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Статья Paper
DOI: 10.6060/mhc130116c
Design and Syntheses of Some New 5-[Benzenesulphonamido]-1r3r4-thiadiazol-2-sulphonamide as Potent Antiepileptic Agent
Mahavir R. Chhajed,a@ Anil K. Shrivastava,b and Vijay S. Tailec
aDepartment of Pharmaceutical Chemistry, Suresh Gyan Vihar University, Jaipur, India bDepartment of Pharmaceutical Sciences, NIMS University, Jaipur, India cDepartment of Chemistry, RTM Nagpur University, Nagpur, India @Corresponding author E-mail: mahavirchhajed@rediffmail.com
A QSAR study was performed on sulphonamide-1,3,4-thiadiazole derivatives using modified version of Allinger MM2 force field in Chem 3D Ultra structural descriptors. The relationship between antiepileptic activity and various descriptors was established by stepwise multiple regression analysis. The analyses have produced well predictive and statistically significant QSAR models which were further cross validated. This study helps to design and to synthesize some expectedly potent compounds. The compounds, 5-[(4-acetamido)benzenesulphonamido]-1,3,4-thiadiazol-2-(N-benzoyl) sulphonamide (8a), 5-[(4-amino)benzenesulphonamido]-1,3,4-thiadiazol-2-(N-benzoyl) sulphonamide (9a) and 5-(4-amino) benzenesulphonamido-1,3,4-thiadiazol-2-sulphonamide (10a) were synthesized from acetazolamide by modified Schotten-Bouman synthesis method and tested for antiepileptic activity by maximal electroshock induced seizures (MES) and subcutaneous pentylenetetrazole (scPTZ) induced seizure models in mice and have shown significant activity. These compounds were further subjected to diuretic studies, showing up to 86 % reduction in diuresis compared to acetazolamide.
Keywords: QSAR, antiepileptic activity, diuretic activity, 1,3,4-thiadiazole, acetazolamide.
Разработка и синтез новых 5—[бензилсульфонамидо]—1,3,4— тиадиазол-2-сульфонамидов как противоэпилептических препаратов
М. Р. Чхаед,а@ А. К. Шривастава,ь В. С. Тайле'
aSuresh Gyan Vihar University, Джайпур, Индия bNIMS University, Джайпур, Индия cRTM Nagpur University, Нагпур, Индия @E-mail: mahavirchhaied@rediffmail.com
QSAR исследование проводили на производных сулъфонамид-1,3,4-тиадиазола с использованием структурных дескрипторов модифицированной версии силового поля ММ2 Allinger в Chem 3D Ultra. Взаимосвязь противоэпилептической активности и различных дескрипторов была установлена с помощью ступенчатого множественного регрессионного анализа. Получены хорошие предсказательные и статистически значимые QSAR модели, которые в дальнейшем были перекрестно подтверждены. По результатам этого исследования были предложены и синтезированы из ацетазолоамида с использованием модифицированного метода Шоттена-Боумана потенциально перспективные соединения - 5-[(4-ацетамидо)бензолсульфонамидо]-1,3,4-тиадиазол-2-(^бензоил) сульфонамид (8a), 5-[(4-амино)бензолсульфонамидо]-1,3,4-тиадиазол-2-(^бензоил) сульфонамид (9a) и 5-(4-амино)бензолсульфонамидо-1,3,4-тиадиазол-2-сульфонамид (10a). Полученные соединения обладают противоэпилептической активностью в опытах на мышах, а также проявляют себя как эффективное противодиуретическое средство.
Ключевые слова: QSAR модели, противоэпилептическая активность, диуретическая активность, 1,3,4-тиадиазол, ацетазоламид.
