CH638814A5 - 4-DESACETOXY-4ALPHA-HYDROXYINDOLE-DIHYDROINDOLS AND THEIR PREPARATION. - Google Patents

Description

It is an object of this invention to prepare derivatives 4a of Vinca alkaloids active on the destructive plane of cancer cells, having either a different antitumor spectrum, or lessened or different side effects, or both, compared to the products currently sold, vinblastine and vincristine, and to supply 40 the 4-oxo derivatives necessary for their preparation.

To meet these and other objects, the present invention provides an indoledihydro-indole of the formula

5 '

1 2

1 3

I

./

1 1

10'f3

n ~ î

• .9 '”.

19

R

f-

'* —R "

- R

(V)

; i rrc r

'• x 16 1 a' * 1 '

»>. 16. • 1 to '

\ -c-o-oto

/ 9X

i: F ~ r 5f -; -

Vv / v / rR

T1 o

"J 1

V *

CH30

o /

.CHs

\ R

• OH

C-R

II

0

5

638,814

in which:

R2 is a CH3 or CHO group;

when taken independently, R5 is a hydrogen atom, and one of R3 and R4 is OH or H and the other is C2H5, and when R4 and R5 are taken together, they form an a-epoxide ring and R3 is a C2H5 group;

when taken independently, R6 is a hydrogen atom and R7 is OR1 and, when R6 and R7 are taken together, they form a substituent = O (in the latter case, it is a starting material for obtain the therapeutically active compounds);

R1 is a hydrogen atom or an acetyl group;

R is an OCH3 or NH-NH2 group, provided that, if R is -NH-NH2, R1 is H,

and its pharmaceutically acceptable acid addition salts.

The invention also provides a process for the preparation of an indoledihydro-indole having the formula V described above in which:

R2 is a CH3 or CHO group;

when taken separately, Rs is H, and one of R3 and R4 is OH or H and the other is C2H5, and when R4 and Rs are taken together, they form an a-epoxy ring and R3 is a group C2H5;

R6 is a hydrogen atom;

R7 is an OR1 group where R1 is H;

R is OCH3,

which consists in reacting an indoledihydroindole of the above formula in which R, R1, R2, R3, R4 and R5 are as defined above, and where R6 and R7 taken together form a substituent with a reducing agent in a non anhydrous solvent reactive, and to collect the product in the form of its free base or of a pharmaceutically acceptable salt thereof, and optionally to first react an indoledihydro-indole of the above formula where R, R1, R2, R3, R4 and R5 are as above and R6 is OH and R7 is H with an oxidizing agent in the liquid phase.

The invention also provides a pharmaceutical composition which comprises an inert ingredient and, as active ingredient, an indoledihydroindole of formula V described above in which R7 is an OR1 group.

The compounds of formula V thus include, when R2 is CH3, the 4-epihydroxylated or acetoxylated (or 4a-hydroxylated or acetoxylated) derivatives of vinblastine, leurosidine, leurosine and deoxy VLB A and B, which are all known cancer cell destroying agents, and when R2 is CHO, the corresponding derivatives of vincristine, 1-formylleurosine (leuroformine), 1-fomylleurosidine, 4'-deoxy-l-formylleurosidine, and 4'-deoxyvincristine, both the latter being described in French patent application No. 7801262 published, filed on January 17, 1978, and also the 4-oxo derivatives which are intermediates in their preparation.

Also included in the scope of this invention are the pharmaceutically acceptable acid addition salts of the above alkaloid bases, comprising salts derived from mineral acids such as hydrochloric, nitric, phosphoric, sulphuric, hydrobromic, hydroiodic, nitrous acids, phosphorous, etc., as well as the salts of organic acids including aliphatic mono and dicarboxylic acids, phenylalkanoic acids, hydroxyalkanoic and alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Such pharmaceutically acceptable salts thus include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monoacid phosphate, diacid phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, Oxalate, ma-lonate, succhiate, suberate, sebacate, fumarate, maleate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, benzenesulfonate, benzenesulfonate phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, 2-hydroxy-

butyrate, glycollate, malate, tartrate, methanesulfonate, propane-sulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, etc.

The 4-oxo indoledihydro-indole derivatives of formula are prepared

V by oxidizing a 4p-hydroxylated component in which R6 is OH, R7 is H and R is OCH3, using the Moffat reagent [dimethyl sulfoxide (DMSO), dicyclohexylcarbodiimide (DCC) and orthophosphoric acid] or d other similar oxidizing agents. This reagent was originally described by Pfitzner and Moffat, "J. Am. Chem. Soc. " 85, 3027 (1963) 87 5661, 5670 (1965), see also Fieser and Fieser "Reagents for Organic Synthesis", 1, 304, 2 162 (John Wiley and Sons, Inc., 1967).

