Synthesis and bioactivity studies on new 4-(3-(4-Substitutedphenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl) benzenesulfonamides

Abstract

A series of new 4-(3-(4-substitutedphenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl) benzenesulfonamides (7–12) was synthesized starting from 2-(4-substitutedbenzylidene)-2,3-dihydro-1H-inden-1-one (1–6) and 4-hydrazinobenzenesulfonamide. The substituted benzaldehydes from which the key intermediate was prepared by introducing 2- or 4-substituents such as fluorine, hydroxy, methoxy, or the 3,4,5-trimethoxy moieties. The compounds were tested for their cytotoxicity, tumor-specificity and potential as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The 3,4,5-trimethoxy and the 4-hydroxy derivatives showed interesting cytotoxic activities, which may be crucial for further anti-tumor activity studies, whereas some of these sulfonamides strongly inhibited both human (h) cytosolic isoforms hCA I and II.

• Benzenesulfonamide
• carbonic anhydrase/enzyme inhibition
• cytotoxicity
• indane
• pyrazole
• tumor selectivity

Introduction

Cancer is the second cause of death all over the world. Although radiation and surgery are used for the treatment of cancer, chemotherapy is the most widely used therapeutic approach for it. Available anticancer drugs in markets have several problems such as side effects, toxicity, cross resistance, and low selectivity1.

The sulfonamides are an important class of drugs known with antibacterial, anti-carbonic anhydrase, diuretic, anti-diabetic or hypoglycemic, and antithyroid activities2–5. A large number of sulfonamide derivatives have recently been reported to show remarkable antitumor activity both in vivo and/or in vitro. Some of these sulfonamide derivatives are currently being evaluated in clinical trial leading to consider them as novel alternative anti-cancer drugs, devoid of the side effects of presently available pharmacological agents5. Recently, new pyrazolines bearing benzene sulfonamides were synthesized and their anticancer activities were investigated6. In this study it was observed promising anti-proliferative activities with GI₅₀ values less than 2 μM particularly against MOLT-4 (1.94), 5R (1.28) in leukemia cancers, EKVX (1.88) in non-small cell lung cancer, COLO 205 (1.69) in colon cancer for the compound 2f (4–(3-(3-chloro-6-hydroxy-2,4-dimethylphenyl)-5–(4-(dimethylamino)phenyl-4,5-dihydro-1H-pyrazol-1-yl) benzenesulfonamide). In the another literature7, it was reported that substituted pyrazoline compound (4–(5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-4, 5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide) and (1–(4-aminosulfonylphenyl)-3-trifluoromethyl-5-[3,5-di-(tri-fluoromethyl)-phenyl]-4,5-dihydro-pyrazole) showed improved antitumoral activity in the treatment of cancer, especially for colon and/or prostate cancer, although these compounds do not inhibit cyclooxygenase-1 and/or cyclooxygenase-2.

Indane or indanone-bearing compounds had been reported to show their several bioactivities including cytotoxic/anticancer activities8–15, inhibition of β-amyloid plaques, which were stimulated by acetylcholinesterase16, and effects on mitochondrial respiration by inhibition of reactive oxygen species17.

Chalcones are widely used precursor molecules for the preparation of pyrazoles and pyrazolines. Chalcones and their derivatives have several bioactivities such as cytotoxic/anticancer activities18–24, topoisomerase I inhibitory25, carbonic anhydrase I and II inhibitory15,26 activities.

Pyrazolines are prominent nitrogen bearing five membered heterocylic compounds with antimicrobial27, anti-inflammatory28, antihipertansive29 activities. Medicinally important pyrazolines are 1,3,5-trisubstituted derivatives and their antiinflammatory30,31, dual antimicrobial and antiinflammatory27, analgesic and antimicrobial32, and selective COX-2 inhibitory (i.e. Celecoxib)33 activities were reported.

The carbonic anhydrases (CAs) are the metalloenzymes containing zinc ions (Zn²⁺), which classically participate in the maintenance of pH homeostasis. CAs catalyze the reversible hydration of carbon dioxide (CO₂) in two-step reaction to yield bicarbonate (HCO₃⁻) ion and proton (H⁺)34. The inter-conversion of these chemical species is shown in following equation, which however is too slow to meet the physiological needs of most biochemical processes35.

