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1. (WO2008016288) BENZIMIDAZO[1,2-C][1,2,3]THIADIAZOL-7-SULFONAMIDES AS INHIBITORS OF CARBONIC ANHYDRASE AND THE INTERMEDIATES FOR PRODUCTION THEREOF
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BENZIMIDAZO[l,2-C][l,2,3]THIADIAZOL-7-SULFONAMIDES AS INHIBITORS OF CARBONIC ANHYDRASE AND THE INTERMEDIATES FOR PRODUCTION THEREOF

Field of the Invention
The present invention describes novel aromatic and heterocyclic sulfonamide derivatives, potentially useful in biomedicine as active ingredients of pharmaceutical preparations because of their ability to inhibit enzymes participating in disease progression. The invention also relates to new intermediate compounds required for the synthesis of target sulfonamides.
The enzymes in this description of the invention include different metal (mostly zinc) ion-possessing proteins, such as carbonic anhydrases and metalloproteinases.

Background of the Invention
Carbonic anhydrases (CA) are zinc-containing enzymes which catalyze reversible reaction of carbon dioxide hydration. These enzymes participate in essential physiological processes related to respiration, CO2/bicarbonate transport between lungs and metabolizing tissues, pH and CO2 homeostasis, electrolite secretion in many tissues/organs, etc. To date there are 15 carbonic anhydrase isozymes identified in humans - 12 catalytically active and 3 inactive, so called carbonic anhydrase related proteins. The 12 active isoforms have different subcellular localization - 5 of them are cytosolic, 4 - membrane bound, 2 mitochondrial and 1 - secreted. There are two major classes of carbonic anhydrase inhibitors: 1) metal-complexing anions; 2) aromatic and heterocyclic inhibitors possessing sulfonamide group. Sulfonamide-class carbonic anhydrase inhibitors are widely used as therapeutic agents for treatment of various diseases, since the 15 carbonic aanhydrase isozymes are widely distributed in most of the cells, tissues and organs where they are responsible for essential physiological functions. Another similar protein class is metalloproteinases, proteolytic enzymes, which are characterized by increased expression during various steps of cancer progression. Sulfonamide inhibitors have a great potencial for the inhibition of metalloproteinases.
Carbonic anhydrases participate in many essential physiological processes, therefore the increased activity or expression of different CA isoforms results in significant pathological outcomes. Therefore the regulation of CA catalytic activity by means of inhibition or activation proposes a therapeutic perspective.
There are several diseases with the characteristic disbalance of the interconversion between carbonic dioxide and bicarbonate resulting in pH alteration, disturbance of ion transport, fluid secretion, etc. A classical example of such a disease is glaucoma. While most often carbonic anhydrase inhibitors are used as antiglaucoma agents, they are also employed for treatment of such diseases as edema, hydropsy, altitude sickness, upper gut ulcer, chronic kidney deficiency, Parkinson's disease, and epilepsy seizures which are not affected by other drugs. It was noticed that CA inhibitors are effective in the treatment of certain cancers. For instance, it was identified that CA inhibitors suppress the growth of leukemia, melanoma, lung, ovarian, colon, kidney, prostate, breast, and CNS cancer cells (C. Supuran et al. (2000), Eur. J. Med. Chem. 35, 867-874). Namely, the carbonic anhydrases IX and XII are directly related to cancer development. The use of CA IX-specific inhibitor set for detection and treatment of precancer and neoplastic state is described in (WO 2004/048544). It was established that CA inhibitors are useful diuretics for the treatment of patients which suffer from oedema and heart deficiency. It is supposed that inhibition of the CA II activity could be useful for the diminishment of the bone resorption. It was shown in prokaryotes that the carbonic anhydrases are essential for respiration, carbon dioxide transport and photosynthesis. Therefore it was hypothesized that carbonic anhydrase inhibitors could be used as antibiotics. Ethoxzolamide was even used for the treatment of meningitis. It was noticed that carbonic anhydrase inhibitors possess an antimallarial activity. (Merlin, C. Master, M. et al. (2003), J. Bacteriol. 185(21), 6415-24; Pastorekova, S. Parkkila, S. et al. (2004), J. Enzyme InMb. Med. Chem. 19(3), 199-229; WO 2005/107470).
Significant consideration in the patent literature is given to various heterocyclic derivatives of sulfonamides which are carbonic anhydrase inhibitors and are the basis for pharmaceutical agents. For example, various thiophene derivatives are widely analyzed. Thienothiopyrane derivatives as thiophene derivatives condensed to the other rings are described in ( US 7030250, US 5157129, US 5120757, US 5091409 and like). One of the thienothiopyrane derivatives is known as the pharmaceutical agent dorzolamide, which is used alone or in combination with other compounds for glaucoma treatment (US 6316443, 6248735, LT 3368, and like). Thienothiazinesulfonamide is the other class of thiophene sulfonamide derivatives (for instance US 5646142, US 5424448, US 5093332, US 5538966, and like). One of such derivatives, namely brinzolamide is an antiglaucoma agent. Other noted condensed derivatives of thiophene sulfonamides are thienothiadiazine derivatives (US 5510347, US 5464831), thienothiophene derivatives (US 4929549, US 4894390 and like), benzenethiophene derivatives (US 4788192, US 4668697), thienofurane derivatives (US 4798831), thienopyridine derivatives (US 4731368), thienopyrrole derivatives (US 4751231). A number of thiophene sulfonamide derivatives, non-condensed to other rings, were also patented, especially before 1990 (US 5378703, US 5240923, US 4847289, US 4929637 and like). Besides the thiophene derivatives a number of thiazole sulfonamides (for instance US 5519040) were patented. Primarily the benzothiazolesulfonamide derivatives were described (US 5059613, US 4975447 and like). One such compound, ethoxzolamide is antiglaucomal drug. Sulfonamides of thiadiazole class are still widely analyzed in patent literature (US 2004/0146955, US 5242937, US 5225424, US 5055480, US 5010204 and like). Two of thiadiazolesulfonamides are used as pharmaceutical agents - acetazolamide and methazolamide. Other classes of compounds are not analyzed in such a detail.
Various substituted non-condensed benzenesulfonamides are well-known and widely analyzed as CA inhibitors, although not much data is patented (US 2004/0146955; Mincione, F. Starnotti, M. et al. (2005), Biorg. Med. Chem. Lett. 15, 3821-3827; Poulsen, S.-A. Bornaghi, L. F. Healy, P. C. (2005), Biorg. Med. Chem. Lett. 15, 5429-33; US 4687855). Several pharmaceutical agents, for example, dichlorophenamide and indisulam, were based on the above mentioned compounds. Indisulam is presently being tested as an anticancer agent in phase II clinical trials.
During the last several years significant attention was shifted towards benzenesulfonamides which are inhibitors of CA and COX-2 (cyclooxygenase 2) (for instance US 2005/0222251, WO 2004/014352, WO 03/013655 and like). COX-2 inhibitors such as celecoxib, valdecoxib, and deracoxib are used as active materials for pharmaceutical agents. These preparations belong to the class of antiphlogistic non-steroid drugs used for arthritis treatment and painkilling. The antiphlogistic effect of Celecoxib-type substituted pyrazolylbenzenesulfonamides is widely analyzed (WO 95/15316).