In the implementation of the synthesis of 4-oxo derivatives (formula

V in which R6 and R7 taken together form an oxo substituent) with the Moffat reagent, a p-hydroxylated compound of formula V is dissolved in which R6 is OH (in the form of the free base) and the dicyclohexylcarbodiimide in DMSO at the room temperature then phosphoric acid is added. The reaction mixture is stirred until complete dissolution (5 to 10 min). The reaction flask is then capped and left at room temperature until the reaction is substantially complete, 5 to 6 h, as shown by TLC analysis of aliquots treated with standard procedures. The desired product is isolated by diluting the reaction product with dilute sulfuric acid, filtering the resulting mixture and washing the precipitated dicyclohexylurea with 1% aqueous sulfuric acid. The filtrate is made alkaline with 14N aqueous ammonium hydroxide and extracted into an organic solvent immiscible with water, such as methylene dichloride or ether. The organic extracts are combined, dried and the solvent is removed by evaporation. The residue containing the desired 4-oxo derivative is purified by chromatography, gradient elution chromatography, or pH gradient extraction. The latter mode is preferred.

In carrying out the aforementioned oxidation, using the Moffat reagent, it is common to use an excess of reagent. For example, use a 2- to 10-fold excess of dicyclohexylcarbodiimide and a 2-5-fold excess of orthophosphoric acid. Dimethyl sulfoxide is used both as a reactant and as a solvent, and is therefore always present in an excess of 10 to 20 times. It is preferred to use a 6 to 7 fold excess of dicyclohexylcarbodiimide in moles, and a 2.5 to 3 fold excess in moles of orthophosphoric acid per mole of starting material. The reaction can be carried out at temperatures between 20 and 50 ° C, but it is preferred to use a temperature of about 20 ° C. As expected, the higher the temperature, the faster the reaction. However, temperatures above about 25 ° C can give rise to excessive amounts of decomposition products. Furthermore, the reaction does not take place quickly at 20 ° C and times of 5 to 7 hours are necessary for substantial completion of the oxidation procedure. Longer durations will obviously be necessary if lower reaction temperatures are used.

Another oxidizing agent is the combination of N-chloro-succinimide and dimethyl sulfide, used with a tertiary amine such as triethylamine. Other oxidizing agents which can also be used will present themselves to the mind of a person skilled in the art.

The compounds of formula V, in which R is OCH3, R6 and R7 are a substituent = O and R2 is CHO, can be prepared by low-temperature oxidation of the corresponding compound in which R2 is CH3, by the procedure of Jovanovics et al. ., US patent No. 3899493.

The starting substances useful for preparing the 4-oxo derivatives of this invention are prepared, that is to say the substances represented by formula V in which R is OCH3, R6 is OH and R7 is H, by hydrolysis of the acetates in corresponding C-4. The compounds of formula V where R2 is CH3 and R6 is an acetoxy group and R7 is H are known compounds; vinblastine is described in the U.S. patent. No. 3097137, leurosidine in the patent of

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638814

6

USA. No. 3205220, Leurosine in the U.S. Patent No. 3370057 and the deoxy VLB A and B in "Tetrahedron Letters", 783 (1958). The corresponding compounds, in which R2 is CHO, are also known and include vincristine, also described in the U.S. Patent. No. 3205220, leuroformine in Belgian patent No. 811110, 4'-deoxyvincristine and 4'-deoxy-1-formylleurosidine in French patent application No. 7801262 filed on January 17, 1978. The remaining compound is prepared, 1-demethyl-1-formylleurosidine, by low temperature oxidation of leurosidine with chromic oxide, according to the procedure of the US patent No. 3899493. The corresponding 4-deacetylated derivatives are prepared from the above Vinca alkaloids, by hydrolysis under acidic or basic conditions. Har-

grove, "Lloydia", 27, 340 (1964) first prepared 4-deacetyl-vinblastine by acid hydrolysis of vinblastine using large volumes of absolute methanol saturated at 0 ° with anhydrous hydrochloric gas. It uses a contact time of approximately 6 h. s Acid hydrolysis also seems to be the procedure of choice for preparing 4-deacetyldoxy VLB B and A. However, it has been found that an alkaline hydrolysis procedure for the preparation of 4-deacetylated derivatives of leurosine and vinblastine. According to this procedure, sodium carbonate in methanol at reflux is used as the hy-io drolysis medium.

The alkaloid bases of formula V of this invention are prepared, in which R6 is H, R7 is OR'1, R 'is H, R is OCH3 and R2 is CH3 or CHO, by reducing a compound of formula

5 '

7 'e' / \ /

il- T — L <4f

/ V ° X \ /;

12'I il iî9 'T8'

1S1 d, 6 'JKvl,'

R "

v / x: /

m

1 4 '

N I

H

(III)

9 / t "'

CHsO

:. Fi t15 flïS — T BT

-Ke. 11. L 4,1—0

W

JCH3

CHs in which R3, R4 and Rs have the same meaning as above, with a compound of formula LiAlH (t-butyloxy) 3 in dry THF (tetrahydrofuran). An excess of reducing agent is generally used in this reaction. Other non-reactive anhydrous solvents such as diethyl ether can be used in place of THF.

