Synthesis, molecular modeling, and biological evaluation of 4-[5-aryl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl] benzenesulfonamides toward acetylcholinesterase, carbonic anhydrase I and II enzymes

Article abstract of DOI:10.1111/cbdd.13149

In this study, 4-[5-aryl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl] benzenesulfonamides were synthesized, and inhibition effects on AChE, hCA I, and hCA II were evaluated. K_i values of the compounds toward hCA I were in the range of 24.2?±?4.6-49.8?±?12.8?nm, while they were in the range of 37.3?±?9.0-65.3?±?16.7?nm toward hCA II. K_i values of the acetazolamide were 282.1?±?19.7?nm and 103.60?±?27.6?nm toward both isoenzymes, respectively. The compounds inhibited AChE with K_i in the range of 22.7?±?10.3-109.1?±?27.0?nm, whereas the tacrine had K_i value of 66.5?±?13.8?nm. Electronic structure calculations at M06-L/6-31?+?G(d,p)//AM1 level and molecular docking studies were also performed to enlighten inhibition mechanism and to support experimental findings. Results obtained from calculations of molecular properties showed that the compounds obey drug-likeness properties. The experimental and computational findings obtained in this study might be useful in the design of novel inhibitors against hCA I, hCA II, and AChE.

Full text of DOI:10.1111/cbdd.13149

DR CEM YAMALI (Orcid ID : 0000-0002-4833-7900)
Article type : Research Article
Synthesis, molecular modeling and biological evaluation of 4-[5-aryl-3-(thiophen-2-yl)-
4,5-dihydro-1H-pyrazol-1-yl] benzenesulfonamides towards acetylcholinesterase,
carbonic anhydrase I and II enzymes
Cem Yamali1, Halise Inci Gul1*, Abdulilah Ece2, Parham Taslimi3, Ilhami Gulcin3
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University,
Erzurum, 25040, Turkey
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Biruni University, Istanbul,
34010, Turkey
3Department of Chemistry, Faculty of Arts and Sciences, Ataturk University, Erzurum,
25040, Turkey
Correspondence author:
*Prof. Dr. Halise Inci Gul
Ataturk University,
Faculty of Pharmacy,
Department of Pharmaceutical Chemistry,
25240, Erzurum/TURKEY
Tel: +90 4422315219
Fax: +90 4422315201
E-mail: incigul1967@yahoo.com or incigul@atauni.edu.tr
Running Title: Bioactivities of pyrazoline-benzenesulfonamides
ABSTRACT
In
the
present
study,
4-[5-aryl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl]
benzenesulfonamides were synthesized and inhibition effects on AChE, hCA I and hCA II were
evaluated. Ki values of the compounds towards hCA I were in the range of 24.2±4.6-49.8±12.8
nM while they were in the range of 37.3±9.0-65.3±16.7 nM towards hCA II. Ki values of the
Acetazolamide were 282.1±19.7 nM and 103.60±27.6 nM towards both isoenzymes,
respectively. The compounds inhibited AChE with Ki in the range of 22.7±10.3-109.1±27.0 nM,
whereas the Tacrine had Ki value of 66.5±13.8 nM. Electronic structure calculations at M06-
L/6-31+G(d,p)//AM1 level and molecular docking studies were also performed to enlighten
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13149
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inhibition mechanism and to support experimental findings. Results obtained from calculations
of molecular properties showed that the compounds obey drug likeness properties. The
experimental and computational findings obtained in the present study might be useful in the
design of novel inhibitors against hCA I, hCA II, and AChE.
KEYWORDS: Carbonic anhydrase, acetylcholinesterase, pyrazoline, sulfonamide, docking,
modeling
1. INTRODUCTION
Enzymes have attracted more interests as targets for drug discovery and development because
of their important roles in many diseases. Among therapeutic agents in market today, almost
half of them carry out their therapeutic effects by inhibiting or activating enzymes. Medicinal
chemists are focused on the identification and optimization of drug candidates that act as
enzyme inhibitors or activators.[1]
-
Carbon dioxide (CO2) and bicarbonate (HCO3 ) are essential compounds in living
organisms[2]. Carbonic anhydrase (CAs, EC 4.2.1.1), a zinc-containing enzyme, catalyzes a
-
reversible reaction between CO2 hydration and HCO3 dehydration. CAs are present in many
organisms and have an essential role in pathological and physiological events including pH
regulation, bone resorption, carboxylation reactions, synthesis of bicarbonate, calcification,
osteoporosis, glaucoma, cancer, and neurological disorders.[3-5] Sulfonamides, especially
aromatic primary sulfonamides, have been reported as the most attractive scaffolds for the
development of new CA inhibitors.[5] Sulfonamide derivatives such as acetazolamide,
methazolamide, ethoxzolamide and celecoxib are used as CA inhibitors (CAIs) in many
studies as reference drugs (Figure 1). [6,7]
Alzheimer’s disease (AD) is a chronic progressive neurodegenerative disorder which
is seen in people over 65 years old.[8] The number of people suffering from AD is increasing
everyday and it is estimated that the number of patients will reach up to 100 million by the
year 2050.[9] Annual cost of the treatment of AD patients is about $100 billion.[8] At present,
there is no way to eradicate the AD. The drugs available on the market provide only palliative
treatment and reduce the speed of disease.[10]
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Cholinergic inhibition is one of the most essential way used for the patients with
AD.[10] Acetylcholinesterase (AChE) is one of the most crucial enzymes for nerve response
and function. It catalyzes the hydrolysis of acetylcholine to choline and acetic acide. AChE is
widely distributed in a variety of nerve muscle tissue in the brain.[8] Tacrine, donepezil,
rivastigmine and galantamine are amongst the drugs that are currently being used in clinics as
acetylcholinesterase inhibitors (Figure 1).[10]
Pyrazolines, as one of the most important heterocyclic compounds in medicinal
chemistry, have various biological activities such as anti-Alzheimer[11,12], anti-Parkinson[13]
and antidepressant.[14,15] They show their effects by inhibiting or activating several enzymes
such as cholinesterases, monoamine oxidases and carbonic anhydrases. Ucar et al. have
reported several N-substituted pyrazoline derivatives as monoamine oxidases-B and
cholinesterase inhibitors with the promising activity, which can be useful for the
development of new drug candidates for Alzheimer and Parkinson diseases.[13] In addition,
the compounds having sulfonamide core were also found as promising compounds, which
can be considered to develop new potent AChE inhibitors since they presented valuable
[16,17]
results in AChE inhibition tests.
