Derivatives anthra[2,1-d]isothiazol-3,6,11-trione

 

The invention relates to new chemical compounds derived from anthra[2,1-d]isothiazol-3,6,11-trione General formula I, where a is the lowest alkylene, R1and R2(independent) - lower alkyl, or R1and R2together with the nitrogen atom form a six-membered saturated, a heterocycle, which may optionally contain a heteroatom such as oxygen atom, and their pharmaceutically acceptable salts. Preferably these compounds inhibit platelet aggregation and reduce the activity of a number of tiltability enzymes, such as reverse transcriptase of human immunodeficiency virus (HIV) and caspase-3. This invention can be used in biochemistry, physiology and medicine. 3 C.p. f-crystals, 1 table.

The invention relates to new chemical compounds derived from anthra[2,1-d]isothiazol-3,6,11-trione General formula Iwhere a is the lowest alkylene, R1and R2(independent) - lower alkyl, or R1and R2together with the nitrogen atom form a six-membered saturated, a heterocycle, which may optionally contain a heteroatom such as oxygen atom, and their pharmaceutically acceptable salts.

Preferably, outow, in particular, such as the reverse transcriptase of human immunodeficiency virus (HIV) and caspase-3.

This invention can be used in biochemistry, physiology and medicine.

The results of numerous studies indicate that increased platelet aggregation plays a key role in the pathogenesis of many cardiovascular diseases such as arterial thrombosis, myocardial infarction, unstable angina). Compounds capable of preventing the activation of platelets, which may be caused by endogenous inducers, such as adenosine-5'-diphosphate (ADP), currently widely used for effective influence on the pathological changes of hemostasis (M. D. Mashkovsky. Medicines, T. 1, Torching, Kharkov, 1998 , pages 462-475 [1]; B. A. Sidorenko, D. C. Transfiguration. Clinical use of antithrombotic drugs, M., 1998 [2]).

It is known that certain bicyclic compounds containing atoms of nitrogen and sulphur and is used as substances in pharmaceuticals production, have the ability to inhibit platelet aggregation.

Thus, the known 5-[(2-chlorophenyl)methyl]-4,5,6,7-tetrahydrothieno[3,2-C]pyridine (is s (f Storey. "The P2Y12 receptor as a therapeutic target in cardiovascular disease". Platelets 2001, v. 12, 4, p. 197-209 [3]; U.S. patent 4963559, 514-301, op.1990 [4]; [1] S. 474).

It is shown that the calcium channel blocker - D-CIS-3-acetoxy-2,3-dihydro-5-[2-(dimethylamino)ethyl] -2-(2-methoxyphenyl)- 1,5-benzothiazepin-4(5H)-she hydrochloride (diltiazem) formula IIIhas antithrombotic action (A. R. Dehpour, T. Samadian et al. "Effects of diltiazem and verapamil on ADP-induced rabbit platelet shape change and aggregation" Gen. Pharmacol., 1995, v.26, 6, p.1295-1299 [5]).

The above compounds in vitro inhibit platelet aggregation caused by ADP at a concentration of >300 μm.

In biochemical studies, have found wide application of low molecular weight inhibitors tiltability enzymes, the so - called "SH-label". These include such compounds as N-ethylmaleimide, 4-nitro-7-chlorobenz-2,1,3-oxadiazole, 5,5'-dithiobis(2-nitrobenzoic acid) and others (e.g., E. Scoffone, A. Fontana "Identification of specific amino acid residues". Mol. Biol. Biochem. Biophys. , 1970, v.8, p. 185-210 [6]; K. K. Han, A. Delacourte, B. Hemon "Chemical modification of thiol group(s) in protein: application to the study of anti-microtubular binding drugs," OMRS. Biochem. Physiol. In 1987, v.88, 4, p.1057-1065 [7]; A. F. Carne. "Chemical modification of proteins. " Methods Mol. Biol., 1994, v.32, p.311-320 [8]). As a rule, these compounds have relatively high reactivity towards Tilsa urgent search for new effective inhibitors tiltability enzymes.

There are various 1,2-glandelinians derivatives of 9,10-anthraquinone having antitumor action (for example, A. L. Jones, I. E. Smith. "Navelbine and the anthrapyrazoles." Hematol. Oncol. Clin. North Am., 1994, v. 8, 1, p.141-152 [9]; H. Gogas, J. L. Mansi. "New drugs. The anthrapyrazoles. " Cancer Treat. Rev., 1996, v.21, 6, p.541-552 [10]; P. Chang, C.-F. Chen. "Antineoplastic anthraquinones. II. Design and synthesis of 1,2-heteroannelated anthraquinones". J. Heterocycl. Chem., 1996, v.33. p.367-371 [11]).

The ability of these compounds to inhibit platelet aggregation, as well as their influence on the activity AGAINST HIV and caspase-3 have not been studied.

Known derivatives of anthraquinone - mitoxantrone formula IV and pesantren formula Vinhibiting platelet aggregation under the action of ADP (data not shown) and collagen (IC50~40-140 and 14-23 μm, respectively, depending on the concentration of the inducer of aggregation) (P. Frank, R. F. Novak. "Mitoxantrone and bisantrene inhibition of platelet aggregation and prostaglandin E2 production in vitro. " Biochem. Pharmacol, 1985, v.34, 19, p.3609-3614 [12]). The impact of these compounds on the activity AGAINST HIV and caspase-3 was not investigated.

