Benzimidazo[1,2-c][1,2,3]thiadiazol-7-sulfonamides as inhibitors of carbonic anhydrase and the intermediates for production thereof

The present invention relates to novel aromatic and heterocyclic compounds 5 sulfonamide derivatives which can be used in biomedicine as pharmaceutical active ingredients because of their inhibition of enzymes involved in disease progression. The invention also relates to novel intermediates required for the synthesis of target sulfonamides.

In the description of the present invention, enzymes include various metal (usually zinc) ion-containing proteins such as carbonic anhydrase and metalloproteinases.

Carbonic anhydrase (CA) is a zinc-containing enzyme that catalyses the reversible hydration of carbon dioxide. They are involved in essential physiological processes related to respiration, CCh / bicarbonate transport between lungs and metabolizing tissues, pH and CO2 homeostasis, electrolyte secretion in many tissues / organs, etc. To date, 15 forms of carbonic anhydrase yeast have been identified in the human body, with different cellular localization - 8 cytosolic forms, 4 membrane-bound 2 mitochondrial and 1 secreted. Two major classes of carbonic anhydrase inhibitors are known: 1) metal complexing anions and 2) sulfonamide-containing aromatic and heterocyclic inhibitors. Sulfonamide class CA inhibitors are widely used as therapeutic agents in the treatment of various diseases. This is because 15 carbonic anhydrase isoforms are found in many cells, tissues and organs where they perform important physiological functions. Another similar class of proteins, metalloproteinases, are proteolytic enzymes which, like carbonic anhydrase, are overexpressed at various stages of cancer development. Sulfonamide inhibitors have great potential also for inhibiting these metalloproteinases.

Because carbonic anhydrase is involved in many essential physiological processes, unregulated expression of CA isoforms leads to important pathological consequences. Therefore, regulation of CA activity through inhibition or activation offers interesting therapeutic perspectives.

Several diseases have been identified which are characterized by disruption of the carbon dioxide and bicarbonate exchange process leading to pH changes, disruption of ion transport, fluid secretion, etc. A classic example of such a disease would be glaucoma. And while CA inhibitors are commonly used to treat glaucoma, they have a much broader range of action. They can be used for edema, tetanus, altitude sickness, upper gastrointestinal ulcers.

chronic kidney failure, Parkinson's disease, epileptic seizures not affected by other medicines. In addition, CA inhibitors have been shown to be effective in the treatment of some cancers. For example, CA inhibitors have been shown to inhibit the growth of leukemia, melanoma, lung, ovarian, colon, kidney, prostate, breast, CNS cancer cells (C. Supuran et al. (2000) Eur. J. Med. Chem. 867-874). Specifically, carbonic anhydrase IX and XII are directly associated with cancer development; The use of kits based on specific inhibitors of CA IX is mentioned for the treatment and detection of anticancer and neoplastic states (WO 2004/048544). CA inhibitors have also been found to be useful diuretics in the treatment of patients suffering from edema and heart failure. In particular, inhibition of CA II activity is thought to be beneficial in reducing bone resorption. In addition, carbonic anhydrase in prokaryotes has been shown to be important for respiration, carbon dioxide transport and photosynthesis. Therefore, the idea was raised that carbonic anhydrase inhibitors could be used as antibiotics; ethoxazolamide (ethoxazolamide) has even been used to treat meningitis. Carbonic anhydrase inhibitors have also been reported to exhibit antimalarial effects (Merlin, C. Master, M. et al. (2003), J. Bacteriol. 185 (21). 6415-24; Pastorekov, S. Parkkila, S. et al. ( 2004), J. Enzyme Inhib. Chem. 19 (3): 199-229; WO 2005/107470).

The patent literature focuses on various heterocyclic sulfonamide derivatives, which are CA inhibitors and based drugs. Of these, the main focus is on thiophene sulfonamide derivatives. Thiophene derivatives condensed with other rings are contemplated: for example, thienothiopyran derivatives (US 7030250, US 5157129, US 5120757, US 5091409, etc.). One of the thienothiopyran derivatives is the known drug dorzolamide (dorzolamide), used alone or with other compounds for the treatment of glaucoma (U.S. Pat. No. 6,316,443, 6,248,735, LT 3368, etc.). Thienothiazine sulfonamides are another class of thiophene sulfonamide derivatives (e.g., U.S. Pat. No. 5,646,142, U.S. Pat. No. 5,444,448, U.S. Pat. Other known condensed thiophene sulfonamide derivatives are thienothiadiazine derivatives (US 5510347, US 5464831), thienothiophene derivatives (US 4929549, US 4894390, etc.), benzthiophene derivatives (US 4,788,192, US 4,668,697), thienopyridine derivatives (US 4,731,368), US 4798831), thienopyrrole derivatives (US 4751231). There are also a number of patents, especially prior to 1990, that have not been condensed with other rings of thiophene sulfonamide derivatives (US 5,378,703, US 5,240,923, US 4,847,289, US 4,929,637). In addition to thiophene derivatives, a number of thiazolosulfonamides have been patented (for example, US 5519040). Benzothiazole sulfonamide derivatives (US 5059613, US 4975447 et al.), Of which one compound (ethoxazolamide, ethoxazolamide) is a drug for the treatment of glaucoma, have been the most studied. Of the thiadiazole sulfonamides, two drugs, acetazolamide and methazolamide, have been synthesized, and sulfonamides of this class are quite extensively studied in the patent literature (US 2004/0146955, US 5,242,937, US 5,225,480, US 5,010,204, etc.).

Other classes of compounds are less widely studied.

The various substituted non-fused benzenesulfonamides are known and widely studied as

CA inhibitors, although not much patented data is available (US 2004/0146955; Mincione, F. Stamotti, M. et al. (2005), Biorg. Med. Chem. Lett. 15, 3821-3827; Poulsen, S.-A. Bomaghi, LF Healy, PC (2005), Biorg. Med. Chem. Lett. 15, 5429-33; US 4687855). Among these compounds are derived several medicinal products, such as dichlorophenamide (dichlorophenamide) and indisulam (indisulam), which are in the second phase of clinical trials as an anticancer agent.

