CH631990A5 - Process for preparing a dimeric indole-dihydroindolecarboxamide - Google Patents

Description

The invention relates to a process for the preparation of dimeric indole-dihydroindolcarboxamides, namely 4-deacetylcarbocamide derivatives of VLB, leurosidine, 60 leurocristine and 1-desmethyl-l-formyl-leurosidine, which are effective antineoplastic active ingredients.

Various naturally occurring alkaloids that can be obtained from Vinca rosea have been shown to be useful for the treatment or control of experimental malignant ulcers in animals. These include leurosine (US Pat. No. 3,370,057), vincaleukobla-stin (vinblastine or VLB, which abbreviation is also used below) (US Pat. No. 3,097,137), leurosidine (vinro-

631990

sidin) and leurocristine (vincristine or VCR, which abbreviation is also used below) (US Pat. No. 3,205,220), deoxy-VLB “A” and “B” and 4-deacetyl-leu-rosin hydrazide (Tetrahedron Letters ( 1958) 783); 4-deacet-oxy-vinblastine (U.S. Patent 3,954,773); 4-deacetoxy-3 '-hydroxy-5 vinblastin (U.S. Patent 3,944,554); Leurocolombin (U.S. Patent 3,890,325) and Vincadiolin (U.S. Patent 3,887,565). Two of these alkaloids, namely VLB and leurocristine, are now available on the market as medicines to fight malignant ulcers, particularly leukemia and related human diseases. Of these commercially available compounds, leurocristine is an extremely effective and useful agent for the treatment of leukemia, but at the same time it is the smallest antineoplastic alkaloid from Vinca rosea. is

The chemical modification of the alkaloids obtained from Vinca did not appear promising. First of all, the molecular structures in question are very complicated and it is difficult to develop chemical reactions that only address a certain group of the molecule. Furthermore, alkaloids which do not have the desired chemotherapeutic properties have been obtained from Vinca rosea fractions, and an examination of their structure has led to the conclusion that these compounds are closely related to the active alkaloids. 25th

The antineoplastic effect thus appears to be limited to very specific structures, so that the prospects of obtaining more active drugs by changing these structures are comparatively small. The successful modifications of the physiologically active 30 alkaloids include the preparation of dihydro-VLB (US Pat. No. 3,352,868) and the replacement of the acetyl group in the C-4 position (at carbon atom No. 4 of the VLB ring system, to which reference should be made to the numbering of the structural formula below) by higher alkanoyl groups or unrelated acyl groups (US Pat. No. 3,392,173). Some of these derivatives are suitable for extending the life of mice suffering from P1534 leukemia. One of the derivatives in which the C-4 acetyl group of VLB is replaced by a chloroacetyl group has proven to be a useful intermediate for the preparation of structurally modified VLB compounds in which the C-4 acetyl group of VLB is replaced by an N, N-dialkylglycyl group is replaced (US Pat. No. 3,387,001). An intermediate compound, namely 4-deacetyl-VLB, was formed in the chemical reactions leading to these latter derivatives. This C-4 hydroxy intermediate was reported by Hargrove (Lloydia, 27 (1964) 340) to be a toxic material that has little chemo-therapeutic activity against the PI 534 murine leukemia system in vivo.

A series of C-3 carboxamide derivatives of the indoldihydroindoles were produced which, as it turned out, have a significant activity against tumors transplanted to mice in vivo (BE-PS 813 168).

A number of compounds are currently available which have an action against one or more neoplastic diseases. They show a very specific behavior in their activity. Because neoplastic diseases often develop resistance to an active ingredient, these new compounds enable additional clinical weapons to combat the wide range of neoplastic diseases. They also enable alternative medicines to treat diseases that have developed resistance.

The invention relates to a process for the preparation of dimeric indole-dihydroindolcarboxamides of the general formula I.

5 'R'

4 '

3 '

11 '8't

/ \ io '\ 19'V /'

:: {f — ii9 'i

R "

iî '

I.

H

J C-O-CHa

I "

I 0

(I)

v \

I 14

CHa-

,: T ~~ U P

l / \, 3 w \ /

ji n. T

VYo ^

17th I.

CHs-CHa

OH

!

'R4 6-N ^

II \ 5

5

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in the

R4 is a group of the general formula

- (CH2) m-HC:

\ 7

where m 1, 2 or 3,

R6 is a group of the formulas -CHO,

-O-jj-Ci-Cn-alkyl, O

-O-C-Cz-Cv-alkenyl, -O-Ci-Cs-alkyl,

1

-NH-jj-Ci-Cs-alkyl,

O

5 or-S-Y,

in which Y represents a hydrogen atom, a C 1 -C 3 -alkyl group or a bond which connects the sulfur atoms of two radicals of the general formula I in which Y represents a bond, and io R7 represents a hydrogen atom; or

R6 and R7 each represent groups of the formula -0-Ci-C3-alkyl;

R5 is a hydrogen atom;

one of the groups R2 and R3 represents a hydrogen atom or a 15 hydroxyl group and the other an ethyl group; and R1 represents a methyl group or a formyl group; as well as their pharmaceutically acceptable salts, which is characterized in that a dimeric indole-dihy-droindole-carboxazide of the general formula II

R "

7 '6y \

»• s.r—?

., / v ° 'Vv /

»• r ~ n.

I.

H

-C-O-CHs II

0

(II)

8th

CHs-O-

i y \

■ 14 1 ° T i r

I • iii i19 ü6

t15 fl — T 1

he II 4I

V / E

CHe-CHs

1 7

w reacts with an amine of the general formula R4R5NH, in which formulas the groups R1 to R7 have the meanings given above, with the proviso that the group Y of the group R6 is different from a bond, and the product of the general formula I in the form of the free amine or a pharmaceutically acceptable salt.

The carboxamides produced according to the invention are limited to the carboxamides of Vinca leukoblastin, leurocristine, leurosidine and 1-desmethyl-l-formyl-leurosidine and their deoxy "A" and "B" analogues and the pharmaceutically acceptable salts of these bases.

Vincaleukoblastin, leurocristin and leurosidine are naturally occurring compounds. 1-Desmethyl-l-formyl-leurosidine and some of the deoxy analogs «A» and r1!