It is shown that 1,2-benzisothiazol-3-one and 2-amino derivatives of General formula VI

where R=H or NH2,
have the ability to inhibit platelet aggregation in vitro caused by adenosine diphosphate and collagen." J. Med. Chem., 1985, v.28, 11, p.1661-1667 [13]; P. Vicini, C. Manotti et al. "Synthesis and antiplatelet effects of 2-amino-1,2-benzisothiazolin-3-one." Arzneim.-Forsch./Drug Res., 1997, v.47, 11, p. 1218-1221 [14]).

The influence of benzisothiazolinone General formula VI activity AGAINST HIV and caspase-3 was not investigated.

Known derivatives anthra[2,1-d]isothiazol-3,6,11-trione General formula VII

where R is H, methyl, benzyl, phenyl or substituted phenyl,
as a by-product of chemical synthesis (F. A. Ernst vikne, S. G. Zlotin. "Synthesis of linear and angular anthraquinonoisothiazole-3-ones, their S-oxides and S, S-dioxides. " Russ. Chem. Bull., 2001, v.50, 9, R. 1657-1662 [15] ). Pharmacological and biochemical properties of these compounds have not been studied.

It is known that the number of derivatives isothiazol-3-one have an inhibitory effect on the activity of some tiltability enzymes, in particular p56 (lck) tyrosinemia protein kinase (J. M. Trevillyan, X. G. Chiou et al. "Inhibition of p56 (lck) tyrosine kinase by isothiazolones." Arch. Biochem. Biophys. , 1999, v.364, 1, p.19-29 [16]), telomerase (N. Hayakawa, K. Nozawa et al. "Isothiazolone derivatives selectively inhibit telomerase from human and rat cancer cells in vitro." Biochemistry, 1999, v.38, 35, p.11501-11507 [17]), as well as prevent the formation stromelysin of postremission (E. C. Amer, M. A. Pratta et al. "Isothiazolones interfere with normal matrix metalloproteinase activation and inhibit cartilage proteoglycan degradation. " Biochem J. , 1996, v.318, pt.2, p.417-424 [18]). It is established that this effect may be obeseity isothiazol-cycle education thioldisulfide connection with the protein [16, 17, 18].

Closest to the compounds of the present invention in structure and properties is 2-[3-(piperidino-1)propyl]-1,2-benzisothiazol-3-one (VIII

which inhibits the aggregation of platelets exposed to collagen or adenosine-5'-diphosphate (ADP) ([13] - prototype).

This connection, according to published data, has not sufficiently distinct pharmacological properties (IC50=57 μm in vitro). His influence on the activity tiltability enzymes have not been studied.

The aim of the invention is the creation of new heterocyclic compounds in the series annelated derivatives isothiazolone with improved pharmacological properties.

This goal is achieved by the new derivatives anthra[2,1-d]isothiazol-3,6,11-trione the above General formula I, where a is the lowest alkylene, R1and R2(independent) - lower alkyl, or R1and R2together with the nitrogen atom form a six-membered saturated, a heterocycle, which may optionally contain a heteroatom such as oxygen atom, and their pharmaceutically acceptable salts. Preferably these compounds inhibit platelet aggregation and reduce the activity of a number of thiol is, for example, containing functionally important residues cysteine and representing pharmacological interest. HIV (RNA-dependent DNA polymerase) catalyzes the formation of proviral DNA in the matrix of viral RNA, promoting HIV replication, and is currently one of the key targets in chemotherapy of AIDS (for example, H. Jonckheere, J. Anne, E. De Clercq. "The HIV-1 reverse reduced (RT) process as target for RT inhibitors." Med. Res. Rev., 2000, v.20, 2, p. 129-154 [19]). The caspase-3 (effector cysteine proteases that are activated during apoptosis) catalyzes the splitting of proteins after aspartic acid residues (G. M. Cohen "Caspases: the executioners of apoptosis." Biochem. J., 1997, v.326, pt.1, p.1-16 [20]). Compounds that inhibit this enzyme may have a cytoprotective effect and are considered as potential pharmacological agents for the treatment of myocardial infarction, cerebral ischemia, Alzheimer's disease, osteoarthritis, liver cirrhosis and other diseases (for example, T. Rudel. "Caspase inhibitors in prevention of apoptosis." Herz, 1999, v.24, 3, p.236-241 [21]; N. Guttenplan, C. Lee, W. H. Frishman. "Inhibition of myocardial apoptosis as a therapeutic target in cardiovascular disease prevention: focus on caspase inhibition." Heart Dis., 2001, v.3, 5, p.313-318 [22]; H. J. Rideout, L. Stefanis. "Caspase inhibition: a potential therapeutic strategy in neurological diseases." Histol. Histopathol., 2001, v.l6, 3, p.895-908 [23]).