Sulfonamide derivatives of heterocyclic system benzimidazo[l,2-c][l,2,3]thiadiazole are sulfonamides of the new class where sulfonamide group is attached to the benzene ring of the condensed three-ring system. As far as known to the authors of this invention, there are no mention in the literature about any three-ring condensed heterocyclic system with sulfonamide group attached to the benzene ring of this condensed system and such compound activity towards CA inhibition.
There are descriptions in the literature of other three-ring condensed heterocyclic systems with sulfonamide group attached to the heterocyclic ring and such compound possession of CA inhibitory activity (US 5681834, US 5334591, US 5308842, US 5235059, US 5175284 and like).
Despite the fact that a large number of different sulfonamides have been synthesized to date, the available pharmaceutical agents created on the basis of these sulfonamides have a number of shortcomings. One of the main shortcomings is the non-selective inhibition of all carbonic anhydrases throughout the whole body. This results in various unexpected side effects, mostly because of non-specific inhibition of all CA isoforms and their toxicity.
Presently clinically used CA inhibitors, when acting non-specifically, cause a number of side-effects. Especially toxic are systemic inhibitors. They cause electrolyte disbalance, drowsiness, head-ache, depression, apathy, malaise, irritability, nervousness, fatigue, gut irritability, anorexia, nausea, thirst, obstruction, muscle weakness, tremor, hyper- and hypoglycaemia, kidney pain, disuria, bone marrow depression, metabolic acidosis and other.
Therefore, the creation of isoform-specific or organ-selective sulfonamide inhibitors is still an important task.

Summary of the Invention

This invention describes new sulfonamides with general structural formula (I)



(D where R is H, Cl, SCH3, SO2CH3, morpholine, thiophenyl, N(CH3)2, piperidine, N-methylpiperazine, pyrrolidine, and the sulfonamide group H2NO2S- is in 7 position or the 5, 6, or 8 position.
The objects of the invention are also the non-toxic, pharmaceutically acceptable salts of the sulfonamides of general formula (I). They include all salts which retain activity comparable to original compounds and do not attain any harmful and undesirable effects. Such salts are obtained from compounds with general structural formula (I) and basic nitrogen, by mixing their solution with pharmacologically acceptable non-toxic organic and inorganic acids, such as hydrogen chloride, butane diacid, citric acid, tartaric acid, phosphoric acid, sulphuric acid and other.
The examples of the implementation of the invention are compounds with sulfonamide group (H2NO2S-) in the 7th position.
Examples of the invented compouds are selected compounds from the group comprising:
3-chlorobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide;
3-morpholinbenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide;
3 -phenylthiobenzimidazo [ 1 ,2-c] [ 1 ,2,3]thiadiazol-7-sulfonamide;
3-methylthiobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide;
benzimidazo[ 1 ,2-c] [ 1 ,2,3]thiadiazol-7-sulfonamide;
3-methylsulfonylbenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide,
where all above listed compounds exhibit CA inhibitor properties.
The new intermediate compounds described below which could be used for the synthesis of sulfonamides with general formula (I) are also the subject of the invention.

Detailed Description of the Invention

New compounds of the invention can be obtained according to general synthesis schemes A-G:

A) the scheme of 3-chlorobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide (compound 3) synthesis.


Synthesis of the starting material (compound 1) is described in: Tumkevicius, S. Labanauskas, L. Bucinskaite, V. Brukstus, A. Urbelis, G. (2003), Tetrahedron Lett. 44, 6635-38. Electrophylic substitution at the thiadiazole 1 goes to the 7th position; therefore using chlorsulfonic acid the sulfonyl chloride 2 is obtained. Chlorine atom of the sulfonyl chloride group of the compound 2 is easily exchanged with the amino group by applying aqueous ammonia in tetrahydofurane, obtaining the compound 3 with general structural formula (I).