Besides, sulfonamides are known with several
bioactivities such as diuretic, antigloucoma, anticonvulsant, and antiobesity. [18,19,20]
Pharmacophore hybrid approach is one of the most effective method in medicinal
chemistry to produce new drug candidates.[21] It includes the combination of two or more
pharmacophore groups in a single molecule to obtain synergetic effect. It also provides a
modified selectivity profile with improved pharmacokinetic and pharmacodynamic
restrictions, reduction of undesirable side effects, having dual or multiple modes of action,
and less drug-drug interactions.[21] This method has been successfully used by several
researchers and has had very remarkable results with diseases having multifactorial profiles
such as Alzheimer´s disease, Parkinson´s disease, cancer, inflammation, and hypertension.[21]
In the present study, it was aimed to synthesize compounds TP1-10, 4-[5-aryl-3-
(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl] benzenesulfonamides, bearing sulfonamide and
pyrazoline moieties, which are known to have CA and/or AChE inhibitory properties in a
single molecule. Aryl part was changed in the design as nonsubstituted/substituted phenyl or
thiophen-2-yl to see the effects of different aromatic rings and substituents having different
electronic nature on AChE, hCA I and hCA II enzymes. In addition, molecular modeling
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studies were also considered as a value added tool in understanding the inhibition mechanism
at molecular level and to enlighten the interactions of the synthesized compounds with their
corresponding target.
2. METHODS AND MATERIALS
2.1 Chemistry
The chemical structures of the compounds TP1-10 were confirmed by the Nuclear Magnetic
1
13
Resonance (NMR) spectra H NMR (400 MHz) and C NMR (100 MHz) (Varian Mercury
Plus spectrometer, Varian inc., Palo Alto, California, U.S.). Chemical shifts (δ) are reported
in ppm and coupling constants (J) are expressed in hertz (Hz). Mass spectra (HRMS) for the
compounds were taken using a 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 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) and are uncorrected.
2.2 General synthesis method of the pyrazolines TP 1-10, Scheme 1
First, chalcones were synthesized via Claisen-Schmidt reaction as described in literatures. [22-
28] 2-Acetylthiophene (2 g, 16 mmol) and the appropriate aromatic aldehyde (1.7 g for 1, 2.0
g for 2, 2.2 g for 3, 2.0 g for 4, 2.2 g for 5, 3.0 g for 6, 2.8 g for 7, 1.8 g for 8, 2.6 g for 9, 2.6
g for 10, 16 mmol) were dissolved in ethanol (10 mL) and an aqueous solution of potassium
hydroxide (10 mL, 40% w/v) was added dropwise into the reaction flask. Reaction mixtures
were stirred for 3-6 hours at room temperature and reactions were followed by thin layer
chromatography (TLC) using dichloromethane:methanol (4.8:0.2) solvent system. After
completion of the reaction, the reaction mixture was poured into the water (25 mL) and then
acidified with hydrochloric acid solution (10%) to pH=3-4. The solid obtained was filtered,
washed with water and ethanol for several times. The purity of the chalcones (Compounds 1-
10, Scheme 1) was checked by TLC and then they were used in the synthesis of pyrazolines
(Compounds 1-10, Scheme 1) without further purification.
At the second step, a solution of the appropriate chalcone (0.5 g, 2 mmol) in ethanol
(30 mL) with glacial acetic acid (0.1 ml) was heated until chalcone dissolved and then p-
hydrazinobenzene sulfonamide hydrochloride (0.5 g, 2 mmol) was added into the reaction
mixture and finally, the mixture was refluxed for 22 h. [22-26] The reactions were monitored by
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TLC plates using chloroform:methanol (5 mL:2 drops) as solvent system. After completion
of the reaction, the mixture was poured into the water (50 mL). The solid compounds
obtained were filtered and dried. The compounds TP1 (ethanol), TP3 (methanol/chloroform),
TP4 (methanol/chloroform), TP7 (ethanol/dichloromethane), TP8 (ethanol/chloroform), TP9
(ethanol/acetone) were purified by crystallization from suitable solvent. On the other hand,
the compounds TP2, TP5, TP6, and TP10 were first purified by using column
chromatography (with chloroform) and then crystallization (dichloromethane/diisopropyl
1
ether), respectively. The chemical structures of the pyrazolines were confirmed using H
13
NMR (400 MHz) and C NMR (100 MHz) and HRMS spectra. Physical and spectral data
for the compounds TP 1-10 are listed below.
2.2.1
4-(5-Phenyl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide
(TP1)[29]
1
Light yellow solid. Mp 212-214 °C. Yield 11%. H NMR (400 MHz, DMSO-d6, δppm) 7.65
(dd, J=5.1, 1.1 Hz, 1H, ArH), 7.55 (d, J= 9.1 Hz, 2H, ArH), 7.35-7.31 (m, 3H, ArH), 7.26-
7.22 (m, 3H, ArH), 7.10 (dd, J=4.8, 3.7 Hz, 1H, ArH), 7.00 (s, 2H, -SO2NH2), 6.97 (d, J=8.8
Hz, 2H, ArH), 5.62 (dd, J= 12.0, 5.0 Hz, 1H, pyrazoline ring), 3.96 (dd, J=17.4, 12.0 Hz, 1H,
pyrazoline ring), 3.17 (dd, J=17.4, 5.0 Hz, 1H, pyrazoline ring). 13C NMR (100 MHz,
DMSO-d6, δppm) 146.6, 146.3, 142.1, 135.7, 133.7, 129.8, 129.3, 129.0, 128.7, 128.4, 127.9,
126.4, 112.6, 63.0, 44.4. HRMS (ESI-MS) m/z Calculated for C19H16N3O2S2 [M-H]-
382.0577; found 382.0590.
