HU198946B - Process for producing new bisindole derivatives with antitumour action and pharmaceutical compositions comprising same - Google Patents

Abstract

The present invention relates to a novel compound of formula (I): wherein R1 is hydrogen or acetyl, R2 is hydrogen, R3 is ethyl, R4 is hydroxy, or R1 is hydrogen or acetyl, R2 and R3 are taken together to form a radical. O-bridge, R 4 is ethyl, and A is in the ω-position to produce C 2-10 -alkyl substituted by hydroxyl or C 2-5 -alkanoyloxy. The new compounds have antitumor activity. 198946 B Scope of the description: 5 pages, Figure 2 -1-

Description

The present invention relates to a process for the preparation of a novel compound of formula (I) wherein

R, hydrogen or acetyl,

R _{2 is} hydrogen,

R _{3 is} ethyl,

R _{4 is a} hydroxyl group, or

R 1 is a hydrogen atom or an acetyl group,

R ₂ and R ₃ together are an -O- bridge,

R4 is ethyl,

A is C2-C10 alkyl substituted at hydroxy or substituted with C2-C5 alkanoyloxy.

Compounds of similar structure to the compounds of the present invention are disclosed in U.S. Pat. Hungarian Patent Application Serial No. 4,679,198. These compounds are bonded to the N 1 atom of the bisindole backbone -CH ₂ -OR - wherein R is an alkyl group.

It has been found that when these compounds are reacted with alcohol under appropriate conditions, a variable OA group may be attached to the -CH ₂ - group attached to the N _r atom of the bisindole backbone.

The process of the present invention comprises the step of providing a compound of formula II wherein R _1; R ₂ , R ₃ , R ₄ and R 5 are as defined above, OX is a leaving group within which O is oxygen - with an alkanol of formula (III) wherein A is as above in the presence of an acid, preferably is reacted in a halogenated hydrocarbon at a temperature below 20 ° C, the reaction mixture is basified, optionally deacetylated with the metal alcoholate to give the compound of formula I, wherein R ₂ , R ₃ , R ₄ , R 5 and A are as defined above. or by acetylating the resulting compound of formula (I) wherein R _b R ₂ , R ₃ , R ₄ and R ₅ are as defined above, and A is a C 2 -C 10 alkyl substituted with hydroxy at the ω position, to give The compound of formula (I) is isolated if desired converted to an acid addition salt.

The compounds of formula (I) obtained by the above process show significant cytostatic activity and are less toxic than the known vinblastine-type bis-indole alkaloids, which are also commercially available. In this way, they can be active ingredients in pharmaceutical compositions for the treatment of cancer.

For the purpose of the activity tests, water-insoluble test substances were prepared by injecting 1 to 1 drop of Tween80 and physiological saline. The injection solutions were administered intraperitoneally.

The effect of the new compounds was i.p. Transplantable tumors (P388 murine leukemia) are described below.

The P388 leukemia was maintained in DBA / 2 inbred mice and in experiments 6-6 BDRi hybrid mice were ^six groups 10 tumorsejtteL'állat ip. transplanted. At 24 hours after transplantation, start with new compounds daily for ip. and animals are monitored daily for weight and condition. The treatment was continued for 5 days and then treated (once daily) for a further 3 days after a 2 day rest period. The effect on the treated animals is expressed as a percentage (T / C%) of the control group's average lifespan expressed in days.

As can be seen from Table 1 below, the derivatives tested significantly prolong the lifespan of P388 leukemic mice at a given dose range.

The advantageous properties of the two compounds highlighted in Table I over vincristine are that they are effective at a single high dose, while no dose of vincristine (up to 1.5 mg / kg up to the toxic region) is effective when administered once to this tumor model.

A further advantage of the compounds is that neither the hind limb and bladder paralysis, known to be vincristine, have been encountered with vincristine for several days after treatment with effective doses.

