DE3427383C2 - - Google Patents

Description

Avaron and its hydroquinone derivative Avarol are natural products, found in the sea sponge Dysidea avara [L. Minale, R. Riccio and G. Sodano, Tetrahedron Letters 1974, 3401-3404; S. de Rosa, L. Minale, R. Riccio and G. Sodano, J. Chem. Soc. Perkin I, 1408-1414 (1976)]. About the biological Effect was previously known that Avarol in very high concentrations tration (130 µM) sea urchin embryos impaired [Cariello et al; Comp. Biochem. Physiol. 65c, 37-41 (1980)]. In Comp. Biochem. Physiol. 71B, 281-283 (1982) report Cariello et al. about the growth-inhibiting effects of Avarol on various Microorganisms. Then the MIC is either included (so for Streptococcus faecalis) or over 100 µg / ml. With this Bacteria becomes an inhibition zone of approximately the same size as through the comparison compound penicillin G only 17 times Active ingredient amount of Avarol achieved.

Derivatives are in the literature u. a. 3'-methylamino and 4′-methylamino-avaron [G. Cimino, S. de Rosa, S. de Stefano, L. Cariello and L. Zanetti, Experientia 38, 896 (1982)] and Avarol Dimethyl Ether and Avarol Diacetate [p. de Rosa, L. Minale, R. Riccio and G. Sodano, J. Chem. Soc. Perkin I, 1408- 1414 (1976)]. The methylamino-avarone affect Sea urchin embryo cells only in ten times higher concentration in the same way as Avarol. Experientia 38, 896 (1982) provides information on physical data of methylamino avarone Derivatives and indicates the possibilities of a general Use of the biological effectiveness of this compound; however, there is no quantifying data. Other biological effects of Avaron derivatives were previously unknown.

It has now been found that Avarol, Avaron, Avarol diacetate as well the avaron and avarol derivatives of the general formula I

in either

• a) X¹ and X² are oxo, each have a double bond between C-1 'and C-6' and between C-3 'and C-4' and
  • α) R¹ is hydrogen and at the same time R² alkylamino with 2 to 4 carbon atoms or
  • β) R² is hydrogen and R¹ is also alkylamino having 2 to 4 carbon atoms, or
• b) the uncondensed ring is aromatic, R¹ and R² Is hydrogen and X¹ and X² are both either (i) Acyloxy each having 3 to 6 carbon atoms or (ii) one of the Radicals X¹ and X² hydroxy and each with acyloxy 2 to 6 carbon atoms or (iii) X¹ with X² is a bis forms acyloxy bridge with 4 to 6 carbon atoms, pronounced antitumor, antibacterial, antiviral and antifungal  Have properties that they are used to treat Cancer and infectious diseases appear suitable to let.

The invention also relates to processes for the production of compounds according to claim 1, characterized in that are that in each case in a manner known per se for the preparation of compounds of group a) Avaron of formula II

with alkylamine hydrochloride of the general formula RNH₂ · HCl, in which R has the meaning given for R¹ or R² in claim 1, and, if desired, the compounds thus obtained are converted into salts with physiologically tolerated acids, or
for the preparation of compounds of group b) Avarol of the formula III

depending on the target product either with a carboxylic acid chloride or anhydride with 2 to 6 carbon atoms in the carboxylic acid residue or with a dicarboxylic acid chloride or anhydride with 4 to 6 carbon atoms in the dicarboxylic acid residue.

As a compound of the general formula RNH₂ · HCl come into question Ethyl, propyl, isopropyl, n-butyl, isobutyl and tert.-o butylamine hydrochloride (reaction 1).  

Response 1

Response 2

For the preparation of the Avaron derivatives with alkylamino Groups by introducing the substituent -NHR in 3′- or The components are left in the 4'-position in the presence of pyridine react with each other in 50% ethanol [cf. Cimino, S. de Rosa, S. de Stefano, L. Cariello and L. Zanetti, Experientia 38, 896 (1982)]. The resulting isomer mixtures 3'- and 4'-alkylamino-avarones can be separate by column chromatography on silica gel.