In recent years, there has been interest in benzenesulfonamides, which are inhibitors of CA and COX-2 (cyclooxygenase 2) (e.g., US 2005/0222251, WO 2004/014352, WO

03/013655, etc.). These benzenesulfonamides also include COX-2 inhibitors, which are used as active substances in the medicinal products celecoxib (celecoxib), valdecoxib (valdecoxib), deracoxib (deracoxib). These products belong to the class of non-steroidal anti-inflammatory drugs and are used to treat arthritis and to relieve pain. Celecoxib-type substituted pyrazolylbenzenesulfonamides are quite extensively studied in anti-inflammatory activity (WO 95/15316).

The sulfonamide derivatives of the heterocyclic system, benzimidazo [1,2-c] [1,2,3] thiadiazole, are a new class of sulfonamides wherein the sulfonamide group is attached to the benzene ring of this fused three-membered system. As is known to the authors of the present invention, a three-membered fused heterocyclic system in which the sulfonamide group is attached to a benzene ring and such compounds have CA inhibition is not mentioned in the literature.

Condensed heterocyclic tricyclic systems having a sulfonamide group and CA inhibition have been described in the literature, but the sulfonamide group is attached to a heterocyclic ring (US 5681834, US 5334591, US 5308842, US 5235059, US 5175284, etc.).

Despite the fact that many different sulfonamides have been synthesized, the present pharmaceutical preparations have many disadvantages. One of the major drawbacks is that inhibition of carbonic anhydrase throughout the body and the use of sulphonamide inhibitors result in a variety of unexpected side effects, mostly due to their non-specific action on all CA isoforms and their toxicity.

The non-specific CA inhibitors currently used for clinical purposes cause various side effects. Systemic depressants are particularly toxic. They cause electrolyte imbalance drowsiness headache depression apathy anxiety nervousness fatigue, dizziness gastrointestinal irritability anarexia nausea thirst, constipation muscle weakness tremor hyper- and hypoglycemia renal pain and bone marrow dysuria

Therefore, the development of sulfonamide inhibitors specific for different isoforms or organ-selective remains a very important and important task.

The present invention provides novel sulfonamides having the general structural formula (I)

<img file="LT5504B_D0001.tif" />

wherein R is H, Cl, SCH3, SO2CH3, morpholine, thiophenyl, N (CH3) 2, piperidine, N-methyl15-piperazine, pyrrolidine, and wherein the sulfonamide group H2NO2S- is located at the 7 or 5, 6, 8 position.

The invention also relates to pharmaceutically acceptable salts of sulfonamides of general formula (I). These are all salts that do not lose activity relative to the parent compounds and do not produce harmful and undesirable effects. Such salts are obtained from compounds of the general structural formula I and basic nitrogen by mixing their solution with pharmacologically acceptable non-toxic organic and inorganic acids such as hydrochloric acid, butanoic acid, citric acid, tartaric acid, phosphoric acid, sulfuric acid and the like.

An exemplary embodiment of the present invention is said sulfonamides wherein the group H2NO2S- is present

7th position.

Specific examples of compounds of the invention are compounds selected from the group consisting of: 3-chlorobenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide 3-morpholinobenzimidazo [1,2-c] [1,2, 3] thiadiazole-7-sulfonamide 3-phenylsulfanyl-benzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide

3-methylsulfanylbenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide benzimidazole-1,2-c] [1,2,3] thiadiazole-7-sulfonamide and 3-methylsulfonylbenzimidazo [1,2-c] ] [1,2,3] Thiadiazole-7-sulfonamide having inhibition of carbonic anhydrase.

The present invention also provides novel intermediates as described below which may be used to prepare sulfonamides of general formula (I).

The novel compounds of the invention are prepared according to the following general synthesis schemes AG:

A) Scheme for preparation of 3-chlorobenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (compound 3).

<img file="LT5504B_D0002.tif" />

The synthesis of the starting material (compound 1) is described by: Tumkevicius, S. Labanauskas, L.

Bucinskaite, V. Brukstas, A. Urbelis, G. (2003), Tetrahedron Lett. 44, 6635-38. As the electrophilic substitution of thiadiazole 1 to position 7, treatment of thiadiazole 1 with chlorosulfonic acid affords the sulfonic chloride 2. The chlorine atom of the sulfonic chloride group of compound 2 is readily substituted with an amino group under ammonia in tetrahydrofuran to give the target sulfonamide 3 of general formula (I).

B) Scheme for preparation of 3-morpholine- and 3-phenylsulfanylbenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamides (compounds 4,5).

<img file="LT5504B_D0003.tif" />

The sulfonamides 4 and 5 are obtained from compound 3 by treatment with morpholine and thiophenol in ethanol. In the same manner, compounds of the general formula (I), substituted by RN (CH3) 2, piperidine, N-methylpiperazine, pyrrolidine groups can be obtained.

C) Scheme for preparation of 3-methylsulfanylbenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (Compound 9).

<img file="LT5504B_D0004.tif" />

First, thiadiazole (compound 1) is converted to thionium by action of thiourea 6. Synthesis of thionium 6 5 is described - Tumkevicius, S. Labanauskas, L. Bucinskaite, V. Brukstas, A. Urbelis, G. (2003), Tetrahedron Lett. 44, 6635-38, but the preparation of compound 6 using thiourea is not described in this article. The resulting thion 6 is reacted with methyl iodide to give the methylated derivative 7. Treatment of thiadiazole 7 with chlorosulfonic acid gives the sulfonic chloride

8th The chlorine atom of the sulfonic chloride group of compound 8 is readily converted to the amino group by treatment with ammonia in tetrahydrofuran to give the sulfonamide 9 of general formula (I).

D) Scheme for preparation of benzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (compound 12).

<img file="LT5504B_D0005.tif" />

Thiadiazole 1 is first treated with aqueous sodium iodide and acetic acid in 2-butanone.