Compounds of formula V, in which R2 is CHO, R is OCH3 and R7 is OR1, are prepared by oxidation at low temperature with a chromic acid-acetic acid mixture of the corresponding compound, in which R2 is a methyl group. The reaction conditions used are generally those of Jovanovics (see the references cited above). The compounds are prepared, in which R is NH — NH2, R2 is CHO or CH3 and R7 is OR1, by reacting the hydrazine hydrate with the corresponding compound in which R is a methoxy group, which has been prepared by the procedures indicated above. The compounds are prepared, in which R2 is a methyl or formyl group, R is a methoxy group and R1 is an acetyl group, by acetylation of the corresponding compound in which R1 is H. The following compounds illustrate the field of this invention: 4-deacetoxy-4a-acetoxyleurosine 4-deacetoxy-4a-acetoxyvinblastin 4-deacetoxy-4a-acetoxyleuroformin

CH

3 *

C — O-CH3

II 0

4'-deoxy-4-deacetoxy-4a-acetoxyleurosidine 4'-deoxy-4-deacetoxy-4a-hydroxyvinblastine 4'-deoxy-4-deacetoxy-4a-hydroxy-1-formylleuròsidine 50 4-deacetoxy-4a-acetoxyvincristine 4- deacetoxy-4a-hydroxyleurosine 4'-deoxy-4-deacetoxy-4a-hydroxyvincristine

55

This invention is further illustrated by the following specific examples.

Example 1

4-deacetoxy-4-oxovinblastine

A reaction mixture is prepared by adding 3.0739 g of 4-60 deacetylvinblastin and 5.8929 g of dicyclohexylcarbodiimide to 20 ml of DMSO. 1.2185 g of orthophosphoric acid (anhydrous) dissolved in 5 ml of DMSO are added. The reaction flask is capped and stirred until dissolution is complete. The reaction mixture is kept at room temperature (18-22 ° C) for 65 h, then it is poured into a mixture of ice and 1% aqueous sulfuric acid. The precipitated dicyclohexylurea is removed by filtration and the filter cake is washed with 1% aqueous sulfuric acid. The aqueous layer is made alkaline with hydroxide

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638,814

of 14 N aqueous ammonium. This aqueous alkaline mixture is extracted several times with methylene dichloride. The methylene dichloride extracts are combined and dried, the solvent is removed by evaporation under vacuum, which leaves a residue containing 4-deacetoxy-4-oxovinblastine (2.6534 g). The residue is dissolved in 1% aqueous sulfuric acid, the resulting solution is filtered and the filtrate is made alkaline with 14 N NaOH as above. Extraction with CHC12 followed by drying and removal of the solvent provides a residue. Thin layer chromatography of an aliquot on silica gel using a 1: 1 solvent mixture of ethyl acetate and ethanol shows that the residue is essentially a single stain substance. The residue is combined with 298 mg of a similar residue from a chromatographic separation in a prior test, and the combined material is dissolved in 400 ml of 0.1 M citric acid buffer (pH 2, 35) and subjected to an extraction procedure with a pH gradient. The acid solution is extracted with 400 ml of benzene and the pH is adjusted to 30. The solution is again extracted with 400 ml of benzene, the pH is raised again, etc. The following table gives the pH of the solution in column 1 and, in column 2, the weight of substance in the fraction after removal of the benzene. Each fraction was analyzed by thin layer chromatography as before to determine the presence of 4-deacetoxy-4-oxovinblastine.

Table 1

layer pH

Weight in mg of aqueous extract product

2.35

205.8

(rejected)

3.0

167.1

3.5

270.6

. 4.0 4.5

671.4] 740.1

(united)

5.0

257.7

5.5

123.0

6.0

66.7

6.5

26.7

7.0

23.8

7.5

12.8

(rejected)

8.0

22.7

9.0

17.8

The fractions taken at pH 3.0-5.0 are combined with a filtrate from a crystallization of 4-deacetoxy-4-oxovinblastine obtained beforehand by chromatography, and the combined fractions are dissolved in 400 ml of buffer. 0.1 M citric acid as before. A second extraction with a pH gradient is carried out as above, but in some cases more than one extraction is carried out with a 400 ml portion of benzene. Table 2 gives the results of this extraction. Column 1 gives the pH of the aqueous layer, column 2 the number of extracts with 400 ml portions of benzene, and column 3 the weight in mg of the product extracted after evaporation of the solvent.

Table 2

pH of the aqueous layer

Number of extractions

Weight in mg of the extracted product

2.35

3

252.4 (rejected)

2.75

2

97.1

3.15

1

114.2

3.55

1

276.1

4.00

3

1154.2

Table 2 (continued)

pH of the aqueous layer

Number of extractions

Weight in mg of the extracted product

4.5

1

213.2

5.0

1

89.7

5.5

1

32.3

6.0

1

18.7

8.0

1

13.5

The fraction is crystallized at pH 4.0, weighing 1.1542 g, in a methanol and water mixture. 854.3 mg of crystalline 4-deacetoxy-4-oxovinblastin are obtained. The NMR and infrared spectra are in agreement with the proposed structure.