2.2.2 4-(3-(Thiophen-2-yl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide
(TP2)
1
Beige solid. Mp 198-199 °C. Yield 14 %. H NMR (400 MHz, DMSO-d6, δppm) 7.64 (d,
J=4.8 Hz, 1H, ArH), 7.54 (d, J= 8.8 Hz, 2H, ArH), 7.30 (d, J=3.3 Hz, 1H, ArH), 7.14-7.01
(m, 5H, ArH), 6.97 (d, J=9.1 Hz, 2H, ArH), 6.99 (s, 2H, -SO2NH2), 5.57 (dd, J= 12.0, 5.0 Hz,
1H, pyrazoline ring), 3.94 (dd, J=17.5, 12.0 Hz, 1H, pyrazoline ring), 3.14 (dd, J=17.5, 5.0
13
Hz, 1H, pyrazoline ring), 2.48 (s, 3H, -CH3). C NMR (100 MHz, DMSO-d6, δppm) 146.6,
146.3, 139.1, 137.6, 135.7, 133.6, 130.3, 129.2, 128.9, 128.6, 127.8, 126.3, 112.6, 62.8, 44.4,
21.3. HRMS (ESI-MS) m/z Calculated for C20H18N3O2S2 [M-H]- 396.0734; found 396.0733.
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2.2.3
4-(5-(4-Methoxyphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (TP3) [29,30]
Light yellow solid. Mp 188-190 °C, 160-162°C[30]. Yield 22 %. 1H NMR (400 MHz, DMSO-
d6, δppm) 7.63 (dd, J=4.9, 1.1 Hz, 1H, ArH), 7.55 (d, J= 9.1 Hz, 2H, ArH), 7.30 (dd, J= 3.7,
1.1 Hz, 1H, ArH), 7.14 (d, J= 8.8 Hz, 2H, ArH), 7.10 (dd, J=4.9, 3.7 Hz, 1H, ArH), 7.00 (s,
2H, -SO2NH2), 6.98 (d, J=8.8 Hz, 2H, ArH), 6.87 (d, J=8.4 Hz, 2H, ArH), 5.56 (dd, J= 12.0,
5.0 Hz, 1H, pyrazoline ring), 3.92 (dd, J=17.5, 12.0 Hz, 1H, pyrazoline ring), 3.68 (s, 3H, -
OCH3), 3.15 (dd, J=17.5, 5.0 Hz, 1H, pyrazoline ring). 13C NMR (100 MHz, DMSO-d6,
δppm) 159.3, 146.6, 146.3, 135.8, 133.9, 133.6, 129.3, 128.9, 128.6, 127.8, 127.6, 115.2,
112.6, 62.6, 55.8, 44.5. HRMS (ESI-MS) m/z Calculated for C20H18N3O3S2 [M-H]- 412.0683;
found 412.0689.
2.2.4
4-(5-(4-Fluorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (TP4)
1
Light yellow solid. Mp 209-210 °C. Yield 30 %. H NMR (400 MHz, DMSO-d6, δppm) 7.65
(d, J=5.3 Hz, 1H, ArH), 7.57 (d, J=8.8 Hz, 2H, ArH), 7.32 (dd, J=3.3 Hz, 1H, ArH), 7.27 (dd,
J= 8.8, 5.3 Hz, 2H, ArH), 7.16 (t, J=8.8 Hz, 2H, ArH), 7.10 (dd, J= 4.8, 3.7 Hz, 1H, ArH),
7.02 (s, 2H, -SO2NH2), 6.96 (d, J=8.8 Hz, 2H, ArH), 5.65 (dd, J= 12.0, 5.0 Hz, 1H,
pyrazoline ring), 3.95 (dd, J=17.6, 12.0 Hz, 1H, pyrazoline ring), 3.17 (dd, J=17.6, 5.0 Hz,
13
1H, pyrazoline ring). C NMR (100 MHz, DMSO-d6, δppm) 162.1 (1JCF=243.4 Hz), 146.6,
146.1, 138.2 (4JCF=2.6 Hz), 135.6, 133.8, 129.4, 129.2, 129.1 (3JCF=7.6 Hz), 128.6, 127.9,
116.7 (2JCF=21.4 Hz), 112.6, 62.3, 44.3. HRMS (ESI-MS) m/z Calculated for C19H17N3O2FS2
[M+H]+ 402.0741; found 402.0740.
2.2.5
4-(5-(4-Chlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (TP5)[30]
1
Beige solid. Mp 189-191 °C, 136-138°C[30]. Yield 10 %. H NMR (400 MHz, DMSO-d6,
δppm) 7.65 (dd, J=5.0, 1.1 Hz, 1H, ArH), 7.57 (d, J=8.8 Hz, 2H, ArH), 7.39 (d, J=8.6 Hz, 2H,
ArH), 7.31 (dd, J= 3.7, 1.1 Hz, 1H, ArH), 7.24 (d, J=8.6 Hz, 2H, ArH), 7.10 (dd, J= 5.0, 3.7
Hz, 1H, ArH), 7.02 (s, 2H, -SO2NH2), 6.97 (d, J=8.8 Hz, 2H, ArH), 5.66 (dd, J= 12.0, 5.0 Hz,
1H, pyrazoline ring), 3.96 (dd, J=17.5, 12.0 Hz, 1H, pyrazoline ring), 3.19 (dd, J=17.6, 5.0
13
Hz, 1H, pyrazoline ring). C NMR (100 MHz, DMSO-d6, δppm) 146.7, 146.1, 140.9, 135.5,
133.9, 132.9, 129.8, 129.5, 129.1, 128.6, 128.4, 127.9, 112.6, 62.3, 44.1. HRMS (ESI-MS)
m/z Calculated for C19H17N3O2S2Cl [M+H]+ 418.0445; found 418.0445.
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2.2.6
4-(5-(4-Bromophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (TP6)
1
Light green solid. Mp 215-217 °C. Yield 16 %. H NMR (400 MHz, DMSO-d6, δppm) 7.65
(dd, J=5.0, 1.1 Hz, 1H, ArH), 7.57 (d, J=8.9 Hz, 2H, ArH), 7.53 (d, J=8.6 Hz, 2H, ArH), 7.31
(dd, J= 3.5, 1.1 Hz, 1H, ArH), 7.18 (d, J=8.6 Hz, 2H, ArH), 7.10 (dd, J= 5.0, 3.5 Hz, 1H,
ArH), 7.02 (s, 2H, -SO2NH2), 6.97 (d, J=8.9 Hz, 2H, ArH), 5.64 (dd, J= 12.0, 5.0 Hz, 1H,
pyrazoline ring), 3.95 (dd, J=17.5, 12.0 Hz, 1H, pyrazoline ring), 3.18 (dd, J=17.5, 5.0 Hz,
1H, pyrazoline ring). 13C NMR (100 MHz, DMSO-d6, δppm) 146.7, 146.1, 141.4, 135.5,
133.9, 132.8, 129.4, 129.3, 129.1, 128.7, 127.9, 121.4, 112.7, 62.4, 44.2. HRMS (ESI-MS)
m/z Calculated for C19H17N3O2S2Br [M+H]+ 461.9940; found 461.9939.
