HUT76925A - Bis-indole derivatives, process for the preparation thereof and pharmaceutical compositions 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

The present invention relates to a compound of formula (I)

Bis-indole compounds having a cytostatic effect and their acid addition salts in the formula

R 1 is methyl or boil 1 group.

R 2 is C 1-4 alkyl. C 3 -C 4 alkenyl, C 2 -C 4 alkyl containing halogen.

The present invention also relates to fcalmodulin anti-tumor pharmaceutical compositions containing said novel compounds for the preparation of said novel compounds, to a process for the preparation of said pharmaceutical compositions, and to a pharmaceutical treatment process using said compositions.

A structure close to the chemical formulations of the present invention is described in U.S. Patent No. 861,417. Belgian and 2,027,184;

English patent specification. The compounds are referred to herein as having antitumor activity. These compounds contain various substituents at the C17 position on the O-acetyl and acetyl groups.

While the above-mentioned Belgian patent application protected molecules at the 4-position of the aspidospermidine ring are CH, O-acetyl. or N-a1-ki1-substituted-urethane, the new molecules we produce contain hydrogen at the 4-position.

The general formula (I) and its acid addition salts are novel and have a valuable cytostatic effect.

The novel compounds of the present invention are prepared from 4'-deacetoxyvinca 1eucubicl 1, which is present in the Catharantus roseus (GDon) plant (60-200 mg / kg), wherein R 1 is methyl, or can be prepared by a known oxidation process.

4'-Deacetoxyhydroxy-1-vinca 1eukoblaststin (see U.S. Patent No. 1,737,379, wherein R 1 is a boil-1 group in formula II) can be prepared by reaction with 1-yl isocyanate 1.

Thus, according to the invention, this is the case. hoev compounds of formula II are indifferent. 4 to 10 molar equivalents of isocyanate in aprotic solvents. Reaction temperature is 30-80 ° C. Organic solvent as solvent. preferably toluene. dioxane. ethyl acetate was used.

The nvers bis-indole compounds obtained in the reaction are purified by column chromatography on silica gel.

The sulfuric acid salts of the aqueous compounds (1: 1) are separated with 5% alcoholic sulfuric acid. such that after addition of an equivalent of sulfuric acid, the resulting salts are precipitated with ether. According to our procedure, the salts of sulfuric acid (1: 1) with respect to the starting 4'-desacetoxyvincaleucoblastin

We can produce it with 18-30% production.

The compounds of the present invention or their pharmaceutically acceptable salts can be used as valuable cytostatic agents in edible formulations. solid vaev in solution form.

The present invention has the general formula (I)

A particular value of the novel compounds is that the bisindole-a-1, which is known hitherto and is used as a medicament, is a soluble drug such as vinblastine. show a novel mechanism of action other than known mechanism of action of vincristine.

An eevik compound within the scope of the compounds of formula (I) wherein R 1 is methyl. R 2 is chloroethyl - abbreviated as 3- (β-chloroethyl) -3-spiro-2. 4 "-dioxo-5-desacetoxy-4'oxazo1idino vinb1asztin. which is referred to below as K.AR-2, is demonstrated by several methods to demonstrate its significant cytostatic effect.

For efficacy studies, the sulfate salt of CAR-2 was dissolved in physiological saline and administered intraperitoneally.

I · Carrier studies

The assays were plated on P388 murine leukemia tumors compared to the effects of the well-known vinblastine (VLB) and vincristine (VCR).

The method is described below:

P388 leukemia was maintained in DBA / 2 in 11 mouse mice and in groups of 6-6 B1) F1 hvbrid mice in the experiments. transzp1antά1juk. Dose: 10 ⁶ tumor cells / µl. Begin at 24 hours after transplantation, and administer daily with the new compound. and the body weight and condition of the animals are monitored daily.

The treatment was continued for 8 days, once daily.

The effect on the treated animals is expressed as a percentage (= T / C%) of the mean control life of the control group in days.