According to a preferred embodiment of the process according to the invention, the following can be done:

The starting compound (II) is reacted with a large excess of an alcohol (III). Among the compounds of formula (11), it is preferred to use the formula wherein -OX is a -OCH ₃ or -O-CH ₂ H ₅ easily leaving group. Such compounds are described in U.S. Pat. No. 181,745, already mentioned in the introduction. s. Hungarian patent specification. Compounds such as those described in U.S. Patent No. 181,746 may also be used. s. Hungarian Patent Specification No. 5,198,198 also discloses compounds containing, in particular, X is alkenyl, cycloalkyl or phenylalkyl. Of course, the latter are more difficult to produce, more expensive starting materials than the simpler, easy-to-produce -O-alkyl derivatives.

The alcohol of formula (III) is preferably employed in an amount of 30 to 50 molar equivalents relative to the compound of formula (II).

The reaction of the compounds of the formulas II and III is carried out in an organic solvent. Solvents include, for example, ether-type solvents such as diethyl ether, tetrahydrofuran or dioxane, or chlorinated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, benzene, or a benzene homologue (toluene, xylene) or other solvents, such as ethyl acetate, acetone or dimethylformamide, or the alcohol (III) used as the reagent itself. Most preferred of the solvents listed are chlorinated hydrocarbons which are used in anhydrous form.

The reaction is preferably carried out at pH 3-5 in the presence of an acid. Mineral acids such as hydrochloric acid, sulfuric acid, or a Lewis acid such as boron trifluoride diethyl etherate are particularly suitable for pH adjustment.

The reaction temperature is ex £ ® F5 cm of the solvent of choice

Dose mg / kg ip / T / C% w

Ό

Ό

CX co a

v>

i

I &

J8 &

»· Z

rf ^ * 4

I

Ji

Z

Λ a

r4 <a í

z a

s

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HU 198946 Β is selected based on the freezing point and the boiling point, and is usually worked at temperatures between - 60 'C and + 25' C.

After completion of the reaction, the reaction mixture is adjusted to pH 7-9 with, for example, ammonium hydroxide or saturated potassium carbonate.

The product is recovered from the reaction mixture by extraction and / or evaporation and, if desired, purified by chromatography and / or crystallization. Chromatography is performed on partially deactivated alumina or fine silica gel adsorbent.

The process according to the invention may also be carried out by further transforming the product of formula (I) obtained. This may be achieved by deacetylation to form a 17-deacetyl derivative or by acetylation of the hydroxyl group of the acetic anhydride.

The invention is illustrated by the following examples.

Example 1

N-desmethyl-N- (2-hydroxyethyl-methyl) vinblastine

N-desmethyl-N- (methoxymethyl) -vinblastine crude product (1.6 g), abs. To a 0 ° C solution of dichloromethane (70 mL) was added ethylene glycol (2 mL), followed by vigorous stirring. adjust the pH of the reaction mixture to 2-3. The n átacetálozást monitored by TLC (CH ₂ Cl ₂ -MeOH 3.20, _Rf DEFAULT. Material> product). (Dark red oil precipitates from the mixture during acidification.)

At the end of the reaction (20 min / 0 'C), the acid was neutralized with saturated potassium carbonate solution (20 mL) and then at pH 9.5 (concentrated).

NH 4 OH), the aqueous phase was extracted with dichloromethane (2 x 20 mL). The combined organic layers were washed with water (2 x 15 mL) and then dried (MgSO ₄ ) in vacuo. 1.6 g of an oily product are obtained, which is purified by column chromatography.

280 mg of pure N- (2-hydroxyethyloxymethyl) vinblastine are obtained.

Mp 228-230 ° C (amorphous); C ₄₈ H ₆₂ N ₄ On; + 28 ° (c = 1, CHCl ₃ ), + 40 '(c = 1, CHCl 3);

Ir (KBr) 730, 1030, 1100, 1200-1240, 1370, 1460, 1495, 1605, 1730, 2900, 3400 cm ^-1 .

Example 2

N-desmethyl-N- (2-hydroxyethyl-yloxymethyl) -leurozin

N-desmethyl-N- (methoxymethyl) -leurosine crude product (1.6 g), abs. dichloromethane (100 mL) and ethylene glycol (1.4 mL) at 0 ° C with vigorous stirring. acidified to pH 2-3 with ethereal hydrochloric acid. (During the reaction, a red oil precipitates as an emulsion.) The re-acetation is followed by TLC (CH ₂ Cl ₂ -MeOH 20/3, R _f : starting material> product). The reaction time is 10-20 minutes at 0 ° C. At the end of the reaction, the acid was neutralized with saturated potassium carbonate solution (20 mL), and the aqueous portion was extracted with dichloromethane (2 x 15 mL).

