WO1996031518A1 - Novel tumor-inhibiting bisindole derivatives with calmodulin-liberator effect and pharmaceutical composition containing them - Google Patents

Abstract

The invention relates to novel spiro-2',4'-dioxo-oxazolidino-bisindole derivatives of formula (I), wherein: R1 means a methyl or formyl group; and R2 stands for a C1-4 alkyl, C3-4 alkenyl or halogenated C2-4 alkyl group, as well as acid addition salts thereof. Furthermore, the invention relates to the preparation of the above new compounds as well as pharmaceutical compositions containing the new compounds as active ingredients. The new compounds and compositions have a cytostatic antitumoral effect involving a new mechanism of action. In addition, these compounds show a calmodulin-liberator effect.

Description

NOVEL TUMOUR-INHIBITING BISINDOLE DERIVATIVES WITH CALMODULIN-LIBERATOR EFFECT AND PHARMACEUTICAL COMPOSITION CONTAINING THEM

The invention relates to novel cytostatic bisindole derivatives of formula (I),

wherein

R\ means a methyl or formyl group: and

R-2 stands for a Cι_4 alkyl, C3.4 alkenyl or halogenated C2-4 alkyl group, as well as acid addition salts thereof.

Furthermore, the invention relates to: a process for preparation of the above new compounds and their acid addition salts; calmodulin-liberating, antitumoural pharmaceutical compositions containg the above new compounds and a process for preparing these pharmaceutical compositions; as well as a method of therapeutical treatment by using these compositions.

Compounds structurally closely related to those of the invention are disclosed in the Belgian patent specification No. 861,417 and British patent specification No. 2,072,184. In these patent specifications, an antitumoural action is attributed to the compounds. These compounds bear various substituents on the 0-acetyl or acetyl groups in position C-17. While the molecules protected in the above-said Belgian patent specification contains an OH, 0-acetyl or N-alkyl substituted urethane group in position 4 of the aspidospermidine ring, the novel molecules prepared by us contain hydrogen in position 4.

Both the compounds of formula (I) and their acid addition salts are new and possess a valuable cytostatic effect.

The new compounds according to the invention can be prepared by reacting an alkyl isocyanate of formula R₂-N=C=0, wherein R.2 stands for a Cι_4 alkyl, C3_4 alkenyl group, with 4'-(deacetoxy)vincaleukoblastine, wherein in the formula (II)

Rl stands for methyl group, occurring in an amount or 60-200 mg/kg in Catharantus roseus plant; or from 4'-deacetoxy-foιτnyl-vincaleukoblastine, respectively, wherein Rj in the formula (II) is formyl group, which in turn can be prepared by the known oxidation process of 4'- (deacetoxy)vincaleukoblastine (see Hungarian patent specification No. 173,379).

Thus, according to the invention, a compound of formula (II) is brought into reaction with 4-10 molar equivalents of an isocyanate in an inert aprotic solvent at 20-80°C temperature. Suitable solvents are organic solvents, preferably toluene, dioxane and ethyl acetate.

The crude bisindole compounds obtained in the above reaction were purified by column chromatography on silica gel. Sulfates of the compounds (of 1:1 ratio) were prepared with an 5% solution of sulfuric acid in ethanol in such a way that, after addition of an equivalent amount of ethanolic sulfuric acid, the salt obtained was precipitated by adding diethyl ether. According to the process of invention, the sulfate salts of 1:1 ratio can be prepared in a yield of 18-30% related to the starting 4'- (deacetoxy)vincaleukoblastine.

Due to their valuable cytostatic effect, the compounds of the invention and their pharmaceutically acceptable salts can be used in pharmaceutical compositions, in solid form or in solution.

A particular value of the compounds of formula (I) according to the invention lies therein that they possess a new mechanism of action different from the known mechanism of action of bisindole derivatives known and used as medicaments up to the pressent such as that of vinblastine or vincrisrine.