Introduction
About 0.5-1% of the world's population is affected by epilepsy, one of the most common neurological disorders, and characterized by recurrent seizure attacks. The drugs that are widely used for treating epileptic seizures have failed to adequately control seizures and have unfavourable adverse effects such as ataxia, hepatotoxicity, gingival hyperplasia and megaloblastic anaemia. The restrictive treatments of epileptic seizures in patients have led to researches to discover new agents with more effectiveness and lesser toxicity.[1-2]
Acetazolamide, a heterocyclic sulphonamide; 1,3,4-thiadiazole derivative, has carbonic anhydrase inhibitor activity and clinically used as diuretic agent and also used in treatment of absence seizures. It is also useful as an adjunct in the treatment of tonic-clonic, myoclonic, and atonic seizures, particularly in women whose seizures occur or are exacerbated at specific times in the menstrual cycle. However, its usefulness is transient often because of rapid development of tolerance.[3] The problem with acetazolamide therapy as an antiepileptic agent is that, it produce diuresis and electrolyte imbalance as an adverse effect.
At least 14 different carbonic anhydrase (CA) isoforms were isolated in higher vertebrates. Several important physiological and physico-pathological functions are played by many CA isozymes, which are strongly inhibited by aromatic and heterocyclic sulphonamides. hCA I and hCA II inhibitors involved in the anticonvulsant activity shown by many sulphonamide drugs with CA inhibitory properties.[4-7]
Among the few reports in the literature our attention was drawn to the earlier discovery by Robin Jr. R.O. and co-workers[8-9] reported that, the only sulphonamides, unsubstituted on the sulphonamide nitrogen in acetazolamide are highly active as carbonic anhydrase inhibitors. The 1,3,4-thiadiazole and its derivatives possess wide variety of activities.[10-25] Furthermore, 1,3,4-thiadiazole nucleus itself exhibit anticonvulsant activity.[23] This increases our interest to design compounds from QSAR study and synthesize the compounds accordingly, having better therapeutic index for epilepsy treatment and very low diuretic activity.
In hope of getting anticonvulsant response of 1,3,4-thiadiazole nucleus itself, substitution of sulphonamide nitrogen at second position and chemically modifying fifth position acetyl group in to aromatic substituted sulphonamide group, that modification of sulphonamido group by some other chemical moiety or any substitution on sulphonamide nitrogen atom yielded structural analogues with loss of diuretic activity and will produce significant anticonvulsant activity.
We report herein designing, synthesis, antiepileptic activity and reduction in diuretic activity of acetazolamide derivatives; 5-[(4-acetamido)benzenesulphonamido]-1,3,4-
thiadiazol-2-(V-benzoyl) sulphonamide (8a), 5-[(4-amino) benzenesulphonamido]-1,3,4-thiadiazol-2-(V-benzoyl)-sulphonamide (9a) and 5-(4-amino)-benzenesulphonamido-1,3,4-thiadiazol-2-sulphonamide (10a).
QSAR Study
Several 1,3,4-thiadiazole and sulphonamide derivatives were selected[26] for the study (Figure 1 and Table 1) with the hope to obtain better antiepileptic agents with low dieresis. All physicochemical parameters of each compound from the
Table 1. CA-IV inhibition data for sulphonamides and 1,3,4-thiadiazole derivatives used in this study.
