Synthesis, cytotoxicity and carbonic anhydrase inhibitory activities of new pyrazolines

Article abstract of DOI:10.1080/14756366.2016.1217852

A series of polymethoxylated-pyrazoline benzene sulfonamides were synthesized, investigated for their cytotoxic activities on tumor and non-tumor cell lines and inhibitory effects on carbonic anhydrase isoenzymes (hCA I and hCA II). Although tumor selectivity (TS) of the compounds were less than the reference compounds 5-Fluorouracil and Melphalan, trimethoxy derivatives 4, 5, and 6 were more selective than dimethoxy derivatives 2 and 3 as judged by the cytotoxicity assay with the cells both types originated from the gingival tissue. The compound 6 (4-[3-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl] benzene sulfonamide) showed the highest TS values and can be considered as a lead molecule of the series for further investigations. All compounds synthesized showed superior CA inhibitory activity than the reference compound acetazolamide on hCA I, and II isoenzymes, with inhibition constants in the range of 26.5–55.5 nM against hCA I and of 18.9–28.8 nM against hCA II, respectively.

Full text of DOI:10.1080/14756366.2016.1217852

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis, cytotoxicity and carbonic anhydrase
inhibitory activities of new pyrazolines
Kaan Kucukoglu, Fatih Oral, Tevfik Aydin, Cem Yamali, Oztekin Algul, Hiroshi
Sakagami, Ilhami Gulcin, Claudiu T. Supuran & Halise Inci Gul
To cite this article: Kaan Kucukoglu, Fatih Oral, Tevfik Aydin, Cem Yamali, Oztekin Algul, Hiroshi
Sakagami, Ilhami Gulcin, Claudiu T. Supuran & Halise Inci Gul (2016): Synthesis, cytotoxicity and
carbonic anhydrase inhibitory activities of new pyrazolines, Journal of Enzyme Inhibition and
Medicinal Chemistry, DOI: 10.1080/14756366.2016.1217852
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1217852
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–5
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1217852
!
RESEARCH ARTICLE
Synthesis, cytotoxicity and carbonic anhydrase inhibitory activities of
new pyrazolines
Kaan Kucukoglu1, Fatih Oral1, Tevfik Aydin1, Cem Yamali1, Oztekin Algul2, Hiroshi Sakagami3, Ilhami Gulcin4,5
Claudiu T. Supuran6, and Halise Inci Gul1
,
2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum, Turkey, Department of Pharmaceutical Chemistry,
3
Faculty of Pharmacy, Mersin University, Mersin, Turkey, Division of Pharmacology, School of Dentistry, Meikai University School of Dentistry,
4
Sakado, Saitama, Japan, Department of Chemistry, Faculty of Science, Ataturk University, Erzurum, Turkey, Department of Zoology, College of
5
6
Science, King Saud University, Riyadh, Saudi Arabia, and Neurofarba Departmente Laboratorio di ChimicaBioinorganica, Universita degli Studi di
Firenze, via U. Schiff 6, SestoFiorentino (Florence), Italy
Abstract
Keywords
A series of polymethoxylated-pyrazoline benzene sulfonamides were synthesized, investigated
for their cytotoxic activities on tumor and non-tumor cell lines and inhibitory effects on
carbonic anhydrase isoenzymes (hCA I and hCA II). Although tumor selectivity (TS) of the
compounds were less than the reference compounds 5-Fluorouracil and Melphalan, trimethoxy
derivatives 4, 5, and 6 were more selective than dimethoxy derivatives 2 and 3 as judged by the
cytotoxicity assay with the cells both types originated from the gingival tissue. The compound
6 (4-[3-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl] benzene sul-
fonamide) showed the highest TS values and can be considered as a lead molecule of the series
for further investigations. All compounds synthesized showed superior CA inhibitory activity
than the reference compound acetazolamide on hCA I, and II isoenzymes, with inhibition
constants in the range of 26.5–55.5 nM against hCA I and of 18.9–28.8 nM against hCA II,
respectively.
Carbonic anhydrase, cytotoxicity, dental cells,
pyrazoline, sulfonamide, tumor selectivity
History
Received 4 June 2016
Revised 18 July 2016
Accepted 20 July 2016
Published online 12 August 2016
Introduction
CAs have become remarkable therapeutic targets for the treatment
of a wide range of disorders. Several CA inhibitors show
anticancer7, diuretics8, antiglaucoma9, antiinfective10 and anti-
obesity11 activities.