One of the compounds described in the present invention, which belongs to the scope of formula (I) and bears a methyl group a Ri and a chlorethyl group as R2: is chemically 3"-(β-chlorethyl)-3-sp_ro-2",4"-dioxo-5- oxazolidino-4'-(deacetoxy) vinblastine (hereinafter abbreviated by the code KAR-2), the significant cytostatic effect of which has been proven by several methods.

For pharmacological investigations, the sulfate salt of KAR-2 was dissolved in physiological saline and the solution obtained was administered in intraperitoneal (i.p.) route.

I. Pharmacological investigations under in vivo conditions

These tests were carried out on P388 mouse leukaemia tumour in comparison to the effects of vinblastine (VLB) and vincristine (VCR) known in the therapy.

The method is illustrated in detail hereinafter.

P388 leukaemia was maintained on DBA/2 inbred mice and transplanted i.p. to groups consisting of 6 BDF1 hybrid mice each in these experiments. A dose of 10^ tumour cells/animal each was administered. The daily i.p. treatment with the new compound was started at 24th hour after transplantation. The body weight and condition of the animals were daily registered.

The treatment was continued for 8 days by treating the animals daily once.

The effect achieved on the treated animals was expressed as percentage of the life span related to the average life span (in days) of the control group (T/C%). Based on the possibility of administering high doses as observed during the experiments, the tests were repeated by using single doses, too. The results are summarized in Table 1.

Table 1

From Table 1 the advantageous property of KAR-2 can be seen that it is active even in a single high dose whereas no dose (including the toxic range, too) for the known vincistine could be found to achieve an effect after one single administration on this tumour model.

An other advantage of the compound KAR-2 consists in that no paralysis of bladder or lower extremities indicating neurotoxic effects were observed even during and after treatments with efficient doses for several days, whereas these adverse effects are known for vincristine.

II. Pharmacological investigations under in vitro conditions

Based on the promising in vivo results, the investigations were repeated by measuring the inhibitory effect on the tubulin polymerization commonly accepted. For the sake of comparison, in this series of measurements, the scope of known and effective medicaments was supplemented with vinorelbine as well as two physiologically inactive monomeric indole alkaloids, namely cataranthine and vindoline.

The expertiments were carried out by using solutions of water-soluble salts of the compounds in physiological saline and on the other hand, tubulin isolated from bovine brain as described hereinafter in more detail. 1. Inhibition of the polymerzation of tubulin

The polymerization of tubulin to microtubules was monitored by measuring the turbidity at 350 nm on a spectrophotometer. The concentration of tubulin purified by us from bovine brain was found to be 10 μM (lmg/ml). This polymerization is inhibited to various extent by a number of antimitotic agents, e.g. indole alkaloids. The inhibition by the compounds was characterized by their effects exerted on the initial rate of polymerization. The extent of inhibition was defined as the dose (IC50) dirmmshing the rate of polymerization to its half value. This value was determined by probit analysis based on the dose/response curves.

The inhibitory effect of the compounds tested on the polymerization of tubulin in two different experimental systems is illustrated in Table 2. [The method of messurement was published by A. Lehotzky et al.: J. Biol. Chem. 268. 10888(1993).]

Table 2

Compound IC50 IC 5θ/IC5θ(vinblastine) nM

Vinblastine 38 1

Vincristine 58 1,53

Vinorelbine 20 0,53

Cataranthine >100000 >2600

Vindoline >30000 >790

KAR-2 10 0.26

It has been proven by the investigations that the polymerization of tubulin was inhibited more effectively by KAR-2 than compounds known and therepeutically used. The reproducibility of the method used has been confirmed also by the measurement data obtained on the physiologically inactive compounds.

Based on our experiments carried out under both in vivo and in vitro conditions, it is presumable that the compound investigated acts through a new mechanism of action unknown till now.

2. Inhibition of the formation of a tubulin immunocomplex

Several immunochemical methods are known to detect the binding of certain compounds to tubulin as well as to determine the binding constant

[see: F. Engwall: Methods Enzvmol. 70. 419 (1980); a well as K. Liliom et.al.:

J.Immunol. Methods 143. 119(1991)]. Molecules interacting with tubulin can be identified and a rapid screening of a high number of molecules can be accomplished by this sensitive method demanding only a small amount of substance.