Given the high dose levels observed in the studies, the studies were repeated, even at single doses.

The results are as follows Table (1)> an f ο e 1 a 1 i u k together. 1. spreadsheet Daily dose (me / kg) T / C% (p. administration) 8 days VLB VCR INJURY-2 8 x 0.1 165 1 65-220 - 8x0.2 177-221 183-234 - 8 x 0.4 1 70 167-207 146 8 x 0.7 t ox. tox. 8x1 tox. Tox. 1 74 8x8 Tox. tox. 1 70 Eating dose 1 x 40 1 7 3 1 x 60 207 1x80 1 9 1

The table below shows the damage to the property of the damage 2-veel. according to which it is effective at a single high dose, no dose of the known vincristine (up to the toxic region) can be found to be effective once administered in this tumor mode.

A further benefit of renal joint is that no paralysis of the hind limb and bladder known to occur with vincristine has been observed during and after several days of treatment with effective doses.

11. In v itro s h o w h e s h a n d research

Based on encouraging in vivo results, the assays were repeated with measurements of generally accepted tubulin polymerization inhibition. In the series of measurements (for comparative purposes) we have extended the range of known effective drugs to vinorelbine1. and two physiologically inactive monomer indo 1-a 1 kaoidda1. catharanthine 1 and vindoline.

Assays are performed with a solution of the chemical compounds in aqueous saline solution and in bovine isolate tubulin, as described below.

I. Inhibition of Tubulin Polymerization

Polymerization of tubulin into microtubules was followed by turbidity measurement at 350 nm. spectrophotometer. The concentration of tubulin we purified from bovine brain was 10 µM (1 mg / ml). Polymerization is also inhibited to varying degrees by a number of antithiotic agents, including indole alkaloids. Inhibition of the compounds was characterized by their effect on the initial rate of polymerization. The degree of inhibition is given by the dose (IC 50). at which the rate of polymerization is halved. The value of this was determined by dose-response curves based on probit analysis.

The inhibitory effect of the test compounds on tubulin dimerization is shown in Table 2 in two different experimental systems. lThe measurement methodology Lehotzkv A.

et al., J. Biol. Chem. 268: 10888 (1093). 1

... t á b 1 tsp

Ve evü1e t

I Cs ο / I Cs o (vinblastine) v i nb1 sa t in v i n k r i s z t i n v i nore1b i n k arant in v i ndo 1 in

DAMAGE-2> 100,000> 30,000 l .53

0.53> 2600> 790

0:26

The water beats it out. that CAR-2 is more effective at inhibiting tubulin dimerization than known guo-amine compounds. The reproducibility of the methodology is also supported by measured data of physiologically inactive compounds. ¹ In vitro and in vitro experiments suggest that the test compound is novel. it acts through a mechanism of action that is not yet known.

2. Inhibition of tubulin immune comp1 ex

Several immunochemical methods are known for detecting the binding of certain compounds to tubulin and for determining the binding strength [See Ensvall, F., Methods Enzymol. 70, 419 (1980) or K. Liliom et al., J. Immunol. Methods. .1.4.3 · 119 (1991) 1. The sensitive low-volume method allows the identification of tubulin-interacting molecules and the rapid screening of a large number of molecules.

In our experiments, tubulin as an antigen is bound to a solid phase. The tubulin cured on a special plastic surface retains its immunochemical properties. It is thus able to specifically bind to the antibody raised against it. All those chemicals. which bind to tubulin. can influence the formation of the immune complex. Using a given compound at various concentrations, the degree of perturbation effects can be used to determine the IC50 value of the compound and the dissociation constant of the tubulin-drug complex.

After immobilization of the tubulin to the vehicle, it is incubated with the antibody-containing solution. in the presence or absence of the molecule to be tested. The amount of immune complex formed is then detected. This can be done directly by labeling the antibodies. High signal gain is available. if the antibody is raised on the primary antibody and conjugated to an enzyme that provides an easily measurable absorbance signal.