The reaction yields a thiadiazole 10 which reacts with chlorosulfonic acid to form the sulfonic chloride 11. The chlorine atom of the sulfonic chloride group of compound 11 is readily converted to the amino group by treatment with ammonia in tetrahydrofuran to give the sulfonamide 12.

corresponding to general formula (I).

Ί

E) Scheme for preparation of 3-methylsulfonylbenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (Compound 15).

<img file="LT5504B_D0006.tif" />

First, the thiadiazole 7 is oxidized with acetic acid to give a mixture of 10 and 13. The mixture of compounds 10 and 13 is separated by chromatography. The thiadiazole 13 reacts with the chlorosulfonic acid to form the sulfon chloride 14. The chlorine atom of the sulfonate group of compound 14 is readily converted to the amino group by treatment with ammonia in dioxane to give the sulfonamide 15 of general formula (I).

F) Scheme for preparation of 3-dimethylaminobenzimidazo [1,2-c] [1,2,3] thiadiazole-6-sulfonamide (Compound 22).

<img file="LT5504B_D0007.tif" />

(5 (6) -Bromo-1H-benzimidazol-2-yl) methanol (16a, b) was synthesized according to the procedure described in the literature: Khan, MK Mohammady, A. Fauzia, AY (1972), J. Sci. and Ind. Res.

15, 11-12. (5 (6) -Bromo-1 H -benzimidazol-2-yl) methanol (16a, b) was aminated with 0-hydroxylamine sulfonic acid according to the procedure for water-soluble benzimidazoles:

rio) KapcKHH, A. Φ. Ky3tMeHK0, BB; ByMŪep, AA ΠετροΒ, 3. C. TepexoBa, MH Hmama, H. JI. HaHaBHH, IM (1989), XTC, 2,221. During the amination, a mixture of the two bromo substituted isomers 17, 18 was formed. Compounds 17, 18 could not be separated because their solubilities and Rf are the same. Treatment of sulfinyl chloride with a mixture of (1-amino-5-bromo- and 1-amino-6-bromo-1H-benzimidazol-2-yl) methanols (17, 18) gave a mixture of bromo-substituted thiadiazole derivatives 19, 20 which was separated by chromotography. Thiadiazole 22 can be obtained from compound 21 according to procedures for the preparation of aromatic sulfonyl chlorides and sulfonamides from their halides (Pandya, R. Murashima, T. Tedeschi, L. Barrett, AGM (2003),

J. Org. Chem., 68,8274-8276; Graham, SLScholz, TH (1986), Synthesis, 1031-1032;

Hamada, T.Yonemitsu, 0. (1986), Synthesis, 852-854).

G) Scheme for preparation of 3-dimethylaminobenzimidazo [1,2-c] [1,2,3] thiadiazole-5- and 8-sulfonamides (compounds 30.32).

<img file="LT5504B_D0008.tif" />

In a similar manner to 3-dimethylaminobenzimidazo [1,2-c] [1,2,3] thiadiazole-6-sulfonamide (22), thiadiazole derivatives having a sulfonamide group at the 5 and 8 positions can be obtained. In this case, the starting compound used is 3-bromobenzene-1,2-diamine (23).

obtained according to the procedure described in the literature: Sunder, S. Peet, NP (1979), J. Heterocycl.

Chem., 6, 33-37. 3-Bromobenzene-1,2-diamine (23) can be condensed with glycolic acid to form 4 (7) -bromo-1H-benzimidazol-2-yl) methanol analogous to 4 (7) -bromo-2 -methyl-1 H-benzimidazole under conditions (Dandegaonker, Recanker, (1961),

J. Karnatak. Univ. 6, 25, 29, 30). The following steps are carried out in the same manner as in the preparation of 35 dimethylaminobenzimidazo [1,2-c] [1,2,3] thiadiazole-6-sulfonamide (22) and the sulfonamides 30 and 32 of general formula (I) can be obtained.

To illustrate the properties of the resulting compounds, the following description is given:

FIG. 1 shows representative results of a calorimetric isothermal titration study of the binding of bovine II carbonic anhydrase to pentafluorobenzenesulfonamide (A) and compound of the invention (B);

FIG. 2 illustrates the stabilization of bovine II carbonic anhydrase (b-CAII) at different concentrations of the compound of the invention by fluorescence;

FIG. 3, which shows the melting point of bovine II carbonic anhydrase (b-CAII) (T_m) depending on the concentration of the compound of the invention (L_t) by fluorescence.

Specific examples of the preparation of the compounds of the invention, including specific intermediates for synthesizing the compounds of the invention (not described in the literature), are provided below. The following examples are provided to illustrate the scope of the invention without limiting the scope of the invention.

example. Intermediate 3-Chlorobenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonium chloride (Compound 2).

3 ml of CISO3H, cooled to -5 [deg.] C., are mixed with 0.3 g (1.43 mmol) of 3-chlorobenzimidazo [1,2-c] [1,2,3] thiadiazole (1) in portions, prepared according to known procedures. The reaction mixture was then stirred at room temperature for 24 h and poured onto ice. Extract with chloroform, wash the chloroform layer with H2O and dry over Na2SO4. Chloroform is evaporated on a rotary evaporator. The resulting compound is crystalline with a bright yellow color.

Yield: 0.38 g (86%), m.p. t. 184-185 ° C.

1 H NMR Spectrum (300 MHz, CDCl 3): 8.1 (1H, d, J = 9Hz, 5-H), 8.23 ​​(1H, dd, J = 2 and 9

Hz, 6-H), 8.94 (1H, d, J = 2Hz, 8-H).

The crude material obtained is used in the next step.

example. Preparation of 3-chlorobenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (Compound 3).

To a solution of 0.04 g (0.13 mmol) of 3-chlorobenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonic chloride (compound 2 according to the preceding example) and 2 ml of tetrahydrofuran is added 0.1 ml of 25% NH3 aqueous solution. The reaction mixture is stirred for 15 min, the resulting crystals are filtered, washed with aqueous NaHCO 3 and H₂Oh, crystallizes from acetic acid. The resulting compound is crystalline, orange in color.

