Synthesis and biological evaluation of new pyrazolebenzene-sulphonamides as potential anticancer agents and hCA I and II inhibitors

Cancer is a disease characterized by the continuous growth of cells without adherence to the rules that healthy normal cells obey. Carbonic anhydrase I and II (CA I and CA II) inhibitors are used for the treatment of some diseases. The available drugs in the market have limitations or side effects, which bring about the need to develop new drug candidate compound(s) to overcome the problems at issue. In this study, new pyrazole-sulphonamide hybrid compounds 4-[5-(1,3-benzodioxol-5-yl)-3-aryl-4,5-dihydro-1 H -pyrazol-1-yl]benzenesulphonamides (4a - 4j) were designed to discover new drug candidate compounds. The compounds 4a - 4j were synthesized and their chemical structures were confirmed using spectral techniques. The hypothesis tested was whether an introduction of methoxy and polymethoxy group(s) lead to an increased potency selectivity expression (PSE) value of the compound, which reflects cytotoxicity and selectivity of the compounds. The cytotoxicity of the compounds towards tumor cell lines were in the range of 6.7 – 400 µM. The compounds 4i (PSE2 = 461.5) and 4g (PSE1 = 193.2) had the highest PSE values in cytotoxicity assays. Ki values of the compounds were in the range of 59.8 ± 3.0 - 12.7 ± 1.7 nM towards hCA I and in the range of 24.1 ± 7.1 - 6.9 ± 1.5 nM towards hCA II. While the compounds 4b, 4f, 4g, and 4i showed promising cytotoxic effects, the compounds 4c and 4g had the inhibitory potency towards hCA I and hCA II, respectively. These compounds can be considered as lead compounds for further research.

compound (34) showed remarkable cytotoxicity with potency-selectivity expression (PSE) and tumor specificity (TS) (10.5 and 9.5) values towards OSCC lines [15]. Other studies also supported that pyrazoline-sulphonamides hybrid compounds are good candidates to develop new anticancer drugs [13 -15]. Besides this, we reported a large library of methoxy substituted pyrazoline derivatives since this group attracted our attention with their cytotoxic properties against OSCC lines in our previous study [16]. Many other studies reporting on the valuable anticancer properties of several mono-or poly-methoxylated chemicals towards OSCC cell lines compared to substituents other than methoxy are available [16 -18].
The hybrid approach is one of the strategies to obtain a compound or a drug with increased activity in medicinal chemistry for new drug development [19].
Of the pharmacophores used, sulphonamide has a very well-known carbonic anhydrase (CA) inhibitory effect. CA is an enzyme that catalyzes the reversible hydration/dehydration of CO 2 /HCO 3 − [20,21] and has various roles in physiological events such as carbon dioxide and bicarbonate transport processes, respiration, pH balancing, and CO 2 homeostasis [22,23]. There are 16 isoforms (hCA I-XVI) that have different localizations [24,25], of which CAs, CA I and CA II are the abundant forms. The hCA I isoform is associated with retinal and cerebral edema, and the inhibition of CA I may help cure such conditions [22, 26 -38]. The physiologically dominant isoform is hCA II, which is another enzyme that is associated with several disease such as epilepsy, edema, glaucoma, and altitude sickness [22, 26 -37]. Furthermore, it has also emerged in the past few years that these enzymes can be used as potential targets for designing antiinfective drugs with a novel mechanism of action [39 -41]. Of α-class carbonic anhydrases, CA IX and CA XII are the ones that are related to tumors. In cancer cases, CA IX levels especially increase.