In the present experiments, a horseradish peroxidase antibody conjugate was used. Properly chosen and

Under 9 controlled conditions, the magnitude of the signals is directly proportional to the amount of immune complex produced.

The inhibitory effect of the test compounds on the tubulin-antibody interaction and the dissociation constants of the bis-indo-1α-kaolinoid complexes of the tubulin are shown in Table 3.

. spreadsheet

Ve gness ICsο, uM kd. M kd / KD (vinblastine vinblastine 3 7 4.5 1 vincristine 33 4.0 0.89 vinorelbine 50 5.0 1:11 catharantine > 500 - - v i ndo 1 i n 240 30.0 6.67 INJURY-2 24 3.0 0.6 '

(Kd in the table means dissociation constant)

In addition to the known physiologically active compounds, physiologically inactive substances (cataranthine, vindoline) were used again in the comparative studies. which are related and contain several similar functional groups. Studies have shown that kAR-2 binds to tubulin with greater affinity than vinblastine.

»· · ·» '»* 4 • · · 4« · β · · »*«, • «« «·« », ·· · ·,

- 10 3. Selectivity of inhibition

The selectivity of the inhibition was determined. The method is described below.

Certain natural materials, such as so-called "soap" As a result of microtubule associated proteins or certain glycolytic enzymes (e.g., major sphofructokinase), microtubules assemble into bonds, resulting in altered stability and dynamics.

Bundle formation is well nvomone-traced at 350 nm by the turbidity measurement1 already described. and differential centrifugation, since the increased molecular weight bundles settle better at lower speeds than single-stranded inubube tubes. In our experiments, we compared the effects of vinblastine and KAR-2 on tubulin polymerization and kinase bundle formation.

In our studies, we found a difference in the effect of vinblastine and damage-2 on bundle formation, as shown in Table 4.

Vinblastine is both a polymerization of tubulin. both inhibit bundle formation by the enzyme, whereas KAR-2 specifically inhibits microtubule formation only. (Bundle formation is dependent on ATP concentration, so the effect of the compounds may depend on the metabolic state of the cells.)

Table 4

Sample - · ·· · - ................................. ·· Tubulin in the precipitate (%) Compound t Hiking (%) MT - 25 9 0 MT + PFK - 100 94 MT vinblastine <5 1 2 MT + PFK inb1 ast in 37 48 MT INJURY-2 <5 6 MT + PFK INJURY-2 8 1 92

MT = microtubule: PFK = phosphofructokinase.

Concentration of each species:

MT = 10 µM: PFK = 2 µM: vinblastine = 2 µM:

DAMAGE 2 = 2 µM:

Temperature: 37 ° C.

From the table it can be seen that KAR-2 selectively inhibits tubulin ρο1i mer ionization depending on the organization of the microtubules.

4. Effect on cytoskeleton

DAMAGE-2, tested at doses of 10 µg / ml or higher on the Sf9rovar jse line and mammalian CHO cells, after 2 hours of in vitro incubation, induces depolymerization and disappearance of acit op 1 azrnitic mycotubes, which is immunohistochemically and electron microscopically. follow.

Under these conditions, tubulin precipitates in the form of paracrystals in the cytoplasm of cells. At a concentration of 1 µg / ml, the depot polymerization effect is partial: the number of microtubules is reduced but not completely disappeared. At a concentration of 0.1 µg / ml, no depot dimerizing effect was observed by morphological methods.

To our knowledge, CHAR-2 enters the cell and targets cytoskeleton as its primary target.

5. Anti-kalmodu1in (side effect)

Antimitotic molecules with different chemical structures act by different mechanisms. Their action is not specific to cancer cells, which severely hampers chemotherapy treatment. However, they are not exclusively targeted at the microtubule price system, which is a source of unwanted side effects. Igv, for example, bis-indole alkaloids are also known to have calmodulin antagonistic activity. Of particular importance is the fact that calmodulin also plays an important role in regulating the dynamics of the tubulin / microtubule system. Its cellular localization is also partly shared with the major microtubule 1 us organization center (centrosome). Calmodulin antagonists, by binding to ka iodine H n, inhibit the interaction of ka iodine I n proteins with other proteins and, by virtue of their effect, perturb many organization and metabolic processes.

