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Synthesis, carbonic anhydrase I and II inhibition studies of the 1,3,5-trisubstituted-pyrazolines

Abstract 4-(3-(4-Substituted-phenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl) benzenesulfonamides (9–16) were successfully synthesized and their chemical structures were confirmed by 1H NMR, 13C NMR, and HRMS spectra. Carbonic anhydrase I and II inhibitory effects of the compounds were investigated. Ki values of the compounds were in the range of 316.7 ± 9.6–533.1 ± 187.8 nM towards hCA I and 412.5 ± 115.4–624.6 ± 168.2 nM towards hCA II isoenzymes. While Ki values of the reference compound Acetazolamide were 278.8 ± 44.3 nM and 293.4 ± 46.4 nM towards hCA I and hCA II izoenzymes, respectively. Compound 14 with bromine and compound 13 with fluorine substituents can be considered as the leader compounds of the series because of the lowest Ki values in series to make further detailed carbonic anhydrase inhibiton studies.


Introduction
Carbonic anhydrases (CAs) are a ubiquitous metalloenzyme catalyzing the reversible conversion of carbon dioxide to bicarbonate. CAs are present in prokaryote and eukaryote life forms. The enzyme is found in many tissues such as gastrointestinal tract, nervous system, kidneys, lungs, skin, and eyes. CAs play key roles in a number of physiological and pathological processes such as ions and gas exchanges, pH regulation, photosynthesis, calcification, and biosynthetic reactions such as gluconeogenesis, lipogenesis, and ureagenesis 1 . The mammalian enzymes belonging to a-CA family consist of 16 active members, in which several are cytosolic (CA I-III, CA VII, and CA XIII), five are membrane-bound (CA IV, CA IX, CA XII, CA XIV, and CA XV), two are mitochondrial (CA VA and VB), and one (CA VI) is secreted in saliva and milk. Out of 16 different isoforms of a-class of human-associated CAs, hCA I is found together with hCA II in erythrocytes. hCA II is the most widely distributed isoform in the eye, kidney, central nervous system, and inner ear, and is a drug target for clinically used diuretics, antiglaucoma drugs, and anticonvulsants. Targeting a particular CA is often associated with treatment of particular diseases such as CA II, IV, and XII are the targets for antiglaucoma agents; CA VA and VB for antiobesity agents; CA IX and XII for antitumour agents or diagnostic tools for imaging hypoxic tumours [2][3][4][5] .
The sulfonamides are an important class of the pharmaceutical compounds with a wide spectrum of biological activities such as anticancer 6,7 , CA inhibitory 8,9 , antibacterial 10 , antihypertensive 11 , antiinflammatory 12 , and antiprotozoal 13 activities among others.
On one hand, Acetazolamide (5-amino-1,3,4-thiadiazole-2-sulfonamide, AZA), which is used systematically to reduce intraocular pressure by lowering the humor formation in the eye, has sulfonamide moiety 14 . Although AZA is in clinical use, it has several side effects such as numbness and tingling in the fingers and toes, taste alterations, blurred vision, kidney stones, and an increase in urination 14 .
The most extensively made modifications on 2-pyrazolines were diaryl/hetroaryl group substitutions at 3 and/or 5 positions. Celecoxib and Valdecoxib, which are in clinical use, were developed as cyclooxygenase-2 (COX-2) specific inhibitors. They are also known with their potent CA inhibitory activities 19 . Both compounds possess a benzenesulfonamide group linked to a fivemembered substituted heterocyclic ring. The presence of the -SO 2 NH 2 moiety seems not to be necessary for COX-2 inhibition but it is essential for the CA inhibition 19 . These two drugs have shown interesting isoform selective CA inhibitory effects. Their X-ray crystal structures were in a complex form with hCA II 19 .
Recently, our research group focused on the synthesis of the compounds having 2-pyrazoline and sulfonamide pharmacophores in a single molecule and investigated their several bioactivities such as cytotoxic and/or CA inhibitory activities based on the results of previous studies [7][8][9]17 . In the present study, it was aimed to synthesize 4-[3-(4-substitutedphenyl)-5-phenyl-4,5-dihydro-1Hpyrazol-1-yl] benzenesulfonamides to investigate their carbonic anhydrase inhibitory effects on hCA I and II isoenzymes.