Ò-N3

II

0

"B" were synthesized, although they were not found on-ss in nature. Vincaleukoblastin and Leurocristin are used clinically to treat neoplastic diseases in humans.

The compounds obtained according to the invention can be generically referred to as derivatives of vincaleukoblastin (vinblastine «or VLB) if R1 is a methyl group, R2 is a hydroxyl group and R3 is an ethyl group. In the derivatives of leurocristine (vincristin or VCR) R1 stands for a formyl group, R2 for a hydroxyl group and R3 for an ethyl group. In the derivatives of leurosidine, R1 is a methyl group, R2 is an ethyl group and R3 is a hydroxyl group. In the derivatives of 1-desmethyl-l-formyl-leurosidine, R1 stands for a formyl group, R2 for an ethyl group and R3 for one

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Hydroxyl group. The deoxy analogs of the above compounds in which one of the groups R2 or R3 represents a hydrogen atom and the other represents an ethyl group are called analogues "A" if R2 is a hydrogen atom and analogs "B" if R3 is a Stands for hydrogen atom.

The term "Ci-C3-alkyl" used herein includes methyl, ethyl, n-propyl and isopropyl groups.

The term "Ci-Cn-alkyl-CO" stands for an alkanoyl group derived from an alkanoic acid having 2 to 18 carbon atoms, i.e. e.g. for an acetyl group, a propionyl group, an isobutyryl group, a stearyl group, a palmitoyl group, a laryl group, a myristoyl group, a caproyl group (Ce), an isovaleroyl group, a capryloyl group (Cs), a capryl group (Cio) and the like. The term “C2-C7-alkenyl-CO” means an unsaturated acid group with 3 to 8 carbon atoms, such as an acrylyl group, a crotonyl group, a methacrylyl group, an allylacetyl group, a vinyl acetyl group, a tiglyl group, a 2-methyl-2-hexenoyl group , a 2-octenoyl group and the like.

Examples of groups for the nitrogen-containing radical of the C-3 carboxamido group in the various modified dimeric indole dihydroindole alkaloids of the above general formula are: the acetaldehyde amide group, the 2-methoxypropylamide group, the 2-acetyloxyethyl amide group, the 2-butyryloxyethyl amide group, the 2-ethoxyethyl amide -group, the 2-dimethoxyethylamide group, the 2-acrylyl-oxyethylamide group, the pyrolidinylamide group, the 2-mer-captoethylamide group, the 3-methylmercaptopropylamide group, the N-2-n-propylmercaptopropylamide group, the 4-acetylamino-n-propylamide group and the like.

Non-toxic acids for preparing the pharmaceutically acceptable acid addition salts of the amine bases are, in particular hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, Jodwassersäure, nitrous acid, phosphorous acid and the like, and nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted Alkancar-acids, hydroxyalkanecarboxylic and alkane dicarboxylic acids, aromatic acids, aliphatic and aromatic sulfonic acids etc. These pharmaceutically acceptable salts thus exclude above all the sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides , Iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, the salts of butyne-1,4-dicarboxylic acid and the Hexin-1 , 6-dicarboxylic acid, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, terephthalates, benzenesulfonates, toluenesulfonates, chlorobenzenesulfonates, xylenesulfonates, phenylacetates, phenylpropylates, pheolate, pylate, pylate, pylate, pentylates, pentylates, pentylates, pentylates, pentylates, pentylates, pentylates, pentylates, pentylate , Malates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates and similar salts.

Examples of compounds that can be produced according to the invention are:

4-deacetyl-deoxy-VLB "A" C-3 N-2-mercaptoethyl-carboxamide,

4-deacetyl-deoxy-VLB "B" C-3 N-2-mercaptoethyl-carboxamide,

4-deacetyl-deoxy-VCR "A" C-3 N-2-mercaptoethyl-carboxamide,

4-deacetyl-deoxy-VCR "B" C-3 N-2-mercaptoethyl

carboxamide,

4-deacetyl-deoxy-VLB "A" C-3 N-2-methylmercapto-ethylcarboxamide,

4-deacetyl-deoxy-VLB "B" C-3 N-2-methylmercapto-ethylcarboxamide,

4-deacetyl-deoxy-VCR "A" C-3 N-2-methylmercapto-ethylcarboxamide,

4-deacetyl-deoxy-VCR "B" C-3 N-2-methylmercapto-ethyl carboxamide,

4-deacetyl-deoxy-VLB "A" C-3 N-2-methoxyethyl-carboxamide,

4-deacetyl-deoxy-VLB "B" C-3 N-2-methoxyethyl-carboxamide,

4-deacetyl-deoxy-VCR "A" C-3 N-2-methoxyethyl-carboxamide,

4-deacetyl-deoxy-VCR "B" C-3 N-2-methoxyethyl-carboxamide,

4-deacetyl-VCR C-3 N-2-mercaptoethylcarboxamide, 4-deacetyl-VCR C-3 N-2-methylmercaptoethylcarboxamide,

4-deacetyl-leurosidine C-3 N-2-mercaptoethylcarboxamide, 4-deacetyl-leurosidine C-3 N-2-methylmercaptoethyl carboxamide,

4-deacetyl-l-desmethyl-l-formyl-leurosidine C-3 N-2-mercaptoethyl-carboxamide,

4-deacetyl-l-desmethyl-l-formyl-leurosidine C-3 N-2-methylmercaptoethylcarboxamide,

4-deacetyl-VCR C-3 N-2-methoxyethylcarboxamide, 4-deacetyl-leurosidine C-3 N-2-methoxyethylcarboxamide.

The derivatives obtained according to the invention are those in which the carbomethoxy group in the 3-position (C-3) of certain known indole dihydroindole alkaloids, which can be obtained either from plants or by partial synthesis, is converted into a carboxamide group. Not all of these derivatives are typically made using a single process. For example, the compounds of general formula I can be prepared as follows:

Treatment of VLB, leurocristine, leurosidine, 1-desmethyl-l-formyl-leurosidine and their deoxy analogues with hydrazine gives the corresponding hydrazide. If starting materials with an intact 4-acetyl group are used as the starting point for this reaction, the product generally obtained is a mixture of compounds in which the C-3 carbomethoxy group has been converted into a carboxhydrazide group, but also in which the C-4 acetyl group completely or partially removed. For purification, the C-4 deacetyl derivatives prepared in this way are separated by chromatography. In general, the reaction is preferably carried out by starting from a 4-deacetyl derivative of VLB, leurocristine, leurosidine or 1-desmethyl-1-formyl-leurosidine.