In this study, the first aim was to synthesize pyrazolebenzene-sulphonamide hybrid compounds bearing mono-or polymethoxy (di/tri) group(s). The chemical structure of the compounds is 4-[5-(1,3-benzodioxol-5-yl)-3-aryl-4,5-dihydro-1H-pyrazol-1-yl]benzenesulphonamide (4a -j, Figure 2). Secondly, the compounds (4a -j) were tested on oral squamous cell carcinoma (OSCC) and normal oral cells to find new anticancer drug candidate compounds. As a final step, it was planned to investigate the CA inhibitory effect of the compounds on hCA I and hCA II. Since CA I and II are the widely available forms of CAs, we had the opportunity facility to study them as sulphonamides are very well-known inhibitors of CAs. If impressive results are obtained is on hCA I/ II, inhibition tests towards cancer-related CA IX and CA XII isoenzymes can be considered in future studies.

Chemistry
The chemical structures of the final compounds 4a -j were confirmed by nuclear magnetic resonance (NMR) spectra; 1 H NMR (400MHz), 13 C NMR (100 MHz) (Varian Mercury Plus spectrometer, Varian Inc., Palo Alto, CA, U.S.) and mass spectra (HRMS) (Shimadzu Corporation, Kyoto, Japan). Chemical shifts (δ) are reported in ppm and coupling constants (J) are expressed in hertz (Hz). Mass spectra (HMRS) for the compounds were taken using a liquid chromatography ion trap-time of flight tandem mass spectrometer (Shimadzu Corporation) equipped with an electrospray ionization (ESI) source, operating in both positive and negative ionization modes. Shimadzu's LCMS Solution software was used for data analysis. Melting points were determined using an Electrothermal 9100/IA9100 instrument (Bibby Scientific Limited, Staffordshire, UK), which is uncorrected. Reactions were monitored by thin layer chromatography (TLC) using silica gel 60 HF254 (Merck KGaA). A solvent mixture of chloroform: methanol (4.8: 0.2) was used as a thin-layer chromatography (TLC) solvent system. DMSO-d 6 (Merck) was used as a NMR solvents.

Carbonic anhydrase enzyme assay
Human CA isoforms (hCAI and hCAII) were purified by the sepharose -4B -L -tyrosine sulfanilamide affinity segregation method as reported [57,58]. The Bradford technique was used to measure protein concentrations at 595 nm [59]. Inhibitory effects of the compounds were investigated by measuring the esterase activity according to Verpoorte et al. [60] as described in previous studies [61 -63]. The hCA activity was determined by measuring the conversion of the p-nitrophenyl acetate substrate to p-nitro phenolate at 348 nm by the spectrophotometer (UV -VIS Spectrophotometer, UV mini-1240, Shimadzu Corporation) [64]. Acetazolamide (AZA) was used as a control drug. The Lineweaver-Burk plot was used to calculate inhibition constants (K i ) of the compounds [65] by using the following equation: V max , maximal velocity; VI max , maximal velocity of inhibitor; I, inhibitor; K i , the inhibitor constant. Lineweaver-Burk graphics are presented as a supplementary file.
First, the question that whether the compounds have cytotoxic/anticancer properties should be answered. The cytotoxicities of the compounds towards tumor cell lines were in the range of 6.7 -400 µM ( Table 1). This shows that the compounds had anticancer properties. The compounds having more potent cytotoxicity than 5-FU and their times of potency (shown in the parenthesis) were as follows towards cell lines: As previously mentioned in the introduction, novel anticancer drugs that show less side effects and higher selectivity towards cancer cells urgently need to be developed [1,3,5]. Normal cells surround tumor cells in humans. Consequently, candidate compounds that are planned to be used in future clinical applications should show higher cytotoxicity against tumor cells rather than normal cells. The selectivity index (SI) value reflects this property. The SI values of the compounds  were calculated towards a specific cell line as described before [55] and presented in Table 1. The SI figure being higher than 1 reflects the selectivity of the tested compound toward tumor cells, rather than a normal cell. The tumor selectivity (TS 1 and TS 2 ) of each compound was calculated as described in previously reported literature procedures [55] and these figures are presented in Table 1. Based on the TS 1 values, 4f and 4g which have 2,5 and 2,4-dimethoxy substituents had the highest TS 1 values in a series with 22.4 and 22.2, respectively. On the other hand, the 3,4,5-trimethoxy substituted compound 4i (TS 1 : 19.6) had the second highest TS 1 value. Among mono-methoxy compounds, the para-methoxy compound 4b had a TS 1 value of 18 and was the third highest. As expected, they were in accordance with the literature findings [16 -18]. The second calculation (TS 2 ) considered the differences of sensitivity between the malignant (Ca9-22) and non-malignant (HGF) cells derived from the same tissue (gingiva). According to TS 2 values obtained, 4i (3,4,5-trimethoxy) had the highest TS 2 value of 43. This result was followed by 4f (2,5-dimethoxy), 4g (2,4-dimethoxy), and 4b (4-methoxy).