Yield: 0.03 g (80%), m.p. t. 242-243 ° C.

1 H NMR Spectrum (300 MHz, DMSO-D₆): 7.49 (2H, s, NH₂), 8.01 (2H, s, 5-H and 6-H), 8.62 (1H, s, 8-H).

¹³C NMR spectrum (75 MHz, CDCl3)₃): 112,00, 121,59, 125,77, 127,34, 127,37, 136,18, 154,13,154,83.

Mass spectrum (m / z,%): M⁺ 288.100%, M⁺ 290,50 %.

C₈H₅C1N₄O₂S₂. Calculated: C 33.28%, H 1.75%, N 19.4 0%. Found: C, 33.39%; H, 1.8%; N, 19.63%.

example. Preparation of 3-morpholinobenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (Compound 4).

0.02 g (0.069 mmol) of 3-chlorobenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (Compound 3 according to the previous example), 0.012 g (0.14 mmol) of morpholine and 20 ml of ethanol the mixture is refluxed for 1.5 h then cooled to room temperature. The resulting crystals are filtered, washed with cold H₂Oh and crystallizes from acetic acid. The resulting compound is crystalline, orange in color.

Yield: 0.02 g (85%), m.p. 253-254 ° C.

1 H NMR Spectrum (300 MHz, DMSO-D₆): 3.9 (8H, s, (CH₂)₄), 7.41 (2H, s, NH₂), 7.88 (2H, s, 5-H and 6-H), 8.44 (1H, s, 8-H).

^I3C NMR Spectrum (75 MHz, CDCl 3): 50.38, 65.79, 111.6, 121.27, 124, 126.4, 135.15, 146.94, 151.7, 154.23.

C,₂H |₄N₅O₃S₂. Calculated: C 42.47%, H 3.86%, N 20.63%. Found: C, 42.56%; H, 3.94%; N, 20.47%.

example. Preparation of 3-phenylsulfanylbenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (Compound 5).

0.1 g (0.35 mmol) of 3-chlorobenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (Compound 3 according to Example 2), 0.039 g (0.35 mmol) of thiophenol and 50 mL the ethanol mixture is refluxed for 1.5h, then cooled. The ethanol is evaporated on a rotary evaporator, the resulting crystals are washed with NaHCO3&gt; aqueous solution and H₂Oh, crystallizes from acetic acid. The resulting compound is crystalline, orange in color.

Yield: 0.02 g (85%), m.p. 225-226 ° C.

Ή NMR spectrum (300 MHz, DMSO-D₆7.3-7.6 (5H, m, SC)₆H₅), 7.65 (2H, s, NH₂), 7.96 (2H, s, 5-H and 6-H), 8.59 (1H, s, 8-H).

¹³C NMR spectrum (75 MHz, CDCl3)₃): 112, 121,54, 125,43, 127,13, 129,9, 130,9, 131,39,131,97,132,51, 136,15, 154,76,155,7.

Ci₄HioN₄0₂S3. Calculated: C 46.39%, H 2.78%, N 15.46%. Found: C, 46.26%, H

2.87%, N, 15.37%.

example. Preparation of the intermediate benzimidazo [1,2-c] [1,2,3] thiadiazole-3-thione (compound 6).

A mixture of 0.5 g (2.38 mmol) of 3-chlorobenzimidazo [1,2-c] [1,2,3] thiadiazole (1) obtained according to a known procedure is boiled in 0.4 g (5.26 mmol) of thiourea and 20 ml of methanol, 5h. The reaction mixture is then cooled to room temperature, the crystals formed are filtered off, washed with cold methanol. The resulting crystals are dissolved in 0.2M NaOH, filtered and acidified to pH 5 with acetic acid; the resulting crystals are filtered and crystallized from dioxane. The resulting compound is crystalline with a bright orange color.

Yield: 0.34 g (69%), m.p. t. 236-237 ° C.

example. Preparation of Intermediate 3-Methylsulfanylbenzimidazo [1,2-c] [1,2,3] thiadiazole (Compound 7)

A mixture of 0.2 g (0.96 mmol) of benzimidazo [1,2-c] [1,2,3] thiadiazole-3-thione (compound 6 according to the previous example), 40 ml of methanol and 0.2 g (1.4 mmol) of iodomethane is boiled 0.5h. The solvent is evaporated on a rotary evaporator, the resulting crystals are washed with NaHCC&gt; 3 aqueous solution and H₂Oh, crystallizes from H₂O / ethanol (2: 1) mixture. The resulting compound is crystalline, orange in color.

Yield: 0.18 g (86%), m.p. t. 120-121 ° C.

1 H NMR Spectrum (300 MHz, DMSO-D₆/ CC1₄): 2.97 (3H, s, SCH₃), 7.26 (1H, t, J =

8Hz, 7-H). 7.51 (1H, t, J = 8Hz, 6-H), 7.77 (1H, d, J = 8Hz, 5-H), 8.09 (1H, d, J = 8Hz, 8-H) .

¹³C NMR spectrum (75 MHz, DMSO-D₆/ CC1₄): 18,49, 113,25, 120,33, 121,18, 127,66,

128,21, 134,79, 153,04, 153,63.

C₉H₇N₃S2. Calculated: C 48.85%, H 3.19%, N 18.99%. Found: C, 48.93%, H, 2.96%, N, 18.89%.

example. Preparation of intermediate 3-methylsulfanylbenzimidazo [1,2-c] [1,2,3] thiadiazole-75 sulfonium chloride (compound 8).

Immersed in C1SO when cooled to -5 ° C₃H under stirring forms 0.1 mg (0.45 mmol) of 3-methylsulfanylbenzimidazo [1,2-c] [1,2,3] thiadiazole (compound 7 according to the previous example). The reaction mixture was then stirred at room temperature for 24 h and poured onto ice. Extract with chloroform, wash the chloroform layer with H₂Oh and dries Na₂SO4. Chloroform is evaporated on a rotary evaporator. The resulting compound is crystalline with a bright yellow color.

Yield: 0.12 g (86%), m.p. 190 ° C.