The C-4 deacetyl C-3 carboxhydrazide derivatives are converted into the corresponding azides by treatment with nitrous acid, nitrosyl chloride, nitrogen tetroxide, amyl nitrite or a similar reagent using conventional procedures. The C-3 azides prepared in this way are then reacted with a suitable amine. The azide-amine conversion is accomplished by the Stoll and Huffman method (Helv. Chim. Acta, 26 (1943) 944 and U.S. Patents 2,090,429 and 2,090,430). In the examples of the present invention, the reaction is carried out in methylene dichloride. However, other suitable solvents that do not react with the azide can be used, such as chloroform, acetonitrile, acetone, benzene and toluene. Compounds that have an aldehyde amide group of the formula

6

s

10th

15

20th

25th

30th

35

40

45

50

55

60

65

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Have -NH-CH2-CHO, is preferably obtained by 18 '-descarbomethoxy-VLB C-3 carboxhydrazide, acidic hydrolysis of the corresponding acetalamides of the formula already described by Neuss et al. (Tetrahedron -NH-CH2- (O-C1-C3 alkyl) 2. The compounds, their amide letters (1968) 783). The thin-layer chromatographic group has an ester function, as in the case of the group investigation shows that the next fractions 4-Des - NH- (CH2) n-OAc, in which n 1, 2 or 3 and Ac a Ci-Cn-5 acetyl- VLB C-3 carboxhydrazide contain, so that this alkyl-CO group or a C2-C7-alkenyl-CO group fractions are combined, whereupon the solvents mean, are preferably obtained by esterifying a vacuum. The solid that forms melts

Hydroxyamids, which contains the group -NH- (CH2) n-OH, with decomposition at about 219 to 220 ° C. In this way, a suitable acid anhydride of the formula AC2O, in which Ac prepared 4-deacetyl-VLB C-3 carboxhydrazide has the meanings given above in IR —. In a similar 10 spectrum, a carbomethoxy absorption band at 1725 to way, compounds are obtained in which R6 is a group 1735 cm-1 and differs from that by Alk-X, where X is a group -NH-CO-Ci-Ca-alkyl, Neuss et al. (loc. cit.) described 18'-descarbomethoxy by acylating an aminoalkylamide group of the formula compound, and at 1690 cm- 'in the IR spectrum has an -NH-Alk-NH2 with an acid anhydride. The azide reacts to bands which can be assigned to the hydrazide function in the presence of a base, preferably in the presence of a 15. The molecular spectrophotographically determined molecular pyridine, with a compound of the formula NH2 (CH2) nSH weight is 768, which corresponds to the value theoretically calculated for the molecular formula to form a mixture of the N-2-mercaptoalkylcarbox-C43H56N6O7, amide and the bis (N -2-alkylcarboxamide) disulfide. The NMR spectrum has the strongest resonance at 8 3.6

One can use the hydrazides to produce the corresponding ones by the methyl group of the C-18 carbomethoxy

Use appropriate azides, which in turn are caused for direct manufacturing functions.

other amides can be used. More like that

One can acylate the hydroxyalkylamides and the amino preparation alkylamides (carefully) to give the corresponding carbalkoxy-4-deacetyl-VLB C-3 carboxazide amides or acylamidoalkylamides. The Acetal-Man prepares a solution of 678 mg 4-deacetyl-VLB

amides are naturally hydrolyzed with an acid to form the 25 C-3 carboxhydrazide (from preparation 1) in 15 ml of anhydrous corresponding acetaldehydamides. Methanol. Then about 50 ml of aqueous 1N chlorine water

An alternative and the currently preferred method to seric acid and cools the solution obtained to about 0 ° C. Preparation of a primary amide from the hydrazide comprises then about 140 mg of sodium nitrite are added and the use of a method based on the reaction mixture formed by Ainsworth for 10 minutes under (US Pat. No. 2,756,235) according to which the hydrazide coexists 30 Maintain a temperature of around 0 ° C. When Raney nickel is split by hydrogenation. Adding the sodium nitrite discolors the solution

The derivatives that can be produced according to the invention become kelrot-brown. The reaction mixture is then prepared simply by referring to the excess cold aqueous 5% sodium

Designated carbon atom newly formed group. For example, umbicarbonate solution alkaline. The aqueous is extracted, the compound is obtained by replacing the methyl solution three times with methylene dichloride. It can be seen that the ester group of VLB in the 3-position of the molecule (C-3) through the 4-deacetyl-VLB C-3 formed in the above reaction receives an amide group only as VLB C-3 carboxamide carboxazide passes into the methylene dichloride layer, and not as VLB C-3 descarbomethoxy C-3 carboxamide Although the solution of 4-deacetyl-

designated. vinblastin C-3 carboxazid in methylene dichloride without further

The compounds which can be prepared according to the invention are used in purification, if an aliquot of the form of their free bases and of the white carboxamides is treated or as follows to characterize the azide:

yellow-brown amorphous solids. However, it is preferred

if possible, the carboxamides in the form of their anionic one evaporates the methylene dichloride, the azide being isolated in salts with non-toxic acids and remaining in a crystalline-amorphous state. You wash the azide backing. These salts are high-melting, white, crystalline with ether and filter the suspension obtained. That or amorphous, water-soluble solids. remaining yellow-brown powder has the following

The following examples serve to further explain characteristic physical characteristics:

the preparation of the compounds described.