CAs have six genetically and distinct enzyme families: the α-, β-, γ-, ɛ-, ζ- and η-CA. Mammals, including humans, generally contain α-CAs, the most popular CA family. Until now, sixteen different α-CA isoenzymes have been identified in various tissues and organs with different expression levels, kinetic and molecular properties and oligomeric rearrangements34. According to the known cellular localization, some of them are cytosolic (CA I, CA II, CA III, CA VII and CA XIII), other CA isoenzymes are membrane bound (CA IV, CA IX, CA XII and CA XIV), two of CAs are mitochondrial (CA VA and CA VB) and one of CAs is salivary (CA VI)36. CA XV is not synthesized in humans and other primates and is abundantly found in rodents and other vertebrates as an isoform. Three acatalytic forms are also reported and named as CA related proteins (CARP), CARP VIII, X and XI, which are found in the cytosol37.

The two important CA isozymes (CA I and CA II) are present at higher concentrations in the cytosol in erythrocytes. hCA I, and II have various medical applications and shows optimal activity at physiological pH and temperatures. Carbonic anhydrase inhibitors (CAIs) have many clinical usages of major diseases such as diuretics, antiglaucoma, gastroduodenal ulcers, anti-obesity drugs, acid-base disequilibria, and antiepileptic. CAIs are useful for the treatment of some neurological disorders such as idiopathic intracranial hypertension38,39. The inhibition and activation mechanisms of CAs are well-understood processes at the molecular level. Usually most classes of CAIs bind to the metal center thus causing disruption of the CO₂ hydration reaction3. The classical CAIs are the primary sulfonamides, RSO₂NH₂, which are in clinical use for more than seventy years as diuretics and systemically acting anti-glaucoma drugs34.

The aim of this study was to design and synthesize new compounds including pyrazoline, sulfonamide, and indane pharmacophores all together to investigate their cytotoxicities, potential carbonic anhydrase inhibition properties to find out a leader compound/s for further studies.

Materials and methods

Melting points were determined using an Electrothermal 9100 (IA9100, Bibby Scientific Limited, Stone, UK) instrument and are uncorrected. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were obtained using a Varian Mercury Plus spectrometer (Varian Inc., Palo Alto, CA). Chemical shifts (δ) are reported in ppm. Mass spectra were undertaken on an HPLC-TOF Waters Micromass LCT Premier XE (Waters Corporation, Milford, MA) mass spectrometer using an electrospray ion source (ESI). All reactions were carried out in CEM Discover microwave synthesis systems (CEM, Matthews, NC).

General procedure for the synthesis of 2–(4-substitutedbenzylidene)-2,3-dihydro-1H-inden-1-one (1–6)

Aqueous solution of sodium hydroxide (10% w/v, 10 mL) was added into the ethanol (6 mL) solution of 1-indanone (20 mmol) and suitable substitute benzaldehyde (20 mmol) (Scheme 1). The mixture was stirred overnight at room temperature and then it was poured on ice-water (100 mL) in the beaker. The mixture was neutralized with hydrochloric acid (10% w/v, 10 mL). The colored precipitate formed was filtered and crystallized from water- ethanol for the compounds (1–6)13–15,17,40,41. Chemical structure of the compounds 1–6 were confirmed by ¹H NMR, ¹³C NMR, HRMS and the literature reported melting points of the compounds. Data are not presented here.

Scheme 1. 4–(3-(4-substitutedphenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl) benzenesulfonamide 7–12. (i) aq. NaOH 10%, EtOH, r.t, 12 h; (ii) 4-hydrazinobenzensulfonamide hydrochloride, EtOH, 100 °C, 200 Watt, 3-7 barr, 10'-120'. R: H (1, 7), 4-OCH₃ (2, 8), 2-OCH₃ (3, 9), 3,4,5-(OCH₃)₃ (4, 10), 4-F (5, 11), 4-OH (6, 12).