As physiologically acceptable acids for salt formation are z. B. hydrochloric acid, hydrobromic acid, Sulfuric acid, phosphoric acid, tartaric acid and maleic acid.

For the production of acarol derivatives of the general Formula I in which X¹ and X² are acyloxy and hydroxy, Avarol is acylated with an acyl chloride of the general type formula

or with a carboxylic anhydride of the general formula

in each case according to the desired end product, at the hydroxyl of C-2 'and / or C-5'. Here come too Acid chlorides or acid anhydrides of dibasic acids, such as e.g. B. succinic acid, in question, with one molecule of Avarol  only one molecule of the dibasic acid derivative is omitted (Bisacyloxy derivative).

As compounds of the general formula

come for example, straight-chain acyl chlorides such as Acetyl chloride, propionyl, n-butyryl, n-valeroyl and Caproyl chloride and branched chain acyl chlorides such as Isobutyryl, isovaleroyl, ethyl-methyl-acetyl and Trimethylacetyl chloride.

As compounds of the general formula

come for example, straight-chain acid anhydrides such as Propionic acid, butyric acid, valeric acid and Caproic anhydride and branched-chain acid anhydrides such as Isobutyric acid, valeric acid, ethyl methyl acetic acid and Trimethyl acetic anhydride (reaction 2) [s. O.].

To prepare the acylated avarol derivatives the components together in the presence of pyridine react (S. de Rosa, L. Minale, R. Riccio and G. Sodano, J. Chem. Soc. Perkin I, 1976, 1408-1414; Organic, VEB German Publishing House of Sciences, 13th edition, Berlin 1974, pp. 441-446.  

The invention further relates to medicinal products with a Content of a compound according to claim 1.

Finally, drugs are provided that are caused by contains avarol, avaron and avarol diacetate Marked are.

The compounds mentioned can be used with the usual Solvents, auxiliaries and carriers for tablets, Coated tablets, capsules, drip solutions, suppositories, injection and process infusion preparations for therapeutic purposes peroral, rectal or parenteral application use to find.

The compounds of the general formula I and Avarol, Avaron and Avarol diacetate have valuable pharmacological properties. The anti-tumor effect was in vitro on the L5178y and L1210 mouse lymphoma cell system proven. These cells were in suspension culture held as already described by us [W. E. G. Müller and R.K. Zahn, Cancer Res. 39, 1102 (1979)]. The ED₅₀ Concentrations of the compounds (inoculation: 4000 Cells / ml; Incubation time: 72 hours) were for the two Tumor cell strains in the range of 0.7 to 6.7 µM, with Avaron,  Avarol, the corresponding amino derivatives and the avarol esters demonstrated the greatest effectiveness. The mode of action could to be clarified by installation studies: In the presence of the Connections at their ED₅₀ concentration becomes the Incorporation of [3 H] thymidine into the DNA, of [3 H] uridine into the RNA, from [3 H] phenylalanine in proteins and from [3 H] mannose promoted in glycoproteins. This effect could go through autoradiographic methods [K. Habel and N.P. Salzmann, Fundamental Techniques in Virology, Academic Press, New York, 1969, p. 242] as a partial synchronization of the cells be cleared during the G₂-M phase.

In cytological examinations by Giemsa staining and Acridine orange staining of the mitosis chromosomes [R. Rigler, Acta Physiol. Scand. 67 (suppl. 267), 1 (1966)] was found to the compounds have the mitotic index of L5178y cells influence. The pharmacological effects of Avaron and its derivatives is that of a mitotic poison. Also biochemical studies proved this Mechanism of action: Avaron and its derivatives inhibit the Microtubule polymerization in the substoichiometric range, as performed by viscometric studies S. D. MacLean-Fletcher and T. D. Pollard [J. Cell Biol. 85, 414-428 (1980)]. The inhibition of Microtubule polymerization takes place at the level of Protofilament elongation. By higher concentrations of Compounds is next to the inhibition of microtubules Polymerization during the mitosis phase also during the  other cell cycle phases inhibited. Chromosome aberrations occur after administration of therapeutic doses of Avaron and its derivatives.