2.2.7 4-(3-(Thiophen-2-yl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (TP7) [30]
1
Beige solid. Mp 198-200 °C, 260-262 °C[30]. Yield 40 %. H NMR (400 MHz, DMSO-d6,
δppm) 7.72 (d, J=8.1 Hz, 2H, ArH), 7.65 (d, J=5.1 Hz, 1H, ArH), 7.57 (d, J=8.8 Hz, 2H, ArH),
7.44 (d, J= 8.1 Hz, 2H, ArH), 7.31 (d, J=3.7 Hz, 1H, ArH), 7.11-7.09 (m, 1H, ArH), 7.02 (s,
2H, -SO2NH2), 6.98 (d, J=8.8 Hz, 2H, ArH), 5.76 (dd, J= 12.1, 4.8 Hz, 1H, pyrazoline ring),
4.00 (dd, J=17.6, 12.1 Hz, 1H, pyrazoline ring), 3.23 (dd, J=17.6, 4.8 Hz, 1H, pyrazoline
ring). 13C NMR (100 MHz, DMSO-d6, δppm) 146.7, 146.1, 135.4, 133.9, 129.6, 129.4, 129.3,
129.2, 128.3, 127.3, 126.8, 112.7, 112.5, 62.5, 44.1. HRMS (ESI-MS) m/z Calculated for
C20H17F3N3O2S2 [M+H]+ 452.0709; found 452.0708.
2.2.8 4-(3,5-Di(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl benzenesulfonamide (TP8)
[30]
Light brown solid. Mp 226-227 °C, 218-220°C[30]. Yield 22 %. 1H NMR (400 MHz, DMSO-
d6, δppm) 7.67 (dd, J=5.0, 1.1 Hz, 1H, ArH), 7.59 (d, J=9.1 Hz, 2H, ArH), 7.38-7.36 (m, 2H,
ArH), 7.13 (dd, J=3.7, 1.2 Hz, 2H, ArH), 7.10 (d, J= 9.1 Hz, 2H, ArH), 7.04 (s, 2H, -
SO2NH2), 6.93 (dd, J=5.2, 3.4 Hz, 1H, ArH), 5.99 (dd, J= 11.5, 4.5 Hz, 1H, pyrazoline ring),
3.92 (dd, J=17.4, 11.5 Hz, 1H, pyrazoline ring), one of the signal of protons on pyrazoline
13
ring was observed under solvent peak. C NMR (100 MHz, DMSO-d6, δppm) 147.0, 146.4,
144.9, 135.6, 134.2, 129.4, 129.2, 128.7, 127.8, 127.7, 127.5, 126.3, 113.1, 59.2, 44.4. HRMS
(ESI-MS) m/z Calculated for C17H14N3O2S3 [M+H]+ 390.0399; found 390.0403
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2.2.9
4-(5-(2,4-Dimethoxyphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (TP9)
1
Light green solid. Mp 205-206 °C. Yield 35 %. H NMR (400 MHz, DMSO-d6, δppm) 7.62
(dd, J=5.1, 1.1 Hz, 1H, ArH), 7.56 (d, J=9.1 Hz, 2H, ArH), 7.30 (dd, J=3.7, 1.1 Hz, 1H,
ArH), 7.09 (dd, J= 5.1, 3.7 Hz, 1H, ArH), 7.00 (s, 2H, -SO2NH2), 6.90 (d, J=8.8 Hz, 2H,
ArH), 6.73 (d, J=8.4 Hz, 1H, ArH), 6.62 (dd, J= 2.3 Hz, 1H, ArH), 6.39 (dd, J=8.4, 2.3 Hz,
1H, ArH), 5.62 (dd, J=12.0, 5.0 Hz, 1H, pyrazoline ring), 3.89 (dd, J=17.6, 12.0 Hz, 1H,
pyrazoline ring), 3.86 (s, 3H, -OCH3), 3.69 (s, 3H, -OCH3), 3.05 (dd, J=17.6, 5.0 Hz, 1H,
13
pyrazoline ring). C NMR (100 MHz, DMSO-d6, δppm) 160.8, 157.8, 147.1, 146.2, 135.9,
133.4, 129.1, 129.0, 128.8, 127.9, 120.8, 112.3, 112.2, 105.6, 99.7, 57.8, 56.5, 55.9, 43.3.
HRMS (ESI-MS) m/z Calculated for C21H22N3O4S2 [M+H]+ 444.1046; found 444.1042.
2.2.10
4-(5-(3,4-Dimethoxyphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (TP10)
1
Beige solid. Mp 168-170 °C. Yield 12 %. H NMR (400 MHz, DMSO-d6, δppm) 7.63 (dd,
J=5.1, 1.1 Hz, 1H, ArH), 7.55 (d, J=8.8 Hz, 2H, ArH), 7.30 (dd, J=3.7, 1.1 Hz, 1H, ArH),
7.09 (dd, J= 5.1, 3.7 Hz, 1H, ArH), 7.01 (s, 1H, ArH), 6.99 (d, J=8.8 Hz, 2H, ArH), 6.91 (s,
2H, -SO2NH2), 6.85 (d, J=8.4 Hz, 1H, ArH), 6.63 (dd, J=8.4, 2.2 Hz, 1H, ArH), 5.51 (dd,
J=12.0, 5.1 Hz, 1H, pyrazoline ring), 3.89 (dd, J=16.9, 12.0 Hz, 1H, pyrazoline ring), 3.69 (s,
13
3H, -OCH3), 3.66 (s, 3H, -OCH3), 3.16 (dd, J=16.9, 5.1 Hz, 1H, pyrazoline ring). C NMR
(100 MHz, DMSO-d6, δppm) 149.7, 148.8, 146.7, 146.5, 135.7, 134.3, 133.6, 129.2, 128.9,
128.7, 127.8, 118.0, 112.8, 112.7, 110.3, 63.0, 56.1, 56.0, 44.5. HRMS (ESI-MS) m/z
Calculated for C21H22N3O4S2 [M+H]+ 444.1046; found 444.1043.