The complex test system developed by us, which is an immunoassay (ELISA), can be advantageously used to visualize the anticalmodulin effect of bis-indo-1α-kaolinoids. enzyme kinetics and fluorescence anisotropy measurements. The ELISA method is based on the same principle and methodology as described in Experimental Summary 2, but this time instead of tubulin, calniodulin is used as an antigen. Enzyme kinetic and anisotropy assays are suitable. to study the effect of the compounds on the interaction of calmodulin with a specific binding enzyme (in this case, phosphofructokinase). During the binding of the enzyme to ka Imodu1in, the change in the molecular weight of calmodulin (labeled with a fluorescent dye) can be determined by anisotropy measurement (anisotropy is directly proportional to the molecular weight); Molecules having calmodulin antagonistic activity, such as vinblastine or vinorelbine. they eliminate the triggers caused by calmodulin.

Table 5 summarizes the results obtained with the 3 different methods described above.

For comparison with the classical anticalmodu1 in. data on trifluoperazine (TFP) [see Lewin R.M. et al., Pharmac. Exp. Ther. 208, 454 (1979).

It can be seen that KAR-2 is virtually ineffective under the given concentration conditions, while vinblastine is present. vincristine. in particular, vinorelbine exhibits significant anti-calmodulin activity.

Table 5

Vegvü1e t ELI SA PFK activity F1uorescence i a method method anisotropy method (a (b) (c) Inhibitory effect ..% Dam 1 hour effect. % Barrier 1h effect. % TFP 94 69 8 7 Vinb1last in 34 6 1 3 9 Winkrist in 4 3 6 2 NS V i η o r é 1 b i n 68 83 48 V i n d ο 1 i n <5 0 NS catharantine <5 0 NS INJURY-2 18 7 3

Concentric Racers:

a) calmodulin = 2.5 µg / ml; antibody = 7.5 ue / ml:

compounds = 250 µM:

b) calmodulin - 3 µM: PFK = 2 µM: chemical compounds = 20 µM:

c) calmodulin = 1.5 µM: PFK = 2 µM:

compounds = 20 µM. NS = not measured

It can be seen from the above that, unlike other bis-indole compounds, CAR-2 does not exhibit anti-cat 1modulin activity.

6. Damage-2 binds to ka 1modu1

The results described in Section 5 showed that.

that CHAR-2 does not exert anti-calmodulin activity. therefore, the compound was tested for binding to calmodulin. To determine this, we developed a direct binding test. based on fluorescence energy transfer measurements. Λ

The damage-2 compound emits fluorescent light when excited. _However} if the arm-two appropriate fluorescent dye EKKE 1 J ~ 5-di-methyloxy bound to 1 am in-naphthalene-l-sulfonyl kloriddafjjelzett kaimodu1 inhoz, the emitted fluorescent translated indications on calmodulin excitation instead of energy emission (energy transfer) . After the energy transfer occurs only within the damage module 2 modulator in complex. Thus, the amount of complex emitted can be determined from the extent of the emitted light, and thus the dissociation constant of the KAR-2-calmodulin complex.

We found that CHAR-2 binds to ka1modu 1 in and that the calmodulin-CHAR-2 complex has a dissociation constant of 2 µM.

Unlike vinb1 astinna 1. the formation of the bond is not dependent on calcium.

Based on our studies, it has now been surprisingly discovered that CHAR-2 exhibits a substantially different mechanism of action from bis-indole alkaloids of similar structure.