Of particular importance for the use of the Links to cancer chemotherapy is likely be the fact that it has a cytostatic activity non-tumor cells in culture in therapeutic applicable concentrations are missing. This result was obtained from experiments with lymphocyte cultures: spleen Lymphocytes were from six week old NMRI mice won; the erythrocytes were removed from the suspension Removed ammonium chloride treatment. The spleen lymphocytes were in RPMI 1640 medium / 20% fetal calf serum in one Density of 1.5 × 10⁷ cells / ml for 72 hours in the presence of 2 µg / ml Concanavalin A kept. 18 hours before the end of the Experiment [3 H] thymidine was added. With incubation with 10 µM of the connections no impairment of the DNA synthesis rate measured. In the presence of 15 µM only a 15% inhibition of the incorporation rate of [3 H] - Thymidine in the DNA.

The anti-tumor effects of Avaron and its derivatives has also been demonstrated in vivo on whole animals. The The test was carried out as previously described [W. E. G. Müller, R. K. Zahn, A. Maidhof, H. C. Schröder, M. Bachmann and H. Umezawa, J. Antibiotics 37, 239 (1984)].  

NMRI mice vaccinated with L5178y lymphoma were ill with this tumor and then for five days e.g. B. with 0.3 mg / kg Avaron i. p. were treated per day treated tumor mice survived significantly. The untreated animals were 50% dead after 18 days 50% of the treated animals even survived the 26th day.

The subacute toxicity of Avaron and its derivatives is a five-day i. p. treatment of mice with Values between 35 and 75 mg / kg so favorable that a Use of the process products in the chemotherapy of Cancer is expected.

The antibacterial effects of Avaron and its Derivatives have been established in vitro tests. She was with determined by the agar dilution method [B. E. Scully, K. Jules and H. C. Neu, Antimicr. Ag. Chemother. 23, 907 (1983)]. The Bacteria were in an amount of 10⁵ colony forming units used. The minimum inhibitory concentration (MIC) z. B. for Avarol with 0.4 µM at Cocci such. B. Micrococcus pyrogenes, with 0.9 µmM for gram-negative Bacteria such as B. Escherichia coli and with 1.3 µM in gram-positive Bacteria such as Bacillus cereus and 0.7 µM Propionibacterium acnes determined. This high in vitro Activity is in the range of in vitro antitumor activity. Because of the low subacute toxicity in mice in vivo there is a favorable chemotherapeutic index (TI = LD₅₀  in the mouse, divided by the sensitivity to germs), e.g. B. Avarol between 100 and 140 depending on the microorganism.

Avaron and its derivatives have an antiviral effect in BHK cell Cultures with type I or type II herpes simplex virus have been infected. Virus concentrations (multiplicity infection) was plaque forming with 0.5 to 1.5 units "/ cell. 2 hours after infection, the Cultures washed and the process products added. 24 hours p. i. the virus titer (p. f. u. / 0.2 ml) certainly. With a concentration of z. B. Avarol of 1 µM became the Type I propagation from 9 × 10⁴ (control) to 1 × 10³ (treated cultures) and the type II propagation of 6 × 10⁵ (Control) to 7 × 10³ (treated cultures) reduced.

The antifungal effect was demonstrated in vitro using Saccharomyces cerevisiae determined by the broth dilution method (E. Lennette [ed.], Manual of Clinical Microbiology, ed. By American Society of Microbiology, Washington 1980, pp. 648- 649). The minimum inhibitory concentration was e.g. B. for Avarol 0.7 µM determined; the 50% inhibitory concentration is 1.6 µM.  