UV spectrum: Âmax = 269 mji (e = 16 700); Shoulder with preparation about 290 m | i .; 309 mji (e = 7100);

4-deacetyl-VLB C-3 carboxhydrazide IR absorption spectrum: the maximum at 1690 cm-1

In a closed reaction vessel, heating (carboxhydrazide) is not present, while the

4-deacetyl-VLB in anhydrous ethanol with an excess maximum at 1730 cm-1 is not affected. Further shot of anhydrous hydrazine for about 18 hours results in a sharply defined maximum at 2135 cm-1, which is about 60 ° C. The reaction vessel is cooled and opened, which is characteristic of the carboxazide function.

takes the content and evaporates the volatile constituents The mass spectrogram shows a molecular ion m / e of the content in a vacuum. The remaining residue, 708, which contains a loss of 71 units (H, CON3) of the 4-deacetyl-VLB C-3 carboxhydrazide, becomes clearer compared to the molecular weight that is taken up with methylene chloride, the methylene chloride solution formula C43H53N7O7 = washes 779 is calculated.

solution with water, separates the layers, dries the organic 60

phase and pulls the methylene chloride through Example 1

evaporate in vacuo. The residue formed is dissolved in 4-deacetyl-VLB C-3 N-ethylcarboxamide in a chloroform / benzene mixture (1/1) and chromato. The procedure of preparation 2 is followed by graphing on silica gel. To develop the chromato- starting from 900 mg of 4-deacetyl-VLB C-3 carboxhydrazide grams, a benzene / chloroform / triethyl-6s solution of 4-deacetyl-VLB C-3 carboxazide in methyl-

amine solution (100/50 / 7.5). The initial chromatographic dichloride. The methylene dichloride solution is dried and phical fractions contain unreacted 4-deacetyl - the volume is reduced to about 20 ml.

VLB. It turns out that the other fractions are 4-deacetyl. Then the solution of the azide is converted into methylene.

631990

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dichloride into a flask equipped with a drying tube and a stirrer. 50 ml of anhydrous ethylamine are added and the reaction mixture is stirred for about 2 hours at room temperature. Evaporation of the volatile constituents in vacuo gives a yellow-brown colored amorphous powder which is chromatographed on silica gel. The chromatogram is developed with a solvent mixture of ethyl acetate and anhydrous ethanol (3/1). The fractions which show the presence of 4-deacetyl-VLB C-3 N-ethylcarboxamide in the thin-layer chromatographic investigation are combined and the solvent of the combined fractions is evaporated off in vacuo. 450 mg of a yellow-brown colored amorphous powder are obtained with the following characteristic physical characteristics:

Mass spectrum: m / e = 781 (which corresponds to the empirical formula C45H59N5O7);

IR spectrum: absorption maxima at 1730 cm1 (ester), 1670 cm * 1 (amide) and 3420 cnr1 (N-H of the amide);

NMR spectrum: 8 = 1.18 (triplet, β-methyl group of the ethyl amide group), 8 = 3.28 (quartet, a-methylene group of the ethyl amide group), 8 = 3.59 (singlet, methyl ester).

By dissolving the above amorphous powder in anhydrous ethanol and adjusting the pH to about 4.0 with 2% sulfuric acid in anhydrous ethanol, 4-deacetyl-VLB C-3 N-ethyl carboxamide sulfate is prepared. Evaporation of the solvent in vacuo gives a water-soluble yellow-brown powder which contains 4-deacetyl-VLB C-3 N-ethylcarboxamide sulfate.

Example 2

4-deacetyl-VLB C-3 N-β-butyryloxyethyl carboxamide

4-Deacetyl-VLB C-3 N-β-butyryloxyethylcarboxamide, which has the following physical characteristics, is prepared according to the procedure of Example 1:

IR spectrum: bands at 3420 cnr1, 1735 cm- 'and 1680 cm1;

Mass spectrum: molecular ion M + = 867, which is consistent with the empirical formula C49H65N5O9.

Example 3

4-deacetyl-VLB C-3 N- (2-hydroxyethyl) carboxamide

According to the procedure of Example 1, 4-deacetyl-VLB C-3 N- (2-hydroxyethyl) -car-boxamide is prepared by reacting 4-deacetyl-VLB C-3 carboxazid with ethanolamine. The material is in the form of a yellow-brown amorphous solid with the following physical characteristics:

IR spectrum: bands at 3420 cnr1 (NH), 1732 cm-1 (COO) and 1670 cm * 1 (CON).

Mass spectrogram: Molecular ion M + = 797, which corresponds to the empirical formula C45H59N5O8.

The corresponding sulfate salt, which is a water-soluble, yellow-brown colored amorphous powder, is prepared in the manner described above.

Example 4

4-deacetyl-VLB C-3 N- (2-acetoxyethyl) carboxamide Acetylation is used to prepare 4-deacetyl-VLB C-3 N- (2-acetoxyethyl) car-boxamide from the N-hydroxyethyl carboxamide. The product is obtained in the form of a yellow-brown amorphous powder with the following physical characteristics:

IR spectrum: bands at 3420 cmr1 (NH), 1740 cnr1 (COO) and 1670 cnr1 (CON).

Mass spectrogram: Molecular ion M + = 839, which corresponds to the empirical formula GwHsiNsOs.

The NMR spectrum agrees with the structure, in particular due to the additional peak at 8 1.91 (acetylmethyl group).

Example 5

4-deacetyl-VLB C-3 N- (2-aminoethyl) carboxamide

The 4-deacetyl-VLB C-3 N- (2-aminoethyl) -carboxamide, which is formed in the form of a yellow-brown amorphous powder and has the following physical characteristics, is prepared in the above manner:

pka = 6,8,9,0 and 4,6.

IR spectrum: bands at 3420 cm1 (NH), 1730 cm * 1 (COO) and 1670 cnr1 (CON).

Mass spectrogram: Molecular ion M + = 796, which corresponds to the empirical formula C45H60N6O7.

The sulfate salt is prepared, which is in the form of a yellow-brown amorphous powder.

Example 6

4-deacetyl-VLB C-3 N-2-dimethoxyethylcarboxamide

According to the procedure of Example 1, 4-deacetyl-VLB C-3 N-2-dimeth-oxyethylcarboxamide is prepared by reacting 4-deacetyl-VLB C-3 carboxazide with 2-dimethoxyethylamine. The amide prepared in this way has the following physical characteristics:

IR spectrum: absorption maxima 1665 cm1 (amide) and 1730 cm-1 (carboxyl group).