Derivatives anthra[2,1-d]isothiazol-3,6,11-trione General formula is achene, were synthesized by methods based on known reactions, such as acylation of the primary amine with the acid chloride substituted anthracenemethanol acid [15], aromatic nucleophilic substitution of the nitro group on allylthiourea (S. G. Zlotin, P. G. Kislitsin et al. "Synthetic utilization of polynitroaromatic compounds. 1. S-derivatization of 1-substituted 2,4,6-trinitrobenzenes with thioles. " J. Org. Chem., 2000, v.65, 25, p. 8439-8443 [24] ), cyclization of amides o-(alkylthio)rankabove acids in isothiazolone under the action of sulfurylchloride (Japan patent 07330745 [95330745] , C07D 275/04, 1995; Chem. Abstr. 1996, v.124, 289523j [25]; European patent 657438, C07D 275/04, on. 1995 [26]) and alkylation of amines by alkylhalogenide (E. K. Harvill, R. M. Herbst, E. G. Schviner. "Halogenoalkyltetrazoles and aminoalkyltetrazoles", J. Org. Chem., 1952, v. l7, 12, p.1597 [27]).

In particular, the derivative anthra[2,1-d]isothiazol-3,6,11-trione General formula I, where a is the lowest alkylen, and AndCH2and R1and R2have the above values, obtained by the method lies in the fact that amine containing a primary amino group, of General formula IX
H2N-And-NR1R2(IX),
where A, R1and R2have the above values,
acelerou the acid chloride of 1-nitro-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid in a mixture of benzene/water received by carbonator potassium and titilate sodium in a medium of dimethylformamide (DMF), obtained the corresponding N-substituted 1-ethylthio-9,10-dioxo-9,10-dihydroanthracene-2-carboxamid enter into interaction with sulfurylchloride in the medium of anhydrous methylene chloride, followed by separation of the desired product as free base or pharmacologically acceptable salts (e.g. hydrochloride, fumarata or citrate) (method A, scheme 1, see the end of the description).

Derivatives anthra[2,1-d]isothiazol-3,6,11-trione General formula I, where a is the lowest alkylen, and AndCH2, R1and R2(independent) - lower alkyl or together with the nitrogen atom form a six-membered saturated, a heterocycle, were also obtained in an alternative way, namely, that aminoalkylsilane General formula X
H2N-A-Hal (X),
where a has the above meanings and Hal is chlorine atom or bromine,
taken in the form of a salt, acelerou the acid chloride of 1-nitro-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid in a mixture of benzene/water in the presence of potassium carbonate obtained the corresponding N-substituted 1-nitro-9,10-dioxo-9,10-dihydroanthracene-2-carboxamid treated with potassium carbonate and timedelta sodium in DMF medium obtained the corresponding N-substituted 1-methylthio-9,10-dioxo-9,10-digit is, the donkey and the resulting intermediate 2-(haloalkyl)anthra[2,1-d] isothiazol-3,6,11(2H)-Trion subjected to reaction with the amine of General formula XI
HNR1R2(XI),
where R1and R2have the above values,
in DMF medium in the presence of potassium carbonate, and optionally the alkali metal iodide such as potassium iodide, at a temperature of 50-55oWith subsequent isolation of the desired product as free base or pharmacologically acceptable salt (method B, scheme 2, see the end of the description).

The source of the acid chloride of 1-nitro-9,10-dioxo-9,10-dihydro-anthracene-2-carboxylic acid was obtained in a known manner by treatment of the appropriate acid chloride tiomila in an environment of absolute benzene at the boiling temperature of the reaction mixture [15].

Derivatives anthra[2,1-d]isothiazol-3,6,11-trione General formula I, where A= CH2and R1and R2have the above values were obtained in a known manner by the reaction of manniche condensation derived isothiazol-3-one with secondary amines in the presence of formaldehyde (E. D. Weiler, G. A. Miller "Isothiazoles VII: N-Hydroxyalkylation and Mannich Reaction of 4-Isothiazolin-3-ones." J. Heterocycl. Chem., 1976, v.l3, 5, p.1097-1098 [28]).

In particular, the original anthra[2,1-d]isothiazol-3,6,11(2H)-Trion introduced in use values, in the presence of an aqueous solution of formaldehyde at 45-50oWith subsequent isolation of the desired product as free base or pharmacologically acceptable salt (method B, scheme 3, see the end of the description).

The original anthra[2,1-d] isothiazol-3,6,11(2H)-Trion was obtained in a known manner by treatment of 1-methylthio-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide by sulfurylchloride in the medium of anhydrous methylene chloride [15].

The invention is illustrated by the following examples.

Example 1. 2-[2-(Dimethylamino)ethyl] anthra[2,1-d] isothiazol-3,6,11(2H)-trione hydrochloride (4) (method A).

a) N-[2-(Dimethylamino)ethyl] -nitro-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide hydrochloride (2).


To a suspension of the acid chloride of 1-nitro-9,10-dioxo-9,10-dihydro-anthracene-2-carboxylic acid [15] (0,30 g, 0.95 mmol) in heterophase system benzene/water (1: 1, by vol.) under intensive stirring of 0.11 ml (0,088 g, 1.00 mmol) 2-(dimethylamino)ethylamine. The mixture is stirred for 2 h at 25oC, the precipitate is filtered off, washed successively with water (2 x 10 ml), 10% Hcl (2 x 10 ml) and ethanol (2 x 10 ml) and dried in air. Obtain 0.31 g of compound 2 (yield 82%). So pl. 282-285oC.