Cancer is one of the most important causes of deaths in the
world1. Various classes of drugs such as alkylating agents,
antimetabolites, antibiotics, hormones and plant alkaloids are
used in the traditional cancer chemotherapy2. Although selective
toxicity is preferable in cancer treatment, the drugs used in clinics
have several side effects such as resistance development to the
anticancer agents in addition to low selectivities3.
Carbonic anhydrase (CA, EC 4.2.1.1) a zinc-dependent
metalloenzyme that catalyzes the reversible hydration of CO2.
Sixteen different a-CA isoforms have been isolated and
characterized so far in mammals: CA I, CA II, CA III, CA VII,
CA XIII are cytosolic; CA IV, CA IX, CA XII, CA XIV and CA
XV are associated with the cell membrane; CA VA and CA VB
are in mitochondria and CA VI is secreted into saliva and milk4.
The CAs contribute many physiological and pathological
processes such as pH homeostasis, electrolyte secretion in various
tissues and organs, gluconeogenesis, lipogenesis, ureagenesis,
bone resorption calcification and tumorigenicity5–7. Thus, the
Pyrazole/pyrazolines are an important class of heterocyclic
compounds containing three carbon atoms and two nitrogen atoms
in adjacent positions. These heterocyclic compounds can be seen
in nature as alkaloids, vitamins, pigments and constituents of
plant and animal cells widely12. Medicinal chemists have great
attention of pyrazolines and substituted pyrazolines because of
their various biological activities such as antimicrobial, anticon-
vulsant, anticancer, anti-inflammatory, antitubercular, antiviral,
analgesic and antidepressive activities13–17. Sulfonamide is an
important functional group in medicinal chemistry having a wide
range of bioactivities such as cytotoxic, carbonic anhydrase
inhibitory, antibacterial, antimalarial, antihypertensive, antiviral,
anti-inflammatory and diuretics activities8,13,18–25
.
In this study it was aimed to synthesize new compounds
bearing pyrazoline and sulfonamide pharmacophores and to test
their carbonic anhydrase inhibitory effects on hCA I and II
isoenzymes. It was also planned to investigate their cytotoxic
activities towards four cancer cell lines (Ca9–22, HSC-2, HSC-3,
and HSC-4) and three non-tumor cells (HGF, HPLF and HPC) to
see whether any of compounds synthesized is/are tumor-specific
cytotoxin/s.
Address for correspondence: Assist. Prof. Dr. Kaan Kucukoglu and Prof.
Dr. Halise Inci Gul, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Ataturk University, Erzurum, Turkey. E-mail: kucukoglu-
k35@hotmail.com (K.K.); incigul1967@yahoo.com (H.G.)

2
K. Kucukoglu et al.
J Enzyme Inhib Med Chem, Early Online: 1–5
Experimental
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was
applied with a Bio-Rad Mini Gel system (Mini-PROTEAN Tetra
System, China) after purification of both CA isoenzymes28.
Materials and methods
Chemical structure of the compounds were determined by 1H Briefly, it was performed in acrylamide for the running (10%) and
NMR (400 MHz) and 13C NMR (100 MHz) spectroscopies using a the stacking gel (3%) contained SDS (0.1%), respectively.
Varian Mercury Plus spectrometer (Varian Inc., Palo Alto, CA). Activities of CA I and II isoenzymes were determined according
Chemical shifts (d) are reported in ppm and coupling constants (J) to the method of Verpoorte et al29. The increasing in absorbance
are expressed in hertz (Hz). Mass spectra for the compound 1 of reaction medium was spectrophotometrically recorded at
were taken using a liquid chromatography ion trap-time of flight 348 nm (Shimadzu, UV-VIS Spectrophotometer, UVmini-1240,
tandem mass spectrometer (Shimadzu, Kyoto, Japan) equipped Kyoto-Japan). Also, the quantity of protein was determined at
with an electrospray ionization (ESI) source, operating in both 595 nm according to Bradford method30. Bovine serum albumin
positive and negative ionization mode. Shimadzu’s LCMS was used as standard protein. The IC50 values were obtained from
Solution software was used for data analysis. Mass spectra for activity (%) versus compounds plots. For calculation of Ki values,
the compounds 2–6 were undertaken on a HPLC-TOF Waters three different concentrations were used. The Lineweaver–Burk
Micromass LCT Premier XE (Milford, MA) mass spectrometer curves were drawn and calculations were realised31.