Mass spectrogram (m / e): molecular ion at 841, further peaks at 782,651,500,355 and 154.

NMR spectrum: 8 = 4.42 (triplet), 8 = 3.41 (doublet, Ci-2H) and 8 = 3.36 to 3.45 (6 methyl ether-hydrogen atoms).

By dissolving the free base prepared in the above manner in methanol and by adding a 2% solution of sulfuric acid in methanol, the sulfate salt is prepared, which is evaporated to dryness by evaporation of the resulting solution in the form of a yellow-brown colored, amorphous, water-soluble Receives powder.

Example 7

4-deacetyl-VLB C-3 N- (2-methylmercaptoethyl) carboxamide

1.8 g of 4-deacetyl-VLB C-3 carboxhydrazide are converted into the azide according to the procedure of preparation 2, for which purpose 100 ml of hydrochloric acid and 180 mg of sodium nitrite in anhydrous methanol are used. The solution of the azide in methylene dichloride is reacted according to the procedure of Example 1 with 4 g of methyl mercaptoethyl amine. The reaction mixture is stirred overnight at room temperature. Then the solution is washed once with water, dried over sodium sulfate, filtered and the solvent is evaporated in vacuo. The residue is applied to a column loaded with silicon dioxide and eluted with an ethyl acetate / methanol mixture (3/1) as the eluent. The fractions containing the N- (2-methylmercaptoethyl) carboxamide are combined and the solvent is evaporated off in vacuo. The title compound is obtained in a yield of 540 mg. The carboxamide has the following characteristic physical characteristics:

Elemental analysis: S = 3.47% (calculated 3.87%).

IR spectrum: absorption maxima 1740 and 1675 cnr1.

5

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NMR spectrum: S = 2.12 (-SCHa), 8 = 2.80 (-NCHs), 8 = 3.58 (-COOCH3) and 8 = 3.78 (ArOCHs).

Mass spectrum (m / e): 827.841 (transmethylation), 486 (half of the vindoline molecule), no peak at 813.

The sulfate salt was prepared in the usual way. s

Example 8

4-deacetyl-VLB C-3 N- (3-methylmercaptopropyl) carboxamide

According to the procedure of Example 1, 10 4.0 g of 4-deacetyl-VLB hydrazide are converted into the azide with 200 ml of hydrochloric acid and 400 mg of sodium nitrite. 5 g of methyl mercaptopropylamine are added to the solution of the azide in methylene dichloride and the solution is stirred overnight at room temperature using the method described in Example 1 15

described conditions. The solution is washed once with water, dried over sodium sulfate, filtered and evaporated. The residue is applied to a column coated with silicon dioxide, whereupon it is eluted with a methylene dichloride / ethyl acetate / methanol mixture (1/1/1) 20 as eluent. The appropriate fractions are combined and the solvent is removed in vacuo. The product is obtained with a yield of 1.86 g. The carboxamide has the following characteristic physical characteristics: 25

30th

Elemental analysis: S = 3.71% (calculated 3.80%).

IR spectrum: absorption maxima at 1720 cm-1 and 1660 cm-1.

NMR spectrum: 8 = 2.08 (-SCH3), 8 = 2.79 (1-NCHs), 8 - 3.58 (-COOCH3) and 8 = 3.76 (-ArOCHs).

Mass spectrum (m / e): 841.855 (transmethylation), 500 (half of the vindoline molecule).

The sulfate salt is prepared in the usual way.

35

Example 9

4-deacetyl-VLB C-3 (N-2-mercaptoethyl) carboxamide and bis [4-deacetyl-VLB C-3 (N-2-ethylcarboxamide)] disulfide

2 40 12 g of 4-deacetyl-VLB C-3 carboxhydrazide are converted into the azide by the procedure of preparation. Subsequently, 68.2 g of 2-mercaptoethylamine hydrochloride are dissolved in the minimal amount of water and the acidic solution formed is made alkaline with a concentrated aqueous sodium hydroxide solution. The free 2-mercaptoethylamine base formed in this way, which is insoluble in the alkaline layer, separates out and is extracted with ethyl acetate.

The aqueous layer is further extracted with ether and methylene dichloride. The organic extracts are combined in this way and the solvents are removed by evaporation. The remaining amine is dissolved in the minimal amount of methylene dichloride and added to a solution of the azide formed in the above manner in 500 ml of methylene dichloride. The reaction mixture is heated to 100 ° C. for 5 ss minutes and then cooled. Then 20 ml of pyridine are added and the mixture is stirred overnight at room temperature. An excess of 5% of an aqueous sodium bicarbonate solution is then added and the aqueous and organic layers are separated. The organic layer 60 is washed three times with water and then dried. The solvent is removed by evaporation in vacuo. The residue, which is a mixture of 4-deacetyl-VLB C-3 (N-2-mercaptoethyl) carboxamide and bis [4-deacetyl-VLB C-3 (N-2-ethylcarboxamide)] disulfide, used in the above 65 reaction was formed, separated, chromatographically on silica using a methylene dichloride / ethyl acetate / methanol solvent mixture

(1/1/1) containing 2% triethylamine. Two fractions are obtained, one with an Rf value of 0.5 and a second with an Rf value of 0.25. Both fractions have practically identical physicochemical properties:

Mass spectrum (m / e): 827 (molecular ion + transmethylation), 486.

IR spectrum: bands at 1730 and 1670 cnr1 (in chloroform).

The NMR spectra are practically identical.

The structure of the materials is differentiated by the following criteria:

The faster moving material of the fraction with an Rf value of 0.5 (in 66% aqueous dimethylformamide) has three titratable groups with pK values of 5,3,7,38 and 11,8.