An NMR spectrum1H (DMSO-d6,-); 7,92-8,00 (m, 2 H, H-6, H-7); 8,09-8,23 (m, 2 H, H-5, H-8); 8,43 (d, 1 H, H-3, J=7,7); and 8.50 (d, 1 H, H-4, J=7,7); 9,54 (t, 1 H, NHCH2-, J=4,9); of 10.73 (ush, C, 1 H, N2H+).

Found (%): 56,73; N 4,51; Cl 8,64; N 10,51. C19H18ClN3O5.

Calculated (%): 56,51; N 4,49; Cl 8,78; N 10,41.

b) N-[2-(Dimethylamino)ethyl] -1-ethylthio-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (3)

To a suspension of amide 2 (0.74 g, to 1.83 mmol) in DMF (10 ml) was added potassium carbonate (0.27 g, 1.9 mmol) and stirred for 30 min at 25oWith, then add teletilt sodium (0.2 g of 2.38 mmol) and stirred for further 12 h at 25oC. the Reaction was poured into 10% hydrochloric acid (50 ml) and stirred for 30 minutes the precipitation is filtered off, washed with water (3 x 15 ml), recrystallized from a mixture of tetrahydrofuran (THF)/ethanol (1:2) and dried. Get 0,57 g of compound 3 (yield 81%). So pl. 161 - 162oC.

An NMR spectrum1H (DMSO-d6,, M. D., J, Hz): 1,10 (t, 3 H, SCH2CH3, J=7,7); of 2.25 (s, 6 H, -NMe2); 2,45 of $ 2.53 (m, 2 H, -CH2NMe2); 2,89 (kV, 2 H, SCH2CH3, J= 7,7); 3,34-of 3.43 (m, 2 H, NCH2-); 7,73 (d, 1 H, H-3, J=7,7); 7,86-of 7.97 (m, 2 H, H-6, H-7); 8,12-8,21 (m, 2 H, H-5, H-8); of 8.15 (d, 1 H, H-4, J=7,7); 8,59 (t, 1 H, NHCH2-, J=5,5).

Found (%): From 66.05; H Of 5.82; N 7,44; S To 8.41. C21


To a suspension of amide 3 (0,57 g, 1,49 mmol) in absolute CH2CL2(8 ml) under vigorous stirring add sulfurylchloride (0.15 ml, of 1.84 mmol). After a quick dissolve the precipitate in 5 to 8 seconds from the red solution begins with the deposition of the reaction product. The reaction mass is stirred for 1.5 h at 20oC, diluted with petroleum ether (50 ml), the precipitate is filtered off, washed with petroleum ether (3 x 15 ml), recrystallized from dimethyl sulfoxide (DMSO) and dried. Get to 0.23 g of compound 4 (yield 39%). So pl. 317-319oC.

An NMR spectrum1H (DMSO-d6,, M. D., J, Hz): 2,50 (s, 6 H, -NMe2); 3,42-of 3.53 (m, 2 H, -CH2NMe2); or 4.31 (t, 2 H, NCH2-, J=4,9); 7,93-of 8.04 (m, 2 H, H-8, H-9); 8,14-8,30 (m, 2 H, H-7, H-10); 8,21 (d, 1 H, H-4,J=7,7); of 8.37 (d, 1 H, H-5,J=7,7).

Found (%): 58,77; N To 4.52; Cl Of 8.95; N 7,21; S 8,07. With19H17lN3S.

Calculated (%): 58,68; N To 4.41; Cl 9,12; N 7,20; S 8,25.

Example 2. 2-[2-(Dimethylamino)ethyl] anthra[2,1-d1]isothiazol-3,6,11(2H)-Trion (5).


Hydrochloride 4 (0.5 g, 1,29 mmol) is suspended in 10 ml of water and with stirring was added sodium acetate (0.12 g, of 1.36 mmol), after which the entire precipitate goes into solution. The product is extracted from the water etilize is uume, the residue is dried and obtain 0.24 g of compound 5 (yield 53%). So pl. 168-170oC.

An NMR spectrum1H (CDCl3,, M. D., J, Hz): is 2.37 (s, 6 H, -NMe2); by 2.73 (t, 2 H, -CH2NMe2, J= 5,3); 4,06 (t, 2 H, NCH2-, J=5,3); 7,81-to $ 7.91 (m, 2 H, H-8, H-9); compared to 8.26 is 8.38 (m, 2 H, H-7, H-10); 8,30 (d, 1 H, H-4, J=6,5); 8,42 (d, 1 H, H-5, J=6,5).

Found (%): 64,92; N 4,51; N 8,02; S 9,12. With19H16About3S.

Calculated (%): 64,76; N 4,58; N 7,95; S 9,10.

Example 3. 2-[2-(Dimethylamino)ethyl] anthra[2,1-d] isothiazol-3,6,11(2H)-trione fumarate (6).