In our experiments tubulin as antigen was coupled with a solid phase. Tubulin bound on the surface of a specific plastic retains its immunochemical properties such as the capability of coupling with an antibody produced against it. Each compound coupled with tubulin can infiience the formation of an immunocomplex. When using a given compound in various concentrations, the IC50 value of the compound and the dissociation constant of the [tubulin-drug] complex can be determined from the extent or perturbing effects.

After immobilization on the carrier, tubulin was incubated with the solution containing the antibody in the presence or absence of a molecule to be tested. Then, the amount of immunocomplex was determined. This could be carried out directly, by labelling the antibodies. A high-grade signal amplification could be achieved by producing antibodies against the primary antibodies and conjugating them with an enzyme affording an absorption signal which is easy to measure.

A horse-radish perozidase-antibody conjugate was used in the present experiment. Under suitably selected and controlled conditions, the extent of the signals is in direct proportion to the quantity of the immunocomplex formed.

The inhibitory effect of the compounds tested on the interaction of tubulin with the antibody as well as the dissociation constants of the complexes of tubulin with the bisindole alkaloids are shown in Table 3.

Table 3.

K_ = dissociation constant

In addition to the compounds with known physiological action, structurally related, psysiologically inactive substances (cataranthine, vindoline) bearing several similar functional groups were also used in the comparative investigations. It has been proven that KAR-2 binds to tubulin with a greater affinity than that of vinblastine. 3. The selectivity of inhibition

The selectivity of inhibition was determined by using a method described hereinafter.

Under effect of certain native substances, e.g. the so-called microtubule- associated proteins or some glycolytic enzymes (e.g. phosphofructokinase), the microrubules are assembled in bundles resulting in an alteration of their stability and dynamics.

The formation of bundles could easily be traced by measuring the turbidity at 350 nm described above or by differentiating centrifugation since the bundles having an increased molecular weight were better settled even a lower revolution number than the one-thread micotubules did. In our experiments, the effect of vinblastine and that of compound KAR-2, respectively on the tubulin polymerization and on the other hand, on bundle fomation induced by kinase were compared.

According to our investigations, a diffenrence was observed between the effect of vinblastine and that of KAR-2 on the bundle formation as it can be seen from Table 4. Vinblastine inhibited both the polymerization of tubulin as well as the bundle formation caused by the enzyme; whereas KAR-2 specifically inhibited only the formation of microtubules. (The bundle formation depends on the concentration of ATP therefore, the effects of the compounds may depend on the metabolic state of the cells.)

Table 4

Sample Compound Turbidity Tubulin in the % precipitate

%

MT - 25 90

MT + PFK 100 94

MT vinblastine <5 12

MT + PFK vinblastine 37 48

MT KAR-2 <5 6

MT + PFK KAR-2 81 92

MT: microtubtule

PFK: phosphofructokinase

Concentrations of the invidual species:

MT = 10 μM; PFK = 2 μM; vinblastine = 2 μM; KAR-2 =2 μM; Temperature: 37°C. It is obvious from Table 4 that KAR-2 selectively inhibits the polymerization of tubulin in a mannar depending on the organization of microrubules.

4. Effect on the cytoskeleton

When tested on Sf9 insect cell line or mammalian CHO cells at 10 μg/ml or in higher range, KAR-2 induced the depolymerization and disappearance of cytoplasmatic microtubules after 2-hour incubation in vitro, which could well be followed by electron microscopy.

Under these conditions tubulin was precipitated in the form of paracrylstals in the cytoplasm of the cells. The depolymerizing effect was partial: in 1 μg/ml concentration, the number of microtubules was diminished but they were not completely eliminated. No depolymerizing effect could be observed in a concentration of 0.1 μg/ml.

According to our results KAR-2 gets in to the cell and its primary target is the cytoskeleton.