2.3 Biological activity
2.3.1 Purification and inhibition studies of carbonic anhydrase (CA) I and II isoforms
In the current study, CA isoforms (hCA I and II) using sepharose-4B-Lyrosine-sulfanilamide
affinity chromatography were purified in a single purification stage using fresh human blood
erythrocytes and the erythrocytes samples were centrifuged at 13000 rpm for 30 min and the
buffy buffy coat and plasma were discarded. Also, pH of the buffer was slowly adjusted to
8.7, by using solid Tris.[22-25] Additionally, after centrifugation, supernatant was transferred to
the Sepharose-4B-L-Tyrosine-sulphanilamide affinity column that was previously
prepared.[31] The enzymes from the column were then spectrophotometrically measured at
280 nm. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) was
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examined for registration of both isoforms purity.[32] Human CA isoforms activity
designation was observed spectrophotometrically (Shimadzu, UV-VIS Spectrophotometer,
UVmini-1240, Kyoto, Japan).[33] In this test, variations in absorbance were obtained during 3
min at 30 ºC. p-Nitrophenylacetate (NPA) molecule was used as substrate and transformed by
both isoforms to p-nitrophenolate ion.[32] For description of protein quantity at the
purification stages, Bradford process was used.[34] The bovine serum albumin molecule was
used as control factor for this definition, which was done at 595 nm. For obtaining the
inhibition results of each isoform, the compounds TP1-10 and an activity (%) [TP1-10]
graph was drawn. To calculate Ki values, three diverse [TP1-10] concentrations were studied.
Acetazolamide (AZA) was used as a reference compound.
2.3.2 Determination of Acetylcholinesterase (AChE) activity
The inhibitory efficacy of the compounds TP1-10 on AChE activity was tested following the
spectrophotometric process of Ellman test.[35] Acetylthiocholine iodide (AChI) was used as
substrates.[36,37] For the mensuration of the AChE activity, 5,5’-dithio-bis(2-nitro-
benzoic)acid compound (DTNB, D8130-1G, Sigma-Aldrich, Steinheim, Germany) was
applied.[38] Briefly, 50 µL DTNB and 100 µL of Tris/HCl solution (1M, pH 8.0), 750 mL of
sample solution dissolved in distilled water at disparate concentrations and 50 µL AChE
(5.32 × 10-3 U) solution were incubated and mixed for 15 min at 30 ºC. Finally, the reaction
was started by adding 50 µL of AChI. The enzymatic hydrolysis of this substrate which
produces a yellow 5-thio-2-nitrobenzoate anion as the result of the product of thiocholine
with DTNB, was recorded spectrophotometrically at a wavelength of 412 nm.[37] Tacrine
(TAC) was used as a reference compound.
2.4 Computational section
2.4.1 Molecular docking
The ligand preparation and protein preparation steps are necessary to ensure that the
structures meet minimum requirements for further computational calculations. LigPrep tool
(Schrödinger Release 2016-4: LigPrep, Schrödinger, LLC, New York, NY, 2016) interfaced
with Maestro module of Schrödinger suite was used for the preparation of all the molecules
including the reference compounds, Tacrine and Acetazolamide. All possible tautomer and
ionization states at pH 7.0 ± 2.0 were generated. The compounds were then minimized using
optimized potential liquid simulations 3 (OPLS3) force field.[39]
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High-resolution protein crystal structures of acetylcholinesterase (AChE), carbonic
anhydrase (CA) I, and II (PDB Ids: 1DX6, 2NMX and 3HS4, respectively) were downloaded
from RCSB Protein Data Bank and used for molecular docking. All X-ray crystal structures
were in complexed with a native ligand. As a first step, protein Preparation Wizard
(PrepWizard, Schrödinger Release 2016-4: Schrödinger Suite 2016-4 Protein Preparation
Wizard; Epik, Schrödinger, LLC, New York, NY, 2016; Impact, Schrödinger, LLC, New
York, NY, 2016; Prime, Schrödinger, LLC, New York, NY, 2016)[40] in Maestro of
Schrödinger software package (Schrödinger Release 2016-4, Maestro. Schrödinger, LLC,
New York, NY, 2016) were used to prepare crystal structures. Bond orders were assigned and
hydrogens were added to the structures. The native ligands in all structures were kept for
defining the binding sites and in case of carbonic anhydrases; zinc metal was also retained
being a key moiety in the active site. All water molecules and heteroatoms were then
removed. The final structures were optimized and finally minimized using OPLS3 force field.
This helps to avoid steric clashes between the atoms.
A grid that represents the binding pocket was generated using the co-crystallized
native ligands as reference. Default settings were applied in each case. Glide SP (standard
precision) module of Schrödinger Suite[41,42] was used to dock the synthesized compounds
into the active site of the crystal structures.
2.4.2 Calculation of physicochemical and ADME properties
QikProp module of Schrodinger (Schrödinger Release 2016-4, QikProp. Schrödinger, LLC,
New York, NY, 2016) was used to calculate some molecular descriptors commonly used in
absorption, distribution, metabolism and excretion (ADME) analysis. Frontier molecular orbital
(HOMO and LUMO) energy values and dipole moments were calculated using Wave function’s
Spartan’16 parallel suite (Spartan 16, Wavefunction Inc., Irvine CA). As a first step, the
geometry of all compounds was optimized at AM1 level. Frequency calculations were also
calculated to verify that this optimized geometry is a real minimum on the potential energy
surface with no imaginary frequencies. Single point energy calculation was computed at M06-L
level using the popular polarized basis set, 6-31+G(d,p), which adds p functions to hydrogen
atoms and d functions on heavy atoms.
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3. RESULTS
3.1 Chemistry
The
compounds
TP1-10,
4-[5-aryl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl]
benzenesulfonamides, were synthesized successfully. Aryl part was changed as phenyl
(TP1), 4-methylphenyl (TP2), 4-methoxyphenyl (TP3), 4-fluorophenyl (TP4), 4-
chlorophenyl (TP5), 4-bromophenyl (TP6), 4-trifluoromethylphenyl (TP7), thiophen-2-yl
(TP8), 2,4-dimethoxyphenyl (TP9) and 3,4-dimethoxyphenyl (TP10). The compounds TP2,
TP4, TP6, TP9 and TP10 were reported for the first time in this study. The compounds TP1,
TP3, TP5, TP7 and TP8 were registered in literatures as racemates and their several
bioactivities such as antidiabetic, anticancer, anti-inflammatory and COX inhibitory activities
were reported.[29,30] The compounds synthesized in this study were also obtained as pure
racemates and they were directly used for CA and AChE inhibiton studies.