To a suspension of triona 5 (0.1 g, 0.28 mmol) in 5 ml of absolute methanol was added to 2.1 ml of 2% solution of fumaric acid (0,033 g, 0.28 mmol) in absolute methanol. The mixture was stirred at 35o1 h, the solvent is distilled off in vacuum, the residue is dried. Gain of 0.13 g of compound 6 (yield 98%). So different. >270oC.

An NMR spectrum1H (DMSO-d6,, M. D., J, Hz): 2,45 (s, 6 H, -NMe2); 2,74-2,96 (m, 2 H, -CH2NMe2); 4,08 (t, 2 H, NCH2-, J=4,5); 6,59 (s, 2 H, (CH)2(COOH)2); 7,93-8,08 (m, 2 H, H-8, H-9); 8,14-8,31 (m, 2 H, H-7, H-10); 8,21 (d, 1 H, H-4, J=7,7); of 8.37 (d, 1 H, H-5, J=7,7).

Found (%): 59,10; N Or 4.31; N 5,93; S Of 6.78. C23H20N2O7S.

Calculated (%): 58,97; N 4,30; N 5,98; S 6,84.

Example 4. 2-[2-(Dimethylamino,1 g, 0.28 mmol) in 5 ml of absolute methanol was added to 3.7 ml of 2% citric acid solution (0,058 g, 0.28 mmol) in absolute methanol. The mixture was stirred at 35o1 h, the solvent is distilled off in vacuum, the residue is dried. Obtain 0.15 g of compound 7 (yield 98%). So different. >270oC.

An NMR spectrum1H (DMSO-d6,, M. D., J, Hz): 2,62 (s, 6 H, -NMe2); to 2.65 (s, 4 H, (BUT)C(CH2)(COOH)3); 3,05-is 3.21 (m, 2 H, -CH2NMe2); 4,08-4,24 (m, 2 H, NCH2-); 3,19 (s, 2 H, (BUT)C(CH2)(COOH)3); 7,93-8,08 (m, 2 H, H-8, H-9); 8,14-of 8.27 (m, 2 H, H-7, H-10); 8,17 (d, 1 H, H-4, J=7,3); a 8.34 (d, 1 H, H-5, J=7,3).

Found (Percent): From To 55.42; H 4,12; N 5,23; S 5,91. C25H24N2O10S.

Calculated (%): 55,14; N. Of 4.44; N 5,14; S Of 5.89.

Example 5. 2-[2-(4-Morpholinyl)ethyl]anthra[2,1-d]isothiazol-3,6,11(2H)-Trion (11) (method B).

a) N-(2-Chloroethyl)-1-nitro-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (8).


To a suspension of the acid chloride of 1-nitro-9,10-dioxo-9,10-dihydro-anthracene-2-carboxylic acid [15] (0,30 g, 0.95 mmol) in heterophase system benzene/water (1:1, by vol.) under vigorous stirring successively added 2-chloroethylamine hydrochloride (0.12 g, of 1.03 mmol) and potassium carbonate (0.3 g, 2,17 mmol). The mixture is stirred for 2 h at 25oWith, sucked, industrial is t 0.28 g of compound 8 (yield 84%). So pl. 259-262oC.

An NMR spectrum1H (DMSO-d6,, M. D., J, Hz): 3,59 (m, 2 H, -CH2Cl); 3,74 (m, 2 H, NHCH2-); 7,92-8,02 (m, 2 H, H-6, H-7); 8,10-of 8.25 (m, 2 H, H-5, H-8); 8,17 (d, 1 H, H-3, J=7,7); 8,48 (d, 1 H, H-4,J=7,7); 9,34 (t, 1 H, NHCH2-, J=4,4).

Found (%): 57,01; N 3,21; Cl 9,74; N Of 7.96. With17H11lN2About5.

Calculated (%): 56,92; N 3,09; Cl 9,88; N 7,81.

b) N-(2-Chloroethyl)-1-methylthio-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (9).


To a suspension of amide 8 (0.5 g, of 1.39 mmol) in DMF (8 ml) was added diameterat sodium (0.12 g, 1,71 mmol) and stirred for 10 h at 20oC. the Reaction was poured into 10% model HC1 (30 ml) and stirred for 30 minutes the precipitation is filtered off, washed with water (3 x 15 ml), recrystallized from a mixture of THF/ethanol (1: 2) and dried. Get 0,37 g of compound 9 (yield 75%). So pl. 193-195oC.

An NMR spectrum1H (DMSO-d6,, M. D., J, Hz): to 2.42 (s, 3 H, -S3); 3,62 (m, 2 H, -CH2CL); with 3.79 (m, 2 H, NHCH2-); 7,76 (d, 1 H, H-3, J=7,2); a 7.85-7,98 (m, 2 H, H-6, H-7); 8,10-8,21 (m, 2 H, H-5, H-8); to 8.14 (d, 1 H, H-4, J=7,2); 8,98 (t, 1 H, NHCH2-, J=5,4).

Mass spectrum (ES, 70 eV), m/z (IRel,%): 361 [M]+/Cl37(4.1 per cent), 359 [M]+/Cl35(14,7%), 346 (11,9%, [M]+/CL37-Me), 344 (28,3%, [M]+/Cl35-Me), 32/Cl35-Cl35-H), 308 (100%, [M]+-Cl-Me-H).