As seen in both calculations poly-methoxylated compounds had higher TS value than mono derivatives' . The selectivity order changed as tri ˃ di ˃ mono or di ˃ tri ˃ mono. These findings also supported in our previous reports [16 -18], thus the methoxylated compounds can be considered for new anticancer drug designs.
The desired properties for a lead compound are being both markedly cytotoxic and selectively toxic for tumors. To identify lead compounds of the study PSE (PSE 1 and PSE 2 ) values were calculated as shown in Table 1 [55]. PSE1 reflects general cytotoxicity and selectivity potency of the compound towards all cells used, whereas PSE 2 seems more specified since it was considered for the same origin cells. When the compounds tested towards OSCC lines were considered in terms of PSE 2 values of the compounds. The best poly-methoxylated compounds were 4i (with 3,4,5-trimethoxy, PSE 2 = 461.5) ˃ 4f (with 2,5-dimethoxy, PSE 2 = 425.1) ˃ 4g (with 2,4-dimethoxy, PSE 2 = 405.4) while the best mono-methoxylated derivative was 4b (with 4-methoxy, PSE 2 = 205). On the other hand, the non-substituted or non-metoxylated derivative 4a had a PSE 2 value of 19.3. The reference anticancer drug 5-FU had a PSE 2 value of 206, which is a similar value to 4-methoxy derivative 4b's.
The introduction of the third methoxy group into the structure in compound 4i (3,4,5-trimethoxy) increased the PSE 2 value, which reflects cytotoxicity and selectivity. Increases in PSE 2 were 2.3, 45.7, and 23.7 times in 4i compared to monomethoxylated compounds 4b, 4c, and 4d. Increases were 1.1 and 1.12 times in 4i compared to dimethoxylated compounds 4f and 4g, respectively.
In addition, an introduction of an electron-donating hydroxy group which is a hydrogen bonding donor group to para positions of phenyl with a meta methoxy substituent in compound 4j, increased the PSE 2 value 2.6 times in 4j comparing to 4c (3-methoxy). This was also found to be a useful modification for the increase in PSE 2 value.
In a previous study, 4-(5-(3,4-dimethoxyphenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl) benzenesulphonamide compounds' having free methoxy groups on its chemical structure were reported as cytotoxic against OSCC [13]. The compounds' CC 50 values were in the range of 22 -200 μM, while their TS values were 0.7 and 3.4. When we compared our compound 4b that has piperonal moiety, which is a cyclic form of 3,4-dimethoxy groups in the previous compound [13], it can be expressed here using a piperonal structure, therefore making it a useful modification since it increased the tumor specificity of compound 4b (TS: 18 and 28.6) by 8 -25 times towards OSCC. These significant outcomes indicate that a piperonal moiety may be used as a favorable group for designing new bioactive compounds in future studies.
The methoxy group is an electron-donating group and can form hydrogen bonds with enzymes. This is an important in the bioactivity of many compounds and drugs. Increases in the bioactivity of the compounds may be attributed to; proper interaction of the compound with the active site of the enzyme, the stability of this complex formed, and the adequate concentration of the biomacromolecules of the compound at the active site of the compound, which depends on the pharmacokinetic properties of that compound. The other factor affecting the cytotoxic potency can be the type of cell line used and the mechanism of action of the compound for the activity at issue. Furthermore, decreases in cytotoxicity may be attributed to the low stability of the compounds, which affect the concentration of the compound at the active side, or improper position of the compound at the active side which limits proper interaction. Unchanged bioactivity can bring the mind ineffectiveness of some groups to realize the activity in question partition coefficient of the compounds can also direct compound travel and its effect. Additionally, the size of molecules can be considered as an affecting factor since the behavior of the molecule in cells can be affected differently.