General procedure for the synthesis of pyrazoline derivatives (7–12)

A solution of 2–(4-substituted benzylidene)-2,3-dihydro-1H-inden-1-one (1–6, 1.00 mmol) and 4-hydrazinobenzensulfonamide hydrochloride (1.10 mmol) in ethanol (50 mL) was heated in (100 °C, 200 Watt, 3–7 barr) for 10–120 min [20 min, 3 barr (7), 60 min, 7 barr (8, 11); 30 min, 7 barr (9); 10 min, 7 barr (10); 120 min, 3 barr (12)]. The reactions were monitored by TLC. When the reaction was stopped, the volume of the reaction mixture was concentrated to the half and the precipitate formed was filtered, washed with cold ethanol, and the compounds were purified by crystallization from ethanol to obtain 7–12. Chemical structures of the compounds 7–12 were confirmed by ¹H NMR, ¹³C NMR, and HRMS.

4–(3-Phenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl)benzenesulfonamide (7)

M.p. 243–246 °C. Yield: 8.4% ¹H NMR (400 MHz, CDCl₃, ppm) δ 7.76 (d, 1H, Ar-H, J = 8.4 Hz), 7.67 (d, 2H, Ar-H, J = 9.1 Hz), 7.34–7.19 (m, 6H, Ar-H), 7.03 (bs, 4H, Ar-H), 5.59 (d, 1H, C₃-H, J = 10.9 Hz), 4.28–4.21 (m, 1H, C_3a-H), 2.91 (dd, 1H, C₄-H_a, J = 15.9, 8.7 Hz), 2.17 (dd, 1H, C₄-H_b, J = 15.9, 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃, ppm) δ 163.9, 151.7, 148.1, 134.3, 131.0, 130.7, 129.8, 128.9, 128.4, 128.3, 127.9, 127.3, 126.6, 122.9, 112.5, 67.5, 55.2, 29.9; Mass spectrum: 390.12 (M⁺+1); HRMS (ESI-MS) Calc.: 390.1276 for C₂₂H₂₀N₃O₂S [M + H]⁺, found: 390.1281.

4–(3-(4-Methoxyphenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl)benzenesulfonamide (8)

M.p. 172–176 °C. Yield: 19.3% ¹H NMR (400 MHz, CD₃OD, ppm) δ 7.73–7.71 (m, 1H, Ar-H), 7.64–7.62 (m, 2H, Ar-H), 7.35–7.30 (m, 2H, Ar-H), 7.26–7.24 (m, 1H, Ar-H), 7.06 (d, 2H, Ar-H, J = 8.4 Hz), 6.96 (bs, 2H, Ar-H), 6.77 (d, 2H, Ar-H, J = 7.7 Hz), 5.74 (d, 1H, C₃-H, J = 10.9 Hz), 4.29–4.23 (m, 1H, C_3a-H), 3.69 (s, 3H, OCH₃), 2.94 (dd, 1H, C₄-H_a, J = 15.9, 8.9 Hz), 2.14 (dd, 1H, C4-H_b, J = 15.9, 7.4 Hz); ¹³C NMR (100 MHz, CD₃OD, ppm) δ 164.3, 159.6, 152.0, 148.1, 131.6, 131.1, 130.5, 128.5, 127.6, 127.4, 126.5, 126.3, 122.1, 114.1, 112.3, 67.3, 54.9, 54.4, 29.3; Mass spectrum: 420.13 (M⁺+1); HRMS (ESI-MS) Calc.: 420.1382 for C₂₃H₂₂N₃O₃S [M + H]⁺, found: 420.1399.

4–(3-(2-Methoxyphenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl)benzenesulfonamide (9)

M.p. 245–247 °C. Yield: 3.6% ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 7.67–7.65 (m, 1H, Ar-H), 7.56 (d, 1H, Ar-H, J = 9.1 Hz), 7.34–7.28 (m, 2H, Ar-H), 7.21–7.17 (m, 1H, Ar-H), 7.07 (d, 1H, Ar-H, J = 8.1 Hz), 7.01 (s, 2H, Ar-H), 6.87 (bs, 2H, Ar-H), 6.67 (t, 1H, Ar-H, J = 7.5 Hz), 6.44 (d, 1H, Ar-H, J = 7.7 Hz), 5.95 (d, 1H, C₃-H, J = 10.9 Hz), 4.32–4.30 (m, 1H, C_3a-H), 3.89 (s, 3H, OCH₃), 2.99 (dd, 1H, C₄-H_a, J = 16.2, 8.4 Hz), 1.93 (dd, 1H, C₄-H_b, J = 16.2, 7.5 Hz); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 164.8, 157.9, 152.1, 147.3, 132.9, 131.3, 131.0, 129.8, 128.4, 128.0, 127.3, 122.7, 121.4, 121.2, 112.2, 111.8, 62.4, 56.3, 54.2, 29.9; Mass spectrum: 420.13 (M⁺+1); HRMS (ESI-MS) Calc.: 420.1382 for C₂₃H₂₂N₃O₃S [M + H]⁺, found: 420.1373.