B) the scheme of 3-morpholin- and 3-phenylthiobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide (compounds 4, 5) synthesis.


Sulfonamides 4 and 5 are obtained from compound 3 using morpholine and thiophenol in ethanol. The compounds with general formula (I) and substituents R - N(CH3)2, piperidine, N-methylpiperazine, pyrrolidine groups could be synthesyzed in the same way.

C) the scheme of 3-methylthiobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide

(compound 9) synthesis.

2ekv. (NH2)2CS, MeOH CH3I, MeOH





ClSO3H


Thiadiazole is first treated with thiourea leading to thione 6. A different method for synthesis of the thione 6 is described in: Tumkevicius, S. Labanauskas, L. Bucinskaite, V. Brukstus, A. Urbelis, G. (2003), Tetrahedron Lett. 44, 6635-38. However, obtaining the compound 6 through thiourea is not described in the above mentioned manuscript. The obtained thione 6 reacts with methyl iodide producing methylated derivative 7. Reaction of thiadiazole 7 with chlorosulfonic acid yields sulfonyl chloride 8. Chlorine atom of the sulfonyl chloride group of the compound 8 can be readily exchanged by amino group using aqueous ammonia in tetrahydrofurane. The resultant sulfonamide 9 satisfies the general formula (I).

D) the scheme of benzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide (compound 12) synthesis.


Thiadiazole 1 is treated with aqueous sodium iodide and acetic acid in 2-butanone. The result of this reaction is thiadiazole 10 which reacts with chlorsulfonic acid yielding sulfonyl chloride 11. Chlorine atom of the sulfonyl chloride group of compound 11 is readily exchanged by amino group using aqueous ammonia in tetrahydrofurane. This reaction results in compound 12 corresponding to general formula (I).

E) the scheme of 3-methylsulfonylbenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide (compound 15) synthesis.


Methyl sulfonyl derivative 13 is prepared by oxidation of 7. It should be mentioned that oxidation of 7 with hydrogen peroxide in acetic acid yields a mixture of compounds 10 and 13. The mixture of compounds 10 and 13 is separated by flash chromatography. Thiadiazole 13 reacts with chlorsulfonic acid yielding sulfonyl chloride 14. Chlorine atom of the sulfonyl chloride group of the compound 14 is readily exchanged with amino group using aqueous ammonia in dioxane and the resultant sulfonamide 15 corresponds to general formula (I).

F) the scheme of 3-dimethylaminobenzimidazo[l,2-c][l,2,3]thiadiazol-6-sulfonamide (compound 22) synthesis.
Synthesis of (5(6)-bromo-lH-benzimidazol-2-yl)-methanol (16 a, b) is described in Khan, M. K. Mohammady, A. Fauzia, A. Y. (1972), J. ScL and Ind. Res. 1_5, 11-12. Amination of compound (16 a, b) with hydroxylamino-O-sulfonic acid yields a mixture of 1-aminobenzimidazoles 17 and 18, which are inseparable by column chromatography or fractional crystallization. Therefore, they are employed in the next reaction with thionyl chloride expecting that the cyclic products will have different physical properties.




Indeed, compounds 17 and 18 reacts with thionyl chloride to give a mixture of 6-bromo-and 7-bromobenzimidazothiadiazoles 19, 20. Isomers 19, 20 are separated by flash chromatography. Compound 19 undergoes chlorine substitution reaction with dimethylamine to form the compound 21. Thiadiazole 22 can be synthesized from compound 21 in accordance with techniques where the preparation of aromatic sulfonyl chlorides and sulfonamides from their halides is described (Pandya, R. Murashima, T. Tedeschi, L. Barrett, A. G. M. (2003), J. Org. Chem. 68, 8274-8276; Graham, S. L. Scholz, T.H. (1986), Synthesis, 1031-1032; Hamada, T. Yonemitsu, O. (1986), Synthesis, 852-854).

G) the scheme of 3-dimethylaminobenzimidazo[l,2-c][l,2,3]thiadiazol-5- and 8-sulfonamides (compounds 30, 32) synthesis.
Thiadiazoles bearing sulfonamide group in 5 and 8 positions of the ring system can be synthesized in a similar way to compound 22. The synthesis of a starting material 23 is described in: Sunder, S. Peet, N.P. (1979), J.Heterocycl Chem., \6, 33-37. Compound 23 can undergo cyclization reaction with glycolic acid to form 4(7)-bromo-lH-benzimidazol-2-yl) methanol (24 a, b). Cyclization reaction can occur under the same conditions as used for the synthesis of 4(7)-bromo-2-m ethyl- 1/f-benzimidazole (Dandegaonker, Recanker, (1961), J. Karnatak. Univ. 6, 25, 29, 30).


Following steps in the synthesis of compounds 30 and 32 are performed in the same way as for the compound 22.

Brief Description of the Drawings

To illustrate the main characteristics of the new compounds this description contains:
Fig. 1. Typical isothermal titration calorimetry data of bovine carbonic anhydrase binding to: A) pentafluorbenzensulfonamide, B) compound of the invention.
Fig. 2. Stabilization of bovine carbonic anhydrase II (b-CAII) by compound of the invention as determined by the fluorescence-based thermal shift assay.
Fig. 3. Bovine carbonic anhydrase II melting temperature (Tm) dependence on the concentration of an invented compound (Lt), determined by the thermal shift assay.