Compd R aIC,0 (MM) bPic50
(Ia) -nh2 300 -2.47712
(Ib) -ch2nh2 170 -2.23045
(Ic) -(CH2)2NH2 160 -2.20412
(Id) -NHCOCH3 246 -2.39094
(Ie) -NHCOCF3 133 -2.12385
(If) -nhcoch2ch3 232 -2.36549
(Ig) -NHCO(CH2)2CH3 227 -2.35603
(Ih) -NHCOCH(CH3)2 258 -2.41162
(Ii) -NHCO(CH2)3CH3 214 -2.33041
(Ij) -NHCOCH(CH3)3 230 -2.36173
(Ik) -NHCO(CH2)4CH3 63 -1.79934
(Il) -NHCO(CH2)7CH3 66 -1.81954
(Im) -nhcoc6h5 6 5 37 -1.5682
(In) -NHCONHCH- 65 240 -2.38021
(Io) -CH2NHCONHC6H5 2 6 5 105 -2.02119
(Ip) -(CH2)2NHCONHC6H5 75 -1.87506
(Iq) -NH(SO^H5 49 -1.6902
(IIa) -coch3 12 -1.07918
(IIb) -COCH(CH3)3 10 -1
(IIc) -COCH, 65 15 -1.17609
(IId) -COC6F5 65 2 -0.30103
(IIe) 4-NHCOCH3C6H4SO2 3 6 4 2 3 -1.44716
(IIf) 3-F,4-NHCOCH,C,HlSO. 7 3 6 4 2 1 0
(IIg) 3-Cl,4-NHCOCH3C6H4SO2 3 6 4 2 1 0
(IIh) 3-Br,4-NHCOCH3C6H4SO2 3 6 4 2 2 -0.30103
(IIi) 4-NH2C6H4SO2 14 -1.14613
(IIIa) -COCH3 1 0
(IIIb) -CO-2-furyl 6 -0.77895
(IIIc) -COCH, 65 17 -1.23045
(IIId) -COC F 6 5 1.5 -0.17609
aIC50 valus were determined by CA-IV enzyme inhibition. bnegative logarithmic value of IC50 (in moles) [pIC50 = -log10 IC50].
0 N —N n H3C\
r, /^V " 1! If o N—N
W o H 5 2 RN S S"NH2
o
(la-q) (lla-i) (llla-d)
Figure 1. 1,3,4-Thiadiazole and sulphonamide derivatives selected for the study.
series were calculated and subjected to stepwise, multiple and sequential regression analysis with respect to biological activity (Table 2). Correlation of each parameter was generated with biological activity and is summarised in Table 3. The number of developed models was high, so further analysis was bases on statistically significant parameters, namely correlation coefficient (R), it's square (R2), variance ratio (F), cross-validation method (Q2) standard deviation based on predicted residual sum of squares (Spress) and standard deviation of error of prediction (SDEP). The parameters used in the model were almost independent, which can be seen from the Pearson correlation matrix (Table 4, Figure 2).
Chemistry
5-Amino-1,3,4-thiadiazol-2-[N-(substituted benzoyl)] sulphonamides (4a-d) was prepared by hydrolysis of the benzoylated acetazolamides (3a-d), which was prepared from the acetazolamide (1) by benzoylationwith substituted benzoyl chlorides (2a-d). Substituted 4-acetamidobenzenesulphonyl chlorides (7a-c) were prepared according to the literature method27 from the substituted acetanilides (6a-c) by sulphonation with chlorosulphonic acid. The crude product was used in the next step immediately. Modified Schotten-Boumann synthesis method[26-27] was used to synthesized
Table 2. Descriptors Calculated for the QSAR Study.