The slower moving fraction with an Rf value of 0.25 has only two titratable groups, which appear at pK values of 5.2 and 7.5. The fraction with an Rf value of 0.5 thus has an additional titratable group, which is the sulfhydryl group of the C-3 amide. The sulfhydryl group is of course not present in the disulfide, which is thus the compound with the Rf value of 0.25. Furthermore, the I3C-NMR analysis indicates that both fractions in the range from 173.6 to 173.8 have peaks which can be assigned to a secondary amide carbon atom (vindesine, a C-3 carboxamide and also a primary amide has a peak at 176.7). Both samples show many identical peaks and only two additional peaks, namely in the fraction with an Rf value of 0.5 at 42.3 and 24.2 and in the fraction with an Rf value of 0.25 at 38.0 and 37.6. An interpretation of this 13C NMR spectra indicates that the former peaks are attributed to the carbon atoms in the substituted side chain (mercaptoethylamide) and the latter to the same state of affairs, with the difference that the sulfur atom is substituted (as in the case of a disulfide). The osmotically determined molecular weight of the fraction with an Rf value of 0.25 is 1770 (calculated 1624), which in turn corresponds to the disulfide structure. The sulfide analysis gives a value of 0.8 for the fraction with an Rf value of 0.5 and a value of 0 for the fraction with an Rf value of 0.25.

Example 10

4-deacetyl-VLB C-3 N-acetaldehyde carboxamide

The 4-deacetyl-VLB C-3 N-2-dimethoxyethylcarboxamide prepared in the procedure of Example 6 is dissolved in aqueous hydrochloric acid. The reaction mixture is then left to stand at room temperature for 4 hours and then made alkaline with 14N aqueous ammonium hydroxide solution. The amide, which is insoluble in the alkaline solution, separates out and is extracted with methylene dichloride. The methylene dichloride layer is separated off, dried and the solvent is removed by evaporation. Chromatography of the remaining powder on silica gel using an ethyl acetate / ethanol solvent mixture (3/1) as eluent gives a purified 4-deacetyl-VLB C-3 N-acetaldehyde-carboxamide with the following characteristic physical characteristics:

Rf value: = 0.43 (the Rf value of the dimethylacetal is 1.50).

IR spectrum: bands at 3420 cm- '(NH), 1735 cm-' (carboxyl group) and 1675 cnr1 (carboxamide group).

NMR spectrum: 8 = 7.78 (triplet, amide hydrogen atom), 8 = 9.67 (aldehyde hydrogen atom)

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10th

The sulfate salt is prepared by dissolving the above amide in absolute ethanol and adjusting the pH of the solution formed to 5.0 with 2% sulfuric acid in absolute ethanol. Evaporation of the solvent to dryness gives the sulfate salt in the form of a yellow-brown amorphous powder.

Example 11

4-deacetyl-VLB C-3 N-2-acetylaminoethylcarboxamide

A solution of 1600 mg of the 4-deacetyl-VLB C-3N-2-aminoethylcarboxamide formed in Example 5 in 30 ml of methylene dichloride is prepared, and the solution is mixed with 5 ml of pyridine. Then 200 mg of acetic anhydride are added, the reaction vessel is closed and the reaction mixture is stirred for 24 hours at room temperature. Then methanol is added in order to react the excess anhydride. The volatile constituents are removed by evaporation and the 4-deacetyl-VLB C-3 N-2-acetylaminocarboxamide remaining as a residue is dissolved in methylene dichloride. The methylene dichloride layer is washed several times with dilute aqueous ammonium hydroxide solution and then with water. The methylene dichloride layer is dried and the methylene dichloride is evaporated. Chromatography of the residue formed on silica gel using an ethyl acetate / methanol solvent mixture (1/1) gives purified 4-desacetyl-VLB C-3 N-2-acetylaminoethylcarboxamide with the following physical characteristics:

Mass spectrogram (m / e): molecular ion = 838, which corresponds to the empirical formula C47H62N6O8.

IR spectrum: bands at 3429 cm-1 (NH), 1735 cm'1 (carboxyl group) and 1670 cnr1 (amide group).

NMR spectrum: peaks at ô = 4.17 and 1.965 (acetyl hydrogen atoms of the β-amino group)

Example 12

4-deacetyl-VLB C-3 N-2-acrylyloxyethylcarboxamide

A solution is prepared which contains 1100 mg of the 4-deacetyl-VLB C-3 N-2-hydroxyethylcarboxamide prepared according to Example 3 in 50 ml of benzene. Then 150 mg of acrylyl chloride are added. The reaction vessel is closed and the reaction is carried out at room temperature for 18 hours. Then open the reaction vessel and add 200 mg of acrylyl chloride. The reaction vessel is closed again and kept at room temperature for a further 10 hours. The reaction vessel is then opened and the reaction mixture is worked up in such a way that the organic solution is brought into contact with dilute ammonium hydroxide solution in order to remove the excess acid chloride. The organic layer is then dried and the solvents are evaporated. Chromatography of the resulting residue, which contains 4-deacetyl-VLB N-2-acrylyloxyethylcarboxamide, with an ethyl acetate / ethanol solvent mixture (3/1) over silica gel gives the purified amide (27 mg) in the form of a yellow-brown amorphous powder with the following physical characteristics:

Mass spectrogram (m / e): molecular ion = 851, which corresponds to the empirical formula C48H51N5O9.

IR spectrum: bands at 3427 cnr1 (NH), 1730 cm * 1 (carboxyl group) and 1675 cnr1 (amide group).

Example 13

4-deacetyl-VLB C-3 N-2-stearoyloxyethyl carboxamide

Using 2 g of 4-deacetyl-VLB C-3 N-2-hydroxyethylcarboxamide, 4-desacetyl-VLB C-3 N-2- is prepared according to the procedure of Example 12, but using stearic anhydride instead of acrylyl chloride. stearoyloxyethyl carboxamide, the molecular ion of which occurs at 1063, which corresponds to the empirical formula s C63H93N5O9, while further peaks can be found in the mass spectrometer at 1004,651,355 and 154.

The sulfate salt is prepared in the usual way using anhydrous ethanol. The sulfate salt 10 (151 mg) obtained is in the form of a yellow-brown amorphous powder which is insoluble in water.