Embodiments of the Invention
Represented below are specific examples of invention compounds, synthesis, including intermediate compounds required for objective compound synthesis (when there is no data of such synthesis in the literature). These examples are presented only for illustrative purpose of the invention; they do not limit the scope of the invention.

Example 1. Production of intermediate compound 3-chlorobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonyl chloride (compound 2).
The 3-chlorobenzimidazo[l,2-c][l,2,3]thiadiazole (compound 1) (0.3 g, 1.43 mmol), prepared according to known methodology, is added slowly with stirring to ClSO3H (3 mL) at - 50C. The reaction mixture is allowed to warm to room temperature and remains under stirring for 24 h. The excess of acid is then hydrolyzed with ice. The product is extracted with chloroform. The combined chloroform layers are washed with H2O, dried over anhydrous

Na2SO4, yielding chlorosulfonyl compound after evaporation. The resultant compound is crystalline and has a bright yellow color.
Yield: 0.38 g (86%), mp 184-1850C.
1H NMR (300 MHz, CDCl3) δ ppm 8.1 (IH, d, J = 9 Hz, CH(5)), 8.23 (IH, dd, J = 2 and 9 Hz, CH(6)), 8.94 (IH, d, J = 2 Hz, CH(8)).
The crude material is used for next step without further purification.

Example 2. Preparation of 3-chlorobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide (compound 3).
To a solution of 3-chlorobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonyl chloride (compound 2) (0.04 g, 0.13 mmol) in tetrahydrofurane (2 mL) is added drop wise with stirring an aqueous NH3 solution (0.1 mL, 25%). After addition the reaction mixture is stirred at ambient temperature for 15 min. The solid that precipitates is filtered, washed (NaHCO3/H2O, H2O). Recrystallization is accomplished from acetic acid. The resultant compound is crystalline and has orange color.
Yield: 0.03 g (80%), mp 242-2430C.

1H NMR (300 MHz, DMSO-d6) δ ppm 7.49 (2H, s, NH2), 8.01 (2H, s, CH(5), CH(6)), 8.62 (IH, s, CH(8)).
13C NMR (75 MHz, DMSO-(J6) δ ppm 112.00, 121.59, 125.77, 127.34, 127.37, 136.18, 154.13, 154.83.
MS (+ESI) (m/z, %): (M+H)+ 289, 100%, (M+H)+ 291, 50%.
Analysis (C8H5ClN4O2S2): calcd: C 33.28%, H 1.75%, N 19.40%; found: C 33.39%, H 1.8%, N 19.63%.

Example 3. Preparation of 3-morpholinbenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide (compound 4).
The mixture of 3-chlorobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide (compound 3) (0.02 g, 0.069 mmol), morpholine (0.012 g, 0.14 mmol), and ethanol (20 mL) is refluxed for 1.5 h. The reaction mixture is cooled to room temperature and the precipitate is filtered, washed (cold H2O). Recrystallization is accomplished from acetic acid.
Yield: 0.02 g (85%), mp 253-2540C.
1HNMR (300 MHz, DMSO-d6) δ ppm 3.9 (8H, s, (CH2)4), 7.41 (2H, s, NH2), 7.88 (2H, s, CH(5), CH(6)), 8.44 (IH, s, CH(S)).
13C NMR (75 MHz, DMSO-d6) δ ppm 50.38, 65.79, 111.6, 121.27, 124.00, 126.4, 135.15, 146.94, 151.7, 154.23.
Analysis (Ci2Hi4N5O3S2): calcd C 42.47%, H 3.86%, N 20.63%; found: C 42.56%, H

3.94%, N 20.47%.

Example 4. Production of 3-phenylthiobenzimidazo[l,2-e][l,2,3]thiadiazol-7-sulfonamide (compound 5).
The mixture of 3-chlorobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide (compound

3) (0.1 g, 0.35 mmol), thiophenol (0.039 g, 0.35 mmol), and ethanol (50 mL) is refluxed for 1.5 h. The reaction mixture is cooled to room temperature, ethanol is evaporated and the resultant precipitate is filtered, washed (NaHCO3/H2O, H2O). Recrystallization is accomplished from acetic acid. The resultant compound is crystalline and has orange color.
Yield: 0.02g (85%), mp 225-2260C.

1H NMR (300 MHz, DMSO-d6) δ ppm 7.3-7.6 (5H, m, SC6H5), 7.65 (2H5 s, NH2), 7.96 (2H, s, CH(5) and CH(6)), 8.59 (IH, s, CH(8)).
13C NMR (75 MHz, DMSO-d6) δ ppm 112, 121.54, 125.43, 127.13, 129.9, 130.9, 131.39, 131.97, 132.51, 136.15, 154.76, 155.7.
Analysis (Ci4HiON4O2S3): calcd C 46.39%, H 2.78%, N 15.46%; found: C 46.26%, H

2.87%, N 15.37%.