Sr. No. Descriptor Abbreviations Unit Type
1 Heat of formation HF kcal/mol Thermodynamic
2 Boiling Point BP Kelvin Thermodynamic
3 Critical Pressure CP Bar Thermodynamic
4 Critical Volume CV cm2/mol Thermodynamic
5 Critical Temperature CT Thermodynamic
6 Henry's law constant HLC Thermodynamic
7 Ideal gas thermal capacity IGTC J/mol K Thermodynamic
8 Log p LP Thermodynamic
9 Melting Point MP Kelvin Thermodynamic
10 Molar Refractivity MR cm3/mol Thermodynamic
11 Standard Gibbs free energy GBS kJ/mol Thermodynamic
12 Connolly accessible area CAA Angstrom S2 Steric
13 Connolly molecular surface area MS Angstrom S2 Steric
14 Connolly solvent excluded volume CSEV Angstrom S2 Steric
15 Ovality Steric
16 Principal Moment of Inertia-X PMI- X Steric
17 Principal Moment of Inertia-Y PMI- Y Steric
18 Principal Moment of Inertia-Z PMI- Z Steric
19 Dipole Moment D Debye Electronic
20 Dipole Moment - X axis DX Debye Electronic
21 Dipole Moment - Y axis DY Debye Electronic
22 Dipole Moment - Z axis DZ Debye Electronic
23 Electronic energy EE eV at 0 0C Electronic
24 Total energy TE eV Electronic
25 HOMO energy HOMO eV Electronic
26 LUMO energy LUMO eV Electronic
27 Repulsion energy RE eV Electronic
28 Bending energy eb kcal/mol Thermodynamic
29 Charge-charge energy Ec kcal/mol Thermodynamic
30 Charge-dipole energy Ed kcal/mol Thermodynamic
31 Dipole-dipole energy DDE kcal/mol Thermodynamic
32 Non-1,4- Van der Waals energy NVDWE kcal/mol Thermodynamic
33 Stretch energy SE Thermodynamic
34 Stretch bend energy SBE kcal/mol Thermodynamic
35 Torsion energy TOE kcal/mol Thermodynamic
36 Total energy TE kcal/mol Thermodynamic
37 Van der Waals energy VDE kcal/mol Thermodynamic
Table 3. Descriptors, observed, calculated and predicted CA-IV inhibition activity data of compounds of training set.
Descriptors pIC
Compd HLC LUMO VDWE aObs. bCal. LOO
(Ia) 2.4950 -1.4370 8.2817 7.6353 7.61418 3.228892
(Ib) 2.3305 -0.3276 3.1643 6.6177 6.60267 2.656621
(Ic) 1.7620 -0.6241 4.4457 6.8029 6.80039 2.734382
(Id) 1.7620 -0.6029 6.0813 6.7930 6.79324 2.756201
(Ie) 2.3305 -0.8487 1.3968 6.9469 6.92539 1.930475
(If) 2.3305 -0.8488 1.3991 6.9285 6.92513 1.737577
(Ig) 2.3305 -0.8377 2.7005 6.9248 6.90509 1.952210
(Ih) 2.3305 -0.8317 3.7304 6.8950 6.87545 1.953405
(Ii) 2.3305 -0.8289 3.8343 6.9654 6.94487 1.951018
(Ij) 2.3305 -0.5802 6.5330 6.6646 6.66516 1.588046
(Ik) 2.3305 -0.5625 7.1177 6.6904 6.68507 1.610167
(Il) 2.3305 -0.8294 5.4088 6.9343 6.94855 1.440459
(Im) 2.3305 -1.2295 8.0392 6.4709 cro cro
(In) 2.3305 -0.7771 4.3643 7.0677 7.09076 1.652787
(Io) 2.3305 -1.8565 4.7188 8.0725 7.98791 1.762105
(Ip) 2.3305 -0.9266 3.1096 7.3222 7.27296 0.889326
(Iq) 1.7620 -0.9547 4.8646 7.3505 7.29577 1.256420
(IIa) 1.7620 -0.8601 6.5776 6.9636 6.97175 1.278754
(IIb) 1.7620 -0.9373 8.5701 7.0206 7.06207 2.440922
(IIc) 2.3305 -1.0746 8.1744 7.2723 7.27390 1.662270
(IId) 1.7620 -0.9003 10.0460 7.1909 7.21692 1.771029
(IIe) 1.7620 -0.8147 11.3092 7.1254 7.19688 1.963489
(IIf) 2.3305 -1.3112 7.8988 7.4437 7.48955 2.344829
(IIg) 2.3305 -2.0134 9.1096 8.1297 8.08186 2.253290
(IIh) 1.7620 -0.7769 10.8721 6.9047 6.91702 2.134695
(IIi) 7.3867 -1.4747 1.1745 8.5932 8.57619 2.476107
(IIIa) 7.3196 -1.6986 1.0305 8.8500 8.88264 2.698101
(IIIb) 7.5169 -1.6800 2.1094 8.8937 8.91858 2.698101
(IIIc) 7.7864 -1.7131 2.3540 9.1877 9.05720 2.298853
(IIId) 4.8975 -1.2736 3.0051 8.0309 8.09029 2.476107
"Observed value
bCalulated (Cal) and predicted (LOO) values of pIC from Model II croCompound removed as outlier
Table 4. Pearson Correlation Matrix for descriptors influencing inhibition of CA-IV.