Found (%): 60,12; N 3,93; Cl 9,72; N 3,90; S 8,86. C18H14ClNO3S.

Calculated (%): 60,08; N 3,92; Cl 9,85; 3,89 N; S 8,91.

a) 2-(2-Chloroethyl)anthra[2,1-d]isothiazol-3,6,11(2H)-Trion (10).


To a suspension of amide 9 (0.5 g, of 1.39 mmol) in absolute CH2CL2(8 ml) under vigorous stirring was added SO2Cl2(0.15 ml, of 1.84 mmol). The precipitate almost immediately dissolved and after 5-8 seconds from red solution begins with the deposition of the reaction product. The reaction mass is stirred for 2 h at 20oC, diluted with petroleum ether (30 ml), the precipitate is filtered off, washed with petroleum ether (3 x 10 ml), recrystallized from DMSO and dried. Obtain 0.33 g of compound 10 (yield 69%). So pl. 261-264oC.

An NMR spectrum1H (DMSO-d6,, M. D., J, Hz): 4,00 (t, 2 H, CH2Cl, J=4,7); 4,24 (t, 2 H, NCH2-, J=4,7); 7,93-8,03 (m, 2 H, H-8, H-9); 8,13-of 8.27 (m, 2 H, H-7, H-10); 8,18 (d, 1 H, H-4, J=5,9); at 8.36 (d, 1 H, H-5, J=5,9).

Found (%): 59,67; N 3,01; Cl To 10.62; N 4,37; S 9,51. C17H10ClNO3S.

Calculated (%): 59,39; N 2,93; Cl 10,31; N 4,07; S Was 9.33.

g) 2-[2-(4-Morpholinyl)ethyl]anthra[2,1-d]isothiazol-3,6,11(2H)-Trion (11).


To a suspension of isothiazolone 10 (0,30 g, 0,8 the number of potassium iodide (5-10 mg). The reaction mixture was stirred at 50-55oC for 48 h, poured into water, stirred for 30 min at 20oWith deposited precipitate is filtered off and chromatographic on a column of silica gel (L 40/100; eluent a mixture of toluene/ethyl acetate 2:1, then ethyl acetate). The fraction containing the target product, evaporated under reduced pressure, the residue is dried in air. Obtain 0.10 g of isothiazolone 11 (yield 31%). So pl. 208-212oC.

An NMR spectrum1H(CDCl3,, M. D., J, Hz): 2,53-of 2.72 (m, 4 H, -CH2Och2-); and 2.79 (t, 2 H, -CH2M(CH3)2-, J=4,8); 3,74-a 3.87 (m, 4 H, -CH2NCH2-); 4,08 (t, 2 H, NCH2-, J=4,8); 7,79-of 7.96 (m, 2 H, H-8, H-9); by 8.22-of 8.47 (m, 4 H, H-7, H-10, H-4, H-5).

Mass spectrum (ES, 70 eV), m/z (IRel,%): 394 (0,3%) [M]+308 (2,8%, [M]+-N(CH2CH2)2O), 294 (of 9.21%, [M]+-H2CN(CH2CH2)2O), 281 (6,1%, [M]+-H2CCHN(CH2CH2)2O), 264 (5,1%), 238 (15,8%), 113 (74,2%, H2CCHN(CH2CH2)20), 100 (100%, H2CN(CH2CH2)2O).

Found (%): 64,11; N To 4.62; N 7,22; S 8,17. C21H18N2O4S.

Calculated (%): 63,94; N 4,60; N 7,10; S 8,13.

Example 6. 2-[(Diethylamino)methyl]anthra[2,1-d]isothiazol-3,6,11-Trion(13), 2-[(1-piperidinyl)methyl] anthra[2,1-d] isothiazol-3,6,11-Trion (1 is ethylamino)methyl]anthra[2,1-d]isothiazol-3,6,11-Trion (13).

Isothiazolin 12 (0.3 g, 1.06 mmol) [15] is stirred in DMSO at 45-50oWith until dissolved, then the solution sequentially added diethylamine (0.15 ml, 0.11 g, 1.5 mmol) and 38% aqueous solution of formaldehyde (0.3 ml, 4.1 mmol). The reaction mass is stirred at 45-50oC for 6 h, the solution is cooled to 20oWith and leave it for 3 hours the Precipitated crystalline precipitate is filtered off, washed with ethanol (3 x 10 ml), dried on air. Obtain 0.14 g of the product 13 (yield 35%). So pl. 158-160oC.

An NMR spectrum1N (l3,, M. D., J, Hz): 1,20 (t, 6 H, N(CH2CH3)2, J=7,2); 2,80 (kV, 4 H, N(CH2CH3)2); is 4.85 (s, 2 H, NCH2NEt2); 7,81-a 7.92 (m, 2 H, H-8, H-9); 8,25-8,46 (m, 4 H, H-4, H-5, H-7, H-10).

Found (%): 65,72; N 5,01; N 7,72; S 8,68. C20H18N2O3S.

Calculated (%): 65,55; N. Of 4.95; N Of 7.64; S 8,75.

b) 2-[(1-Piperidinyl)methyl]anthra[2,1-d]isothiazol-3,6,11-Trion (14).