Example 2

Other preparation of 4-deacetoxy-4-oxovinblastine

A solution is prepared containing 2.47 g of 4-deacetyl-vinblastine in 37 ml of toluene and 7.4 ml of methylene dichloride. 1.29 g of N-chlorosuccinimide is suspended in 30 ml of anhydrous toluene, and the suspension is stirred under a nitrogen atmosphere while cooling. 1.06 ml (0.9 g) of dimethyl sulfide is added to this suspension dropwise. Then the mixture is stirred for an additional 15 min. The solution of 4-deacetylvinblastine is then added dropwise over 5 min at 0 ° C. to the mixture of N-chlorosuccinimide and dimethyl sulfide in toluene. (The following volumes of solvents are used: 15 times the weight of the starting substance for toluene by weight / volume, 3 times the weight of the starting substance by weight / volume for N-chlorosuccinimide and an excess of sulfide of dimethyl.) After stirring for 5 to 6 h at 0 ° C, 2.02 ml (1.47 g) of triethylamine is added and the reaction mixture is stirred for another half hour at room temperature, then it is keep overnight in the refrigerator. Thin layer chromatography indicates that all of the starting material, 4-deacetylvinblastin, has been consumed, and that the reaction mixture consists essentially of a single stain material. The reaction mixture is diluted with ether and methylene dichloride, the resulting organic layer is washed three times with water and then dried. Removal of the solvents in vacuo provides a yellow solid comprising 4-deacetoxy-4-oxovinblastine, 2.39 g. This substance is chromatographed on 240 g of silica of activity I. The chromatograph is developed with 400 ml portions of a 1: 1 mixture of methylene dichloride and ethyl acetate containing 9.13.5 , 20, 30 and 45% methanol added. After collecting 700 ml of eluent, 20 ml fractions are taken. Fractions 21-45, when they are combined and the solvents are evaporated, give 1.28 g of a yellow-brown solid, 4-deacetoxy-4-oxovinblastine.

4-oxo-4-deacetoxy-leurosidine, 4-oxo-4-deacetoxyvincristine, 4-oxo-4-deacetoxy-1-demethyl-1-formylleurosidine and 4-oxo-4 are prepared in a similar manner. -desacetoxyleurosine, 4-oxo-4-deacetoxy-1 -leuroformin, 4-oxo-4-deacetoxy-4'-deoxy VLB, 4-oxo-4-deacetoxy-4'-deoxyleurosidine, and derivatives 1- corresponding formylates, by oxidizing the corresponding 4-deacetylated derivative with the Moffat reagent or a similar oxidizing agent.

Example 3

Preparation of 4-deacetoxy-4a-hydroxyvinblastine

A solution is prepared from 76.6 mg of 4-deacetoxy-4-oxovinblastine and 0.90 ml of anhydrous tetrahydrofuran (THF). All the equipment used to form the solution and carry out the reaction is dried in an oven. 76.2 mg of LiAlH (t-BuO) 3 are added to the preceding solution. The reaction mixture is stirred at room temperature in a sealed flask under a nitrogen atmosphere for about 22 h. Then a solution of 0.11 g of

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ammonium sulfate and 0.17 ml of water. Infusory earth is then added and the reaction mixture is filtered on the infusory earth. The filter cake is washed with THF. The clear colorless filtrate is concentrated in vacuo and the resulting residue, comprising 4-deacetoxy-4a-hydroxyvinblastin formed in the preceding reaction, is dissolved in methylene dichloride. Elimination of methylene dichloride gives 78.7 mg of a white solid, which a TLC shows is essentially a substance with a stain. The residue is chromatographed on 20 g of Woelm silica gel using a 2: 1 mixture of benzene and chloroform containing 6,9,13,5,20, 30 and 45% methanol for each 30 ml portion of eluent . 10 ml fractions are taken. Fractions 7 to 12 are combined, which give, after evaporation of the solvents, 55.1 mg of a bright yellow solid, of which a TLC shows that it is essentially a substance with a stain. Recrystallization from ethanol gives 28.8 mg of flaky white crystals. The 4-deacetoxy-4a-hydroxy-vinblastine thus prepared has the following physical characteristics:

RPM spectrum at 100 MHz: 5 (COCl3) 8.23 (broad s, 1, NH of indole), 7.43-7.61 (m, 1, H at C „), 7.01-7, 22 (m, 3, H in Ci2-_i4 <), 6.59 (s, 1, H in C14), 6.05 (s, 1, H in CI7), 5.66-5.77 (m, 2, H in C67), 4.02 (d, J = 3, 1, H in C4), 3.88 (s, 3, OCH3 in C15), 3.75 (s, 3, CÓ2CH3 in C3), 3.73 (s broad, 1, H in C2), 3.59 (s, 3, C02CH3 in C18.), 2.85 (s, 3, N — CH3), 0.98 and 0.87 (2t , J = 7, 6, H in C2iav).

