by standard methods to vinblastine as a free base. This free base, obtained as a residue by evaporation of the dried ether solution, was dissolved in 200 ml of anhydrous methanol. Anhydrous liquid ammonia in 300 ml was added and the reaction mixture was sealed and heated at 100 ° G for 60 hours. The reaction vessel was opened and the contents removed and evaporated to dryness in vacuo. The resulting residue was chromatographed again and the fractions containing 4-desacetyl-vinblastine C-3 carboxamide were combined and the solvent was evaporated therefrom in vacuo to give a residue of purified 4-desacetyl-vinblastine C-3 carboxamide as a free base. Nmr and Ir spectra of the solid confirmed the above structure. The free base showed bands in the infrared spectrum at 1687 cm ”1, characteristic of the amide function. The molecular weight of the free base was determined by mass spectroscopy to 753 consistent with the theoretical value calculated for C C HH NN NO,, 600 mg of the above residue was converted to sulfate salt according to the method described in Example 1. Evaporation of the reaction mixture to dryness gave 4-desacetyl-vinblastine C-3 carboxamide sulfate, which crystallized from an ethanol-isopropyl solvent mixture and melted at 250 ° C during decomposition.

The salt was soluble in water.

4-Desacetyl-vinblastine C-3 carboxamide was converted to vinblastine C-3 carboxamide by the method described in Example 1 Bom follows * 2.8 g of 4-desacetyl-vinblastine C-3 carboxamide as free base acetylene with a mixture of anhydrous pyridine and acetic anhydride. The reaction mixture was kept at room temperature for 3 days. The volatiles were removed by evaporation in vacuo and the residue of the resulting residue was dissolved in methylene chloride. The methylene chloride solution was washed with water, dried and evaporated to dryness in vacuo to give a residue of vinblastine C-3 carboxamide. The amide was purified by chromatography 10 Htstl over silica gel using ethyl acetate-ethanol (1: 1) solvent mixture as eluent. The fractions containing vinblastine C-3 carboxamide were combined and the solvent removed therefrom by evaporation in vacuo to give a residue of vinblastin C-3 carboxamide as a free base. The free base had a characteristic amide band in the infrared spectrum at 1700 cm ”\. The molecular weight of mass spectroscopy was 795, which corresponded to the theoretical value calculated for the formula C ^ HH₂N₂Og. Vinblastine C-3 carboxamide sulfate was prepared by the method of Example 1 and crystallized from o ethanol, m.p. 250 C.

Example 3 4-Desacetyl-vihblastine C-3 carboxyhydrazide

Following the method of Example 1, 4-desacetyl-vinblastine was heated in anhydrous ethanol with excess anhydrous hydrazine o in a sealed reaction vessel at 60 ° C for 18 hours. The reaction vessel was cooled and opened and the contents removed, removing the volatiles in vacuo. The residue containing 4-desacetyl-vinblastine C-3 carboxhydrazide was dissolved in methylene chloride and the methylene chloride solution washed with water, separated and dried and the methylene chloride removed in vacuo.

The resulting residue was dissolved in 1: 1 chloroform: benzene-solvent mixture and chromatographed over silica gel. The benzene chloroform triethylamine eluant in Example 1 was used to induce the chromatogram. The first chromatographic fractions contained unreacted 4-desacetyl-vinblastine.

Other fractions contained 4-desacetyl-18-descarbomethoxy-vinblastine C-3 carboxhydrazide described in Tetrahedron letters, 1968, 783. The next fractions found to contain 4-desacetyl-vinblastine C-3 carboxhydrazide by thin layer chromatography were combined and the solvents were evaporated in vacuo . The residue melted at 210 - 220 C during decomposition. The 4-desacetyl-vinblastine C-3 carboxhydrazide prepared in this way had a carbomethoxy absorption at 1725 - 1737 cm, thereby separating it from the 18-descarbomethoxy compound from the above reference, and it further had a 1690 cm band n 141511 in the IR spectrum due to the hydrazide function. The molecular weight by mass spectroscopy was 768 according to the theoretical value calculated for C The NMR spectrum contained resonance at 3.6 ppm, representing a methyl group at C-18 c arb ome thoxy functional ion.