In the above described conditions of isothiazolone 12 (0.3 g, 1.06 mmol) and piperidine (0.15 ml, of 0.13 g, 1.5 mmol) get to 0.22 g of the product 14 (yield 55%). So pl. 199 -202oC.

An NMR spectrum1H (CDCl3,, M. D., J, Hz): 1,35-of 1.55 (m, 2 H, N(CH2CH2)2CH2); and 1.56 to 1.76 (m, 4 H, N(CH2CH2)2CH-4, H-5, H-7, H-10).

Found (%): 66,55; N To 4.62; N 7,45; S 8,51. C21H18N2O3S.

Calculated (%): 66,65; N 4,79; N 7,40; S Of 8.47.

a) 2-[(4-Morpholinyl)methyl)]anthra[2,1-d]isothiazol-3,6,11-Trion (15).

In the above described conditions of isothiazolone 12 (0.3 g, 1.06 mmol) and research (0,13 ml of 0.13 g, 1.5 mmol) to obtain 0.26 g of the product 15 (yield 63%). So pl. 230-232oC.

An NMR spectrum1H (CDCl3,, M. D., J, Hz): 2,69-to 2.85 (m, 4 H, N(CH2CH2)2O); 3,65-a 3.83 (m, 4 H, N(CH2CH2)2O); 4,74 (s, 2 H, NCH2N); 7,78-to 7.93 (m, 2 H, H-8, H-9); 8,21-8,46 (m, 4 H, H-4, H-5, H-7, H-10).

Found (%): 63,25; N 4,20; N 7,31; S 8,54. C20H16N2O4S.

Calculated (%): 63,14; N 4,24; N Of 7.36; S 8,43.

Example 7. Inhibition of aggregation of human platelets caused by derivatives anthra[2,1-d]isothiazol-3,6,11-trione.

The effect of the compounds of the present invention on the aggregation of human platelets was studied known turbidimetric method of born. This venous blood collected in the morning from healthy donors, was centrifuged at 450 g at room temperature in plastic dishes for 10 min, using as an anticoagulant sodium citrate. The supernatant, i.e. platelet-rich plasma was collected and centrifuged at 650 g in techenie 2,5-108cells/ml with dilution platelet-poor plasma was added and the resulting suspension in a cuvette with a volume of 0.5 ml. Concentration of ADP was selected in each experiment so that the aggregation was reversible and maximum accounted for 2 min after addition of ADP. In this experiment, the ADP at a final concentration of 12.5 μm initiated platelet aggregation, which was ~60%. The scattering suspension of platelets was measured using aggregometry developed in the laboratory of Bioorganic chemistry of biological faculty of Moscow state University. M. C. University. Study the connection (in the form of an aqueous solution, optionally containing DMSO 0.2%) was added to the sample prior to the introduction of the inductor aggregation (ADP).

The results obtained show that the compounds described in conditions inhibited platelet aggregation. The value of the concentration at which was achieved premaxillae inhibition (IC50for compounds 5, 11 and 14 were 4.5; 7 and 23 μm, respectively. Known analogue, according to the prototype, in similar conditions, had the value of the IC50equal to 57 μm.

Example 8. The impact of derivatives anthra[2,1-d]isothiazol-3,6,11-trione activity thiosalicylic enzymes.

a) Inguinalis the plasmid pBRP-HR and were cultured in a volume of 250 ml. Gene expression FROM induced addition of isopropylthio-D-galactoside. The enzyme was purified in a known manner using affinity chromatography on a column with Ni-N-tetraacetate. Per unit activity FROM the received amount of enzyme catalyzing the incorporation into DNA 1 nmole dNTP for 10 min at 37oC.

The mixture for the determination of enzyme activity contained 50 mm Tris-HCl, pH 8.0; 5 mm.

MgCl2, 75 mm KCl, 10 mm DTT, 0.05% of NP-40, 50 μm deoxynucleoside-5'-triphosphates (dATP, dGTP, dCTP, TTP), 2000000 cpm 32P-ATP, 1 μg of activated DNA I nm (volume of sample 20 µl). After 20-30 min at 37oTo the mixture were applied to the filters Whatman 3MM and washed 3 times with trichloroacetic acid (10, 5 and 5%). The filters were dried and the amount of incorporated label was determined by scintillation counter in toluene scintillator.

The investigated compounds were made in a sample in a 10 mm aqueous solutions (if necessary to increase the solubility was added dimethyl sulfoxide in an amount such that the concentration in the sample did not exceed 1-2%). Preincubation compounds with the enzyme were performed both in the absence and in the presence of DNA, the enzymatic reaction was started by adding to the sample deoxynucleoside-5'-triphosphates.

Poluchennoi concentration in samples equal 50-1000 μm, in the described conditions exert an inhibitory effect on the activity FROM. Thus, the activity of the enzyme in the presence of 500 μm of compound 5 in conditions without prior incubation amounted to 63.2 and 73,6% of the initial value (in the absence and presence of DNA, respectively). Pre-incubation in for 5-20 minutes the inhibitory activity of the compounds was increased, the activity was 55.3 and 45,1% of the initial value (in the absence and presence of DNA, respectively).

b) Inhibition of the activity of caspase-3.