Materials and methods
1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were obtained using a Varian Mercury Plus spectrometer (Varian Inc., Palo Alto, CA). Chemical shifts (d) were reported in parts per million (ppm). HRMS spectra of the compounds were taken by liquid chromatography ion trap-time of flight tandem mass spectrometer (Shimadzu, Kyoto, Japan) equipped with an electrospray ionization (ESI) source, operating in both positive and negative ionization mode. Shimadzu's LCMS Solution software (Shimadzu, Kyoto, Japan) was used for data analysis. Melting points were determined on Buchi 530 (Buchi Labortechnik AG, Flawil, Switzerland).

Biological activity
Carbonic anhydrase enzyme assay The purification of cytosolic CA isoenzymes (CA I and II) were previously described with a simple one-step method by a Sepharose-4B-L tyrosine-sulfanilamide affinity chromatography 23 . The protein quantity in the column effluents was determined spectrophotometrically at 280 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was applied with a Bio-Rad Mini Gel system (Mini-PROTEAN Tetra System, Guangdong, China) after purification of both CA isoenzymes 24,25 . Briefly, it was performed in acrylamide for the running (10%) and the stacking gel (3%) contained SDS (0.1%), respectively. Activities of CA I and II isoenzymes were determined according to the esterase method by Verpoorte et al. 26 . The increase in absorbance of reaction medium was spectrophotometrically recorded at 348 nm (UV-VIS Spectrophotometer, Shimadzu, UVmini-1240, Kyoto, Japan). Also, the quantity of protein was determined at 595 nm according to Bradford method 27 . Bovine serum albumin was used as standard protein. The IC 50 values were obtained from activity (%) versus compounds plots. For calculation of K i values, three different concentrations were used. The Lineweaver-Burk curves were drawn and calculations were realised 28 .

Results and discussion
The compounds designed were successfully synthesized. The chemical structures of the compounds were confirmed by 1 H NMR, 13 C NMR, and HRMS spectra. The CA inhibition effects of the compounds were evaluated towards hCA I and hCA II isoenzymes.
The inhibition values are presented in Table 1. As shown in Table 1, IC 50 values were in the range of 402.9-554.8 nM towards hCA I, while they were in the range of 458.6-620.4 nM towards hCA II. The IC 50 values of the reference compound AZA towards hCA I and hCA II were 985.8 nM and 489.4 nM, respectively. All compounds had lower IC 50 value than AZA toward hCA I, while the compounds 11, 14, and 16 had lower IC 50 value than AZA towards hCA II. According to IC 50 values of the compounds, chlorine-bearing compound 12 and bromine-bearing compound 14 were the most effective compounds towards hCA I while it was hydroxy derivative 16 towards hCA II.
When K i values of the compounds were considered, K i values of the compounds were in the range of 316.7 ± 9.6-533.1 ± 187.8 nM towards hCA I, while K i values were 412.5 ± 115.4-624.6 ± 168.2 nM towards hCA II. The K i values of reference compound AZA were 278.8 ± 44.3 nM and 293.4 ± 46.4 nM towards hCA I and hCA II, respectively. When K i values of the compounds were considered, 12 with chlorine, 14 with bromine, and 15 with nitro substituents, which have very close K i values, are the leader compounds of series towards hCA I, while 14 was the leader compound of the series towards hCA II because of the lowest K i values. All compounds were less effective than AZA on both hCA I and II isoenzymes, since K i value of the compound had higher values than AZA's.
Any substitution rather than hydrogen at the para position of phenyl ring decreased K i value of the substituted compound toward hCA I by comparing with 9, which is non-substituted derivative. This means that substitution at the para position of phenyl ring was a useful modification to increase the effect of compound toward hCA I. Exception was 11, which is a methoxy- substituted derivative. The effect of any substituent on phenyl ring to K i value of substituted compound towards hCA II isoenzyme was similar to hCA I's. It means that any type of substituent on phenyl ring rather than hydrogen was a useful modification to increase the compounds effect on hCA II by lowering the K i values without exception.

Conclusion
In conclusion, the compounds 12, 14, and 15 towards hCA I and 14 towards hCA II seem to be the leader compounds of the series for further investigations.