Following the above method, 4-desacetyl leurocristine (U.S. Patent No. 3,392,173) is reacted with anhydrous hydrazine in anhydrous methanol to give 4-desacetyl-1-deBformyl-leurocristine C-3 carboxhydrazide isolated as an amorphous powder. In the infrared spectrum, there were absorption maxima at 1730 cm (ester), 1670 cm (hydrazide), molecular ion spectrum m / e = 754 (corresponding to C CHH ^ UgO₂), nmr 63.60 (C1Q-methyl), 53.74 (C ^-methyl), (54.05 (C-hydrogen), 56.34 (amide-hydrogen)).

Example 4 4-Pesacetyl-leurocristine C-3 ff-methylcarboxamide

A solution of 900 mg leurocristine and 473 ml of anhydrous methanol was saturated with gaseous hydrogen chloride at ca. 0 C. The reaction vessel was provided with a drying tube and warmed to room temperature. After standing at this temperature for 24 hours, the volatiles were removed by evaporation in vacuo and the remaining residue was dissolved in water. The aqueous solution was basified with 14 N ammonium hydroxide and the alkali-soluble bases present were extracted with methylene chloride. The methylene chloride extracts were combined, dried and evaporated to dryness in vacuo. The resulting residue containing 4-desacetyl-1-desformyl-leurocristine was a light brown amorphous solid. The molecular weight was found to be 754 according to that calculated for c43h54n4? 8 · 1 the infrared spectrum lacked an amide band at 1670 cm cm · · ·, which is characteristic of the 1-formyl group, but there was a strong band at 1730 cm \. is characteristic of ester absorption. Similarly, the NMS spectrum showed peaks at 3.61 ppm and 3.85 ppm characteristic of two carbomethoxy groups; furthermore, there was a peak at 4.11 ppm characteristic of the proton at C-4.

141511 12

A solution was prepared containing 500 mg of 4-desacetyl-1-deformyl-leurocristine prepared as above in 75 ml of anhydrous methanol saturated with methylamine at -78 ° C.

(The amount of methylamine present was about 20 g). The reaction vessel was sealed and heated at 60 ° C for one week. The volatiles were then removed by evaporation in vacuo to give 4-desacetyl-1-desformyl-leurocristine C-3 N-methyl-carboxamide, a light brown amorphous solid. The infrared spectrum of the compound showed a strong amide band at 1668 cm in addition to the ester band at 1730 cm -1.

4-Desacetyl-1-desformyl-leurocristine 0-3 N-methylcarboxamide was formulated with a mixture of 24 ml of 97 $ formic acid and 2 ml of acetic anhydride. The reaction mixture was allowed to stand at room temperature for 24 hours, after which volatiles were removed in vacuo. The residue was dissolved in water and the aqueous solution was basified with 14 N ammonium hydroxide. The nitrogenous bases which were insoluble in the basic solution were extracted with methylene chloride. The methylene chloride extracts were combined, dried and the methylene chloride was then removed by evaporation in vacuo to give 4-desacetyl-leurocristine C-3 lime thylcarboxamide, a light brown solid having the following characteristics:

Molecular weight by mass spectroscopy was 781 consistent with the theoretical value calculated for 4-desacetyl-leurocristine 0-3 N-methylcarboxamide, C

The NMR spectrum had peaks at 3.65 ppm, characteristic of the carbomethoxy group and at 2.79 ppm, characteristic of the lime thylcarboxamide group.

The infrared spectrum contained a strong amide band at 1678 cm, indicating the presence of more than one amide function in the molecule and a characteristic ester band at 1728 cm

Following the method of Example 1, 4-desacetyl-leurocristine C-3 N-methylcarboxamide was converted as a free base to the corresponding sulfate salt with 1 of sulfuric acid. The sulfate was a white amorphous solid powder.