Due to the pronounced antibacterial, antiviral and antifungal activity are the links to Suitable for treating infectious diseases.

Of the examples below, examples 1a to 1d after reaction 1, while Examples 2 to 3 run after reaction.

Example 1 3'-ethylamino-avaron and 4'-ethylamino-avaron

• a) 2.5 g of ethylaminohydrochloride and 5 ml of pyridine are added to a solution of 500 mg of Avarol in 1000 ml of 50% ethanol and, after 20 hours, the ethanol is distilled off under a water pump vacuum. The aqueous residue is extracted with dichloromethane and the concentrated dichloromethane extract is chromatographed on a silica column using dichloromethane as the eluent. The separation into 3'-ethylamino and 4'-ethylamino avarone succeeds. Mp: 105-107 ° C (3′-ethylamino-avaron), or
  M.p .: 106-109 ° C (4′-ethylamino-avaron)

The following were obtained in an analogous manner:

• b) 3'-propylamino- and 4'-propylamino-avaron,
• c) 3'-isopropylamino- and 4'-isopropylamino-avaron,
• d) 3'-n-butylamino and 4'-n-butylamino-avaron.

Example 2 Avarol diacetate

• a) 500 mg of Avarol are dissolved in 20 ml of absolute pyridine and 1 g of acetyl chloride in portions while shaking added. It is worked up in the usual way Dry concentrated and the residue with boiling heptane extracted. The ester crystallizes on cooling. He is recrystallized from hexane. Mp: 62-64 ° C.

The following were obtained in an analogous manner:

• b) avarol dipropionate,
• c) Avarol divalerianate,
• d) Avarol di (trimethyl acetate).

Example 3 Avarol dicapronate

• a) 300 mg of Avarol are dissolved in 25 ml of absolute pyridine and to the solution in portions with shaking 0.6 g of caproic anhydride added. It will be in the usual way worked up, evaporated to dryness and the residue with extracted boiling heptane. It is made from acetone and then recrystallized from hexane. Mp: 107-114 ° C.

The following were obtained in an analogous manner:

• b) Avarol diisovalerate,
• c) avarol-di-ethyl-methyl-acetate,
• d) Avarol succinate.

Claims (4)

1. Avaron and Avarol derivatives of the general formula I in either

• a) X¹ and X² are oxo, each have a double bond between C-1 'and C-6' and between C-3 'and C-4' and
  • α) R¹ is hydrogen and at the same time R² alkylamino with 2 to 4 carbon atoms or
  • β) R² is hydrogen and R¹ is also alkylamino having 2 to 4 carbon atoms, or
• b) the uncondensed ring is aromatic, R¹ and R² are hydrogen, and X¹ and X² both either (i) acyloxy each having 3 to 6 C atoms or (ii) one of the radicals X¹ and X² hydroxy and the other acyloxy each having 2 to 6 carbon atoms or (iii) X¹ with X² forms a bisacyloxy bridge with 4 to 6 carbon atoms.

2. A process for the preparation of compounds according to claim 1, characterized in that in each case in a manner known per se
for the preparation of compounds of group a) Avaron of formula II with alkylamine hydrochloride of the general formula RNH₂ · HCl, in which R has the meaning given for R¹ or R² in claim 1, and, if desired, the compounds thus obtained are converted into salts with physiologically tolerated acids, or
for the preparation of compounds of group b) Avarol of the formula III depending on the target product, either with a carboxylic acid chloride or anhydride with 2 to 6 carbon atoms in the carboxylic acid residue or with a dicarboxylic acid chloride or anhydride with 4 to 6 carbon atoms in the dicarboxylic acid residue. 3. Medicinal product, characterized by the content of one A compound according to claim 1. 4. Medicinal products characterized by the content of Avarol, Avaron or avarol diacetate.