5. The anti-calmodulin(side) effect

Antimitotic molecules of various chemical structures exert their effects through various mechanisms of action. Their effects on the cancer cell are non- specific; this fact limits their roles in the chemotherapeutical treatment. At the same time, their target is not exclusively the microtubular system; and this is the source of undesired side effects. Thus, the calmodulin-antagonistic effect of bisindole alkaloids is also known. This is endowed of particular importance by the fact that calmodulin plays a significant role in the regulation of dynamics of the tubulin/microtubule system. Its cellular localization is partially common with the main organizing centre of microtubule (centrosome). Calmodulin- antagonists inhibit the interaction of calmodulin with other proteins through their binding to calmodulin and in this way, a number of organisation and metabolic processes are perturbed under their effect.

The complex test system developed by us can preferably be used for investigating the anti-calmodulin effect of bisindole alkaloids. This test system consists of immunochemical (ELISA), enzyme-kinetical and fluorescence anisotropy measurements. The ELISA method is based on a principle identical and methods similar to those described in point 2. involving summary of experiment, except that here, calmodulin was used instead of tubulin as antigen. The enzymekinetical and anisotropy studies are useful to investigate the effect of compounds on the interaction of calmodulin with an enzyme (in this case phosphofructokinase) specifically bound thereto. In the course of binding of the enzyme to calmodulin, the change in the molecular weight of calmodulin (labelled by a fluorescent dye) can be determined on one hand, by anisotrpy measurement (the anisotropy being in direct proportion to the molecular weight); and on the other hand, the effect induced by calmodulin can be followed by measuring the enzyme activity. The effects induced by calmodulin are abolished by molecules endowed of a calmodulin-antagonistic effect, such as vinblastine or vinorelbine.

The results obtained by using three different methods expounded ebove, are summarized in Table 5. Data relating to the classical anti-calmodulin trifluoperazine (TFP) [R.M. Lewin et al.: Pharmac. Exp.Ther.208, 454 (1979)] are also given for comparison.

It can be seen that KAR-2 is practically inactive under the given concentration conditions, whereas vinblastine, vincristine and especially vinorelbine show a significant anti-calmodulin effect.

Table 5

Compound ELISA PFK activity Fluorescence method method anisotrophy method

(a) (b) (c)

Inhibitory effect Inhibitory effect Inhibitory effect

% % %

TFP 94 69 87

Vinblastine 34 61 39

Vincristine 43 62 n.m.

Vinorelbine 68 83 48

Vindoline < 5 0 n.m.

Cataranthine < 5 0 n.m.

KAR-2 18 7 3 n.m.: not measured Concentration: a) Calmodulin = 2.5 μg/ml; antibody = 7.5 μg/ml; compounds = 250 μM; b) Calmodulin = 3 μM; PFK = 2 μM; compounds = 20 μM; c) Calmodulin = 1.5 μM; PFK = 2 μM; compounds = 20 μM.

It is obvisous from the above results that, in opposition to other bisindole derivatives, KAR-2 does not exert any anti-calmodulin activity. 6. KAR-2 binds to calmodulin

The results shown in point 5. have shown that KAR-2 did not exhibit any anti-calmodulin activity. Thus, it was investigated if the compound could be bound by calmodulin. For deciding this, a direct binding test was developed on the basis of measurements on the transfer of fluorescence energy. By exitation of KAR-2 fluorescent light is emitted. However, if KAR-2 is bound to calmodulin labelled by a suitable fluorescent dye [namely, 5-(dimethyl__mino) naphtalene-1-sulfonyl chloride], the fluorescent energy emitted is expended on induction of the signal present on calmodulin instead of emission (energy transfer). Since energy transfer is established only within the complex of KAR-2 with calmodulin, the amount of complex and dissociation constant can be determined.

It has been stated that KAR-2 binds to calmodulin and the dissociation constans of [KAR-2-calmodulin] complex is 2 μM. In opposition to vinblastine, the formation of this bond is not calcium cation-dependent.

Based on our investigations we have got the surprising recognition that the compound KAR-2 possessed a new mechanism of action which is significatly different from that of similarly structured bisindole alkaloids known up to the present.