using an electrospray ion source (ESI). Melting points were
determined using an electrothermal 9100/IA9100 instrument Cytotoxicity assay
(Bibby Scientific Limited, Staffordshire, UK) and are uncor-
The cytotoxicity of the compounds were assayed towards human
rected. The reactions were monitored using silica gel HF254–366
oral squamous cell carcinoma cell lines (Ca9–22, HSC-2, HSC-3,
TLC plates (E. Merck, Darmstadt, Germany). IR spectra of the
HSC-4) and human normal oral cells (HGF, HPLF, HPC) as
compounds were taken using a FT-IR spectrometer (Perkin Elmer
described with some minor modifications32–37. All cells were
Spektrum One FT-IR, Bridgeport Avenue Shelton, CT).
cultured in DMEM supplemented with 10% fetalbovine serum
(FBS). All test samples were dissolved in dimethylsulfoxide
Synthesis of chalcone compounds, 1a–6a
(DMSO). Near confluent cells were incubated in 96-microwell
4-Methoxyacetophenone and suitable aldehyde in 1:1 mol ratio (Becton Dickinson, Franklin Lakes, NJ) for 48 h with 0.78, 1.56,
were dissolved in ethanol (10 ml)23. Aqueous NaOH solution 3.12, 6.25, 12.5, 25, 50, 100 or 200 mM of each test compound.
(10%, 20 ml) was added into the reaction flask. Reactions were Any cytotoxicity induced by DMSO (0.002, 0.004, 0.0078,
monitored by TLC. When at least one of the starting compounds 0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5 or 1%) were subtraced
finished, reactions were stopped. Reaction content was poured on from the corresponding treated groups. The viable cell numbers
ice-water and neutralized by HCl (10%). The precipitates obtained were determined by the MTT method. In brief, the treated cells
were washed with cold water and ethanol, filtered, and dried. The were incubated for another 3 h in fresh culture medium containing
purities were checked by TLC and used for the synthesis of 0.2 mg/ml MTT. Cells were then lysed with 0.1 ml of DMSO and
pyrazoline derivatives 1–6 without further purification.
the absorbance at 562 nm of the cell lysate was determined using a
¨
microplate reader (Sunrise Rainbow RC-R; TECAN, Mannedorf,
Synthesis of pyrazoline containing benzene sulfonamides, 1–6
Switzerland). CC50 (the concentrations of the compounds in
micromoles which reduce the viable cell number by 50%) was
determined from the dose–response curve and the mean value of
CC50 for each cell type was calculated from triplicate assays.
A mixture of a suitable chalcone compound (1a–6a) and
4-hydrazinobenzenesulfonamide hydrochloride in 1:1.1 mol ratio
in ethanol (25 ml for 1–3 and 30 ml for 4–6) in the presence of
glacial acetic acid (0.05 ml) was refluxed26. Reactions were
monitored by TLC using CHCl3:MeOH (4.8:0.2) as a solvent
Results and discussion
system. When the reactions were stopped, the precipitate obtained All the compounds studied here are reported for the first time with
was filtered, dried and crystallized from suitable solvent system. their detailed synthesis, spectral analysis, and bioactivities except
Experimental data of the compounds are presented in Table 1. 3 and 6. The synthetic pathway of the compounds are summarized
Spectral data of the compounds are presented in Table 2.
in Scheme 1 and their experimental data (Table 1), spectral data
(Table 2), cytotoxicities of the compounds against human oral
squamous cell carcinoma cell lines and human normal oral cells
(Table 3), the inhibitory potential of the compounds on hCA I and
II isoenzymes (Table 4) are summarized in Tables 1–4.
Biological activity
Carbonic anhydrase inhibition assay
The purification of cytosolic CA isoenzymes (CA I and II) were
Structures of the compounds synthesized were established on
previously described with a simple one-step method by a the basis of spectral data taken from FT-IR, H NMR, 13C NMR
sepharose-4B-L tyrosine-sulphanilamide affinity chromatog- and HRMS. The IR spectra of the compounds showed absorption
raphy27. The protein quantity in the column effluents was bands in the regions 1599–1571 cmꢀ1 corresponding to C¼N
determined spectrophotometrically at 280 nm. Sodium dodecyl stretching bands because of ring closure. Infrared spectra revealed
1
Table 1. Experimental data of the compounds 1–6.