14, 141511

J-D

Example 5 4-Desacetyl-vinblastine C-5 carboxazide

A solution of 678 mg of 4-desacetyl-vinblastine C-3 carboxhydrazide 1 (from Example 3) was prepared in 15 ml of anhydrous methanol. Ca.

50 ml of 1 K aqueous hydrochloric acid was added and the resulting solution cooled to ca. 0 ° C. 140 mg of sodium nitrite was then added and the resulting reaction mixture was stirred for 10 minutes while maintaining the temperature at 0 ° C. The solution turned dark red-brown by the addition of sodium nitrite. The reaction mixture was then made basic by the addition of an excess of 1 shot of a cold 5 # aqueous sodium bicarbonate. The aqueous solution was extracted 3 times with methylene dichloride. 4-Desaeethyl-vinblastine C-3 carboxazide formed by the above reaction passed into the methyl endichloride phase. 3LT

While the methylene dichloride solution is usually used without further purification, a sample was treated to characterize the azide: Evaporation of methylene dichloride gave the azide in an amorphous state. The azide residue was washed with ether and the resulting suspension was filtered. The remaining light powder had the following physical properties * Ultraviolet spectrum lambda ^.,. = 269 ms, (epsilon = 16,700), shoulder at approx. 290 µm, 309 up, (epsilon = 7,100), infrared absorption maximum at 1690 cm (carboxhydrazide) was not present, while the maximum at 1730-1 was not affected. Furthermore, a sharply defined maximum was noted at 2135 cm ”, which characterized the carboxazide function. The mass spectrogram gave a molecular ion m / e = 708, showing a loss of 71 mass units (H, CON · *) from the molecular weight calculated for C. „H_, N_0_ = 779 · 4343 7 7

Example 6 4-Desacetyl-vinblastine C-3 N-Ethyloarboxamifl

A solution of 4-desacetyl-vinblastine C-3 carboxazide was prepared in methylene dichloride solution according to the above method from 900 mg of 4-desacetyl-vinblastine C-3 carboxhydrazide. The methylene dichloride solution was dried and the volume reduced to 20 ml * 14 ms ten

The solution of the azide in methylene dichloride is placed in a flask equipped with a drying tube and a stirrer. 50 ml of anhydrous ethylamine was added and the reaction mixture was stirred at room temperature for approx. 2 hours. Evaporation of volatile constituents in vacuo afforded a light colored amorphous powder which was chromatographed over silica gel * Kroma t o gram t was developed with ethyl acetate aqueous ethanol (3: 1). the interactions containing 4-desacetyl-vinblastine C-3 N-ethylcarboxamide as determined by thin layer chromatography were combined and the solvent removed from the combined fractions in vacuo. 450 mg of a light amorphous powder was obtained with the following physical characteristics: Molecular ion spectrum, m / e = 781 (corresponding to ^ 45 ^ 59 ^ 507) 3420 cm-1 (NH-amide), NMR spectrum: δ in 1.18 (triplet-β-methyl of ethylamide group), methyl ester).

4-Desacetyl-vinblastine C-3 N-ethyl carboxamide sulfate was prepared by dissolving the above-mentioned amorphous powder in anhydrous ethanol and adjusting the pH to 4.0 with 2% sulfuric acid in anhydrous ethanol.

Evaporation of the solvent in vacuo gave a water-soluble light powder consisting of 4-desacetyl-vinblastine C-3 N-ethyl-carboxamide sulfate.

Following the above method, 4-desacetyl-vin-blastin C-3 N-isopropylcarboxamide was prepared. The compound had the following characteristics: molecular ion spectrum, m / e = 795 (corresponding to ^ 46 ^ 51 ^ 5 ^ 7) * infrared spectrum: maxima at 1730 cm (ester), 1660 cm '(amide), NMR spectrum: 41, 16, 11, 22 (doublet-for-isopropylmethyl groups), 4-desacetyl-vinblastine C-3 N-isopropyl-carboxamide sulfate were prepared by the above method and were a water-soluble amorphous powder.