7. Effect of Damage-2 Liberator Since the affinity of Damage-2 ka for 1modulin is comparable to that of the investigated antagonists1, we investigated its effect on the interaction between compounds with anti-calmodin 1 activity and calmodulin. For this purpose, the activity and anisotropy assays already described were used. Table 5 summarizes the relevant results. It is evident that the derivatives of bis zi-ndo 1 s that possess anti-calmodu1 in activity exhibit a synergistic effect on TFP-1, and that KAR-2 exhibits an antagonistic effect on both TFP-1. all vinb 1 astin 1 opposite. That is, it releases' calmodulin from the action of antagonists (liberator effect).

Table 6

Vegvü 1 e t PFK activity ( the ) Dam horse effect. % fluorescence anisotropy (b) Barrier effect. % 'L'FP 69 87 V i n b 1 a s z t i n 61 3 9 vinorelbine 83 48 INJURY-2 7 3 TFP + Vinblastine 94 9 7 l'FP + Vinorelbin NS 94 TFP + DAMAGE-2 41 77 Vinblastine + INJURY-2 4 1 NS

Concepts:

a) calmodulin - 3 µM: PFK = 2 µM: compounds = 20 µM:

b) calmodulin = 1.5 pM: PFK = 2 pM: chemical compounds = = 20 pM. NS - not measured

According to the measured data, KAR-2 behaves like a "liberator," which is the liberator of calmodulin antagonists. that is, they are not antagonistic to otherwise calmodulin antagonist compounds. Our discovery was not expected because the basic framework of the molecules and the structure of the bis-indole are closely related to the structure of compounds known to date and used in human therapy. Based on the surprisingly novel mechanism of action and the cytostatic activity of the harm-2 compound in various in vivo and in vitro experiments, the compounds of the present invention can be used as active ingredients in cancer therapy-enriched pharmaceutical formulations.

Estimated dose for human therapy: 0.1-5 mg / day / kg body weight.

The active ingredient of formula (I) is conventionally administered orally in the pharmaceutical art. formulated into pharmaceutical compositions in admixture with non-toxic inert solid or liquid carriers and / or excipients for parenteral or enteral administration.

Suitable carriers include, for example, water, gelatin, milk sugar, starch, pectin. magnesium stearate, stearic acid. talc, vegetable oils such as peanut oils.

ο 1 curve-ο 1 a i can be applied.

The active ingredient may be in the form of conventional pharmaceutical compositions, in particular solid forms, for example, round or square tablets, dragees, capsules, such as gelatin capsules. pills, suppositories, etc. form it. The amount of solid active ingredient may vary over a wide range, preferably from about 1 mg to about 200 mg.

The compositions may also contain conventional pharmaceutical excipients, such as preservatives, stabilizers, wetting agents, emulsifying agents.

They are prepared by conventional means, for example, in the case of solid preparations, by sieving the component. mixing, granulating and pressing. The formulations may also be subjected to further conventional pharmaceutical techniques such as sterilization in the case of injection.

Preferred embodiments for the preparation of the compounds of the invention are detailed in the following examples.

Example 1

- ((3 - k 1 ó r - e t i 1) - 3 - s ρ i r o - 2, 4 - d i o x o - 5 - o x a ζ ο I i d ί η o - 4 ′ -deacetoxy-vinblastine.

To a base of g (13.28 mmoles) of 4'-deoxybenzene oxalate is added 8 ml of 10.1 g (95.9 mmoles) of β-k 1 -orethyl 1-isocyanate.

The resulting solution was allowed to stand for 2 hours and 200 ml of dry toluene was added. The mixture is heated at 80 "(* for 18 hours.). The mixture is cooled to 2 ° C. 40 ml of water and 25 ml of 25% aqueous ammonia are added. The mixture is stirred at room temperature for 30 minutes. The organic phase is separated and The solution was evaporated to dryness in a water bath at 35-40 ° C under vacuum (20-27 hPa) to give a dry residue on a column of 500 c silica gel (particle size 0.04-0.063 mm) (column diameter 30 mm). (length = 500 mm) eluted with ethyl acetate: ethanol 9: 1 Elution monitored by thin layer chromatography (run: ethyl acetate: ethanol 9: 1) Merck 3552.