1 H NMR Spectrum (300 MHz, CDCl 1)₃): 2.99 (1H, s, SCH₃), 8.05 (1H, dd, J = 0.6 and 9Hz, 5-H), 8.17 (1H, dd, J = 2 and 9Hz, 6-H), 8.90 (1H, dd) , J = 0.6 and 2Hz, 8-H).

The crude material obtained is used in the next step.

example. Preparation of 3-methylsulfanylbenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (Compound 9).

To a solution of 0.04 g (0.125 mmol) of 3-methylsulfanylbenzimidazo [1,2-c] [1,2,3] thiadiazole-7sulfon chloride (Compound 8 according to the previous example) and 2 ml of tetrahydrofuran was added 0.1 ml of 25% aqueous NH3 solution. . The reaction mixture is stirred for 15 min, the resulting crystals are filtered, washed with NaHCO₃ aqueous solution and H₂Oh, crystallizes from acetic acid. The resulting compound is crystalline, orange in color.

Yield: 0.02 g (53%), m.p. t. 260-261 ° C.

1 H NMR Spectrum (300 MHz, DMSO-D₆): 2.99 (1H, s, SCH₃), 7.47 (2H, s, NH₂), 8.00 (2H, s, 5-H and 6-H), 8.58 (1H, s, 8-H).

¹³C NMR Spectrum (75 MHz, DMSO): 18.58, 112.00, 121.47, 125.04, 126.69, 135.95,

138,72,154,00,154,98.

C₉H₈N4O₂S3. Calculated: C 35.99%, H 2.68%, N 18.65%. Found: C, 36.16%, H, 2.62%, N, 18.80%.

example. Preparation of Intermediate Benzimidazo [1,2-c] [1,2,3] Thiadiazole (Compound 10)

0.2g (0.95mmol) of 3-chlorobenzimidazo [1,2-c] [1,2,3] thiadiazole (1) obtained according to known procedure 0.9g (4.7mmol) of Na₂O, 10 ml of a mixture of acetic acid and 50 ml of 2-butanone is boiled for 5 h.

The solvent is then evaporated on a rotary evaporator and the resulting mass is added to Na₂S₂O3 aqueous solution and mix well. The resulting crystals are recrystallized from a mixture of H2O and ethanol (10: 1). The resulting compound is crystalline, orange in color.

Yield: 0.06 g (38%), m.p. t. 158-160 ° C.

1 H NMR Spectrum (300 MHz, CDCl 3): 7.34 (1H, t, J = 8Hz, 7H), 7.6 (1H, t, J = 8Hz, 6H), 7.94 (1H, d , J = 8Hz, 5-H), 8.2 (1H, d, J = 8Hz, 8-H), 8.47 (1H, s, 3-H).

¹³C NMR spectrum (75 MHz, CDCl3)₃): 113,29,116,76, 120,47, 121,15,127,71,128,28, 154,98,155,38.

Mass spectrum (m / z,%): M⁺ 175, 93%, M⁺-S 146, 77%, M⁺-S, CH, N 120, 57%.

CgHsNsS. Calculated: C 54.84%, H 2.88%, N 23.98%. Found: C 54.65%, H 2.37%,

N, 23.60%.

example. Preparation of Intermediate Benzimidazo [1,2-c] [1,2,3] Thiadiazole-7-Sulfon Chloride (Compound 11).

To 0.5 ml of CISO3H cooled to -5 ° C was added 0.06 g (0.34 mmol) of benzimidazo [1,2-c] [1,2,3] thiadiazole (compound 10 according to the previous example) in portions with stirring. The reaction mixture was then stirred at room temperature for 24 h and poured onto ice. Extract with chloroform, wash the chloroform layer with H2O and dry over NaaSCU, then evaporate the chloroform in a rotary evaporator. The resulting compound is crystalline with a bright yellow color.

Yield: 0.06 g (64%), m.p. t. 188-189 ° C.

1 H NMR Spectrum (300 MHz, CDCl 1)₃): 8.1 (1H, dd, J = 0.6 and 9Hz, 5-H), 8.24 (1H, dd,

J = 2 and 9 Hz, 6-H), 8.74 (1H, s, 3-H), 9 (1H, dd, J = 0.6 and 2 Hz, 8-H).

The crude material obtained is used in the next step.

11th example. Preparation of benzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (Compound 12).

To a solution of 0.06 g (0.22 mmol) of benzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfon chloride (compound 11 according to the preceding Example) and 2 ml of tetrahydrofuran was added 0.15 ml of 25% NH3 under stirring. aqueous solution. The reaction mixture is stirred for 15 min, the crystals formed are filtered off and washed

NaHCCb in aqueous solution and H2O, crystallizes from acetic acid. The resulting compound is crystalline yellow.

Yield: 0.039g (70%), m.p. t. 245-246 ° C.

1 H NMR Spectrum (300 MHz, DMSO-D₆): 7.46 (2H, s, NH₂), 7.98 (2H, s, 5-H and 6-H),

8.63 (1H, s, 8-H), 9.3 (1H, s, 3-H).

¹³C NMR Spectrum (75 MHz, DMSO): 112.08, 121.22, 122.69, 125.18, 125.94, 135.37, 155.3, 157.75.

C₈H₆N₄O₂S₂. Calculated: C 37.79%, H 2.38%, N 22.03%. Found: C 37.86%, H 2.41%, N 22.18%.

example. Preparation of intermediate 3-methylsulfonylbenzimidazo [1,2-c] [1,2,3] thiadiazole (13).

To a solution of 0.3 g (1.36 mmol) of 3-methylsulfanylbenzimidazo [1,2-c] [1,2,3] thiadiazole (compound 7 according to Example 6) in 7 ml of acetic acid was added 0.65 g of H₂O₂ 35% and keeps for five days at room temperature. The solvent is then evaporated on a rotary evaporator, the resulting mass is poured into aqueous NaHCO3 solution and mixed well. Obtains a mixture of compounds 10 and 13 in a ratio of 1: 3. The product mixture is partitioned by flash chromatography using a 15 cm high and 1 cm diameter column. Uses Silica gel 60 (0.04-0.063mm), Merck. Use ethyl acetate as eluent. The resulting compound 13 is crystalline, dark red in color.