Example 5. Production of intermediate compound benzimidazo[l,2-c][l,2,3]thiadiazol-3-thione (compound 6).
The mixture of 3-chlorobenzimidazo[l,2-c][l,2,3]thiadiazole (compound 1) prepared according to known methodics (0.5 g, 2.38 mmol), thiourea (0.4 g, 5.26 mmol), and ethanol (20 niL) is refluxed for 0.5 h. The reaction mixture is cooled to room temperature, the precipitate is filtered, washed (cold methanol), dissolved in 0.2 M NaOH. The solution is filtered and acidified with acetic acid to pH ~5. The resultant precipitate is filtered and recrystallization is accomplished from dioxane. The resultant compound is crystalline and has bright orange color. Yield: 0.34g (69%), mp 236-2370C.

Example 6. Production of intermediate compound 3-methylthiobenzimidazo[l,2-c][l,2,3]thiadiazole (compound 7).
The mixture of benzimidazo[l,2-e][l,2,3]thiadiazol-3-thione (compound 6) (0.2 g, 0.96 mmol), methyl iodide (0.2 g, 1.4 mmol), and methanol (40 mL) is refluxed for 0.5 h, solvent is evaporated, the resultant precipitate is filtered, washed (NaHCO3/H2O, H2O). Recrystallization is accomplished from H2O/ethanol (2:1) mixture. The resultant compound is crystalline and has orange color.
Yield: 0.18g (86%), mp 120-1210C.
1H NMR (300 MHz, DMSO-d6/CCl4) δ ppm 2.97 (3H, s, SCH3), 7.26 (IH, t, J= 8Hz, CH(7)), 7.51 (IH, t, J= 8Hz, CH(6)), 7.77 (IH, d, J= 8Hz, CH(5)), 8.09 (IH, d, J= 8Hz, CH(8)).

13C NMR (75 MHz, DMSO-d6/CCl4) δ ppm 18.49, 113.25, 120.33, 121.18, 127.66, 128.21, 134.79, 153.04, 153.63.

Analysis (C9H7N3S2): calcd: C 48.85%, H 3.19%, N 18.99%; found: C 48.93%, H 2.96%, N 18.89%.

Example 7. Production of intermediate compound 3-methylthiobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonyl chloride (compound 8).
The 3-methylthiobenzimidazo[l,2-c][l,2,3]thiadiazole (compound 7) (0.1 g, 0.45 mmol) is added slowly with stirring to ClSO3H (1 mL) at -50C. The reaction mixture is allowed to warm to room temperature and remains under stirring for 24 h. The excess of acid is then hydrolyzed with ice. The product is extracted with chloroform. The combined chloroform layers are washed with H2O, dried over anhydrous Na2SO4. Chloroform is evaporated. The resultant compound is crystalline and has a bright yellow color.
Yield: 0.12g (86%), decomposition at 19O0C.
1H NMR (300 MHz, CDCl3) δ ppm 2.99 (IH, s, SCH3), 8.05 (IH, dd, J= 0.6 and 9Hz, CH(5)), 8.17 (IH, dd, J= 2 and 9 Hz, CH(6)), 8.90 (IH, dd, J= 0.6 and 2Hz, CH(8)).
The crude material is used for next step without further purification.

Example 8. Production of 3-methylthiobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide (compound 9).
To a solution of 3-methylthiobenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonyl chloride (compound 8) (0.04 g, 0.125 mmol) in tetrahydrofurane (2 mL) is added dropwise with stirring an aqueous NH3 solution (0.1 mL, 25%). After addition, the reaction mixture is stirred at ambient temperature for 15 min. The solid that precipitated is filtered, washed (NaHCO3/H2O, H2O). Recrystallization is accomplished from acetic acid. The resultant compound is crystalline and has an orange color.
Yield: 0.02g (53%), mp 260-2610C.
1H NMR (300 MHz, DMSO-d6) δ ppm 2.99 (IH, s, SCH3), 7.47 (2H, s, NH2), 8.00 (2H, s, CH(5) and CH(6)), 8.58 (IH, s, CH(8)).
13C NMR (75 MHz, DMSO-d6) δ ppm 18.58, 112.00, 121.47, 125.04, 126.69, 135.95, 138.72, 154.00, 154.98.

Analysis (C9H8N4O2S3): calcd: C 35.99%, H 2.68%, N 18.65%; found: C 36.16%, H 2.62%, N 18.80%.

Example 9. Production of intermediate compound benzimidazo[l,2-c][l,2,3]thiadiazole (compound 10).
The mixture of 3-chlorobenzimidazo[l,2-c][l,2,3]thiadiazole (compound 1) prepared according to known methodics (0.2 g, 0.95 mmol), NaI-2H2O (0.9 g, 4.7 mmol), acetic acid (10 mL) and 2-butanone (50 niL) is refluxed for 5 h. The solvent is evaporated, the resultant mass is mixed with aqueous Na2S2O3 solution. Recrystallization is accomplished from H2O/ethanol (10:1) mixture. The resultant compound is crystalline and has orange color.
Yield: 0.06g (38%), mp 158-16O0C.
1H NMR (300 MHz, CDCl3) δ ppm 7.34 (IH, t, J= 8Hz, CH(7)), 7.6 (IH, t, J= 8Hz, CH(6)), 7.94 (IH, d, J= 8Hz, CH(5)), 8.2 (IH, d, J= 8Hz, CH(8)), 8.47 (IH, s, CH(3)).
13C NMR (75 MHz, CDCl3) δ ppm 113.29, 116.76, 120.47, 121.15, 127.71, 128.28, 154.98, 155.38.
MS (EI) (m/z, %): M+ 175, 93%.
Analysis (C8H5N3S): calcd: C 54.84%, H 2.88%, N 23.98%; found: C 54.65%, H 2.37%, N 23.60%.