Keleeleel on disc.)

Rf (LOSS-21 = 0.7-0.75

RF (deacetoxyvinb1 asstI I = () .5 - (). 6

The fractions containing the pure component are concentrated. s z onto r a p r o p ο 1 i ti k.

Product: 3.29 ε (30%) pure title base.

¹³ C NMR: ( ⁸ L MS = 0.0 ppm). CDC 1, i: signal bands

6.9 (C H.i): 8.3 (CH 3): 2 8.1 (C fb I: 29.0 (( 1 (2): 30.7 (CH): 34. 1 (CH2) : 35.5 (CH2) : 3 6.0 (Clb) : 3 9.8 (CH2 ) f 39, 9 (Clb ): 41 . 3 (Clb): 4 1 .9 (C) : 4 3.7 (Clb) 48.8 (Cl12) : 51.1 (Clb ): 52.3 (CH a ): 5 2, 8 (Clb ): 5 5. 4 (CH ): 5 5. 8 (C): 5 5.8 (Clb): 64.4 (CH): 64.8 (Clb): 6 9.8 (C): 74.2 (CH): 92.2 (CH); 110.3 (CH): 116.2 (C): 1 18.2 (cil): 118th 5 (CH) : 120. 0 (C) : 121.9 (CH) : 122. 9 (CH) : 12 3. 7 (cil): 125. 7 (C): 12 9.0 (C): 13 1.8 (C): 13 2.2 (CH): 13 4.8 (Cl: 15 0.6 (C): 154.0 (C): 15 7.5 (C): 17 1.8 (C): L e g i e 1 more significant IR bands (cm ¹ ) 3465 , 18 14 .1743. 16 17 1408. 1241, 103 9. 74 1

Mass: quasi-molecule ion mass M + H = 826 by FAB method. example

3- (β-Chloroethyl) -3-fluoro-2.4 ”-dioxo-5-oxazolyldino-4 ′ -dealethoxy-vinblast sulfate

The pure base prepared in Example 1 for 3 s (3.63 mmol) was dissolved in ethanol (5 mL) and added.

Of ethanol containing 0.363 g (3.63 mmol) of sulfuric acid, ⁷ µl -1. Allow to stand for 4 hours in a maid 40 'C water bath

Concentrate to 5-6 ml under vacuum (mmHg). The salt is then precipitated by addition of 20 ml of ether, the salt is filtered off and dried at a maximum temperature of 30 ° C at 13 hPa for 8-15 hours.

Product: 3.05 g (25%) of the sulfate salt of cyanide

Op: above 300 'C (decomposes)

Forea t: C = 1% Water ο 1dat («In ² · '' = +3.3 ': [ct I ^{2 5} nt 5 ⁷ s - +3.6”: f σ. I ^{2 5} h «5 4 6 = = +4.5: I α | ^{2 5} n «4 2 6 = + 12.9: I a | ^{2 5} π · 3 6 5 = + 36.7 °. P é 1 da

- A 1 1 i 1 - 3 -s p i ro-2. 4-Dioxo-5-oxo-4-deacetyl-4'-desac β-oxy-vinh 1 astine (13.28 mmol) for 4'-deacetoxyvinic acid 1 in 10 mL of 9.4 g (113, 25 mins) is added to 1 µl of 1 µl of zocyanate. After standing for 2 hours, dry toluene (200 ml) was added and the mixture heated at 80 ° C for 18 hours. The reaction mixture was cooled to 20 ° C and 40 ml of water and 2 ml of 25% aqueous ammonia were added. Stir for 30 minutes at room temperature.

The organic layer was separated, washed with water (3 x 30 mL), dried over sodium sulfate and evaporated to dryness at 13-27 hPa.