The combined organic layer was washed with water (2 x 15 mL) and then dried (MgSO ₄ ) in vacuo. 1.4 g of an oily product are obtained, which is purified by column chromatography.

Adsorbent: silica gel 0.04 to 0.063 mm, TLC: CH ₂ Cl _2: 20; column 0 30 mm, L: 180 mm, CH ₂ Cl ₂ in MeOH: 2; solution and wash: CH ₂ Cl ₂ : 20 + 80 ml; development: 1% MeOHCH ₂ Cl ₂ : 300 mL, elution: 3% MeOH-CH ₂ Cl ₂ : 200 mL; 5% MeOH-CH ₂ Cl ₂ :: 1100 mL; 7% MeOH-CH ₂ Cl ₂ : 200 mL; 10% MeOH-CH ₂ Cl ₂ : 200 mL.

700 mg (37%) of N- (2-hydroxyethyloxymethyl) -leurosine were obtained in the same manner.

Mp 215-217 ° C (amorphous); C ₄ gH ₆₀ N ₄ O _n ; {u] _D = + 55 '(c = 1, CHCl ₃ ), [α] 54 ₆ ρ = + 75' (c = 1, CHCl ₃ ).

Example 3

17-deacetyl-N-demethyl-N- (2-hydroxyethyl-yloxymethyl) -leurozin

A solution of N-desmethyl-N- (2-hydroxyethyloxymethyl) leurosin (150 mg, 0.17 mmol) in 0.5 N sodium methylate (5 mL) was allowed to stand at room temperature for 24 hours (TLC). : CH ₂ Cl ₂ -MeOH 20/2, Rf: starting material> product). The base was neutralized with equivalent (0.15 mL) glacial acetic acid and the solution concentrated in vacuo. The residue was dissolved in dichloromethane (50 mL) and the solution was partitioned between saturated potassium carbonate (15 mL) and concentrated in vacuo after washing with water (2 x 10 mL) and drying (MgSO ₄ ). The crude product obtained is purified by column chromatography. Physical data of 85 mg of the title product obtained by chromatography:

189-191 ° C (amorphous); C _{4 (} j Hsgn ₄ ° ₁₀ ; M d = +68 '(c = 0.8, CHCl ₃ ), µ 1546 = 6 90' (c = 0.8, CHCl ₃ ).

Example 4

N-desmethyl-N- (2-acetoxyethyl-yloxymethyl) -leurozin

N-desmethyl-N- (2-hydroxyethyloxymethyl) -leurosin (150 mg) abs. A solution of pyridine (2.5 mL) was treated with acetic anhydride (0.5 mL) at room temperature, followed by acetylation on TLC (CH ₂ Cl ₂ -MeOH 20/2, R ₍ product> starting material)). hours / 25 ° C), the solvent was distilled off under vacuum at 1.5 mm Hg / 40 ° C, the residue was dissolved in dichloromethane (40 ml) and extracted at pH 9.5 (concentrated NH ₄ OH). The phase was washed with water (2 x 15 mL) and then dried (MgSO ₄ ) in vacuo to remove traces of pyridine by digestion with petroleum ether (2 x 15 mL) to give 100 mg of crude product which was purified by column chromatography.

58 mg (37%) of N-desmethyl-N- (2-acetoxyethyloxymethyl) leurosin were obtained.

168-170 ° C (amorphous); C5 ₀ H ₆₂ N ₄ O ₁₂ ; h _D - +41 '(c = 0.65, CHCl ₃ ),

μ] 54 ₆ = +58 '(C = 0.65, CHCl ₃ ).