Example 14

Preparation of 4-deacetyl-VLB N-β-methoxyethyl-15 carboxamide

In the manner described in preparation 2, 5.0 g of 4-deacetyl-VLB hydrazide are converted into the azide. Then 10 ml of β-methoxyethylamine are added to the solution of the azide in methylene dichloride and the reaction mixture is stirred for 20 nights at room temperature using the conditions described in Example 1. The solution obtained is washed once with water, dried over sodium sulfate, filtered and evaporated. The residue is applied to a column loaded with silicon dioxide and eluted with a 25% methylene dichloride / methanol / ethyl acetate mixture (1/1/1). The appropriate fractions are combined and the solvent is removed in vacuo. The physical characteristics are determined on 50 mg of the material:

30 IR spectrum: absorption maxima at 3670.3550.3470, 1730 and 1670 cm "1.

NMR spectrum (in CDCb): ô = 2.80.3.34.3.58 and 3.77.

Mass spectrum (m / e): 811.825 (transmethylation), 780.752.571.470.353.154.124 and 122.

35 titration (in 66% dimethylformamide): pKa = 5.35 and 7.38.

The remaining material is converted into the sulfate salt in the usual way. Yield = 1.8 g.

40 Example 15

Manufacture of salts

Other salts, including salts with inorganic anions, such as the chlorides, bromides, phosphates, nitrates and the like, and salts with organic anions, such as the 45 acetates, chloroacetates, trichloroacetates, benzoates, alkylsulfonates, arylsulfonates and the like, are prepared from the Amide bases according to the invention using a procedure analogous to that described in Example 1 for the preparation of the sulfate salt, using a suitable acid in a suitable diluent instead of 50% of the 2% aqueous sulfuric acid used there.

As is readily apparent, the presence of other ester groups and / or amide groups in the 55 indole dihydroindole derivatives of the invention requires special care in the preparation of salts to avoid hydrolysis, transesterification and other reactions that occur at high temperatures and at extremely acidic pH values.

It has been shown that the compounds obtained according to the invention are active in vivo against transplanted mouse tumors. For example, 4-deacetyl-VLB C-3 N-2-butyroxyethyl carboxamide sulfate, 4-deacetyl-VLB C-3 N-2-mercaptoethyl carboxamide sulfate, 4-deacetyl-VLB C-3 show 65 N-2-dimethyloxyethyl carboxamide sulfate, 4-deacetyl -VLB C-3 N-2-acetaldehyde carboxamide sulfate and 4-deacetyl-VLB C-3 N-2-acryloxyethyl carboxamide as well as other compounds according to the invention of the above general

11

631990

Formula this effect. In order to demonstrate the effect of the active pharmaceutical ingredients against these tumors, a test method was used which consists in administering the medicinal product in the usual way by intraperitoneal administration in a given dose during 7 to 10 days after the tumor has been transmitted.

Table I below summarizes the results of various investigations in mice to which tumors have been transmitted which have been successfully treated with the compounds which can be prepared according to the invention. In the following table, the name of the compound is in column 1, the transplanted tumor in column 2, the dose or dose range and the column in column 3

10th

Number of days during which the dose was administered and column 4 shows the percentage inhibition of tumor growth or the percentage prolongation of survival.

The abbreviations given have the following meanings:

ROS = Ridgeway bone sarcoma, GLS = Gardner lymphosarcoma,

B16 = malanoma,

P388 = lymphocyte leukemia and PI 533 = leukemia.

Table I

connection

tumor

Dose (mg / kg x days)

Percentage inhibition

VLB C-3 carboxamide sulfate 4-deacetyl VLBC-3 carboxamide sulfate

4-deacetyl VLB C-3 N-2-butyroxycarboxamide sulfate

4-deacetyl-VLB C-3 N-2-acetoxyethyl carboxamide sulfate

4-deacetyl-VLB C-3 N-2-dimethoxyethyl carboxamide sulfate

4-deacetyl VLB C-3 N-2-acetaldehyde carboxamide sulfate

4-deacetyl-VLB C-3 N-2-acrylyloxyethyl carboxamide sulfate

Bis [4-deacetyl-VLB C-3 (N-2-ethylcarboxamide)] disulfide, sulfate salt

4-deacetyl-VLB C-3 N-2-stearoyloxyethyl carboxamide sulfate 4-deacetyl-VLB C-3 N-2-methoxyethyl carboxamide sulfate

4-deacetyl-VLB C-3 N-2-methylmercaptylethylcarboxamide 4-deacetyl-VLB C-3 N-2-acetylaminoethylcarboxamide sulfate VCR C-3 carboxamide sulfate 4-deacetyl-VCR C-3 carboxamide sulfate

ROS 0.1-0.5 x 10 23-84

GLS 0.05-0.8 x 8 47-100

GLS 0.15-1.0x 10 79-100

ROS 0.15-0.4x 10 49-100

P1534 (J) (S.C.) 0.2-0.25 x 10 36-37

ROS 0.2 x 10 100

GLS 0.1-0.2 x 10 72-100

ROS 0.2 x 10 100

GLS 0.05-0.2 x 10 37-100

GLS 0.1-0.2 x 7 83-100

P388 0.05 x 10 40

P388 0.4 x 10 48

GLS 0.1-0.4x 7-9 84-100

P388 0.4 x 10 48

GLS 0.05-0.4 x 7-10 53-100

GLS 0.2-0.4 x 7-9 100

B16 0.8-1.5 x 3 16- 65

P388 0.6-0.9 x 3 22- 54

GLS 0.2-0.4x 7-9 87-100

B16 0.75 x 10 9 undetermined

surviving animals *

P388 0.9 x 3 42

GLS 0.125-0.5 x 10 92-100 (7th day)

ROS 0.5 x 10 49

GLS 0.2-1.0x 7 36- 75

ROS 1.0 x 10 39

* Indefinite, since 60 days after the end of therapy, surviving 1

Undetermined surviving animals also result from treatment with 4-deacetyl-VLB C-3 N-2-methoxyethyl-carboxamide sulfate.

The compounds obtained according to the invention, like the commercially available active pharmaceutical ingredients leurocristin and VLB, have a toxic effect on the mice when they are administered in doses which are above those at which they cause 100% inhibition of the transplanted tumor. Furthermore, for reasons that are not fully understood, all drugs in a given test, in which control agents are also used, can be toxic at doses at which they normally cause an inhibition of the tumor without toxic side effects. Thus, the results given in Table I above represent the results of typical experiments in which the control agents show the expected results and do not represent an average of all tests.