Infrared spectrum: v (CHCl3) = 3465, 3005,1724, 1616,1500, 1458,1432 cm-1.

Ultraviolet spectrum: A. (EtOH) = 214 (e4.46 x 104), 288.296 nm.

Mass spectrum: m / e 768 (M +), 737, 709, 651, 543, 154.

Example 4

Preparation of 4-deacetoxy-4a-hydroxyvincristine

A solution is prepared from 76.8 mg of 4-deacetoxy-4a-hydroxyvinblastine and 10 ml of acetone. 43.5 μl of an acid solution prepared from 19.9 ml of water and 2.5 ml of 18 M sulfuric acid are added. A fine precipitate is immediately formed. This acid mixture is cooled to approximately - 60 ° C., and an oxidizing solution containing 90 mg of chromic anhydride in 10 ml of glacial acetic acid and 0.1 ml of water is added dropwise over 4 min. . The reaction mixture is stirred at -60 to -50 ° C for 20 min and then cooled to -70 ° C. 2 ml of 14 N aqueous ammonium hydroxide is added and the resulting mixture is poured into 50 ml of a mixture of ice and water. The aqueous layer is extracted three times with chloroform and the chloroform extracts are combined. The combined extracts are washed with dilute sodium bisulfite and then dried. Evaporation of the solvent gives 61.6 mg of a light yellow solid residue. The residue is chromatographed on 20 g of Woelm silica gel of activity I. The chromatogram is developed with 30 ml portions of a 1: 1 mixture of methylene dichloride and ethyl acetate containing 4, 6, 9, 13.5, 20 and 30% methanol. 10 ml fractions are taken. Fractions 15 to 20 are combined, and 34.8 mg of a white solid consisting of 4-deacetoxy-4a-hydroxyvincristine formed in the preceding reaction are obtained.

The residue is dissolved in anhydrous ethanol and 124 microliters of a 2% v / v aqueous sulfuric acid solution in anhydrous ethanol are added, thereby forming 4-deacetoxy-4a-hydroxyvinblastin sulfate . The free base has the following physical characteristics: mass spectrum: m / e 782 (M +), 751,355, 154.

Example 5

Preparation of C-3 carbohydrazide of 4-deacetoxy-4a-hydroxyvinblastine

A reaction mixture is prepared from 76.8 mg of 4-deacetoxy-4a-hydroxyvinblastine, 6 ml of anhydrous hydrazine (97%) and 9 ml of anhydrous methanol. The reaction vessels were dried in an oven and purged with nitrogen. The reaction mixture is heated at 53 ° C for 24 h and then concentrated in vacuo. The resulting residue is dissolved twice in methylene dichloride and the resulting methylene dichloride extract is concentrated in vacuo, giving a white solid residue which has a main spot by TLC and no spot corresponding to the starting substance . The white solid is chromatographed on 22 g of Woelm activity I silica gel. The chromatogram is developed with 30 ml portions of a 2: 1 mixture of benzene and chloroform containing 4, 6, 9, 13.5, 20 , 30 and 45% methanol. 10 ml fractions are taken. The fractions 15-23 are combined and 54.3 mg of the C-3 carbohydrazide of 4-deacetoxy-4a-hydroxy-vinblastine are obtained, having the following physical characteristics:

Mass spectrum: m / e 768 (M +), 737, 709, 651,154.

Infrared spectrum: v (CHCl3) 3430, 3050,1720, 1655, 1615, 1497,1455,1428,1220 cm- '.

Example 6

Preparation of 4-epi VLB (4-deaeoxy-4a-acetoxy VLB)

A reaction mixture containing 440 mg of 4-deacetoxy-4a-hydroxy VLB, 6 ml of acetic anhydride and 6 ml of Pyridine is prepared and allowed to stand at room temperature for 17 h. The volatile constituents are removed in vacuo at 30 ° C. The residual oil comprising the 4-ear VLB formed in the preceding acetylation is dissolved in methylene dichloride. The organic layer is washed with cold water. The aqueous layer is separated and extracted with methylene chloride. This methylene chloride extract is added to the aqueous layer and the combined layers are dried. Evaporation of the solvent gives a residue of 4-epi VLB which is purified by chromatography on 20 g of silica gel (activity I) using a 1: 1 mixture of methylene chloride and ethyl acetate, containing increasing amounts (9, 13.5, 30 and 45%) of methanol as the elution solvent. The fractions are combined, a TLC showing that they contain the 4-ear VLB, and the solvent is removed from the combined extracts. The resulting residue is a yellow solid comprising 14-ear VLB with the following physical characteristics:

RPM spectrum at 100 MHz: 5 (CDC13) 8.41 (wide s, 1, NH of indole), 7.39-7.58 (m, 1, H in Cn), 7.0-7.3 (m, 3, H in CI2M4 <), 6.58 (s, 1, H in Ci4), 6.03 (s, 1, H in C17), 5.49-5.82 (m, 2, H in C6) 7), 4.56 (s, 1, H in Q), 3.79 (s, 3, OCH3 in C15), 3.75 (s, 3, CÒ2CH3 in C3), 3.60 (s , 3, C02CH3 in C18-), 2.90 (s, 3, N-CH3), 1.25 and 0.90 (2t, J = 7, 6, H in C212r).