HLC LUMO VDWE pic50
HLC 1.000
LUMO -0.600 1.000
VDWE -0.560 0.113 1.000
pic50 0.787 -0.792 -0.130 1.000
5-[(4-acetamido) substituted benzenesulphonamido]-1,3,4-thiadiazol-2-[V-(substituted benzoyl)]sulphonamides (8a-g), where the nucleophilic substitution reaction occurs. In this reaction acetone:water mixture (1:1) was used instead of pyridine, sodium hydroxide or any other strong base. Compounds (8a-g) were prepared from the 5-amino-1,3,4-
thiadiazol-2-[V-(substituted benzoyl)]sulphonamides (4a-d) (Scheme 1) and substituted benzene sulphonyl chlorides (7a-c) (Scheme 2). After purification, further, subjected to hydrolysis with aqueous sodium hydroxide (20 %) solution to obtain the compounds (9a-g) while, 5-(4-amino) benzenesulphonamido-1,3,4-thiadiazol-2-sulphonamides
0 12 3
pIC Calculated from Model II
Figure 2. Predictive performance of the model II built on datasets for 29 compounds. The plots show the correlations between predicted (in cross validation) versus experimental log IC values.
(10a-b) were obtained by hydrolysis of (9a and 9g) using 70 % sulphuric acid (Scheme 3).
The FT-IR spectra show S=O stretching at 1070 -1030, sulphonamide stretching at1177-1125 cm-1, sulphonamide N-H stretching at 3385-3265 cm-1, C-S stretching at 712662 cm-1, S=O stretching at 1128-1026 cm-1, but C = O stretching peak at 1687-1513 cm-1 is absent in compounds (10a-b). The 'H-NMR spectra of all compounds indicated expected peaks in the region of 1.249-1.254 8 ppm singlet of Ar-SO2NH, while multiplets of aromatic ring are in the range of 6.6-8.2 8 ppm. Thin layer chromatography (TLC) was run throughout the reaction to optimize the reaction for purity and completion.
Pharmacology
The synthesized derivatives obtained from the reaction sequence were administered orally into mice and evaluated
for their antiepileptic activity in maximal electroshock (MES) [28-29] and subcutaneous pentylenetetrazole (scPTZ)[30] using a dose of 100 mg/kg. The same derivatives were also studied for their diuretic activity in conscious rats, by collecting urine for four hrs. The results of antiepileptic and diuretic activity are shown in Table 5 and Table 6 respectively.
Results and Discussion
The descriptors of 1,3,4-thiadiazole were found to have good correlation with biological activity for designing antiepileptic agent which are summarised in Table 3. Herein the result of QSAR study for antiepileptic activity of the mentioned series are reported.
The Model II tested for 29 compounds as a test set. The predicted activity shows linear relationship with observed activity in the test set (R=0.9310) showing the robustness of the model. Henry's law constant (HLC), lowest unoccupied molecular orbital energy (LUMO) and Van der Waals energy (VDWE) have better correlation with biological activity and have low value of standard deviation. The data show (Model II) overall significant level greater than a 99.9 as it exceeded the tabulated F value and Q2 value found to be greater than 0.5. The equation was validated by leave one out cross validation method and bootstrapping method as an internal validation, which gives statistically significant values.