Compounds
Chalcone mmol
PHBS mmol
Time h
Yield %
Crystallization Solvents
Mp, ꢁC
1
2
3
4
5
6
1.68
1.68
1.68
1.52
1.52
1.52
1.84
1.84
1.84
1.67
1.67
1.67
5
18
4
6
4
53
71
19
67
85
72
Ethanol/Chloroform
Ethanol/Chloroform
Methanol
Methanol
Methanol/Chloroform
Methanol
267–269
197–199
200–202
168–170
242–244
238–240
1
PHBS: 4-Hydrazinobenzensulfonamide hydrochloride.

DOI: 10.1080/14756366.2016.1217852
Activities of new pyrazolines
3
Table 2. Spectral data of the compounds 1–6.
Compounds
1H NMR, 13C NMR, and HRMS
1
1
2
3
4
5
6
4-(5-(2,5-dimethoxyphenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide. H NMR (DMSO-d6, d ppm):
7.69 (d, J ¼ 8.8 Hz, 2H), 7.57(d, J ¼ 9.2 Hz, 2H), 7.02-6.94 (m, 6H), 6.79 (dd, J ¼ 2.9, 9.0 Hz, 2H), 6.35 (d, J ¼ 2.9 Hz, 1H), 5.62
(dd, J ¼ 11.9, 5.0 Hz, 1H), 3.84 (s, 3H, -OCH3), 3.82 (s, 3H,OCH3), 3.77 (s, 3H, -OCH3), 3.04 (dd, J ¼ 17.6, 5.0 Hz, 1H)(One of the
proton peak of the pyrazole ring under the solvent peak). 13C NMR (DMSO-d6, d ppm):160.9, 153.8, 150.9, 150.8, 146.6, 133.1,
130.2, 128.4, 127.9, 125.0, 114.8, 113.3, 112.8, 112.1, 57.8, 56.8, 55.9, 55.8, 42.6. HRMS (ESI-MS) Calc. forC24H26N3O5S
[M + H]+, 468.1588; found 468.1582
1
4-(5-(2,4-dimethoxyphenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1- yl)benzenesulfonamide. H NMR (DMSO-d6, d ppm):
7.69 (d, J ¼ 8.8 Hz, 2H), 7.56 (d, J ¼ 8.8 Hz, 2H), 6.99-6.93 (m, 6H), 6.73 (d, J ¼ 8.8 Hz, 1H), 6.62 (d, J ¼ 2.2 Hz, 1H), 6.38 (dd,
J ¼ 8.4, 2.6 Hz, 1H), 5.58 (dd, J ¼ 11.7, 4.8 Hz, 1H), 3.87 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.69 (s, 3H, -OCH3), 3.01 (dd,
J ¼ 17.4, 4.8 Hz, 1H)(One of the proton peak of the pyrazole ring under the solvent peak). 13C NMR (DMSO-d6, d ppm): 160.9,
160.7, 157.8, 150.8, 146.6, 132.9, 128.3, 127.9, 127.2, 125.2, 121.2, 114.8, 112.1, 105.7, 99.7, 57.5, 56.5, 55.9, 55.8, 42.8. HRMS
(ESI-MS) Calc. for C24H26N3O5S [M + H]+ 468.1593; found 468.1590
1
4-(5-(3,4-dimethoxyphenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide. H NMR (DMSO-d6) d (ppm):
7.69 (d, J ¼ 8.8 Hz, 2H), 7.53 (d, J ¼ 9.0 Hz, 2H), 7.04-6.97 (m, 6H), 6.90 (d, J ¼ 2.2 Hz, 1H), 6.84 (d, J ¼ 8.4 Hz, 1H, Ar-H), 6.63
(dd, J ¼ 8.2, 2.0 Hz, 1H), 5.45 (dd, J ¼ 11.6, 5.3 Hz, 1H), 3.11 (dd, J ¼ 17.2, 2.9 Hz, 1H) (One of the proton of the pyrazole ring and
methoxy peaks under the solvent peak).13C NMR (DMSO-d6) d (ppm): 160.9, 150.4, 149.7, 148.7, 146.9, 134.7, 133.1, 128.4,