4–(3-(3,4,5-Trimethoxyphenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl)benzenesulfonamide (10)

M.p. 266–269 °C. Yield: 41.5% ¹H NMR (400 MHz, DMSO-d₆, ppm) δ 7.71–7.69 (m, 1H, Ar-H), 7.58 (d, 2H, Ar-H, J = 9.1 Hz), 7.36–7.32 (m, 3H, Ar-H), 7.05–7.01 (m, 4H, Ar-H), 5.80 (d, 1H, C₃-H, J = 10.6 Hz), 4.30–4.27 (m, 1H, C_3a-H), 2.97 (dd, 1H, C₄-H_a, J = 16.1, 8.7 Hz) 2.02 (dd, 1H, C₄-H_b, J = 16.1, 7.7 Hz); 9 hydrogen peaks of three methoxy groups were under the peak of solvents DMSO-d₆. ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ 164.6, 153.7, 152.3, 147.8, 137.3, 133.2, 131.2, 131.1 131.0, 128.5, 127.9, 127.4, 122.9, 112.6, 67.4, 60.6, 56.4, 56.5, 54.9; Mass spectrum: 480.15(M⁺+1); HRMS (ESI-MS) Calc.: 480.1593 for C₂₅H₂₆N₃O₅S [M + H]⁺, found: 480.1599.

4–(3-(4-Florophenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl)benzenesulfonamide (11)

M.p. 162–165 °C. Yield: 16.5% ¹H NMR (400 MHz, CD₃OD, ppm) δ 7.73 (d, 1H, Ar-H, J = 4.2 Hz), 7.65 (d, 2H, Ar-H, J = 9.1 Hz), 7.34–7.32 (m, 2H, Ar-H), 7.26 (d, 1H, Ar-H, J = 4.3 Hz), 7.07–6.97 (m, 6H, Ar-H), 5.81 (d, 1H, C₃-H, J = 10.6 Hz), 4.32–4.25 (m, 1H, C_3a-H), 2.97 (dd, 1H, C₄-H_a, J = 15.8, 8.9 Hz), 2.09 (dd, 1H, C₄-H_b, J = 15.8, 7.6 Hz); ¹³C NMR (100 MHz, CD₃OD, ppm) δ 164.2, 151.9, 147.9, 131.9, 130.9, 130.8, 130.6, 129.3, 127.7, 127.5, 126.4, 122.2, 115.6, 115.4, 112.3, 66.9, 54.8, 29.4; Mass spectrum: 408.11(M⁺+1); HRMS (ESI-MS) Calc.: 408.1182 for C₂₂H₁₉N₃O₂SF [M + H]⁺, found: 408.1174.

4–(3-(4-Hydroxyphenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl)benzenesulfonamide (12)

M.p. 267–271 °C. Yield: 8.8% ¹H NMR (400 MHz, CD₃OD, ppm) δ 7.73–7.71 (m, 1H, Ar-H), 7.64 (d, 2H, Ar-H, J = 9.1 Hz), 7.34–7.25 (m, 3H, Ar-H), 7.06 (d, 2H, Ar-H, J = 8.0 Hz), 6.87 (bs, 2H, Ar-H), 6.64 (d, 2H, Ar-H, J = 7.3 Hz), 5.70 (d, 1H, C₃-H, J = 10.6 Hz), 4.28–4.21 (m, 1H, C_3a-H), 2.94 (dd, 1H, C₄-H_a, J = 16.0, 8.9 Hz), 2.21–2.13 (m, 1H, C₄-H_b); ¹³C NMR (100 MHz, CD₃OD, ppm) δ 164.4, 157.1, 152.0, 148.1, 131.5, 131.1, 130.4, 128.5, 127.5, 127.3, 126.4, 125.3, 122.1, 115.4, 112.3, 67.5, 54.9, 29.3; Mass spectrum: 406.12 (M⁺+1); HRMS (ESI-MS) Calc.: 406.1225 for C₂₂H₂₀N₃O₃S [M + H]⁺, found: 406.1214.