Epileptic mediators are membrane based and increasing the lipophillic nature of antiepileptic drug molecule may improve its pharmacokinetic and pharmacodynamic properties. Increase in lipophilicity and minimum electronic interference increases antiepileptic activity suggested by positive value of HLC, VDWE and negative value of LUMO, as former two are molecular properties describing thermodynamic and later one is molecular property describing electronic parameters of the moiety. Normally both of parameters are referred to certain groups, thus substitution of bulkier, lipophillic, aromatic group will contribute to
9 N—N ^
A
H,C N S S-NH, H Ô ' (1)
o _
c|A0 (2a-d)
9 N-N ^
A
H„C N S S-N 3 H ¿H
(3a-d)
R1=H, p-CI, P-N02, m-N02
R1 10% NaOH [j1 o O -~ H,N S S-N
A 2 M u
o n
(4a-d)
Scheme 1.
Acetic anhydride
A
(5a-c)
H„C N
R2=H; 2-CH3, 5-CI; 2-NOa, 5-OCH3
Scheme 2.
(7a-c)
O
H3C fl^Q (7a-c)
O
ii
s-ci o
H?N
h2n
N-N O
VML" &H
(4a-d)
20% NaOH a o
H3C
A
Acetone: water mixture (1:1)
70% H-SO,
N-N
A i? A
HPN—\ N>—S—N^S S
w n h
-NH,
(10a-b)
Compd. No. ri r2 Compd. No. ri r2
8a H H 9b 4-Cl H
8b 4-Cl H 9c 4-NO2 H
8c 4-NO2 H 9d 3-NO2 H
8d 3-NO2 H 9e H 2-CH3, 5-Cl
8e H 2-CH3, 5-Cl 9f 4-Cl 2-CH3, 5-Cl
8f 4-Cl 2-CH3, 5-Cl 9g H 5-OCH3, 2-NO2
8g H 5-OCH3, 2-NO2 10a -- H
9a H H 10b -- 5-OCH3, 2-NO2
Scheme 3.
HLC and VDWE. Furthermore, reduced electron density of -NH2 (amine and sulphonamide) increases the electrostatic attraction for receptor. The LUMO descriptor indicates the strength and orientation behaviour of molecule in an electrostatic field. It is also important in determining the behaviour of the molecule in vicinity of molecule. Based on the above results, some new derivatives of 1,3,4-thiadiazole were designed and synthesized, accordingly as depicted in Scheme 1, 2 and 3.
In this series, all the analogues show more potent anticonvulsant activity while diuretic activity is absent. In the earlier reports it was highlighted that the only sulphonamides, unsubstituted on the sulphonamide nitrogen in acetazolamide[8-9] are highly active as carbonic anhydrase inhibitors. It is also reported that presence of electron rich atom/group attached at the para position of the aryl ring showed increased potency in the MES screen.[31-32]
All the tested compounds were found to exhibit anticonvulsant activity in MES screening; however, compound 8c is more potent while compound 10b shows
potency similar to standard drug (Phenytion). All the synthesized compounds were active in MES screen and showed recovery without any mortality. Compound 8c displayed activity in the MES screen with recovery of animals in 130.30 sec while compound 8g showed 100% incidence of recovery from mortality in the scPTZ test since animals does not produce any clonic convulsions. This compound exhibited rapid onset of action and long duration of activity. The most active compound in the scPTZ test, a test used to identify compound that elevates seizure threshold, were 8d and 8g. Experimental results indicated that our compound exhibited better anticonvulsant activity as compared to diuretics so could be better drugs compared to acetazolamide in treatment of convulsions (Table 5). All the compounds were screened for diuretic activity. In the diuretic study, compounds showed decrease in urine output up to 80% when compared to acetazolamide as reported in Table 6. Generally compounds possessing higher log p value showed higher decrease in diuretic activity. Bulkier compounds are more lipophillic and can cross blood brain barrier to exert their
Table 5. Effect of the synthesized compounds on MES induced and scPTZ induced convulsions in mice.