Example 10. Production of intermediate compound benzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonyl chloride (compound 11).
The benzimidazo[l,2-c][l,2,3]thiadiazole (compound 10) (0.06 g, 0.34 mmol) is added slowly with stirring to ClSO3H (0.6 mL) at -50C. The reaction mixture is allowed to warm to room temperature and remains under stirring for 24 h. The excess of acid is then hydrolyzed with ice. The product is extracted with chloroform. The combined chloroform layers are washed with H2O, dried over anhydrous Na2SO4. Chloroform is evaporated. The resultant compound is crystalline and has a bright yellow color.
Yield: 0.06g (64%), mp 188-1890C.
1H NMR (300 MHz, CDCl3) δ ppm 8.1 (IH, dd, J= 0.6 and 9Hz, CH(5)), 8.24 (IH, dd, J= 2 and 9 Hz, CH(6)), 8,74 (IH, s, CH(3)), δ (IH, dd, J= 0.6 and 2Hz, CH(8)).

The crude material is used for next step without further purification.

Example 11. Production of benzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide (compound 12).
To the solution of benzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonyl chloride (compound

11) (0.06 g, 0.22 mmol) in tetrahydrofurane (2 mL) is added dropwise with stirring an aqueous NH3 solution (0.15 mL 25%). After addition, the reaction mixture is stirred at ambient temperature for 15 min. The solid that precipitated is filtered, washed (NaHCO3/H2O, H2O). Recrystallization is accomplished from acetic acid. The resultant compound is crystalline and has a yellow color.
Yield: 0.039g (70%), mp 245-2460C.
1H NMR (300 MHz, DMSO-d6): δ ppm 7.46 (2H, s, NH2), 7.98 (2H, s, CH(5) and CH(6)), 8.63 (IH, s, CH(8)), 9.3 (IH, s, CH(3)).
13C NMR (75 MHz, DMSO-d6): δ ppm 112.08, 121.22, 122.69, 125.18, 125.94, 135.37, 155.3, 157.75.
Analysis (C8H6N4O2S2): calcd: C 37.79 %, H 2.38%, N 22.03 %; found: C 37.86 %, H 2.41 %, N 22.18 %.

Example 12. Production of intermediate compound 3-methylsulfonylbenzimidazo[l,2-c][l,2,3]thiadiazole (compound 13).
To a solution of 3-methylthiobenzimidazo[l,2-c][l,2,3]thiadiazole (compound 7) (0.3 g, 1.36 mmol) in acetic acid (7 mL) is poured H2O2 (0.65 g, 35%). The reaction mixture is kept at room temperature for five days. The reaction solvent is evaporated and residue is poured into NaHCO3/H2O solution and mixed up carefully. The precipitate contains a mixture of compounds 10 and 13. The compounds are separated by flash chromatography (ethylacetate). The resultant compound 13 is crystalline and has a dark red color.
Compound 10: yield: 0.03g (1%), mp 158-16O0C.
Rp 0.13 (ethylacetate).
3-methylsulfonylbenzimidazo[l,2-c][l,2,3]thiadiazole (compound 13): yield: 0.19g (56%), mp l86-187°C.

Rf= 0.73 (ethylacetate).
1H NMR (300 MHz, CDCl3) δ ppm 3.64 (3H, s, SO2CH3), 7.47 (IH, t, J= 8Hz, 7-H), 7.71 (IH, t, J= 8Hz, CH(6)), 8.03 (IH, d, J= 8Hz, CH(5)), 8.24 (IH, d, J= 8Hz, CH(8)).
13C NMR (75 MHz, CDCl3) δ ppm 44.82, 113.39, 121.68, 122.2, 128.35, 129.86, 141.86, 149.59, 155.6.
Analysis (C9H7N3O2S2): calcd: C 42.67%, H 2.79%, N 16.59%; found: C 42.86%, H 2.75%, N 16.48%.

Example 13. Production of intermediate compound 3-methylsulfonylbenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonyl chloride (compound 14).
The 3-methylsulfonylbenzimidazo[l,2-c][l,2,3]thiadiazole (compound 13) (0.05 g, 0.2 mmol) is added slowly with stirring to ClSO3H (0.6 mL) at -5°C. The reaction mixture is allowed to warm to room temperature and remains under stirring for 24 h. The excess of acid is then hydrolyzed with ice. The solid that precipitated is filtered. The resultant compound is crystalline and has orange color.
Yield: 0.05g (72%), decomposition at 21O0C.
1H NMR (300 MHz, DMSO-d6) δ ppm 3.73 (3H, s, SO2CH3), 8.1 (IH, d, J = 9 Hz, CH(5)), 8 (IH, dd, J = 2 and 9 Hz, CH(6)), 8.42 (IH, s, CH(8)).
The crude material was used for next step without further purification.

Example 14. Production of 3-methylsulfonylbenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide (compound 15).
To a solution of 3-methylsulfonylbenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonyl chloride (compound 14) (0.032 g, 0.09 mmol) in dioxane (2OmL) is added dropwise with stirring an aqueous NH3 solution (0.1 mL, 25%). After addition the reaction mixture is stirred at ambient temperature for 0.5 h. The reaction solvent is evaporated and residue is washed (NaHCO3/H2O, H2O). Recrystallization is accomplished from acetic acid. The resultant compound is crystalline and has orange color.
Yield: 0.018 g (60%), mp 249-25O0C.