Assay for cytotoxicity

The compounds were assayed towards human oral squamous cell carcinoma cell lines (Ca9-22, HSC-2, HSC-3, HSC-4), and human oral normal mesenchymal cells [gingival fibroblast (HGF), pulp cell (HPC) and periodontal ligament fibroblast (HPLF)] based on a literature procedure with some minor modifications42,43. In brief, cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Varying concentrations of the compound in dimethylsulfoxide were added to the medium and incubated at 37 °C for 48 h. The viable cell numbers were determined by the MTT method except for HL-60 cells, the viable cell number of which was counted with a hemocytometer after staining with 0.15% trypan blue. The 50% cytotoxic concentration (CC₅₀) value was determined from the growth curves plotted at different concentrations of each compounds in triplicate wells.

Carbonic anhydrase enzyme assay

The Carbonic Anhydrase (CA) I, and II isoenzymes were purified from fresh human blood erythrocytes using by Sepharose-4B-L-Tyrosine-sulfanilamide affinity chromatography44,45. This method contains the purification of CA isoenzymes via a single step described previously46.

CA isoenzyme activity was determined spectrophotometricaly at 348 nm as described by Verpoorte et al.47. According to this method the absorbance changes were measured during the time of 3 min at 25 °C as p-nitrophenylacetate (PNA) converted to 4-nitrophenylate ion. These type of spectrophotometric determinations are described in detail in our previous studies48.

Bradford method was used to quantify the amount of protein during the purification steps. This spectrophotometric assay has been explained previously49. Bovine serum albumin was used as standard protein50.

After the purification process of the CA isoenzymes, SDS-polyacrylamide gel electrophoresis (SDS–PAGE) has been carried out51. Stacking and resolving gel containing 3% and 10% acrylamide, and 0.1% SDS was used for running the process using a Minigel system (Mini-PROTEAN® system Casting stand, Catalog 1658050, Bio-Rad Laboratories, Inc., China). The method used for visualization of protein has been explained in detail in our previous studies52. According to this method, the gel was fixed then stained with Coomassie Brilliant Blues R-250 later on the gel stained by using standard methods for detecting protein bands that are belong to purified CA isoenzymes53.

The effects of novel benzenesulfonamides (7–12) derivatives were examined using the hydratase activity and recorded in triplicate analysis at each concentration used54. For this purpose, different concentrations of novel benzenesulfonamides (7–12) derivatives were determined in preliminary assays. CA isoenzyme activities were measured in the presence of different quantity of them. The control sample activity in the absence of a novel benzenesulfonamides (7–12) derivatives were taken as 100%55. For each novel benzenesulfonamides (7–12), an activity (%)-[Benzenesulfonamides] was drawn using Excel program. IC₅₀ of each novel benzenesulfonamides (7–12) derivatives was calculated from graphs. IC₅₀ value is a measure of the effectiveness of benzenesulfonamides (7–12) derivatives in inhibiting both CA isoenzymes56. For determination of Ki values, three different benzenesulfonamides (7–12) concentrations were used. Ki values reflect the binding affinity of benzenesulfonamides (7–12) to both CA isoenzymes. In this way, Value is converted to an absolute inhibition constant Ki value. In this experiment, PNA was used as substrate at five different concentrations. Then, Lineweaver–Burk curves were drawn57.

Result and discussion

Condensation between 1-indanone and the appropriate benzaldehyde afforded the compounds 1–6. These compounds reacted with 4-hydrazinobenzensulfonamide hydrochloride to produce pyrazoline derivatives, the compounds 7–12. ¹H NMR, ¹³C NMR, and HRMS spectroscopies confirmed the chemical structures.

The cytotoxicity data (Table 1), hCA I and II inhibition percentages data (Table 2) of the compounds were presented in Tables 1 and 2, respectively.

Table 1. Cytotoxicity, tumor-specificity and Log P values of new 4–(3-(4-substitutedphenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl) benzenesulfonamides (7–12).