Infrared spectrum: v (CHC13) = 3450, 3000.1730, 1225 cm- '.

Ultraviolet spectrum: At (EtOA) = 214 (s4.46 x 104), 288, 296 nm.

Mass spectrum: m / e 810 (M +), 779, 751, 469, 295.154.

The sulfate is prepared in ethanolic sulfuric acid at pH 2.

Leurosidine, deoxy VLB A, deoxy VLB B and leurosine can be subjected to the same series of reactions; that is to say a moderate alkaline hydrolysis to form the 4-deacetylated derivative, the oxidation of the 4-hydroxylated derivative into 4-oxo derivative, the reduction of the 4-oxo derivative by LiAlH (t-BuO) 3 and for obtain the corresponding 4-deacetoxy-4a-hydroxylated derivative. Each of these derivatives can then be oxidized to obtain the corresponding derivative of the vincristine type (compounds in which R2 is CHO); then any of the compounds can be reacted with hydrazine to obtain the corresponding C-3 carbohydrazide.

Any of the 4a-hydroxylated derivatives of this invention can be acetylated, except those in which R is NH-NH2, with acetic anhydride in the presence of a base to obtain the corresponding 4a-acetoxylated derivative.

The compounds of this invention are inhibitors of mitosis in the ovary cells of Chinese hamsters. In particular, 4-deacetoxy-4a-hydroxyvinblastine exhibits mitosis inhibition, roughly corresponding to that which can be seen with vinblastine sulphate or 4-deacetylvinblastine, exhibiting activity at a dose as low as 0 , 01 ng / ml.

The compounds of this invention, represented by formula V in which R7 is OR1, are anti-tumor agents. They show their activity with respect to tumors transplanted in mice.

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For this determination, a protocol is used which comprises the administration of the drug intraperitoneally at a given dose for 7 to 10 d after inoculation with the tumor, or else the first, fifth and ninth days after inoculation .

The following table, Table 3, gives the results of several experiments in which transplanted tumors in mice have been successfully treated with a compound of this invention.

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In Table 3, column I gives the name of the compound, column 2 the transplanted tumor, column 3 the dose or dose interval and the number of days during which this dose was administered, and column 4 the percentage of inhibition of tumor growth or percentage of prolongation of survival time. (GLS is an abbreviation for Gardner's lymphosarcoma; CA 755 is an adenocarcinoma; and B16 is a melanoma.)

drug toxicity. Another method, making it possible to arrive at a therapeutic dose, is based on the surface area of the body, administering a dose of between 0.1 and 10 mg / m2 of body surface area of the mammal every 7 or 14 d.

When a compound of this invention is used clinically, the practitioner will administer the compound initially by the same route and 45 in the same vehicle and presumably against the same types of tumors as that indicated for vincristine and VLB . The doses used will reflect the difference in the doses found in the treatment of experimental tumors in mice, the doses of the compounds of this invention being lower than those used with vincristine and VLB. In clinical trials, as well as with other anti-tumor agents, special attention should be paid to the effect of the compounds of this invention on the ten control tumors described on page 126 of "The Design of Clinical Trials in Cancer Therapy" edited by Staquet (Futura Publishing 55 Company, 1973).

Table 3

Compound

Tumor mg / kg x d

% inhibition or prolongation of survival time

4-deacetoxy-4a-hydroxyvin-blastin

GLS

6.0 x 3.0 x 1.5 x

10 10 10

100 (day 7) 91 (day 7) 35 (day 7)

toxic 78 (day 11) 25 (day 11)

B16

9.0 x 6.0 x 3.0 x

3 3 3

Toxic 90 65

755

3.0 x

10

38

4-deacetoxy-4a-hydr-oxyvincristine sulfate

GLS

6.0 x 3.0 x 1.5 x

10 10 10

100 (day 7) 100 (day 7) 36 (day 7)

toxic 100 (day 11) 29 (day 11)

Carbohydrazide at C-3 of 4-deacetoxy-4a-hydroxyvinblastine

GLS

6.0 x 3.0 x 1.5 x

10 10 10

Toxic 100 (day 7) 97 (day 7)

100 (day 11) 98 (day 11)

B16

9.0 x 6.0 x 3.0 x

3 3 3

Toxic 121 *

67

* Indefinite survivors.