1HNMR (300 MHz5 DMSOd6): δ ppm 3.69 (3H, s, SO2CH3), 7.56 (2H, s, NH2), 8.1 (2H, s, CH(5)5 CH(6)), 8.7 (IH, s, CH(8)).
13C NMR (75 MHz, DMSO-d6): δ ppm 44.96, 112.13, 121.7, 126.44, 126.95, 132.96, 137.24, 152.79, 155.73.
Analysis (C9H8N4O4S3): calcd: C 32.52%, H 2.43%, N 16.86%; found: C 32.61%, H

2.44%, N 16.75%.

Example 15. Production of intermediate compounds (l-amino-5-bromo-lH-benzimidazol-2-yl)methanol (compound 17) and (l-amino-6-bromo-lH-benzimidazol-2-yl)methanol (compound 18).
The 5(6)-bromo-lH-benzimidazol-2-methanol (16 a, b) (5.2 g, 22.7 mmol), prepared to known methodology, is dissolved in a solution of KOΗ (4.8 g, 72.8 mmol) in H2O (40 niL). To the mixture is added under stirring at 400C solution Of NH2OSO3H (6g, 50mmol) in H2O (15 mL), neutralized NaHCO3. Upon the completion of the exothermal reaction the mixture is still incubated at 40-500C for 0.5 h and then cooled to room temperature. The resultant precipitate is filtered and recrystallization is accomplished from water. The mixture of compounds 17 and 18 is obtained in 1 :1 ratio (according to 1H NMR). The mixture of these compounds is crystalline, white.
Overall yield: 3.7g (67%).
1H NMR (300 MHz, DMSO-d6): δ ppm 4.73 (4H, s, 2CH2), 5.43 (IH, s, OH), 5.45 (IH, s,

OH), 6.01 (2H, s, NH2), 6.03 (2H, s, NH2), 7.31 (IH, d, J- 9Hz, ArH), 7.39 (IH, d, J= 9Hz, ArH), 7.46 (IH, d, J- 9Hz, ArH), 7.53 (IH, d, J= 9Hz, ArH), 7.67 (IH, s, ArH), 7.73 (IH, s, ArH).
13C NMR (75 MHz, DMSO-d6): δ ppm 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 (v, cm"1): 3350, 3313, 3184, 3120 NH2.
Analysis (C8H8BrN3O): calcd: C 39.67%, H 3.31 %, N 17.36%; found: C 39.88%, H 3.52%, N 17.46%.

Example 16. Production of intermediate compounds 3-chloro-6-bromobenzimidazo[l,2-c][l,2,3]thiadiazole (compound 19) and 3-chloro-7-bromobenzimidazo[l,2-c][l,2,3]thiadiazole (compound 20).
The mixture of (l-amino-5-brorno-lH-benzimidazol-2-yl)-methanol (compound 17) and (l-amino-6-bromo-lH-benzimidazol-2-yl)-methanol (compound 18) (0.5 g, 2.1 mmol) is refluxed with SOCl2 (5 mL) for 0.5 h. The excess of SOCl2 is evaporated. The residue is washed (NaHCO3ZH2O, H2O). The mixture of products consists of compounds 19 and 20 in proportion 1: 1. The compounds were separated by flash chromatography (dichlormethane: ethy lacetate= 10:1).
Overall yield: 0.36 g (60%) (recrystallization is accomplished from methanol).
20: Yield: 0.15g (25%), mp 193-1940C.
Rf= 0.48 (dichlormethane: ethylacetate=10:l).
1H NMR (300 MHz, DMSO-d6): 7.7 (IH, dd, J= 2 and 9Hz, CH(6)), 7.79 (IH, dd, J= 0.5Hz and 9Hz, CH(5)), 8.46 (IH, dd, J= 0.5 and 2Hz, CH(8)).
13C NMR (75 MHz, DMSO-d6): 112.76, 116.24, 123.03, 126.61, 129.36, 131.66, 151.72,

152.49.
Analysis (C8H3BrClN3S): calcd: C 33.30%, H 1.05 %, N 14.56%; found: C 33.56%, H 1.15%, N 14.63%.
3-chloro-6-bromobenzimidazo[l,2-c][l,2,3]thiadiazole (compound 19) yield: 0,15g (25%), mp l53-154°C.
Rf= 0.59 (dichlormethane: ethylacetate=10:l).
1H NMR (300 MHz, DMSO-d6): 7.45 (IH, dd, J= 2 and 9Hz, CH(7)), 8.07 (IH, d, J= 2Hz, CH(5)), 8.19 (IH, d, J= 9Hz, CH(8)).
13C NMR (75 MHz, DMSO-d6): 115.36, 121.57, 123.38, 123.77, 127.76, 129.38, 152.77, 154.55.
Analysis (C8H3BrClN3S): calc: C 33.30 %, H 1.05 %, N 14.56 %; found: C 33.01 %, H 1.33 %, N 14.36 %).

Example 17. Production of 6-bromo-3-dimethylaminbenzimidazo[l,2-c][l,2,3]thiadiazole (compound 21).