Structures of the compounds synthesized were established on the basis of spectral data
1
1
taken from H NMR, 13C NMR and HRMS. In the H NMR and 13C NMR spectra of the
compounds TP1-10, chemical shift values of the three hydrogen atoms attached to the C-4
and C-5 carbon atoms of the pyrazoline ring and C-4 and C-5 carbon atoms of the pyrazolines
were observed as expected. [25,26] The appearance of three doublets in the range of δ 5.99-3.05
ppm confirmed the formation of pyrazole ring, except TP8 (one of the signals of proton on
pyrazole ring was hidden under DMSO peak). Two carbon atoms of pyrazoline ring (C-4 and
C-5) were appeared around 63.0 ppm and 44.0 ppm. The signal of protons on SO2NH2 group
were observed in the range of δ 7.04-6.99 pm as a singlet. Mass spectra showing [M+H]+ or
[M-H]- peaks further confirmed the synthesis of desired compounds.
3.2 Bioactivity Studies
The inhibition of human CA isoforms I, and II with the compound TP1-10 was reported for the
first time and the inhibition data are shown in Table 1. The inhibitory effects of the compounds
TP1-10 on AChE enzyme are also reported for the first time and the inhibition data are shown in
Table 1. Selectivity ratios of the compounds on enzymes are calculated and data are presented in
Table 3.
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3.2.1 Carbonic anhydrase inhibitory effects
The compounds having sulfonamide pharmacophore have been reported with high binding
affinities for most of CA isoenzymes.[18] In this study, the compounds were designed to have
sulfonamide moiety as one of the pharmacophores. The compounds TP1-10 have shown
better inhibitory activities than the reference drug Acetazolamide (AZA). According to Table
1, Ki values of the compounds towards hCA I were in the range of 24.2±4.6 - 49.8±12.8 nM,
while they were in the range of 37.3±9.0 - 65.3±16.7 nM towards hCA II isoenzyme. Ki
values of acetazolamide (AZA) were 282.1±19.7 nM and 103.6±27.6 nM towards both
isoenzymes, respectively. When Ki values were considered it was difficult to decide about the
lead compound towards hCA I, and II, since Ki values were similar, although they were lower
than Ki values of AZA. It was also noticed that Ki values of the compounds towards hCA I
were less than the Ki values towards hCA II.
3.2.2 Acetylcholinesterase inhibitory effects
Most of the compounds effectively inhibited AChE at nanomolar level. When Ki values of the
compounds considered towards AChE, they were in the range of 22.7±10.3 -109.1±27.0 nM
while the reference drug Tacrine (TAC) had Ki value as 66.5±13.8 nM. Ki values of the
compounds pointed out the compound TP2 with the lowest value, as the most potent
compound while TP10 was the least effective one towards AChE.
3.2.3 Selectivity of the compounds on enzymes
Selectivity Ratio (SR) value reflects the selectivity of a compound towards hCA I or II
isoenzymes. The SR values were calculated by dividing Ki value of the certain compounds to
hCA II or hCA I. Potency Selectivity Ratios (PSR1 and PSR2) of a compound towards hCA I
and II were also calculated. PSR1 values were produced by dividing Ki value of AZA (282.090
nM) to Ki value of a certain compound towards hCA I. PSR2 values were produced by dividing
Ki value of AZA (103.60 nM) to Ki value of a certain compound towards hCA II. In addition,
PSR3 values of the compound towards AChE were also calculated. Reference compound was
TAC. PSR3 values were produced by dividing Ki value of TAC (66.521 nM) to Ki value of a
certain compound towards AChE. Data were presented in Table 3.
Based on the Table 3, SR values of the compounds calculated were between 0.9-1.9.
According to these values, the compounds were found to be 2.3-4.8 fold more selective than
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reference drug AZA towards hCA I isoenyzme but their selectivity towards hCA II were not
higher than AZA.
PSR1 values of the compounds were in the range of 5.7-11.7. The compound TP8
(PSR1=11.7) bearing thiophene is the most selective one towards hCA I. Additionally, having
the SR values between 1.7-2.8, the compounds were found to be more selective towards hCA II
than AZA. The compound TP6 (PSR1=2.8) bearing 4-bromophenyl was the most selective one
towards hCA II. According to PSR1 and PSR2 values, it can be said that compounds were
found to be more selective towards hCA I than hCA II isoenzyme.
On the other hand, except TP3, TP6, TP10, other compounds were effectively inhibited
AChE enzyme. Seven of the compounds had selectivity ratios higher than 1. The compounds
TP2 (PSR3=2.9) bearing 4-mehylphenyl and TP7 (PSR3=2.3) bearing 4-trifluoromethylphenyl
can be considered as lead inhibitor compounds of this study towards AChE enzyme to design
new AChE inhibitors.
3.3 Computational Section
As a first step, we have calculated some molecular descriptors commonly used in absorption,
distribution, metabolism and excretion (ADME) analysis (Table 2). Accordingly, molecular
weight (MW), predicted octanol/water partition coefficient (QplogPo/w), percent human oral
absorption, polar surface area (PSA) and number of violations of Lipinski’s rule of five of the
incorporated ligands were estimated using QikProp module of Maestro molecular modeling
package. All compounds obeys Lipinski’s rule of five[43] which is an indication of the drug-
likeness of a molecule, and PSA values are within the range that Veber et. al. suggested.[44]
Furthermore, several studies report the importance of dipole moment and the energy of
frontier molecular orbitals having important contributions to the AChE inhibitory activity.[45,46]
Hence, we found it necessary to calculate the quantum chemical properties of TP1-10 and of
TAC for comparison, at a high and accurate level (M06-L/6-31+G(d,p)//AM1).[47] The results
agree well with the experimental findings. All active compounds have high dipole moments and
high HOMO energy values in Table 2. The most active compound TP7 has the highest dipole
moment and its HOMO energy value is higher than the reference molecule TAC itself. In fact,
HOMO energy values of all the compounds are higher than TAC.