Following the above method, 4-desacetyl-vinblastine C-3 N-dimethylcarboxamide having the following physical properties was prepared: molecular ion spectrum, m / e = 781 (corresponding to C svarH ^N₂O₂, infrared spectrum: absorption maxima at 1730 cm “^) (ester), 1620 cm 2 (amide), NMR spectrum ό 2.96 (singlet-N-methyl), tf 3.4-6 (singlet-N-methyl). The sulfate salt of 4-desacetyl-vinblastine C-3 N, N-dimethyl-U1ST1 carboxamide was prepared by the above method and was a soluble bright amorphous powder.

Following the above method, 4-desacetyl-vinblastine C-3 N- [2- (N, N-dimethylaminoethyl)] carboxamide was prepared from carboxazide and Ν, Ν-dimethylethylamine, and the product had the following physical properties: Infrared spectrum, absorption maxima at 3410 cm ((NH-amide), 1740 cm ”1 (ester), 1670 cm 1 ((amide), NMR Spectrum: δ ), molecular spectrum, m / e = 824 (corresponding to C47H64N607), pKa = 4.85, 7.0, 8.5. The corresponding sulfate salt was prepared and was a bright water-soluble amorphous powder.

4-Desacetyl-vinblastine C-3 N-benzylcarboxamide was prepared using benzylamine by the above method and the product had the following physical properties: Infrared spectrum: maximum at 3420 cm -1 (NH-amide), 1735 cm 1 (ester), 1675 cm 1 (amide), NMR spectrum: <57 * 32 (aromatic protons), <53.69 (methylene group 1 benzylamide), molecular ion spectrum, m / e = 843 »corresponding to G50H61N5 ° 7 + *1ΐΒΤ & Γβηάθ Bulphate salt was prepared by the above method , and was a water-soluble, light colored powder *

Following the above method, 4-desacetyl-vim-blastin 0-3 N-cyanomethyl carboxamide was prepared by reacting 4-desacetyl-vinblastyl 0-3 carboxazide with cyanomethylamine. The new amide had the following physical characteristics: Infrared absorption maxima at 1690 cm 1 ((amide), 3420 cm 1 ((amide NH), NMR spectrum: ¢ 4.17 (C ^-H), ¢ 2.80 (N- CH 3), d 3.77 (aromatic H), (f 3.69 (methyl ester H), ¢ 54.48 (J = 6Hz), 63.92 (J * = 17Hz), both cyanomethylene. Molecular spectrum m / e = 792 (according to 0 ^ H ^ gNg07).

Following the above method, 1-desformyl-4-desacetyl-leurocristine C-3 carboxhydrazide obtained by the method of Example 3 was reacted with sodium nitrite in dilute hydrochloric acid to give 1-desformyl-4-desacetyl-leurocristine 0-3 carboxazide, which was obtained as a yellow amorphous powder. A methylene dichloride solution of 1-desformyl-4-desacetyl-leurocristine C-3 carboxazide was then reacted with ethylamine to give 1-desformyl-4-desacetyl-leurocristine C-3 N-ethylcarboxamide. The compound was purified by chromatography over silica gel using ethyl acetate aqueous ethanol (1: 1) solvent mixture as eluent. The fractions which by thin layer chromatography were found to contain the N-ethylamide were combined and the combined fractions were evaporated to dryness. 1-Desformyl-4-desacetyl-leurocristine C-3 ethyl-ethylcarboxamide obtained as a yellow amorphous powder with the following physical properties: Infrared —1–1 spectrum: absorption maxima at 1665 cm (amide), 1745 cm and 1730 cm (ester band, 1 hydrogen bonded), 3430 cm (BT-H amide), molecular ion spectrum: m / e = 767 (according to C44H57li5 ° 7) *