127.7, 125.0, 118.0, 114.9, 112.7, 112.5, 110.3, 62.8, 56.1, 56.0, 55.9, 43.9. HRMS (ESI-MS) Calc. for C24H26N3O5S [M + H]+
468.1593; found 468.1599
1
4-(3-(4-methoxyphenyl)-5-(2,3,4-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide. H NMR (DMSO-d6, d
ppm): 7.71 (d, J ¼ 8.8 Hz, 2H), 7.56 (d, J ¼ 9.1 Hz, 2H), 6.99-6.97 (m, 6H), 6.69-6.65 (m, 2H), 5.57 (dd, J ¼ 12.1, 5.1 Hz, 1H), 3.86
(s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.75 (s, 3H, -OCH3), 3.71 (s, 3H, -OCH3), 3.09 (dd, J ¼ 17.6, 5.1 Hz, 1H) (One of the proton
peak of the pyrazole ring under the methoxy peak). 13C NMR (DMSO-d6, d ppm): 160.9, 153.7, 151.2, 150.7, 146.6, 142.6, 133.1,
128.3, 127.9, 127.1, 125.2, 121.4, 114.8, 112.2, 108.6, 61.5, 61.0, 58.3, 56.4, 55.9, 43.1. HRMS (ESI-MS) Calc. for C25H28N3O6S
[M + H]+ 498.1699; found 498.1691
1
4-(3-(4-methoxyphenyl)-5-(2,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide. H-NMR (DMSO-d6, d
ppm): 8.24 (s, 1H), 7.69 (d, J ¼ 8.4 Hz, 2H), 7.54 (d, J ¼ 8.8 Hz, 2H), 6.98-6.95 (m, 4H), 6.72 (s, 1H), 6.49 (brs, 2H), 5.55 (dd,
J ¼ 12.1, 5.6 Hz, 1H), 3.81 (s, 3H, -OCH3), 3.76 (s, 3H, -OCH3), 3.74 (s, 3H, -OCH3), 3.63 (s, 3H, -OCH3), 3.05 (dd, J ¼ 17.4,
5.6 Hz, 1H) (One of the proton peak of the pyrazole ring). 13C NMR (DMSO-d6, d ppm): 160.9, 151.4, 150.8, 149.9, 146.9, 143.4,
132.9, 128.3, 127.8, 125.1, 120.4, 114.9, 112.2, 111.9, 99.3, 79.7, 57.2, 57.1, 56.4, 55.9, 42.7. HRMS (ESI-MS) Calc. for
C
25H28N3O6S [M + H]+ 498.1699; found 498.1701
1
4-(3-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide. H NMR (DMSO-d6, d
ppm): 8.2 (s, 1H), 7.70 (d, J ¼ 8.8 Hz, 2H), 7.56 (d, J ¼ 9.1 Hz, 2H), 7.04 (d, J ¼ 8.8 Hz, 2H), 7.01 (brs, 2H), 6.98 (d, J ¼ 8.8 Hz,
2H), 6.54 (s, 1H), 5.40 (dd, J ¼ 12.0, 5.0 Hz, 1H), 3.77 (s, 3H, -OCH3), 3.65 (s, 3H, -OCH3), 3.10 (dd, J ¼ 17.6, 5.0 Hz, 1H)(Other
methoxy peaks under the solvent peak). 13C NMR (DMSO-d6, d ppm): 160.9, 153.9, 150.6, 147.2, 138.3, 137.1, 133.3, 128.4,
127.8, 124.9, 114.9, 112.5, 103.4, 79.2, 63.5, 60.6, 56.5, 55.9, 43.9. HRMS (ESI-MS) Calc. for C25H28N3O6S [M + H]+ 498.1699;
found 498.1698
Scheme 1. Synthetic pathway of the pyrazoline bearing benzenesulfonamides 1–6. Reagents and conditions. i: EtOH, aq. NaOH (10%), rt. ii: 4-
Hydrazinobenzensulfonamide.HCl, EtOH, glacial CH3COOH, reflux. 1: (R1 ¼ R4 ¼ OCH3, R2 ¼ R3 ¼ H), 2: (R1 ¼ R3 ¼ OCH3, R2 ¼ R4 ¼ H), 3: (R2
¼ R3 ¼ OCH3, R1 ¼ R4 ¼ H), 4: (R1 ¼ R2 ¼ R3 ¼ OCH3, R4 ¼ H), 5: (R1 ¼ R3 ¼ R4 ¼ OCH3, R2 ¼ H), 6: (R2 ¼ R3 ¼ R4 ¼ OCH3, R1 ¼ H).