Compound 10: Yield: 0.03g (1%), m.p. 158-160 ° C.

R f = 0.13 (ethyl acetate).

Yield of 3-methylsulfonylbenzimidazo [1,2-c] [1,2,3] thiadiazole (Compound 13): 0.19 g (56%), m.p. t. 186-187 ° C.

R f = 0.73 (ethyl acetate).

1 H NMR Spectrum (300 MHz, CDCl 3): 3.64 (3H, s, SO)₂CH₃), 7.47 (1H, t, J = 8Hz, 7-H),

7.71 (1H, t, J = 8Hz, 6-H), 8.03 (1H, d, J = 8Hz, 5-H), 8.24 (1H, d, J = 8Hz, 8-H) .

¹³C NMR spectrum (75 MHz, CDCl3)₃): 44,82,113,39,121,68,122,2, 128,35,129,86, 141,86,149,59,155,6.

C₉H7N₃O₂S₂. Calculated: C 42.67%, H 2.79%, N 16.59%. Found: C, 42.86%, H

2.75%, N, 16.48%.

example. Preparation of intermediate 3-methylsulfonylbenzimidazo [1,2-c] [1,2,3] thiadiazole-7sulfon chloride (Compound 14).

To 0.5 ml of CISO3H cooled to -5 [deg.] C., 0.05 g (0.2 mmol) of 330 methylsulfonylbenzimidazo [1,2-c] [1,2,3] thiadiazole (compound 13 according to the previous example) is added in portions.

The reaction mixture was then stirred at room temperature for 24 h and poured onto ice. The crystals formed are filtered off. The resulting compound is crystalline, orange in color.

Yield: 0.05 g (72%), m.p. 210 ° C.

1 H NMR Spectrum (300 MHz, DMSO): 3.73 (3H, s, SO)₂CH₃), 8.1 (1H, d, 9Hz, 5-H), 8 (1H, dd, J = 2 and 9Hz, 6-H), 8.42 (1H, s, 8-H).

The crude material obtained is used in the next step.

14th example. Preparation of 3-methylsulfonylbenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (15).

To a solution of 0.032 g (0.09 mmol) of 3-methylsulfonylbenzimidazo [1,2-c] [1,2,3] thiadiazole-7sulfon chloride (compound 14 according to the previous example) and 20 ml of dioxane is added 0.1 ml of 25% NH₃ aqueous solution. The reaction mixture is stirred for 0.5h, the resulting crystals are filtered off, washed with NaHCO₃ aqueous solution and H₂Oh, crystallizes from acetic acid. The resulting compound is crystalline, orange in color.

Yield: 0.018g (60%), m.p. t. 249-250 ° C.

1 H NMR Spectrum (300 MHz, DMSO-D₆): 3.69 (3H, s, SO₂CH₃), 7.56 (2H, s, NH₂),

8.1 (2H, s, 5-H and 6-H), 8.7 (1H, s, 8-H).

¹³C NMR Spectrum (75 MHz, DMSO): 44.96, 112.13, 121.7, 126.44, 126.95, 132.96,

137,24,152,79,155,73.

C₉HgN₄O₄S₃. Calculated: C 32.52%, H 2.43%, N 16.86%. Found: C 32.61%, H 2.44%, N 16.75%.

15th example. Intermediates (1-amino-5-bromo-1 H -benzimidazol-2-yl) methanol (compound 17) and (1-amino-6-bromo-1 H -benzimidazol-2-yl) methanol (compound 18) ) receipt

To 5.2 g (22.7 mmol) of (5 (6) -bromo-17H-benzimidazol-2-yl) methanol (compound 16a, b), heated to 40 ° C, was prepared according to a known procedure 4.8 g (72.8 mmol). KOH solution

40ml H₂While stirring, 6g (50mmol) of NH was added₂OSO₃H solution 15ml H₂Oh, neutralized NaHCO₃, preventing the temperature from rising above 40 ° C. After the exothermic reaction, the mixture was further heated at 40-50 ° C for 0.5h and cooled to room temperature. The resulting crystals are filtered and crystallized from H₂O. Obtains a mixture of compounds 17 and 18 in a 1: 1 ratio (from * H NMR spectrum). The resulting mixture of compounds is a crystalline white solid.

Overall yield: 3.7g (67%).

1 H NMR Spectrum (300 MHz, DMSO-D₆): 4.73 (4H, s, 2CH)₂), 5.43 (1H, s, OH), 5.45 (1H, s, OH), 6.01 (2H, s, NH)₂), 6.03 (2H, s, NH₂), 7.31 (1H, d, J = 9Hz, ArH), 7.39 (1H, d,

J = 9Hz, ArH), 7.46 (1H, d, J = 9Hz, ArH), 7.53 (1H, d, J = 9Hz, ArH), 7.67 (1H, s, ArH).

7.73 (1H, s, ArH).

¹³C NMR spectrum (75 MHz, DMSO-D₆): 55,81, 55,81, 112,48, 113,46, 114,15,

114,98,121,46,122,01, 124,93, 125,36, 135,73,137,78,139,62, 141,88, 156,17, 156,48.

IR (ν, cm ·): 3350, 3313, 3184, 3120 NH₂.