Example 7 4-Desacetyl-leurosidine C-3 carboxhydrazide

Following the method of Example 3, 1500 mg of leurosidine was reacted with 25 ml of anhydrous hydrazine in anhydrous methanol solution. The reaction mixture was sealed in the reaction vessel and heated to 50 ° C for 12 days. Evaporation of volatile components in vacuo gave 4-desacetyl-leurosidine C-3 carboxhydrazide having the following physical properties: Infrared maxima at 1735 cm (ester), 1660 cm 1 (hydrazide), molecular spectrum, m / e = 768 (according to c H56b6 ° y) 4-Desacetyl-leurosidine C-3 carboxhydrazide converted to the corresponding azide by treatment with sodium nitrite, and the azide o was reacted with methanol saturated with ammonia at -78 ° C to give 4-desacetyl-leurosidine C-3 amide. which was purified by chromatography as above. The compound had the following physical properties: Infrared maxima at 3400 cm (HH-amide), 1740 cm (ester), 1690 cm-1 (amide), molecular ion spectrum, m / e = 753 (in accordance with C 4), 3.57 (C ^-methyl ester), 3 3.78 (C ^, methoxyl), 2.88 (C ^-methyl), <£ 5.78 (C-3 amide hydrogens), The corresponding sulfate salt was prepared by a method comprising neutralizing an ethanol solution of the base with ethanolic sulfuric acid to give a light amorphous powder.

17 14.811

Preparation of salts

Other salts comprising salts with inorganic anions such as chloride, bromide, phosphate, nitrate and the like, and salts with organic anions such as acetate, chloroacetate, trichloroacetate, benzoate, alkyl or arylsulfonates and the like are prepared from amides by a method analogous to that of is listed in Example 1 by replacing the 1 L aqueous sulfuric acid solution with the corresponding acids in a suitable solvent.

As will be appreciated by those skilled in the art, the presence of other esters and / or amide groups in the indole dihydroindole component requires additional precautions in the preparation of salts to avoid hydrolysis, transesterification and other reactions taking place at high temperatures, extremely acidic pHs, etc. . ^

The compounds of this invention have shown antiviral activity in vitro against herpes virus using a culture system in an experiment similar to that described in Siminoff, Applied Microbiology, 9, 66-72 (1961). For example, vinyl blastin C-3 carboxamide gave a 20 mm inhibition zone with a 3 rating and without toxicity a concentration of 125 µg / ml. The same compound was found to be capable of inducing metaphase arrest in "2 cultured Chinese hamster ovary cells at a dose of 2 - 10 µg / ml - 2 x 10 µg / ml. The most effective agent for such stopping was found to be 4-desacetyl-leurocristine C-3 N-methylcarboxamide sulfate, effective at a dose of 10 µg / ml.

In addition, the compounds of this invention have been found to be active against mouse transplanted tumors in vivo. For example, 4-desacetyl-vinblastine C-3 carboxamide sulfate, 4-desacetyl-vinblastine C-3 N-methylcarboxamide sulfate, vinblastine C-3 carboxamide sulfate, vinblastine C-3 N-methylcarboxamide sulfate, 4-desacetyl-vinblastine C-3 Ν, har -dimethylcarboxamide sulfate, 4-desacetyl-leurosidine C-3 carboxamide sulfate, 4-desacetyl-vinblastine C-3 N-benzylcarboxamide sulfate and 4-desacetyl-vinblastine C-3 carboxhydrazide and other compounds within the scope of the present invention have shown such an effect. . Of particular interest, however, is the action of the compounds of the invention in particular 4-desacetyl-cinblastine C-3 carboxamide sulfate and its N-alkyl derivative against Ridgeway osteogenic sarcoma (ROS) and Gardner lymphosarcoma (GLS). When the action of these drugs against tumors is demonstrated, a method comprising administering drugs is usually employed by intraperitoneal route for a given dose 7-10 days after grafting.