NH2 peaks in the regions 3371–3226 cmꢀ1. FT-IR spectra of the times), 5 (7.16 times) and 6 (5.51 times) towards Ca9–22 cell line;
compounds are provided in the Supplementary file. The FT-IR the compound 6 (2.22 times) towards HSC-2 cell line, the
values of the compounds were in accordance with literature compounds 5 (2.35 times) and 6 (5.19 times) towards HSC-3 cell
1
values23. In the H NMR and 13C NMR spectra of pyrazolines, line, the compounds 5 (1.95 times) and 6 (1.77 times) towards
chemical shift values of the three hydrogen atoms attached to the HSC-4 cell line were more cytotoxic than 5-FU. On the other
C-4 and C-5 carbon atoms of the heterocyclic ring and C-3, C-4 hand, the compounds 2 (1.83 times), 4 (1.83 times), 5 (5.23
and C-5 carbon atoms of the pyrazolines were observed at times), and 6 (4.03 times) towards Ca9–22 cell line, the
expected ppms (See Table 2 and Supplementary File). compound 6 (1.24 times) towards HSC-2 cell line were more
Cytotoxic activities of the compounds were tested against four cytotoxic than reference compound Melphalan.
cancer cell lines (Ca9–22, HSC-2, HSC-3, HSC-4) and three non-
Tumor specificity (TS) of the compounds was considered by
tumor normal cells (HGF, HPLF, HPC). The cytotoxicities of the TS values. TS values were calculated by two types of calculation.
compounds were compared with reference drugs 5-Fluorouracil Tumor-specificity (TS) value reflects the selectivity of the
(5-FU) and Melphalan (Table 3). The compounds 2, 4 (2.50 compounds against cancer tissues rather than normal ones.

4
K. Kucukoglu et al.
J Enzyme Inhib Med Chem, Early Online: 1–5
Table 3. Cytotoxicities of the synthesized compounds towards human oral squamous carcinoma cell lines and human normal oral cells.
CC50 (mM)
Human oral squamous carcinoma cell lines
Human normal oral cells
Compounds Ca9-22 (A) HSC-2 HSC-3 HSC-4 Mean (B)
SD
HGF (C)
HPLF
HPC
Mean (D)
SD
TS
1
2
3
4
5
6
Not Tested
22
200
22
7.7
10
D/B
0.4
0.6
0.7
0.5
1.5
C/A
1.0
0.3
3.4
1.4
2.0
200
200
200
41
200
200
200
31
200
200
200
20
155.5
200.0
155.5
24.9
89.0
0.0
89.0
14.3
5.4
22
63
75
11
20
119
120
135
12
46
181
115
12
62.3
121.3
108.3
11.7
50.5
59.0
30.6
0.6
11
14
22
14.3
21
23
21.3
1.5
5-FU
Melphalan
55.1
40.3
24.4
13.6
72.7
11
38.9
10.3
47.8
18.80
–
–
4200
4200
4200
151.0 4175.5
4200
4200
175.2
–
–
44.2 43.6
49.3 44.9
CC50 values refer to the concentrations of the compounds in micromoles which reduce the viable cell number by 50%. Oral squamous cell carcinoma
(OSCC) cell lines used are Ca9–22 (derived from gingiva), HSC-2, HSC-3, HSC-4 (derived from tongue). Normal oral cells used are human gingival
fibroblasts (HGF), human periodontal ligament fibroblasts (HPLF), and human pulp cells (HPC). Tumor-specificity (TS) value is calculated by
dividing the mean CC50 value of each compound against normal cells to mean CC50 value against OSCC. CC50 value was determined from the growth
curves plotted at different concentrations of each compounds in triplicate wells. SD: standard deviation, 5-FU: 5-fluorouracil, mM: micromolar.
Table 4. Carbonic anhydrase inhibitory activities of the compounds on hCA I and II isoenzymes.
IC50 (nM)
KI (nM)
Compounds
hCA I
r2
hCA II
r2
hCA I
hCA II
1
2
3
4
30.1
40.8
46.2
38.5
49.2
36.5
293.0
0.99
0.98
0.92
0.98
0.93
0.94
0.98
24.8
30.1
26.7
23.8
25.7
23.9
146.5
0.96
0.98
0.97
0.98
0.98
0.98
0.98
26.5 4.6
39.7 8.3
55.5 19.4
31.4 7.3
29.9 5.5
34.1 7.3
276.3 98.1
18.9 9.0
20.5 3.7
28.8 6.5
19.9 2.7
25.9 7.0
20.2 3.0
117.8 14.1
5
6
AZA
Acetazolamide (AZA) was used as a standard inhibitor for all hCA I and II isoenzymes.