7. The "liberator" effect of KAR-2

Since the affinity of KAR-2 to calmodulin is commensurable to the antagonists tested, its effect on the interaction of anti-calmodulin agents with calmodulin was studied. The activity and anisotropy tests expounded above used to this purpose.

The results referring thereto are summarized in Table 6. It can be seen that KAR-2 shows an antagonistic effect both against TFP and vinblastine whereas bisindole derivatives having an anti-calmodulin activity exert a synergistic action together with TFP. This means that KAR-2 "liberates" calmodulin from the effect of antagonists ("liberator effect").

Table 6

Compound PFK activity Fluorescence anisotropy

(a) (b)

Inhibitory effect Inhibitory effect

% %

TFP 69 87

Vinblastine 61 39 vinorelbin 83 48

KAR-2 7 3

TPF + Vinblastine 94 97

TPF + Vinorelbine n.m. 94

TPF + KAR-2 41 77

Vinblastin + KAR-2 41 n.m. n.m.: not measured

Concentrations a) Calmodulin = 3 μM; PFK = 2 μM; compounds = 20 μM; b) Calmodulin = 1.5 μM; PFK = 2 μM; compounds = 20 μM.

Based on the results of measurements, KAR-2 behaves as a "liberator". Namely, it is a liberator of the calmodulin antagonists, i.e. the otherwise calmodulin-antagonistic compounds do not exhibit their antagonistic effect. This recognition could not be expected because the basal skeleton and bisindole stucture of the molecules can be brought into a close relation to the structure of compounds known till now and utilized in the human therapeutics. Due to the surprisingly novel mechanism of action and cytostatic effect of KAR-2 proven by various in vivo and in vitro experiments, the compounds according to the invention can be used as active agents (ingredients) of pharmaceutical compositions enriching the therapy of tumour diseases.

The dose estimated for human therapy can be expected to be daily 0.1-5 mg kg of body weight.

After mixing with commonly used, nontoxic, inert solid or liquid carriers and/or additives (auxiliaries) commonly used in the therapy for oral, parenteral or enteral administration, the active agents of formula (I) are transformed to pharmaceutical compositions.

Useful carriers are e.g. water, gelatine, lactose, starch, pectin, magnesium stearate, stearic acid, talc as well as vegetable oils such as peanut oil or olive oil.

The active agent is formulated to the usual pharmacetical compositions, particularly to solid forms such as rounded or cornnered tablets, dragees or capsules, e.g. gelatine capsules, pills, suppositories, etc. The amount of the active agent can be varied between broad limits; it is preferably between 1 mg and 20 mg. Optionally, the compositions may contain usual pharmaceutical additives, e.g. preserving agent, stabilizers, wetting or emulsifying agents.

These compositions can be prepared by using common methods, e.g. sieving, mixing, granulating and compressing the components in the case of solid compositions. The compositions may be subjected to additional usual operations of pharmaceutical techniques, e.g. sterilization for the preparation of injectable solutions.

The preferred embodiments of preparation of the compounds according to the invention are described in more detail in the following non limiting Examples.

Example 1

3"-(β-Chloroethyl)-3-spiro-2",4"-dioxo-5-ox__zolidino-4'- (deacetoxy)vinblastine

After adding 8 ml (10.1 g, 95.9 mmol) of β-chloroethyl isocyanate to 10 g (13.28 mmol) of 4'-(deacetoxy)vincaleukoblastine base, the solution formed was left to stand for 2 hours, then, after adding 200 ml of dry toluene, the reacton mixture was heated at 80°C for 18 hours, then cooled to 2°C, 40 ml of water and 2 ml of 25% aqueous ammonia solution were added and stirred at room temparature for 30 minutes. The organic phase was separated, washed with 3x300ml of water and dried over anhydrous sodium sulfate. After evaporating the solution under reduced pressure of 20-27 hPa in a water bath of 35-40°C temperature. The dry residue obtained was purified by chromatography on a silicagel column consisting of 500 g of silicagel (of 500 mm in length, 30 mm in diameter) by using a 9:1 mixture of ethyl acetate/ethanol. The separation was controlled by thin layer chromatography (TLC; on Merck 3552 Kieselgel plate by using a 9:1 mixture of ethyl acetate/ethanol as developing system.