C₈H₈BrN₃O. Calculated: C 39.67%, H 3.31%, N 17.36%. Found: C, 39.88%, H

3.52%, N, 17.46%.

example. The intermediates 3-chloro-6-bromobenzimidazo [1,2-c] [1,2,3] thiadiazole (compound 19) and 3-chloro-7-bromobenzimidazo [1,2-c] [1,2,3]. yielding thiadiazole (compound 20)

A mixture of 0.5 g (2.1 mmol) of (1-amino-5-bromo-177-benzimidazol-2-yl) methanol (a compound according to the preceding example) and (1-amino-6-bromo-1 H -benzimidazole- 2-yl) methanol (compound according to the previous example) is boiled with 5 ml SOC1₂ 0.5h. Then SOC1₂ the excess is evaporated on a rotary evaporator and the resulting crystals are washed with NaHCO₃ aqueous solution and H₂O. Obtains a mixture of 19 and 20 in a 1: 1 ratio. The product mixture was subjected to vacuum chromotography. The 7cm high and 2.5cm diameter filter is filled with 5cm high silica gel under vacuum and pressure. The solvent is dissolved in a volatile solvent, the silica gel is added as much as the chromatographic substance and the solvent is evaporated off on a rotary evaporator. The dry silica gel with the material is evenly distributed on the prepared column and compressed under vacuum. Then, after the vacuum has been added, the eluent is poured and the fractions collected in 10 ml. The purity of the fractions is checked by thin layer chromatography. Evaporate the solvent on a rotary evaporator. Uses Silica gel 60, Fluka. It uses a mixture of benzene and ethyl acetate in the eluent, gradually increasing the ethyl acetate content. Initially uses benzene and then adds 2% ethyl acetate.

Overall yield: 0.36 g (60%) (crystallizes from methanol).

20: Yield: 0.15 g (25%), m.p. t. 193-194 ° C.

R f = 0.47 (ethyl acetate: benzene = 1: 1).

1 H NMR Spectrum (300 MHz, DMSO-D₆): 7.7 (1H, dd, J = 2 and 9Hz, 6-H), 7.79 (1H, dd, J = 0.5Hz and 9Hz, 5-H), 8.46 (1H, dd, J = 0.5 and 2Hz, 8-H).

¹³C NMR spectrum (75 MHz, DMSO-D₆): 112,76, 116,24, 123,03, 126,61, 129,36,

131,66,151,72,152,49.

C₈H₃BrCIN₃S. Calculated: C 33.30%, H 1.05%, N 14.56%. Found: C 33.56%, H 1.15%, N 14.63%.

Yield of 3-chloro-6-bromobenzimidazo [1,2-c] [1,2,3] thiadiazole (Compound 19): 0.15 g (25%), m.p. t. 153-154 ° C.

R f = 0.52 (ethyl acetate: benzene = 1: 1).

1 H NMR Spectrum (300 MHz, DMSO-D₆): 7.45 (1H, dd, J = 2 and 9Hz, 7-H), 8.07 (1H, d,

J = 2Hz, 5-H), 8.19 (1H, d, J = 9Hz, 8-H).

¹³C NMR spectrum (75 MHz, DMSO-D₆): 115,36, 121,57, 123,38, 123,77, 127,76,

129,38,152,77,154,55.

C₈H₃BrClN₃S. Calculated: C 33.30%, H 1.05%, N 14.56%. Found: C 33.01%, H 1.33%, N 14.36%).

example. Preparation of 6-bromo-3-dimethylaminobenzimidazo [1,2-c] [1,2,31-thiadiazole (Compound 21).

A mixture of 2g (6.9mmol) of 3-chlorobenzimidazo [1,2-c] [1,2,3] thiadiazole (compound 19 according to the previous example), 0.94g of 33% aqueous dimethylamine in 100ml of ethanol is refluxed for 3h, then the solvent is evaporated steamer. The resulting crystals are filtered, washed with cold H2O and crystallized from methanol. The resulting compound is crystalline, orange in color.

Yield: 1.6 g (78%).

example. Preparation of 3-dimethylaminobenzimidazo [1,2-c] [1,2,3] thiadiazole-6-sulfonamide (Compound 22).

To a cooled solution of -70 ° C was added 3ml (7.37mmol) of a solution of 2g (6.7mmol) of 6-bromo-3-dimethylaminobenzimidazo [1,220c] [1,2,3] thiadiazole (Compound 21 in the previous example) and 10ml of tetrahydrofuran. ) Of a 2.5M solution of n-butyllithium in hexane. The mixture is stirred for 6h at -70 [deg.] C., then the SO2 gas is injected into the mixture until the solution shows a basic medium. The solution is then stirred at room temperature for 1 h, added to 10 ml of hexane to give lithium sulfinate, which is filtered off. To a suspension of crude lg (3.5mmol) salt was added 15ml of anhydrous hexane at 0 ° C in portions

A solution of 0.47 g (3.5 mmol) of sulfonyl chloride in 7 mL of anhydrous hexane was filtered off. The resulting crystals are washed with NaHCO₃ solution and extracted with chloroform. The chloroform layer is dried over Na₃SO₄ and chloroform is evaporated on a rotary evaporator. The crude sulfonyl chloride is dissolved in a small amount of tetrahydrofuran and mixed with 0.2 ml of 25% NH₃ aqueous solution. The reaction mixture is stirred for 15 min, the resulting crystals are filtered, washed with NaHCO₃ aqueous solution and H2O, crystallizes from acetic acid.

The resulting compound is crystalline, orange in color.

Yield; 0.6 g (30%).

Inhibition of the activity of carbonic anhydrase (as with all enzymes) is measured by observing the retardation of their catalyzed reaction. Carbonic anhydrases are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide:

CO₂ + H₂O θ HCOj + H *

Inhibition of this reaction can be monitored by measuring the carbon dioxide uptake of bicarbonate formation and pH changes in the medium (Krebs, J. F. and CA. Fierke, (1993), J. Biol. Chem. 268 (2), 948-54). Inhibitors that bind to the active center of carbonic anhydrase compete with the enzyme substrate and inhibit the catalytic reaction. All sulfonamides bind at the active center and inhibit this reaction. In their case, inhibition is equivalent to binding (Chakravarty, S. and KK Kannan, (1994), J. Mol. Biol. 243 (2), 298-309; Lindskog, S. (1997) Pharmacol. Ther. 74 (1), 1-20; Baird, TTJr. et al. (1997), Biochemistry, 36 (9), 2669-78). However, their binding and, at the same time, their inhibition strengths vary greatly. The specificity of the various sulfonamides also varies greatly, that is, to the extent that the same sulfonamide inhibits different carbonic anhydrase (Di Fiore, A. et al. (2005), Bioorg. Med.