When the new compounds of this invention are used as anti-tumor agents, the parenteral or oral route of administration can be used. For oral administration, a suitable amount of a pharmaceutically acceptable salt of a base according to formula II, formed with a non-toxic acid, such as sulfate, is mixed with starch or other excipient, and the mixture is placed. in interlocking gelatin capsules, each containing 7.5 to 50 mg of active ingredients. Likewise, the anti-neoplastic active salt can be mixed with starch, a binder and a lubricant, and compress the mixture to obtain tablets each containing 7.5 to 50 mg of salt. The tablets can be scored if lower or divided doses are to be used. Parenteral administration is preferred, however. For this purpose, isotonic solutions containing 1 to 10 mg / ml of a salt of an indoledihydroindole of formula II such as sulphate are used. The compounds are administered in an amount of 0.01 to 1 mg / kg, and preferably in an amount of 0.1 to 1 mg / kg of the mammalian body weight, once or twice a week, or every two weeks, depending activity and r

Claims (9)

638,814 2 1. Therapeutically active indoledihydro-indole of formula e ' I rrp-. ,at R (V) I H f — C-O-CHs II 0 CHaO- 8 / e \ F ~ ï 7i Wy! ~ C-R II 0 in which: R2 is CH3 or CHO; when taken independently, R5 is H, and one of R3 and R4 is OH or H and the other is C2H5, and when R + and Rs are taken together, they form an a-epoxy ring and R3 is a group C2HS; R6 is H and R7 is OR1; R1 is H or an acetyl group; R is OCH3 or NH-NH2, provided that when R is NH-NH2, R1 is H, and its pharmaceutically acceptable acid addition salts. 2. 4-deacetoxy-4-a-hydrovinblastine according to claim 1. 3. Process for the preparation of an indoledihydro-indole of formula V according to claim 1, in which the symbols have the meaning given to claim 1, R1 being hydrogen, which consists in reacting an indoledihydro-indole of formula V in which R, R1, R2, R3, R4, Rs are as described above and in which R6 and R7, taken together, form an oxo substituent with a reducing agent in an anhydrous non-reactive solvent, and collect the product in the form of the free base or one of its pharmaceutically acceptable salts. 4. The method of claim 3 for preparing a 4-deacetyl-4a-hydroxyindoledihydro-indole of formula V, wherein the reducing agent is LiAlH (t-butyloxy) 3. 5. Method according to one of claims 3 or 4 to prepare 4-deacetoxy-4a-hydroxyvinblastin by reacting 4-deacetyl-4-oxovinblastin with LiAlH (t-butyloxy) 3 in anhydrous tetrahydro-furan with room temperature under nitrogen atmosphere, stirring for 22 h. 6. 4-Oxo-indòledihydro-indole of formula Y in which R6 and R7 together form an oxo group, the other symbols having the 50 meaning given to claim 1, as a product for implementing the method according to claim 3. 7. 4-deacetoxy-4-oxovinblastine according to claim 6. 8. Pharmaceutical composition which comprises an inert ingredient and, as active ingredient, an indoledihydroindole according to the 55 Claim 1 of formula V wherein R7 is OR1. 40 45 Vinca's alkaloids, a group of dimeric indoledihydro-indoles, have achieved considerable prominence as experimental or commercially available chemotherapy drugs for the treatment of susceptible carcinomas, sarcomas and leukemias. These agents are used alone or in combination with other cancer cell destroying agents. As a category, Vinca alkaloids include the compounds that can be obtained from the leaves of Vinca rosea, the derivatives obtained by their chemical modification and, more recently, the dimer alkaloids obtained by coupling two monomeric indoles by a reaction. of modified Polonovski, see Langlois and Potier, "Tetrahedron Letters", 1099 (1976), Potier et al., "JCS Chem. Comm. ", 670 65 (1975), Kutney et al.," Heterocycles ", 3.205 (1975) and Atta-ur-Rahman," Tetrahedron Letters ", 2351 (1976). A majority of known Vinca alkaloids can be represented by the following formula: 3 638,814 R " 11 / ®v io 'i.3 'G' / \ / ¥ 4 '/ - a 'i1S 3 — R5 R , v. I W w, p Oy rrjpY. 13 * v, IJ6 A 18 * 1 (I) S.XeV / \ Z neither i H 1 4 e> ^ BC — 0 ~ CH3 " 0 9 / B -NOT 7 • CHsO 'l10 l I, • 0.1 £ 1 • | /, X \ / »<5 • f S? ** î »13 L- - “0 02 4 # n; HMbX1 / V / P ^ OH 2 C ~ 0— CH? II 0 CH / , CH3 IN I R1, In the preceding formula, when R1 is an acetoxy group, R2 a methyl group, R3 a hydroxy group, R4 an ethyl group and R5 a hydrogen atom, vinblas-tine is represented; when R1 is an acetoxy group, R2 a formyl group, R3 a hydroxy group, R4 an ethyl group and R5 a hydrogen atom, vincristine is represented; when R1 is an acetoxy group, R2 a methyl group, R3 an ethyl group, R4 a hydroxy group and R5 a hydrogen atom, leurosidine is represented; when R1 is an acetoxy group, R2 is a methyl group, R3 and R5 are hydrogen atoms and R4 is an ethyl group, the deoxy VLB A is shown; when R1, R2 