The resulting dry residue was chromatographed (80 x 100 x 1) with 1 µl of opone ethyl acetate.

upper: 2.66 g pure title base (25%) ¹³ C NMR: ( ⁸ Ί MS = 0.0 ppm), CPC13 system with signal bands:

6.9 (CH2): 8.4 (CH2); 28.2 (CH2I; 29.0 (CH2):

_ · · ··· ·; ί. · · ♦ · ·· ·; · ·· ... ·

30.7 (CH 1: 34. 1 (CH ₂ ): 35.1 (CIO): 35. (CH2) : 3 9.9 (C): 4 1.8 (CIO) : 4 3.8 (CIO 5 1.2 (CIO): 5 2.3 (CH3 1 : 5 2. 8 ((.70): 5 5 (OH2) : 5 5.0 <i '): 5 5. 5 (CIO 1 : 5 5 X (C): (CH 1: 64. V (CH2 I : 6 9.9 (C): 74.5 (CH): (<71 1: 1 1 0.3 (OH) : 116. 1 (O) : 118.3 (CH (CH2) : 119, 7 (Cl ; 122 0 (CH): 122.9 (C 12 5.7 (Cl 129.1 (C 1: 1 30.0 (CH): 132. (C): 1 50.5 (C): 154.1. (C): 157.6 (C);

1.8 (C1: VI.9

Most common IR bands (cm ^-1 1:

3467. 1813. 1743, 1617. 1402, 1240, 1129, 1036, 741

Weight: quasi-molecule ion M + H - 804 based on FAB mass spectrum. p é 1 <1 a

3A11il-3-spiro-2,4-dioxo-5-oxazolidine-4'-desacetoxy vinblastine sulfate

Dissolve 2.6 g (3.23 mmol) of pure base in 5 ml of ethanol and add 7 ml of ethanol containing 0.323 g (3.23 mmol) of sulfuric acid. Allow to stand for 4 hours. then concentrated to 4-5 ml on a 40 'C water bath at 80 hPa. The sulfate salt is precipitated with 20 ml of ether and filtered. max. It is dried at 30 ° C (1 Hgmni) for 8-15 hours.

Flesh: 2.7 1 g (22%) of cis inbred parent tree.

'Op .: above 300' C (decomposes)

Rotation: C = 1% water g 1 η ^{2 5} = +1.1: f η 1 ^{2 5} n »s 18 = +1.4: (a 1 ^{2 s} hg 5 46 = = + 1.8; I« 1 ^{2 s} ii t 436 + 7.3 ”: ί α 1 ^{2 5} ti «365 +30.8":

- 22 Example 5

3 (β-Chloroethyl) -3-spiro-2,4-dioxo-5-oxazolidino-4'-deacetoxyvincristine g (11.9 mmol) for 4'-deacetoxyvincristine base 8 mL 10.1 g (95.9 mmol) 2-Chloroethyl isocyanate is added. After standing for 2 hours, dry toluene (200 mL) was added. Heat at 80 ° C for 18 hours.

The reaction mixture was brought to 20 ° C. 40 ml of water and 2 ml of 25% aqueous ammonia were added. Stir at room temperature for 30 minutes. The organic phase was separated

Wash with 3 x 30 mL water and dry over sodium sulfate. The solution was filtered. Evaporate to dryness under vacuum at 13-27 hPa in a 40 'C water bath.

The resulting dry residue was chromatographed on a silica gel column (80-100 times) with ethyl acetate: ethanol = 10: 1.

Product: 3.3 g (29%) of pure title base

13c NMR: (®TMS - 0.0 ppm), typical values measured in DMSO:

7 .0 (CH.a): 8 .0 (CH3): 27.8 (CH2): 29.7 (CH) : 31.6 (CH 2): 34.2 (CH 2): 34.4 (CH 2): 37 3 (CH2 ): 37.5 (CH 2); 41st 2 (CH 2): 41.4 (CH ₂ ); 41.5 (CH): 46.9 (CH 2): 49.4 (CH 2): 50.6 (CH 2 ); 51.9 (CWhen ) *: 5 2.9 (C): 56.2 (CH 2): δ 5.4 (Cl: 56.3 (CH 2) 62.8 (CH) : 63.7 (CH 2): 66.4 (CH): δ 7.3 (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, (CH): 171.9 • · ·