In this work, we used rats of Wistar rats with average weight of 180-200 g For activated caspase-3 animals were injected intraperitoneally with bacterial lipopolysaccharide from E. coli in a dose of 20 µg/kg, 12 h after injection, rats were deceptional and held perfusion of the liver via the portal vein with 0.9% NaCl solution. In further liver homogenized in 5 volumes of buffer (100 mm HEPES, pH 7.4, 140 μm NaCl, 1 mm phenylmethylsulfonyl, 10 μg/ml Aprotinin, pepstatin and leupeptin) and centrifuged for 30 min at 13000g at 4oC. the Obtained supernatant was used for determining the activity of caspase-3.

The activity of caspase-3 was determined by known spectrophotometric method used,4, containing 200 μm Ac-DEVD-pNa and 0.1% CHAPS. The reaction was stopped by cooling the samples on ice. For deposition nerastvorim components, samples were centrifuged at 13000g for 10 min at 4oC. the Absorbance of pNa formed by enzymatic cleavage of Ac-DEVD-pNa was measured at 405 nm.

The compounds were dissolved in water (if necessary) was added DMSO to a final concentration of 10% in the sample). The obtained solutions of the compounds were incubated with supernatants liver in the above buffer. The influence of compounds on the activity of caspase-3 in the liver of rats was determined in two ways incubation: from pre-incubation with supernatants liver at room temperature for 30 min and without pre-incubation. Further, the activity of caspase-3 was determined as described above. As a known inhibitor of the enzyme (control) used 5 μm Ac-DEVD-CHO.

Determination of protein content in samples was carried out according to the method of Bradford.

The results show that the investigated derivatives anthra[2,1-d] isothiazol-3,6,11-trione at final concentrations in the samples, equal 30-300 μm, in the described conditions have inhibitory effect on the activity of caspase-3 in the liver of rats. So, the activity is about values. Pre-incubation for 30 min at 22oWith the inhibitory effect of this compound increased the activity of caspase-3 was 29.7% of the original value).

Example 9. The interaction of the derivatives anthra[2,1-d]isothiazol-3,6,11-trione with low molecular weight thiols
The study of the interaction of derivatives of anthra[2,1-d]isothiazol-3,6,11-trione with low molecular weight thiols (dithiothreitol and glutathione) was performed using spectrometry in the UV and visible spectral regions using the connection 5. The reaction mixture (3 ml) contained 85 μm compound 5, 100 μm glutathione or dithiothreitol, 25 mm Tris-Hcl (pH 7,6) or 100 mm KOH (pH 13) and 5% ethanol.

In the course of the experiment was not observed changes in the optical density of the reaction mixture in the described conditions at pH of 7.6 in the presence of dithiothreitol or glutathione, as well as at pH 13 in the presence of glutathione with the meanings specified wavelengths. At pH 13 in the presence of dithiothreitol there were considerable changes in the optical density of the reaction mixture at wavelengths corresponding to the absorption maxima of compounds 5 in the UV spectrum (see table). These changes in optical density were developed for 20 min and indicate the ATA show in the described conditions at physiological pH values derivatives anthra[2,1-d]isothiazol-3,6,11-trione do not enter into chemical reaction with low molecular weight thiols.

Example 10. Acute toxicity of the derivatives anthra[2,1-d] isothiazol-3,6,11-trione.

Acute toxicity of the compounds of the present invention was determined in a known manner by LD50using outbred mice of both sexes with average weight of 20 g at room temperature; standard diet and water was given ad libitum during the whole experiment; the mobility of the animals is not restricted. Solutions of the compounds in DMSO were injected using a sterile syringe intraperitoneally. After injection the animals were observed for 48 h after which time the mice were additionally observed within 72 h (none of the animals died within any additional period of time). The results obtained indicate that the values LD50for the studied compounds is not less than 30-50 mg/kg For well-known analogue of the definition of acute toxicity have not been conducted.

Derivatives anthra[2,1-d] isothiazol-3,6,11-trione inhibit platelet aggregation in vitro at concentrations lower than comply with the us in biochemical research for studying the structure and mechanism of action tiltability enzymes (for example, of HIV reverse transcriptase and caspase-3).

Thus, the derivative anthra[2,1-d]isothiazol-3,6,11-trione General formula (I) expand the range of effective platelet aggregation inhibitors and inhibitors tiltability enzymes.


Claims

1. Derivatives anthra[2,1-d]isothiazol-3,6,11-trione General formula I

characterized in that A - lowest alkylene; R1and R2(independent) - lower alkyl, or R1and R2together with the nitrogen atom form a six-membered saturated, a heterocycle, which can contain an additional heteroatom such as oxygen atom, and their pharmaceutically acceptable salts.

2. Derivatives anthra[2,1-d]isothiazol-3,6,11-trione under item 1, inhibiting platelet aggregation.

3. Derivatives anthra[2,1-d]isothiazol-3,6,11-trione under item 1 as inhibitors of thiol-dependent enzymes.

4. Derivatives anthra[2,1-d]isothiazol-3,6,11-trione under item 1 as inhibitors of HIV reverse transcriptase and caspase-3.

 

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