and R5 are the same as in the deoxy VLB A (4'-deoxyvinblastine), but when R3 is an ethyl group and R4 is a hydrogen atom, the deoxy VLB B (4'-deoxyleurosidine) is represented; when R1 is an acetoxy group, R2 a methyl group, R3 an ethyl group and R4 and R5 taken together form an α-epoxide ring, the leurosine is represented; and when R1, R3, R4 and R5 are the same as in leurosine, but that R2 is a formyl group, leuroformine (N-formylleurosine) is represented. The aforementioned alkaloids are described in the following publications: leurosine (vinleurosine, U.S. Patent No. 3,377,057), VLB (vincaleucoblastin, vinblastine, U.S. Patent No. 3097137), leurosidine (vinrosidine) and vincristine (leurocristine or VCR) (both in US Patent No. 3205220) and deoxy VLB A and B, "Tetrahedron Letters", 783 (1958). Other alkaloids, which can be obtained from Vinca rosea, include 4-deacetoxyvinblastin (US Patent No. 3,954,773); 4-deacetoxy-3'-hydroxyvinblastine (U.S. Patent No. 3,944,554); leurocolombine (2'-hydroxy VLB, U.S. Patent No. 3,890,325) and vincadioline (3'-hydroxy VLB, U.S. Patent No. 3,875,565). Two of the foregoing alkaloids, VLB and vincristine, are currently sold as drugs for the treatment of malignant tumors, particularly leukemia and related diseases in humans. Among these two latter compounds, vincristine is the most active agent and the most usable in the treatment of leukemias, but it is also the least abundant of the antineoplastic alkaloids of Vinca rosea. Jovanovics et al., In the U.S. patent. N ° 3899493, have developed an elegant oxidation procedure to transform the most abundant alkaloid, VLB, into vincristine, using chromic acid in acetone and acetic acid at around - 60 ° C The same procedure was used to prepare leuroformin (N-formylleurosine) from leurosine, see Belgian Patent No. 811110. Leuroformine is currently undergoing clinical trials in Europe, mainly in the treatment leukemia and multiple myeloma. The chemical modifications that have been made to VLB and vincristine include hydrolysis of the 4-acetoxy group to obtain 4-deacetyl-VLB (DAVLB) or 4-deacetyl-vincristine (DAVCR), then re-esterification with other acyl or amino-acyl groups, see USA patents No. 3392173 and 3387001, and the replacement of the ester function at C-3 with an amide function, see Belgian patent No. 837390. One of the first 4-acylated derivatives, the ester of 4-N, N -dimethylglycine, has undergone a brief clinical trial, and one of the last, vindesine (C-3 carboxylate of deacetyl VLB), is currently being clinically tested against various neoplasms. 50 Other chemical modifications of the VLB molecule, as the hydrolysis and decarboxylation of the carbomethoxy group in position C-18 ', resulted in a loss of anticancer activity, as well as the formation of N-oxides, i.e. pleurosine ( Leurosin N-oxide). The oxidative attack on the VLB in 55 temperatures above - 60 ° C or in neutral or basic solution resulted in the formation of a chemically inactive compound, vinamidine represented by the following formula: (Formula at the top of the next page) 60 Oxidation of VLB with MnO2, in acetone or in CH2Cl2 at room temperature, also gave vinamidine. The same alkaloid has been found in alkaloidal fractions of Vinca rosea leaves, see Tafur et al. "J. Pharm. Sci. ”, 64.1953 (1975), 65 but it is believed that the structure attributed to it in this reference (formula II page 1956) is incorrect and that the preceding structure more faithfully represents the NMR spectral data, IR and mass spectrum obtained in studies 638,814 CHO C2H5! I 0 „• r — y 4'f , 3'i A, 6'1,7.'8'L ' II 1 4 ' H e / 8 \ • -—y 7 • (ii) i '° i ii CH 0! "'3 f' f '" • CH / .CHs OH CHs C-O-CH3 II 0 physico-chemical of the compound. Vinamidine can come from the oxidative attack on the VLB, in which a neighboring 4 ', 5'-dihydroxylated derivative, glycol is formed, which, by further oxidation, is cut between the hydroxy groups (4', 5 'bond) to give an open cycle derivative as in formula II above. 4-deacetyl VLB and other 4-deacetylated compounds, i.e. those in which R1 in formula I is a hydroxy group, are not oxidized to the corresponding 4-oxo derivatives by oxidation by l chromic acid at low temperature from Jovano-vics. Theoretical considerations show the difficulty of the oxidation of the C-4 hydroxyl group to form a carbonyl group. The C-4 carbon containing nucleus is blocked in its position because four of the cyclic carbons are part of other rings in the vindoline portion of the molecule. To undergo oxidation, when passing from a secondary hydroxy group to a carbonyl group, the bond angles of the C-4 carbon should change from the angle of the tetrahedron (108 °) to the angle sp2 (120 °). Such a change, in a condensed cycle environment in which the C-4 carbon is found, would cause considerable cyclical stresses.