CC50 (μM)
Human oral squamous cell carcinoma cell lines										Human normal oral cells
	Ca9-22 (A)	SI	HSC-2	SI	HSC-3	SI	HSC-4	SI	mean (B)	mean SI	HGF (C)	HPLF	HPC	mean (D)	TS(D/B)	TS(C/A)	*Log P
7	19.3 ± 4.7	1.7	37.1 ± 1.3	0.9	8.6 ± 5.2	3.8	4.6 ± 0.6	7.2	17.4	3.4	27.4 ± 24.7	8.1 ± 2.3	63.3 ± 19.8	33	1.9	1.4	3.68
8	18.7 ± 4.7	2.5	41.2 ± 1.8	1.1	32.8 ± 1.6	1.4	34.8 ± 7.8	1.3	31.9	1.5	48.7 ± 15.3	38.0 ± 2.0	52.0 ± 10.3	46,2	1.5	2.6	3.55
9	14.0 ± 2.0	2.4	25.6 ± 1.1	1.3	23.0 ± 0.7	1.5	17.1 ± 2.2	2.0	19.9	1.8	36.0 ± 1.6	36.0 ± 7.5	30.3 ± 6.0	34,1	1.7	2.6	3.55
10	24.7 ± 4.7	2.8	35.2 ± 1.9	1.9	29.4 ± 3.8	2.3	27.8 ± 2.9	2.4	29.3	2.3	97.3 ± 25.1	41.7 ± 3.2	65.3 ± 18.5	68,1	2.3	3.9	3.3
11	21.3 ± 0.6	1.9	35.2 ± 2.0	1.2	26.1 ± 7.8	1.6	19.2 ± 3.3	2.2	25.5	1.7	45.0 ± 2.6	38.3 ± 2.3	41.3 ± 4.6	41,6	1.6	2.1	3.84
12	58.0 ± 20.8	6.9	>400	<1	>400	<1	>400	<1	314.5	<2.5	>400	>400	>400	400	1.3	>6.9	3.29
Average	26	3	>95,7	>1.2	>86,6	>1.9	>83,9	>2.7	73.1	2.2	>109,1	>93,7	>108,7	103,8
5-FU	12.1 ± 0.7	72.3	15.2 ± 2.7	57.6	7.2 ± 1.0	121.6	7.3 ± 0.17	119.9	10.5	92.8	>1000	>1000	626.3 ± 87.9	875.4	83.8	83.3

The CC₅₀ values refer to the concentrations of the compounds in micromoles, which kill 50% of the cells. The letters SI indicate the selectivity index, i.e. the quotient of the average CC₅₀ figures towards HGF, HPC and HPLF nonmalignant cells divided by the CC₅₀ figure of the compound towards a specific tumor cell line.

* Log P values were calculated using ChemDraw Ultra (Version 12.0, Cambridge Soft Corporation, Cambridge, MA).

Table 2. Inhibition of human carbonic anhydrase isoenzymes (hCA I and II) by new 4–(3-(4-substitutedphenyl)-3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl) benzenesulfonamides (7–12).

	IC50 (nM)				KI (nM)
Compounds	hCA I	r^2	hCA II	r^2	hCA I	hCA II
7	644.65	0.9846	514.47	0.9725	547.35 ± 154.2	500.87 ± 122.5
8	502.17	0.9717	391.08	0.9543	456.93 ± 117.3	400.58 ± 172.4
9	510.31	0.9755	463.54	0.9801	458.30 ± 69.20	470.71 ± 136.1
10	413.97	0.9693	330.78	0.9726	324.61 ± 47.16	262.92 ± 72.05
11	535.13	0.9814	418.73	0.9624	328.92 ± 31.02	318.06 ± 120.1
12	498.92	0.9783	410.78	0.9610	550.21 ± 103.2	487.73 ± 214.1
AZA*	656.87	0.9908	610.57	0.9958	460.27 ± 192.8	455.28 ± 146.0

*Acetazolamide (AZA) was used as a standard inhibitor for all hCA.