- 23 (0: 173.9 (0:

Most common IR bands (cnr ¹ ):

3445. 1817. 1744. 1617, 1595. 1411. 1230. 1028. 742

Mass: FAB mass spectrum quasi-molecular ion M + H = 840

Example 6 (B - k 1 h - e l i 1! - 3 s p i r <> - 2, 4 - d ί ο x o - 5 - o x a ζ ο 1 i d ί η o - 4 '

- d e z a c e t o x i v i n k r i s z t i n - s z u 1 f á t

base in 1 .357 2 ( Third 5 7 thyme 1 On 4 volumes á t •• i 1 1 ii i vacuo 5 -6 in 1 - r

2 (3,57 ιηιηό 1) 5. not more than one liter of base is dissolved in ethanol. To the page was added 7 µl I-1 of ethanol containing sulfuric acid. then let it go. The solution is then concentrated to 5-6 ml in a water bath (40 ° C) at 80 hPa, the mother tree salt precipitated by the addition of 20 ml of ether, filtered and dried to max. Dry at 30 ° C under vacuum for 8-15 hours.

Product: 3.05 g (25%) of the title compound Op: 300 'C (dec.)

Rotation: C = 1% water [α] D ^{2 S} - + 0.8 °; fa] ^2S Hg sis => + 1.0 °: fa] ^í5 H «546 = = + 1.2 °: ί C! 1 ^{2 S} H «436 a 1 ^{2 5} ii + 25.9 °:

«. * ··; · ϊ · ϊ. . · *

Claims (5)

Claims A spiro-2,4-dioxooxazolidinobenzoindole compound of formula (I): wherein: Its meaning is methyl iron. Group 1 t. R2 is (C1-C4) alkyl: (C2-C4) alkenyl: (2-4C) halogen-containing alkyl and their acid addition salts. 2. A process for preparing a compound of formula (I) wherein: R 1 is methyl or formyl, R 2 is C 1-4 alkyl: C 2-4 alkenyl; C2-C4 haloalkyl - bis indole compounds and their acid addition salts. characterized in that it is a bis-indole compound of formula II wherein: R 1 is methyl or H-1, a compound of formula (III) wherein R 2 is C 1 -C 4 alkyl: C 2 -C 4 alkenyl; After reacting with a C 2 -C 4 halogen-1-alkyl alkyl isocyanate in an inert solvent, the resulting product is isolated and optionally converted to the acid addition salt with an acid. 3. Anti-tumor pharmaceutical compositions having calmodulin liberator activity. characterized in that the active ingredient is a compound of general formula (I) - wherein: · · ··· ·· ·· · « - 25 -R 1 is methyl or formyl. R2 is C1-C4 alkyl: C2-C4 alkenyl: C2-C4 haloalkyl compound or a salt thereof is non-toxic for parenteral administration commonly used in saline nutrition. inert solid ore mixed with liquid carriers and / or auxiliaries. 4. Elimination for the preparation of anti-tumor pharmaceutical compositions. characterized by it. to a bis-indo-chemical of formula (I) wherein R 1 is methyl or formyl. R 1 is C 1 -C 4 alkyl: C 2 -C 4 alkenyl: C 2 -C 4 haloalkyl - or salts thereof, in admixture with pharmaceutically acceptable non-toxic inert solid or liquid carriers and / or excipients for parenteral or enteral administration. formulations formulated. 5. A method of treating tumor diseases characterized by. that the subject or animal is treated with a bis-indole chemical of formula (I) wherein I R 1 is methyl or boil 1. R2 is C1-C4-alkyl: C2-C4-alkenyl: C2-C4-haloalkyl or a salt thereof, or a pharmaceutically effective dose thereof, in pure form, as an active ingredient in the form of a pharmaceutical composition.