HU 19894ο B

Example 5

N-desmethyl-N- (8-hydroxy-octyloxy) methyl -leurozin

N-desmethyl-N- (methoxymethyl) -leurosine crude product (1.6 g), abs. To a solution of dichloromethane (650 mL) was added 1,8-octane diol (2.29 g, 13 eq.) and after complete dissolution, the reaction mixture was cooled to + 10 ° C and treated with absolute abs. adjust the pH to 3 with ethereal hydrochloric acid. The progress of the reaction was monitored by TLC (CH ₂ Cl ₂ -MeOH 20/2, Rj: starting material> product). At the end of the reaction (20 min / +10 ° C), the acid was neutralized with saturated potassium carbonate solution, and the aqueous solution was extracted with dichloromethane (2 x 20 mL). The combined organic layer was washed with water (2 x 20 mL) and then dried (MgSO ₄ ) in vacuo. The oily residue was dissolved in 10 ml of dichloromethane and after cooling, the precipitated 1,8-octane diol was filtered off with suction and washed with dichloromethane (2 x 5 ml) (1.6 g). The combined filtrate was evaporated and purified by column chromatography.

In this way, 370 mg (17%) of N-desmethyl-N- (8-hydroxyoctyloxymethyl) -leurosine were obtained.

140-144 ° C (amorphous); C 54 H 72 N ₄ O 1 _li; Md = + 46 '(c = 1, CHCl ₃ ), [α] 1546 = + 59 ° (c = 1, CHCl ₃ ).

Example 6

Manufacture of pharmaceutical preparations

weighed in:

0.1 g of N-desmethyl-N- (2-hydroxyethyloxymethyl) -vinblastine hydrochloride

0.03 g of zinc sulfate

0.02 g of propyl paraoxybenzoate

0.13 g of methyl paraoxybenzoate

0.025 g acetic acid (98%)

Sodium steelate (0.06 g) in ethanol (96%) made up to 100 ml with distilled water for injection.

Preparation: Dissolve zinc sulfate, sodium acetate in freshly boiled distilled water and add acetic acid. In this way, the solution will have a pH of 4.45. Dissolve propyl or methyl paraoxybenzoate separately in ethanol and combine this solution with the stock solution. Finally, the active ingredient, dissolved in water, is injected into the system, made up to a final volume of 100 ml, homogenized, sterile-filtered and filled into 1 ml glass vials under nitrogen.

Claims (3)

PATENT CLAIMS A process for the preparation of a novel compound of formula (I) wherein: R 1 is hydrogen or acetyl, R _{2 is} hydrogen, R _{3 is} ethyl, R _{4 is} hydroxy, or R 1 is a hydrogen atom or an acetyl group, R ₂ and R ₃ together are an -O- bridge, R _{4 is} ethyl, A is w-position with hydroxy or 2-5 carbon atoms, alkanoyloxymethyl substituted 2-10 alkyl group - preparation, characterized in that a compound (II) of general formula - wherein R, R _2, R ₃ , R ₄ is as defined above, -OX represents a leaving group within which O represents an oxygen atom, by reaction with an alkanol of formula (III) wherein A is as defined above, the reaction mixture is made basic, if desired The compound of formula I - where R ₂ , R ₃ , R 4 and A are as defined above and R 1 is acetyl - is deacetylated with a metal alcoholate or the resulting compound of formula I - wherein R 1, R ₂ , R ₃ , R ₄ is as defined above, A is C 2 -C 10 alkyl acetic anhydride substituted in the w position with hydroxy and the resulting compound of formula (I) is isolated. (Priority: 06/12/1985) Process for the preparation of N-desmethyl-N- (2-hydroxyethyloxymethyl) -vinblastine according to claim 1, characterized in that a compound of formula (II) - wherein Rj is acetyl, R ₂ is hydrogen, R ₃ is ethyl, R4 is hydroxy, --OX is _-O-CH3 - benzothiazine is reacted with glycol. (Priority: 06/12/1985) 3. A process for the preparation of a pharmaceutical composition comprising as an active ingredient a compound of formula (I) wherein R ₁ , R ₂ , R ₃ , R ₄ and A are as defined in claim 1, characterized in that the active ingredient obtained is mixed with the carrier (s) and / or excipient (s) used in the preparation of the pharmaceutical composition and converted into a pharmaceutical composition.