Rf of KAR-2: 0.7-0.75

Rf of (deacetoxy)vinblastine: 0.5-0.6

The fractions containing the pure component were combined and evaporated to dryness to obtain 3.29 g (30%) of the pure (aimed) title base.

13 C NMR ( δ TMS = 0.0 ppm, CDCI3), characteristic bands:

6. 9 (CH3), 8.3 (CH3), 28.1 (CH2), 29.0 (CH2), 30.7 (CH), 34.1 (CH2), 35.5 (CH2), 36.0 (CH3), 39.8 (CH2), 39.9 (CH2), 41.3 (CH2), 41.9 (C), 43.7 (CH2), 48.8 (CH2), 51.1 (CH2), 52.3 (CH3), 52.8 (CH2), 55.4 (CH ), 55.8 (C), 55,8 (CH3), 64.4 (CH), 64.8 (CH₂), 69.8 (C), 74.2 (CH), 92.2 (CH), 110.3 (CH), 116.2 (C), 118.2 (CH), 118.5 (CH), 120.0 (C), 121.9 (CH), 122.9 (CH), 123.7 (CH), 123.7 (CH), 125.7 (C), 129.0 (C), 131.8 (C), 132.2 (CH), 134.8 (C), 150.6 (C), 154.0 (C), 157.5 (C), 171.8 (C), Most characteristic IR bands (cm-1) are: 3465, 1814, 1743,1617,1408, 1241, 1039, 741

According to the mass spectrum (MS) taken by the Fast Atom Bombardment (FAB) method, the value of quasimolecular ion M+H is 826.

Example 2

3"-(β-Chloroethyl)-3-spiro-2",4"-dioxo-5-oxazolidino-4'- (deacetoxy)vinblastine sulfate

To a solution of 3 g (3.63 mmol) of pure base (prepared according to Example 1) in 5 ml of ethanol, 7 ml of an ethanolic solution containing 0.363 g (3.363 mol) of sulfuric acid added, the mixtures was left to stand 4 hours then evaporated to 5-6 ml under reduced pressure of 20-27 hPa. Subsequently, the salt was precipitated from the mixture by adding 20 ml of ether, the salt was filtered and dried at a temperature at most 30 °C under reduced pressure of 1-3 hPa for 8-15 hours to obtain 3.05 g (25%) of title sulfate, m.p.: above 300°C (with decomposition).

The optical rotatory power was measured in 1% aqueous solution (c= 1, water).

[α] 25_D = +3.3°; [α] 25_Hg578 = +3.6°; [α]25_{Hg 546} = +4.5°; [α] ²⁵Hg426 = +12.9°; [α]25_{Rg 365} = +36.7°.

Example 3

3 "- Allyl-3-spiro-2 " ,4 "-dioxo-5 -oxazolidino-4'-(deacetoxi)vinblastine

After adding 10 ml (9.4 g, 113.25 mmol) of allyl isocyanate to 10 g (13.28 mmol) of 4^,-(deacetoxy)vincaleukoblastine base, the mixture obtained was left to stand for 2 hours, then 200 ml of dry toluene were addad and heated at 80°C for 18 hours. The reacton mixture was cooled to 20 °C, 40 ml of water and 2 ml of 25% aqueous ammonia solution were added. Thereafter, it was stirred at room temperature for 30 minutes.

After separating, the organic phase was washed with 3x30 ml of water, dried over sodium sulfate and evaporated to dryness under a pressure of 13-27 hPa.