The following table, Table I, lists results of several experiments in which mice having transplanted tumors were successively treated with a compound of formula I. In column 1 of the table, the name of the compound is listed; in column 2, the constant tumor; column 3 lists the concentration or range of concentration and number of days this dose was administered, and column 4 indicates the percent inhibition of tumor growth (ROS is an abbreviation for Ridgeway osteogenic Sarcoma, GIS for Gardner lymphosarcoma, and CA 755 is an adeno -caricinoma).

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The compounds of formula I, like leurocristine and vinblastine, are toxic to mice at a dose higher than that at which they provide 100% inhibition of the transplanted tumor.

In addition, for all reasons not fully understood in a given trial, all drugs including the control drugs, such as vinblastine, may exhibit toxicity at a concentration where they usually produce tumor inhibition without toxicity. Therefore, the results are listed in the table. Results from a typical experiment where the control drug yields the expected results and is not an average for all trials.

The compounds of formula I are also active against other transplanted tumors. For example, Mecca lymphosarcoma hair, parenteral administration of 0.25 mg / kg for 9 days of vinblastine 0-5 N-methylcarboxamide sulfate gave a 54% inhibition of growth and using vinblastine C-5 amide a 28% inhibiting growth. At the same concentration, vinblastine itself was completely inactive.

In addition, in studies of CA 755 adenocarcinoma, 4-desacetyl-vinblastine C-5 carboxamide sulfate gave a 67% inhibition of tumor growth, 4-desacetyl-vinblastine C-5 N-methylcarboxamide sulfate, a $ 61 inhibition, and vinblastine C-5 carboxamide sulfate a 49 °. J> inhibition at a concentration of 0.25 mg / kg for 8 days and 72 # inhibition at a dose of 0.5 mg / kg. In a similar experiment, vinblastine gave a 31 # inhibition, while leurocristine at a slightly lower dose of 0.2 mg / kg gave 79 # inhibition with an excellent efficiency factor. Against L 5178 Y lymphocytic leukemia, vinblastine C-3 gave carboxamide sulfate at a concentration of 0.25 mg / kg for 10 days in one experiment. using 5 mice, it provided three safe surviving mice; the life span of the two diseased mice in this experiment was extended by 26 relative to the control mice. At the same trial, vinblastine gave a $ 36 extension, but no safe surviving mice, and provided only a minimal efficacy factor.

As may be expected, the amides and hydrazides in their anti-tumor spectrum from vinblastine leurocristine and leuroside as well as from C-4 Ν, Ν-dialkylglycyl esters of vinblastine vary in the same way> as anti-tumor spectra of these compounds vary within 1 * 1511 , with some being more effective against certain tumors or classes of tumors, and less effective against others.

22

By using amides and hydrazides as antineoplastic agents, one can use either parenteral or oral route when administered.

At the oral dose, a suitable amount of a pharmaceutically acceptable salt or base of formula I, except those compounds in which R is NH-RHg or, formed with a non-toxic acid thoroughly starch or with another diluent, is mixed and the mixture is applied. in a gelatin capsule containing 7.5 ~ 50 mg of active ingredient. Similarly, the anti-neo-plastic salt can be mixed with starch, a binder and a lubricant, and the mixture is pressed into tablets, each containing 7.5 - 50 mg. The tablets can be divided if a lower dose is to be used. By parenteral administration, the intravenous route is preferred. For this purpose, isotonic solutions containing 1 - 10 mg / ml of a salt of an indole dihydroindolamide of formula I except hydrazides and azides are used. The compounds are administered in an amount of 0.1 - 1 mg / kg body weight once a week, depending on both the effect and the toxicity of the drug. Free bases of the compounds of formula I wherein R is NH-NH 4 or N 2 are mixed in suitable dosage forms and similarly administered at similar concentrations.