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In an attempt to get insight into the interaction of the synthesized compounds with the
target receptors, molecular docking simulations were also carried out. Thus, all compounds were
docked into the active sites of the AChE, CA I, and II separately. Docking scores in terms of
binding energies were calculated and are given in Table 2. Despite the fact that the present
docking algorithms fail to discriminate between active and inactive compounds in a high success
rate[48,49], glide docking performed well in ranking the most active compounds especially for
AChE and CAII.
2D ligand interaction diagrams are shown in Figure 2 (a-c). As can be seen from Figure
2a, the R configuration of the most active compound TP7 makes key interactions with the
binding pocket of AChE. However, the S-enantiomer failed to generate any poses. Hence, we
believe that the active form is most probably (R)-TP7. The aromatic sulfonamide moiety of TP7
orient itself deeply into the active site region establishing a hydrogen bond with the glutamic
residue, GLH199, located at the anionic subsite. It also makes π-π contacts with HIS440 (one of
the components of the estaric site) and Phe330 (the aromatic residue situated at the midpoint of
the gorge).
On the other hand, the compounds showed high activities against CAI and CAII and their
IC50 ranges were very close to each other. As can be seen from Table 2, with the exception of
only TP4, the compounds scored higher than the reference compound, AZA against CAI.
Sulfonamide groups in the most active compound TP8, adopts similar orientation with AZA,
making the key interactions with the Thr199 and zinc ion in the catalytic binding site (Figure
2b). Same key interactions exist in the TP1-CAII complex, too (Figure 2c). Besides interaction
with THR199 and zinc ion, the oxygen of the sulfonamide moiety forms an extra hydrogen bond
with backbone amine group of Thr200.
4. DISCUSSION
The aim of a bioisosteric replacement is to design a new molecule with similar biological
properties to the parent compound. The replacement can reduce toxicity, optimize activity of the
[50]
lead molecule, and/or alter pharmacokinetics or the toxicity of the lead molecule.
In present
study, 5th position of pyrazoline ring was substituted with phenyl or substituted phenyl ring
having different electronic nature or thiophene ring which is a bioisoster of phenyl ring. Several
bioactivity data related to inhibitory effects of the compounds on hCA I, hCA II and AChE were
obtained which lead to produce SAR data.
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The cytosolic isoform hCA I was inhibited by all compounds investigated here in
nanomolar range, with IC50 in the range of 25.7-45.8 nM. All compounds were found 6.5-11.6
times more potent than the reference drug AZA (IC50=297.1 nM) according to IC50 values.
When para position of phenyl ring was substituted with methyl group, inhibition potency of the
compound TP2 (IC50=30.1 nM) increased slightly (1.1 times) compared with TP1 (IC50=34.7
nM) which has non-substituted phenyl. When the methyl group was replaced with methoxy
group (TP3, IC50=36.5 nM) the inhibitory activity of the compound slightly decreased
comparing TP1. On the other hand, introduction of dimethoxy groups (TP9, IC50=45.8 nM;
TP10, IC50=40.5 nM) on phenyl decreased the inhibitory activity of the compounds towards
hCA I compared with TP1’s.
When hydrogen at para position of phenyl ring was replaced by a halogen, inhibition
effect increased only in the compound TP6 having bromine substituent (IC50=27.7 nM)
compared with TP1 (IC50=34.7 nM). The general trend for hCA I inhibition potency of the
compounds was in the order of Br (IC50=27.7 nM) > Cl (IC50=34.5 nM) > F (IC50=40.8 nM),
which is inversely correlated by electronegativity of the substituents. It can be said that
electronegativity and size of halogen affected the inhibition capability of the compounds.
Bioisosteric replacement of the phenyl by thiophen ring was found to be a useful modification in
present study to increase the inhibitory effects on hCA I. According to IC50 values, the
compound TP8 (IC50= 25.7 nM) having the thiopene ring was the most effective one. It was
11.6 fold more potent inhibitor than AZA (IC50=297.1nM). In addition, TP8 was the best
inhibitor towards hCA I isoenzyme among others according to IC50 value.
The isoform hCA II was inhibited by the compounds reported in this study at nanomolar
concentration with IC50 in the range of 33.0-57.8 nM. All compounds were found 2.0-3.5 more
potent than the reference drug AZA (IC50=115.1 nM). The compound TP1 having phenyl ring
was found the most potent with IC50=33.0 nM. Introduction of other substituents rather than
hydrogen such as methyl, methoxy, polimethoxy, halogens, trifluoromethyl on various positions
of phenyl ring and replacement of phenyl by thiophen reduced the inhibition effects of the
compounds on hCA II compared with compound TP1.
It could be seen from Table 1 that the compounds inhibited AChE with IC50 values in the
range of 61.6-141.3 nM. Among the series, the compound TP7 bearing trifloromethyl group at
para position of phenyl ring was considered the most potent compound with IC50=61.6 nM
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among others. Substitution of methyl and trifloromethyl groups at para position of phenyl ring in
the compounds TP2 and TP7, or replacement of phenyl by thiophen increased the inhibition
effects of the compounds compared with TP1 and reference drug TAC. However, AChE
inhibitory activity decreased by replacing the hydrogen at para position of phenyl with methoxy,
polimethoxy and halogen.
5. CONCLUSIONS
4-[5-Aryl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl] benzenesulfonamide derivatives TP1-
10 were synthesized and biological activities were evaluated towards AChE, hCA I and hCA II
enzymes. The compounds were found to have inhibitory activities at nanomolar concentrations
against AChE, hCA I, and hCA II isoenzymes. Electronic structure calculations and molecular
docking studies were also performed to enlighten inhibition mechanism and to support
experimental findings. The experimental and computational findings obtained in the present
study might be useful in the design of novel inhibitors against hCA I, hCA II, and AChE.
ACKNOWLEDGEMENTS
The authors thank to Ataturk University BAP office for the financial supports, Ataturk
University, Faculty of Science, Department of Chemistry for NMRs, and Anadolu University,
Faculty of Pharmacy, Department of Pharmaceutical Chemistry for HRMS analysis.
DECLARATION OF INTEREST
The authors declare that they have no competing interests.