First TS calculation was made by dividing the mean CC50 value of inhibitor than AZA on hCA II. When Ki values of the
each compound against three human oral normal cells (Column compounds were considered they were in the range of
D) to the mean CC50 value against four human OSCC cell lines 26.5 4.6–55.5 19.4 nM towards hCA I, while they were in
(Column B, Table 3). Second TS calculation was also calculated the range of 18.9 9.0–28.8 6.5 nM towards hCA II. Ki values
by dividing the CC50 value of each compound against HGF cells of the reference compound AZA were 276.3 98.1 nM and
(Column C) to the CC50 value of each compound against Ca9–22 117.8 14.1 nM towards hCA I and hCA II, respectively.
cell line (Column A, Table 3) since these cells are originated from
Introduction of a methoxy group to 4 positions of 2,5-
the same tissue (gingiva). When TS values were considered, all dimethoxy derivative 1 decreased both hCA I and II inhibition
the compounds had lower TS values than two reference in compound 5, while introduction of a methoxy group to
compounds (5-FU and Melphalan) in both the calculations.
3 positions of 2,4-dimethoxy derivative 2 decreased or had similar
When the first TS calculation of values was considered the inhibitory effect on hCA I and II in compound 4 according to Ki
compound 6 had the highest TS value (1.5) while the second values. On the other hand, introduction to a methoxy group to
calculation of TS pointed out compounds 4 (TS ¼ 3.4), 6 5 positions of compounds 2 and 3 increased hCA I inhibitory
(TS ¼ 2.0) and 5 (TS ¼ 1.4) with higher TS values greater effect in both cases in compounds 5 and 6, while it decreased the
than 1. At the second TS calculation, compound 4 was the most hCA II inhibitory effects in compounds 5 and 6 compared with
tumor selective one. Tri-methoxy derivative compounds 4, 5, and compounds 2 and 3.
6 had superior tumor specificity than di-methoxy derivatives 2
and 3. It seems that addition of one methoxy group made a
Conclusion
valuable contribution to the cytotoxic activities of the compounds
in compounds 4, 5, and 6. This may result from increased In conclusion; although tumor selectivity of the compounds were
possibility to form hydrogen bonding in trimethoxy derivatives.
less than reference compounds 5-FU and Melphalan, trimethoxy
IC50 (the drug concentration causing 50% inhibition of the derivatives compounds 4, 5, and 6 were more selective than
desired activity) of the compounds on hCA I isoenzyme were in dimethoxy derivatives 2 and 3 as judged by the cytotoxicity assay
the range of 30.1–49.2 nM while acetazolamide (AZA) has IC50 with the cells both cell types originated from the gingival tissue.
value of 293.0 nM. It means that the compounds 1–6 [1 (30.1), 2 Compound 6 (4-[3–(4-methoxyphenyl)-5–(3,4,5-trimethoxyphe-
(40.8), 3 (46.2), 4 (38.5), 5 (49.2), 6 (36.5)] were 5.9–9.7 times nyl)-4,5-dihydro-1H-pyrazol-1-yl] benzenesulfonamide) having
more potent inhibitor than AZA on hCA I. On the other hand, the highest TS values in both calculations can be considered as
IC50 values of the compounds on hCA II were in the range of the leading molecule of the series for further investigations in
23.8–30.1 nM while AZA had IC50 value of 146.5 nM. This terms of cytotoxicity. Since all the compounds had shown
means that the compounds 1–6 [1 (24.7), 2 (30.1), 3 (26.7), 4 superior inhibitory activity than reference compound AZA on
(23.8), 5 (25.7), 6 (23.9)] were 4.8–6.3 times more potent hCA I and II isoenzymes, the compounds synthesized can be

DOI: 10.1080/14756366.2016.1217852
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Acknowledgements
The authors thank Dr. Y. Ozkay (Anadolu University, Eskisehir, Turkey)
and Dr. M. K. Sukuroglu (Gazi University, Ankara, Turkey) for HRMS
analysis.
Declaration of interest
The authors report no conflict of interest and are responsible for the
contents and writing of the paper. This study was supported by
the Research Foundation of Ataturk University, Turkey. Grant Number:
2015/078 and 2015/322.
ORCID
Ilhami Gulcin
http://orcid.org/0000-0001-5993-1668
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