Carbonic anhydrase I and II inhibition
hCA I and hCA II inhibition results of the compounds are presented in Table 2 as IC 50 (µM) and K i (µM). When CA inhibitory profiles of the compounds were investigated based on the IC 50 values, the compounds were effective at 6.6 -30.1 µM toward hCA I while they were effective at 9.2 -20.0 µM toward hCA II isoenzyme. The 3-methoxy-bearing compound 4c was the most effective inhibitor on hCA I and hCA II while phenyl-bearing compound 4a had the least effective toward hCA I in terms of IC 50 values. The reference compound, acetazolamide (AZA), had IC 50 values of 16.6 µM and 8.4 µM towards hCA I and II, respectively. The compounds 4b (1.5), 4c (2.5), 4d (1.3), 4e (1.6), 4f (1.2), 4g (2.5), 4h (1.2) times were more potent than AZA towards hCA I while all compounds had less inhibition potential than AZA towards hCA II in terms of IC 50 .
When the inhibition constants (K i ) were considered, K i values of the compounds were in the range of 12.7 ± 1.7 µM -59.8 ± 3.0 µM towards hCA I and in the range of 6.9 ± 1.5 µM -24.1 ± 7.1 µM towards hCA II. The K i values of AZA towards hCA I and hCA II were 30.2 ± 7.8 µM and 4.4 ± 0.6 µM, respectively. When K i values were considered, the compound 4g, which has a 2,4-dimethoxy substituent, towards hCA I and compound 4c, which has a 3-methoxy substituent, towards hCA II had the best inhibition potential. Differences in inhibition potentials of the compounds may result from differences in their chemical structures and also differences in their interactions with the active site(s) of enzymes. In another study conducted by our research group, a series of poly-methoxylated pyrazoline benzene sulphonamides were synthesized and their inhibitory effects on CAs were investigated. All compounds presented superior CA inhibitory activity compared to the reference compound, acetazolamide, on CAs with inhibition constants in the range of 30.1 -49.2 nM against hCA I and of 23.8 -30.1 nM against hCA II in terms of IC 50 values, respectively [13]. Based on the literature findings, to obtain more potent hCA I, II inhibitors, and cytotoxic compounds, pyrazoline type compounds can be derived with poly-methoxylated phenyl rings such as 2,3,4-trimethoxy, 2,4,6-trimethoxy, and 2,4-dimethoxy groups. Additionally, bioisosteric heterocyclic rings such as furan and thiophen can be used instead of phenyl rings. Furthermore, molecular docking studies can be carried out to identify molecular interactions in future research.

Conclusion
A new series of pyrazole-sulphonamides, [4-[5-(1,3-benzodioxol-5-yl)-3-aryl-4,5-dihydro-1H-pyrazol-1-yl] benzenesulphonamide] were synthesized and evaluated their cytotoxicities and carbonic anhydrase inhibitory potencies. The cytotoxicities of the compounds towards tumor cell lines were in the range of 6.7 -400 µM. The compounds 4i (PSE 2 = 461.5) and 4g (PSE 1 = 193.2) had the highest PSE values in cytotoxicity assays. The use of methoxy substituents in different parts of the ring severely affected bioactivity. All compounds presented hCA I and hCA II inhibition potency. The compounds 4c (K i = 6.9 ± 1.5 µM, hCA II) and 4g (K i = 12.7 ± 1.7 µM, hCA I) had the lowest K i values as the best CA inhibitors. The compounds that show impressive bioactivities on the targets can be considered as lead compounds for further studies.