FIGURE LEGENDS AND TABLES
FIGURE 1 Chemical structures of several CA (a) and AChE (b) inhibitors
SCHEME
1
Synthesis of 4-[5-aryl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl]
benzenesulfonamides derivatives, TP 1-10
TABLE 1. Inhibitory effects of the compounds TP 1-10 on AChE, hCA I and hCA II
enzymes
TABLE 2 Docking scores and selected molecular properties of TP1-10 and standard
inhibitors
TABLE 3 Selectivity ratios of TP1-10 and standard inhibitors
FIGURE 2 2D ligand interaction diagrams of AChE-TP7 (a), CA I-TP8 (b) and CA II-TP1
(c) complexes obtained from Glide SP calculations.
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TABLE 1 Inhibitory effects of the compounds TP 1-10 on AChE, hCA I and hCA II enzymes
IC50 (nM)
KI (nM)
hCA II
Compound
TP1
Ar
hCA I
r2
hCA II
r2
AChE
r2
hCA I
AChE
34.7
0.9853
33.0 0.9753 105.4 0.9739 27.8±4.7
43.3 0.9693 80.1 0.9632 24.8±1.8
42.1 0.9595 140.0 0.9453 33.1±4.4
36.5 0.9985 126.3 0.9585 38.0±11.3
40.1±11.1 54.6±1.8
42.5±5.7 22.7±10.3
62.7±24.7 70.7±22.9
38.5±3.8 43.9±20.4
TP2
TP3
TP4
30.1
36.5
40.8
0.9847
0.9838
0.9920
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TP5
TP6
TP7
TP8
34.5
27.7
32.8
25.7
0.9765
0.9770
0.9830
0.9924
57.8 0.9803 141.3 0.9864 34.3±4.3
38.5 0.9685 104.8 0.9666 25.6±3.9
40.5 0.9432 61.6 0.9787 30.1±5.1
53.3 0.9938 69.7 0.9633 24.2±4.6
65.3±16.7 56.1±22.7
37.3±9.0 80.6±37.7
55.2±15.6 28.8±9.2
44.7±8.9 34.3±15.3
45.8
40.5
0.9688
0.9634
46.2 0.9643 116.2 0.9850 49.8±12.8
42.2 0.9554 132.6 0.9836 44.8±11.3
49.4±9.3 52.1±21.4
TP9
109.1±27.
TP10
43.1±9.9
0
AZA
TAC
297.1 0.9889 115.1 0.9719
-
-
282.1±19.71 103.6±27.6
-
-
-
-
-
115.6 0.9862
-
-
66.5±13.8
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TABLE 2 Docking scores and selected molecular properties of TP1-10 and standart
inhibitors
Dipole
Moment
μ (D)b
%Human Rule
Oral of
Absorptiond Five
DScore
(kcal/mol)
EHOMOb
(eV)
PSAc
Å2
Compound
QPlogPo/wa
AChE CA I CA II
-8.50 -6.46 -5.50
-8.92 -6.73 -4.93
-8.88 -6.93 -4.96
-8.89 -6.14 -5.26
-8.69 -6.84 -5.01
-8.61 -6.78 -4.79
-9.39 -6.81 -4.62
-8.72 -6.34 -5.44
-8.70 -6.70 -5.11
-9.17 -6.35 -5.02
-8.85
TP1
TP2
TP3
TP4
TP5
TP6
TP7
TP8
TP9
TP10
TAC
AZA
3.65
3.92
3.72
3.89
4.05
4.14
4.50
3.55
3.75
3.74
2.59
-4.92
-4.87
-4.87
-5.00
-5.01
-5.01
-5.12
-4.95
-4.70
-4.83
-5.13
7.41
7.28
7.34
7.98
8.00
7.94
9.10
7.40
6.70
7.41
3.53
80.65
80.09
88.45
78.40
81.03
81.14
81.14
80.21
99.10
95.23
34.25
95.31
100.00
96.60
+
+
+
+
+
+
+
+
+
+
+
96.34
100.00
100.00
100.00
94.15
96.22
95.75
100.00
-6.33 -7.16
a Logarithm of the partition coefficient of the compound between n-octanol and water (recommended value <5)
b Calculated at M06-L/6-31+G(d,p)//AM1 level with Spartan ‘16 parallel suite.
c Polar surface area (recommended value ≤ 140Å2)45
d Percentage of human oral absorption (<25% is weak and >80% is strong).
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TABLE 3 Selectivity ratios of TP1-10 and standard inhibitors
KI (nM)
SR
PSR1
PSR2
PSR3
Compound
TP1
hCA I
hCA II
AChE
hCA II/hCA I
hCA I
10.1
11.4
8.5
hCA II
2.6
AChE
1.2
2.9
0.9
1.5
1.2
0.8
2.3
1.9
1.3
27.808±4.668 40.142±11.06 54.620±1.821
24.771±1.831 42.538±5.660 22.713±10.33
33.137±4.428 62.727±24.68 70.710±22.95
38.014±11.32 38.470±3.756 43.885±20.42
34.254±4.274 65.302±16.56 56.105±22.70
25.583±3.984 37.314±9.003 80.580±37.67
30.099±5.085 55.181±15.61 28.805±9.211
24.181±4.550 44.704±8.925 34.301±15.25
49.812±12.80 49.426±9.305 52.070±21.42
44.788±11.33 43.138±9.897 109.05±26.95
1.4
1.7
1.9
1.0
1.9
1.5
1.8
1.8
1.0
0.9
TP2
TP3
2.4
1.7
TP4
7.4
2.7
TP5
8.2
1.6
TP6
11.0
9.4
2.8
TP7
1.9
TP8
11.7
5.7
2.3
TP9
2.1
TP10
AZA*
TAC**
6.3
-
2.4
-
0.6
-
282.090±19.71 103.60±27.60
-
0.4
-
-
-
66.521±13.76
-
-
-
Selectivity Ratio (SR) value reflects the selectivity of a compound towards hCA I or II isoenzymes. The SR values were calculated by dividing Ki value of the certain
compounds to hCA II or hCA I. Potency Selectivity Ratios (PSR1 and PSR2) of a compound towards hCA I and II were calculated. PSR1 values were produced by dividing Ki
value of AZA (282.090 nM) to Ki value of a certain compound towards hCA I. PSR2 values were produced by dividing Ki value of AZA (103.60 nM) to Ki value of a certain
compound towards hCA II. In addition, PSR3 values of the compound towards AChE were calculated. PSR3 values were produced by dividing Ki value of TAC (66.521 nM) to
Ki value of a certain compound towards AChE. Data were presented in Table 3.
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.