As is to be expected, the carboxamides which can be prepared according to the invention differ in the spectrum of their antitumor activity from VLB, leurocristine and leurosine and from the C-4 N, N-dialkylglycyl esters of VLB in the same way that the spectra of the antitumor activity differ between these compounds distinguish, some being effective against certain tumors or tumor classes and less effective against others. When the compounds mentioned are used clinically, the doctor will initially use the active substances in the same way and in the same carrier material against the same types of tumors as is the case for leurocristine and VLB. Differences in dosage may of course arise due to the relative activity that leurocristine and the drugs described show in the same experimental tumor in mice. The amides obtained according to the invention show a reduced neurotoxicity compared to leurocristine.

When using the manufactured according to the invention

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12

Carboxamides as antineoplastic agents can be used both parenterally and orally. For oral administration, one mixes e.g. B. a suitable amount of a pharmaceutically acceptable salt of a base of the general formula I above with a non-toxic acid with starch or another carrier material and introduces the mixture into gelatin capsules, each containing 7.5 to 50 mg of the active ingredient. Similarly, the antineoplastic salt can be mixed with starch, a binder and a lubricant and the mixture compressed into tablets, each containing 7.5 to 50 mg of the salt. The tablets can be scored if lower or partial doses are to be used. It is also possible to use isotonic solutions which contain 1 to 10 mg / ml of a salt of an indole dihydroindole carboxamide of the general formula I.

The compounds obtained according to the invention are usually administered in doses of 0.01 to 1 mg and preferably 0.1 to 1 mg / kg of the body weight of the mammal to be treated once or twice a week or every two weeks depending on the activity and toxicity of the Active ingredient administered. Alternatively, if the therapeutic dose is based on the body surface, a dose in the range of 0.1 to 10 mg / m 2 of the mammalian body surface can be administered every 7 or 14 days.

B

Claims (5)

631990 1 R4 h V in the R4 is a group of the general formula R6 - (CH2) m-HC- R7 where m l, 2 or 3, R6 is a group of the formulas -CHO, -O-C-Ci-Cn-alkyl, II O -0-C-C2-C7-alkenyl, O 45 -O-Ci-Cî-alkyl, -NH- | j-Ci-C3-alkyl, so O or-S-Y, ss in which Y represents a hydrogen atom, a C 1 -C 3 -alkyl group or a bond which connects the sulfur atoms of two radicals of the general formula I in which Y represents a bond, and R7 is a hydrogen atom; or 60 R6 and R7 each represent groups of the formula -0-Ci-C3-alkyl; R5 is a hydrogen atom; one of the groups R2 and R3 represents a hydrogen atom or a hydroxyl group and the other an ethyl group; and R1 65 represents a methyl group or a formyl group; as well as their pharmaceutically acceptable salts, characterized in that a dimeric indole-dihydroindolcarbox-azide of the general formula II 631990 5 'R 7 '6y \ t N 4 '' r ~ T 'I • v ^: y ^ CH3 - ~ R " 14 ' (II) II O 8th 9 / \. 10 | ¥ || 7 1ll | 19 HG / \ 13 \ 2 / \ / T15 îl — î T CH3-0— • ^ 6 il \ y> —OH . ! r1! i-N.t, II 0 CH2-CH3 with an amine of the general formula R4RSNH, in which formulas the substituents R1 to R7 have the meanings given above with the proviso that the 35 group Y of the group R6 is different from a bond, and the product of the general formula I in the form of isolated free amine or a pharmaceutically acceptable salt. 1 / \ iB \ / , s fl — Î T "" 06 l y * OH 17, » - CHe-CHa R1 1 O »- N *? "I i» S » 1. Process for the preparation of a dimeric indole dihydroindole carboxamide of the general formula I. 5 'P fiT 4 t \ '9 V / " -i n, t 14 ', l " - -Ft I. H II 0 (I) 8th v \ CH3 — o— 2. The method according to claim 1, characterized in that the reaction is carried out in methylene dichloride. 2nd PATENT CLAIMS 3. The method according to claim 1, characterized in that one carries out the reaction at room temperature. 45 4. The method according to claim 1 for the preparation of dimeric indole-dihydroindolcarboxamides of the general formula I, in which R4 is a group of the general formula R6 like this - (CHa) m-H (X ^ R7 6. The method according to claim 1 for the preparation of 4-deacetyl-VLB C-3 N-2-dimethoxyethyl carboxamide, characterized in that 4-deacetyl-VLB C-3 carboxazide is reacted with 2-dimethoxyethylamine. 7. The method according to claim 1 for the preparation of 4-deacetyl-VLB C-3 N-2-methylmercaptoethylcarboxamide, characterized in that 4-deacetyl-VLB C-3 carboxazide is reacted with 2-methylmercaptoethylamine. 8. The method according to claim 1 for the preparation of 4-deacetyl-VLB C-3 N- (3-methylmercaptopropyl) carboxamide, characterized in that 4-deacetyl-VLB C-3 carboxazide is reacted with 3-methylmercaptopropylamine. 9. The method according to claim 1 for the preparation of 4-deacetyl-VLB C-3 N-2-mercaptoethyl carboxamide, characterized in that 4-deacetyl-VLB C-3 carboxazide is reacted with 2-aminoethyl mercaptan. 10. The method according to claim 1 for the preparation of 4-deacetyl-VLB C-3 N-ß-methoxyethyl-carboxamide, characterized in that 4-deacetyl-VLB C-3 • carboxazide is reacted with β-methoxyethylamine. where m 1, 2 or 3, R6 is a group of the general formula -S-Y, in which Y represents a bond, R7 represents a hydrogen atom and R5 represents a hydrogen atom, characterized in that a dimeric indole-dihydroindole carboxazide of the general formula II is reacted in the presence of a base with an amine of the general formula H2N-Ci-C3-alkyl-S-H. 5. The method according to claim 1 for the preparation of bis [4-deacetyl-VLB C-3 (N-2-ethyl-carboxamide)] - disulfide, characterized in that 4-deacetyl-VLB C-3 carboxazide in the presence of a base reacted with 2-aminoethyl mercaptan.