Mixture of 3-chloro-6-bromobenzimidazo[l,2-c][l,2,3]thiadiazole (2 g, 6.9 mmol) (compound 19), aqueous dimethylamine (0.94 g, 33%), ethanol (100 mL) is refluxed for 3 h. Solvent is evaporated and the residue is washed with cold H2O. Recrystallization is accomplished from methanol. The resultant compound is crystalline and has orange color.
Yield: 1.6 g (78%), mp 176-1770C.
1H NMR (300 MHz, CDCl3): 3.49 (6H, s, N(CH3)2), 7.29 (IH, dd, J= 2 and 9Hz, CH(7)), 7.86 (IH, d, J= 9Hz, CH(8)), 7.98 (IH, d, J= 2Hz, CH(5)),
13C NMR (75 MHz, CDCl3): 42.78, 113.79, 119.89, 122.84, 123.37, 127.23, 145.06, 151.7, 153.95.
Analysis (Ci0H9BrN4S): calc: C 40.42 %, H 3.05 %, N 18.85 %; found: C 40.51 %, H

3.02 %, N 18.87 %).

Inhibition of carbonic anhydrases (also all enzymes) is measured by determining the reduction in the speed of catalysis. Carbonic anhydrases are zinc-containing metalloenzymes, catalyzing the reversible reaction:
CO2 + H2O ^ HCO- + H+
The inhibition of this reaction may be determined by measuring carbon dioxide consumption, bicarbonate appearance and the changes of pH (Krebs, J.F. and CA. Fierke, (1993), J. Biol. Chern. 268(2), 948-54). Inhibitors bind to the active center of carbonic anhydrases, compete with the substrate and diminish the catalysed reaction. All sulfonamides bind to the active center and diminish this reaction. Inhibition is equivalent to binding. (Chakravarty, S. and K.K. Kannan, (1994), J. MoI. Biol. 243(2), 298-309; Lindskog, S. (1997), Pharmacol. Ther. 74(1), 1-20; Baird, T.T.Jr. et al. (1997), Biochemistry, 36(9), 2669-78). However, their binding and inhibitory efficiency varies greatly. Furthermore, the specificity of various sulfonamides, i.e., how much they inhibit particular isozymes, varies greatly. (Di Fiore, A. et al. (2005), Bioorg. Med. Chem. Lett. 15(7), 1937-42; Lee, D.A. 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, R.P. et al. (2005), Epilepsy Res. 63(2-3), 103-12; Simone, G.D. et al. (2005), Bioorg. Med. Chem. Lett. 11(9), 2315-20). Sulfonamide binding to carbonic anhydrases is measured by a number of standard means. Most often used methods are isothermal titration calorimetry, surface plasmon resonance, and ultracentrifugation. (Myszka, D. G. et al. (2003), J. Biomol. Tech. 14(4), 247-69). Specificity is determined by measuring binding constants with various isozymes and also by measuring "true" binding constants (Matulis, D. and MJ. Todd, (2004), Trends in Biocalorimetry).

Example 18. Determination of the specificity of CA binding and inhibition by isothermal titration calorimetry.
The heat evolved upon inhibitor (compound 3, compound 4, and compound 9) binding to bovine carbonic anhydrase II and human carbonic anhydrase I was measured by isothermal titration calorimetry and compared to known carbonic anhydras inhibitors, namely, trifluoromethanesulfonamide (TFMSA), acetazolamide (AZM), etoxzolamide (EZA), and pentafluorobenzenesulfonamide (PFS).
To illustrate the results, Figure 1 shows isothermal titration calorimetry curves of bovine carbonic anhydrase II binding to pentafluorobenzenesulfonamide (A) and 3-morpholinbenzimidazo[ 1 ,2-c] [ 1 ,2,3]thiadiazol-7sulfonamide (4) (B) .
The resultant "observed" binding constants were in the order of 106-107 M"1 for all tested compounds of this invention. However, these constants depend on protonation events involving buffer, zinc, and inhibitor molecules. Therefore, the more precise measure- is the calculated "true" or "intrinsic" binding constants that are not dependent on above mentioned 'protonation events. When the intrinsic binding constants were compared to the literature compounds, the advantage of the invented compounds was obvious. Below is the series of compounds in the order of decreasing their intrinsic binding constant. Numbers above the series show the times difference between the neighboring compounds. The range is due to different activities towards carbonic anhydrase II and I.
l-2x l-2x 4-1Ox 3-5x 2x l-2x
4 > 3 > 9 > EZA > PFS > AZM > TFMSA
As seen from the series, the compounds 4, 3, and 9 are all significantly better than literature compounds and could be potentially used as active ingredients of pharmaceutical agents.

Example 19. Determination of the observed binding constants by the fluorescent thermal shift assay.
Inhibitor binding to carbonic anhytdrases was also measured by the fluorescent thermal shift assay, which employs l-anilino-8-naphthalene sulfonate (ANS) probe that visualizes enzyme thermal stabilization by the inhibitor thus determining the binding constant.
Figure 2 shows the enzyme raw unfolding curves and the increase in Tm upon increasing concentration of inhibitors. Figure 3 shows the Tm increase of bovine carbonic anhydrase II upon increasing concentration of the inhibitor, 3-morpholinbenzimidazo[l,2-c][l,2,3]thiadiazol-7-sulfonamide (4). The curve in Figure 3 shows the simulated curve according to literature model. Resultant data is consistent with the isothermal titration calorimetry data in determining the observed binding constants of the inhibitors.

Newly synthesized sulfonamides of general formula (I)



exhibit a comparable efficiency to the existing inhibitors used as drugs, but the invented compounds exhibit significantly better specificity than the existing compounds promising to solve the problem of non-specific binding of literature inhibitors.