When the cytotoxicity data of the compounds were considered, the first question to be addressed is whether the compounds 7–12 have anti-neoplastic properties. The results portrayed in Table 1 reveal that in general the CC₅₀ of 7–12 are in the range of 4.6–58.0 μM towards Ca9–22, HSC-2, HSC-3, and HSC-4 cells. The potency of the compounds 7–12 towards tumor cell lines was compared with a reference compound 5-Fluorouracil (5-FU). Compounds 7 was more potent than 5-FU towards HSC-4 cells.

The second aspect of these compounds to be considered is whether they are tumor-specific cytotoxins since tumors are surrounded by different types of normal cells. Selectivity index (SI) figures were generated which are quotients of average CC₅₀ values of normal cells and CC₅₀ figure of a compound towards a specific cell line. The results in Table 1 reveal that SI values of greater than 1 were obtained in general. Exceptions were 12 towards HSC-2, HSC-3, and HSC-4 cell lines and 7 towards HSC-2 cells.

When the most-selective compounds (SI) toward tumorous cells were considered, the following sets of combination were found to be the best: 12 (SI: 6.9) towards Ca9–22, 10 (SI: 1.9) towards HSC-2, 7 (SI: 3.8) towards HSC-3, 7 (SI: 7.2) towards HSC-4 cell lines.

Tumor-specificity (TS) value reflects the selectivity of the compounds against cancer tissues rather than normal ones. In this study, two types TS values were calculated. First, TS was also calculated by dividing the mean CC₅₀ value of each compound against three human oral normal cells (Column D) to mean CC₅₀ value against four human OSCC cell lines (Column B) (Table 1). Second, TS was calculated by dividing the CC₅₀ value of each compound against HGF cells (Column C) to the CC₅₀ value against Ca9–22 cell line (Column A), both cells being originated from the same tissue (gingiva) (Table 1). All compounds showed lower TS values than reference drug 5-FU by these two types of criteria for TS. According to TS values obtained by first calculation method, the order of potency of TS values of the compounds was as follows: The compound number (TS value): 10 (2.3) > 7 (1.9) > 9 (1.7) > 11 (1.6) > 8 (1.5) > 12 (1.3). When the second calculation was considered, the order of potency of TS values of the compounds was as follows: 12 (6.9) > 10 (3.9) > 8 and 9 (2.6) > 11 (2.1) > 7 (1.4).

When the esterase assay with 4-nitrophenyl acetate as substrate were applied to the compounds 7–12, all benzenesulfonamide compounds 7–12 behaved as powerful inhibitors against slow cytosolic isoenzyme hCA I with Ki values in ranging of 324.61 ± 47.16 – 550.21 ± 103.2 nM. Compound 10 (Ki: 324.61 ± 47.16 nM), which is 3,4,5-trimethoxy derivative, and compound 11 (Ki: 328.92 ± 31.02), which is 4-fluoro derivative, inhibited hCA I activity more potently than reference drug AZA (Ki: 460.27 ± 192.8), which is used for the treatment of idiopathic intracranial hypertension, cystinuria, glaucoma, altitude sickness, epileptic seizure, periodic paralysis, central sleep apnea and dural estasia. Since hCA I isoenzyme is found in many tissues and involved in retinal and cerebral edema, its inhibition by the compounds 7–12 may be a valuable tool for fighting against these symptoms. On the other hand, the compounds 7–12 demonstrated Ki values ranging between 262.92 ± 72.05 and 500.87 ± 122.5 nM towards hCA II. The compounds 10 (Ki: 262.92 ± 72.05 nM) and 11 (Ki: 318.06 ± 120.1 nM) inhibited hCA II activity more potently than reference compound AZA (Ki: 455.28 ± 146.0 nM), like in the case of hCA I experiment. Since CAII isoenzyme involved in several diseases, such as glaucoma, edema, epilepsy, and altitude sickness, its inhibitory property of 7–12 may be applicable for fighting these diseases.

As a result, the compounds 10, which is 3,4,5-trimethoxy derivative, and 12, which is 4-hydroxy derivative, seem candidate cytotoxic compounds for further studies in terms of tumor-specificity according to two types of TS calculations while the compounds 10 and 11, which is 4-fluoro derivative, seem candidate compounds as both hCA I and II inhibitors for further studies.

Declaration of interest

The authors report no conflict of interest and are responsible for the contents and writing of the paper. This research work was supported by Ataturk University Research Found, Turkey (Project No BAP: 2012/74.