Chem. Lett. 15 (7), 1937-42; Lee, DA and EJ Higginbotham, (2005), Am. J. Health Syst. Pharm. 62 (7), 691-9; Matulis, D. et al. Biochemistry, (2005), 44 (13), 5258-66; Ozensoy, O. et al. (2005), Bioorg. Med. Chem. Lett. 15 (21), 4862-6; Shank, RP et al. (2005), Epilepsy Res. 63 (2-3), 103-12; Simone, GD et al. (2005), Bioorg. Med. Chem. Lett. 15 (9), 2315-20). Binding of sulfonamides to carbonic anhydrase is measured by a number of standard methods.

The most commonly used methods are isothermal titration calorimetry, surface plasmon resonance, ultracentrifugation (Myszka, DG et al. (2003), J. Biomol. Tech. 14 (4), 247-69). Specificity is determined by measuring the coupling with various carbonic anhydrides and by measuring the "true" coupling constants (Matulis, D. and MJ Todd, (2004), Trends in Biocalorimetry).

example. Evaluation of CA inhibition and binding specificity by isothermal titration calorimetry.

Calorimetry of isothermal titration measured the binding of carbonic anhydrase forms of bovine II (bCA II) and human I (hCA I) to sulfonamide inhibitors of the invention 330 chlorobenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (compound 3). , 3-morpholinobenzimidazo- [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (Compound 4), 3-methylsulfanylbenzimidazo [1,2-c] [1,2,3] -thiadiazole-7 -Sulfonamide (Compound 9) during heat release and compared with the known carbonic anhydrase inhibitor trifluoromethanesulfonamide (TFMSA), binding of acetazolamide (AZM), ethoxazolamide (EZA), pentafluorobenzenesulfonamide (PFS) to the same proteins.

To illustrate the results, FIG. Fig. 1 presents specific results of the isothermal titration of bovine carbonic anhydrase to pentafluorobenzenesulfonamide (A) and 3-morpholinobenzimidazo [1,2-c] [1,2,3] thiadiazole-75 sulfonamide (4) (B) by calorimetry.

Observed coupling constants for carbonic anhydrase - sulfonamide ligand binding were found to be quite similar for all compounds tested (10⁶ -10⁷ M '¹) queues. Based on the results obtained, the "true" coupling constants, which are independent of the thermal effects of the protonization-deprotonation reactions occurring during the coupling, were calculated. These results indicate that the studied Ugandans have a distinctive centrally recognized activity of carbonic anhydrase. According to the "true" binding constants of carbonic anhydrase-sulfonamide ligands, the above compounds 3, 4, 9 (7x10⁸ - 6* 10⁹ M '¹). The specificity (specificity) of the studied sulfonamide carbonic anhydrase inhibitors for the active site of the protein is in the following order. The numbers above the&gt; mark indicate how many times the specificity is to the left of the suppressor:

l-2x l-2x 4-10x 3-5x 2x l-2x&gt; 3&gt; 9&gt; EZA&gt; PFS&gt; AZM&gt; TFMSA

As can be seen from the results obtained, the compounds of the invention, namely compounds 3, 4, 9, could be good starting compounds for the development of sulfonamide inhibitors of certain carbonic anhydrase isoenzymes - they bind to carbonic anhydrase sufficiently strongly (Kb._steb ~ 10⁶ M '¹) and uniquely identifies the pocket of the active site of the protein ("true" Kb ~ 10⁹ M '¹).

example. Evaluation of CA inhibition and binding constants by fluorescence.

In addition to isothermal titration calorimetry, the interaction of inhibitors with carbonic anhydrase was investigated by fluorescence using l-aniline-8-naphthalene sulfonate (ANS) as a fluorescence tag, observing an increase in the melting point of the protein in the presence of a protein-binding ligand. The interaction of bovine carbonic anhydrase II (bCAII) with sulfonamide inhibitors according to the invention was investigated, namely, 3-chlorobenzimidazo [1,230c] [1,2,3] thiadiazole-7-sulfonamide (3), 3-morpholinobenzimidazo [1,2-c]. ] [1,2,3] thiadiazole-7-sulfonamide (4), 3-methylsulfanylbenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (9).

For the purpose of illustration, FIG. Figures 2 and 3 show characteristic results of melting of b-CA II with 3morpholine-benzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (4) and determination of the coupling constant. FIG. 2 shows the stabilization of bovine carbonic anhydrase II (b-CAII) at different concentrations of the compound of the invention 3-morpholinobenzimidazo [1,2-c] [1,2,3] thiadiazole-7sulfonamide (4) (0 μΜ, 3,125 μΜ, 6.25 μΜ). , 12.5 μΜ, 25 μΜ 50 μΜ). FIG.

shows the melting point of bovine II carbonic anhydrase (b-CAH) [T_m) depending on the concentration of the compound of the invention 3-morpholinobenzimidazo [1,2-c] [1,2,3] thiadiazole-75 sulfonamide (Lt). The dots represent the experimental data through which the theoretical curve is plotted. Binding constant of 3-morpholinobenzimidazo [1,2-c] [1,2,3] thiadiazole-7-sulfonamide (4) Kb = 4.5 χ 10⁵ M '¹, dissociation constant K &lt;j = 2.2 μΜ, denaturation enthalpy A_uH-rr = 150 kcal / mol, protein concentration P_t = 3.4 μΜ.

The obtained data lead to the conclusion that the coupling constants measured by carbonic anhydrase - sulfonamide ligand binding were quite similar for all compounds of the invention (10).⁵-10⁶M '¹).

Newly synthesized sulphonamides of general structural formula (I)

<img file="LT5504B_D0009.tif" />

have at least the potency of existing carbonic anhydrase inhibitors used as medicaments, but exhibit a significantly higher specificity that can solve the present problem of non-specific inhibition.

1. Benzimidazo [1,2-c] [1,2,3] thiadiazole sulfonamides of general formula (I)

DEFINITION OF INVENTION