The dry residue obtained was subjected to chromatography on a 80- 100- fold amount of Kieselgel column with ethyl acetate to yield 2.66 g (25%) of pure title base. ¹³ C NMR (δχMS⁼⁰ ° PP^m _> CDCL3) characteristic bands:

6.9 (CH3), 8.4 (CH3), 28.2 (CH ), 29.0 (CH2), 30.7 (CH), 34.1 (CH2), 35.1 (CH2), 35.5 (CH3), 35.7 (CH2), 39.9(C), 41.8 (CH2), 43.8 (CH2), 49.0 (CH₂), 51.2 (CH₂), 52.3 (CH3), 52.8 (CH ), 55.0 (C), 55.5 (CH ), 55,7 (C), 55.8 (CH3), 64.4 (CH), 64.9 (CH₂), 69.9 (C), 74.5 (CH), 91.8 (C), 91.9 (CH), 110.3 (CH), 116.1 (C), 118.3 (CH), 118.6 (CH), 119.5 (CH2), 119,7 (C), 122.0 (CH), 122.9 (CH), 123.9 (CH), 125.7 (C), 129.1 (C), 130.0 (CH), 132.0 (C), 132.3 (CH), 134,8 (C), 150.5 (C), 154.1 (C), 157.6 (C), 171.6 (C), 174.9

(C),

Most characteristic IR bands (cm- 1) are: 3467, 1813, 1743,1617,1402, 1240, 1129, 1036, 741

According to the mass spectrum (MS) taken by the Fast Atom Bombardment (FAB) method, the value of quasimolecular ion M+H is 804.

Example 4

3"-Allyl-3-spiro-2",4"-dioxo-5-oxazolidino-4'-(deacetoxi) vinblastine sulfate

After dissolving 2.6 g (3.23 mmol) of pure base in 5 ml ethanol, 7 ml of an ethanolic solution containing 0.323 g (3.23 mmol) of sulfuric acid were added, the mixture was left to stand for 4 hours, then evaporated to 4-5 ml under reduced pressure of 20-27 hPa in a bath of 40°C temperature. Subsequently, the sulfate salt was precipitated by adding 20 ml of ether, the salt was filtered and dried at a temperature not higher than 30°C at 2 hPa pressure for 8-15 hours to obtain 2.75 g (22%) of the title sulfate, m.p.: over 300°C (with decomposition).

The optical rotatory power was measured in 1% aqueous solution (c=l water).

[α] 25_{D = +}ι.ι°_{; [α]} 25_Hg578 = +1.4°; [ ]25_{Rg 546} = +1.8°; M ²⁵Hg436 = +7.3⁰; [α]25_{Hg 365} = +30.8°.

Example 5

3"-(β-Cr_loroethyl)-3-spi^o-2",4"-dioxo-5-oxazolidino-4^,- (deacetoxy)vincristine

After adding 8 ml (10.1 g, 95.9 mmol) of β-chloroethyl isocyanate to 10 g (11.9mmol) of 4'-(deacetoxy)vincristine base, the mixture was left to stand for 2 hours, then 200 ml of dry toluene were added and the mixture was heated at 80°C for 18 hours. Then, the reaction mixture was cooled to 20°C, 40 ml of water and 2 ml of 25 % aqueous ammonia solution were added and the mixture was stirred at room temperature for 30 minutes. After separation, the organic phase was washed with 3 x 30 ml of water and dried over anhydrous sodium sulfate. After filtration, the solution was evaporated to dryness in a water bath of 40°C under reduced pressure of 13-27 hPa.

The dry residue obtained was subjected to chromatography on a column consisting of 80- 100-fold amount of Kieselgel by using a 10:1 mixture of ethyl acetate/ethanol to yield 3.3 g (29%) of pure title base. 13 c NMR (δτMS⁼0O PP^m _> DMSO) characteristic values:

7.0 (CH3), 8.0 (CH3), 27.8 (CH2), 29.7 (CH), 31.6 (CH2), 34.2 (CH2), 34.4 (CH2), 37.3 (CH₂), 37.5 (CH2), 41.2 (CH2), 41.4 (CH2), 41.5 (CH), 46.9 (CH2), 49.4 (CH₂), 50.6 (CH2), 51.9 (CH3), 52.9 (C), 56.2 (CH2), 55.4 (C), 56.3 (CH3), 62.8 (CH), 63.7 (CH2), 66.4 (C), 67.3 (C), (!:), (C), 95.7 (CH), 111.3 (CH), 115.7 (C), 118.0 (CH), 121.2 (CH), 124.1 (CH), 124.4 (CH), 125.7 (C), 127.2 (C), 128.2 (C), 130.7 (C), 133.0 (CH), 135.6 (C), 140.1 (C), 153.6 (C), 156.7 (C), 160.0 (CH), 171.9 (C), 173.9 (C), Most characteristic IR bands (cnrl) are: 3445, 1817, 1744, 1617,1595, 1411, 1230, 1028, 742

According to MS taken by the FAB method, the value of quasimolecular ion M+H is 840.

Example 6

3"-(β-Chloroethyl)-3-spiro-2",4"-dioxo-5-oxazolidino-4'- (deacetoxy)v_ncristine sulfate

To a solution of 3 g (3.57 mmol) of base (described in Example 5) in a 7 ml of ethanol, 7 ml of an ethanohc solution containing 0.357 g (3.57 mol) of sulfuric acid were added, the mixtures left to stand 4 hours then evaporated to 5-6 ml under reduced pressure of 20-27 hPa in wather bath of 40°C. Subsequently, the sulfate salt was precipitated by adding 200 ml of ether, the salt was filtered and dried at a temperature not higher than 30°C under reduced pressure for 8-15 hours to give 3.05 g (25%) of title product, m.p.: over 300°C (with decomposition).

The optical rotatory power was measured in 1% aqueous solution (c= 1, water).

[ ] 5_D = +0.8°; [α] 25_Hg578 = +1.0°; [α]25_{Hg 546} = +1.2°; ²⁵Hg436 = +5.8°; [α]25_{Hg 365} = +25.9°.

Claims

Claims

1. Spiro-2",4"-dioxo-oxazolidino-bisindole derivatives of formula (I),

wherein

Rj means a methyl or formyl group: and

R2 stands for a C1.4 alkyl, C3.4 alkenyl or halogenated C2-.4 alkyl group, as well as acid addition salts thereof.

2. Process for the preparation of bisindole derivatives of formula (I)

wherein

Rl means a methyl or formyl group: and

R2 stands for a C1.4 alkyl, C3_4 alkenyl or halogenated C2-.4 alkyl group, as well as acid addition salts, which comprises reacting a bisindole coumpond of formula (II),

a

wherein R2 stands for a Cj_4 alkyl, C3..4 alkenyl or halogenated C2.4 alkyl group, in an inert solvent, then isolating the product obtained and, if desired, fransforming it to an acid addition salt by an acid.

3. Antitumoural pharmaceutical composition with calmodulin-liberator effect, which c o m p r i s e s as active ingredient a compound of formula (I), wherein

R\ means a methyl or formyl group: and

R2 stands for a C}_4 alkyl, C3_4 alkenyl or halogenated C2-.4 alkyl group, or a salt thereof in an admixture with inert, non-toxic, solid or liquid carriers and/or auxiliaries commonly used for enteral or parenteral aministration in the therapy.

4. Process for the preparation of an antitumoural pharmaceutical composition, which c o m p r i s e s mixing as active ingredient a bisindole derivative of formula (I), wherein

Rj means a methyl or formyl group: and

R2 stands for a Cj_4 alkyl, C3_4 alkenyl or halogenated C2.4 alkyl group, or salt thereof wirth non-toxic, inert, solid or liquid carriers and/or auxiliaries commonly used for enteral or parenteral adrninistration in the therapy and fransfoπning the mixture into a pharmaceutical composition.

5. Method for treatment of tumoural diseases, characterized by introducing to the body of a patient or mammal to be treated one or more therapeutically active dose(s) of a bisindole derivative of formula (I), wherein

Rl means a methyl or formyl group: and

R2 stands for a Cι_4 alkyl, C3.4 alkenyl or halogenated C2-4 alkyl group, or salt thereof alone or in the form of a pharmaceutical composition containing it as active ingredient.