MES Test scPTZ Test
Time Duration (in Sec) Time Duration (in Sec)
Treatment Dose (mg/kg) flexion extension Clonus stupor Recovery Onset of clonic convulsions in sec Incidence of protection against mortality (%)
Solvent Control 5.87 ±0.56 54.15±5.61 15.75±1.02 147.2±6.05 204.4 142.4±12.9 00
Phenytoin 100 3.23±0.49*** 10.20±0.67*** 9.23±1.78* 60.03±3.88*** 126.2 A 100
Acetazolamide 100 4.03±0.78* 12.46±1.62* 11.29±1.55* 85.19±4.02*** 182.67 487.17±29.08 16.6
8a 100 3.83±0.86* 10.45±0.79*** 8.83±0.51*** 62.86±3.80*** 143.72 664.4 ± 74.1* 66.6
8b 100 3.97±0.93* 10.56±1.19* 10.76±0.64*** 58.85±3.76*** 137.43 NT -
8c 100 3.05±0.77* 11.35±1.54* 9.80±1.54* 63.50±5.29*** 113.30 369.16±23.64 16.6
8d 100 4.15±0.96* 10.48±0.77*** 11.00±1.02* 70.96±4.83*** 130.30 779.9 ± 52.7* 83.3
8e 100 2.92±0.67* 11.49±0.88*** 11.11±0.81*** 65.30±4.00*** 129.51 570.16±39.64*** 66.66
8f 100 3.50±0.93* 11.04±1.01* 12.24±1.53* 71.71±3.66*** 158.64 503.16±27.30* 50
8g 100 2.80±0.59* 10.88±1.05* 11.22±0.71*** 68.15±4.23*** 132.67 A 100
9a 100 4.07±0.97* 11.55±1.43* 11.61±1.32* 67.47±3.01*** 172.10 440.16±40.97 16.6
9b 100 3.02±0.62* 10.90±1.00* 9.65±1.88* 62.02±6.53*** 155.33 NT -
9c 100 3.57±0.66* 11.29±0.94*** 10.12±0.85*** 64.85±3.08*** 163.98 396.84±41.87 33.33
9d 100 3.42±0.81* 12.47±1.61* 10.46±1.16* 59.06±4.70*** 164.40 NT -
9e 100 3.52±0.64* 11.09±0.76*** 9.95±1.27* 63.81±3.76*** 157.67 NT -
9f 100 3.62±0.86* 11.45±0.81*** 11.46±1.63* 67.85±5.99*** 144.71 780.0±12.4 66.6
9g 100 3.52±0.55* 12.60±1.53* 10.97±1.41* 67.82±6.46* 176.20 NT -
10a 100 4.13±0.71* 13.13±1.12*** 11.69±1.18* 66.64±4.33*** 134.87 629.73±32.7 66.6
10b 100 4.07±0.79* 11.44±1.37* 10.39±1.22* 55.96±3.09*** 127.29 667.6±95.0 50
Values are expressed as mean±SEM, n=6, P<0.05* and <0.001*** as compared to control group, A: Absence of convulsions, NT: Not tested
Table 6. Diuretic activity of the synthesized compounds.
Treatmenta Body weight of five rats Normal saline intake (mL) Urine output (mL)b % Output T/C T/S % Diuretic activityc % Reduction in Diuretic Activityd
Control 730 36.5 7.2 19.73 1.0
Acetazolamide 660 33 20.7 62.73 3.18 1.0 100 0
8a 690 34.5 3.8 11.01 0.56 0.184 18.4 81.6
8f 542 27.1 2.9 10.7 0.54 0.14 14 86
9e 769 38.5 5.3 13.77 0.697 0.256 25.6 74.4
9h 680 34 4.1 12.06 0.569 0.198 19.8 80.2
10a 687 34.4 12.4 36.047 1.72 0.599 59.9 40.1
a The animals of each groups received normal saline (5 mL/100 g p.o.). Except the control group, the animals of each group received the
dose of 45 mg/kg. n=6,
b Urine volume collected in 4 hr,
c Diuretic activity = (T/S)x100,
d Compared to standard
T/C = ratio of test to control,
T/S = ratio of test to standard
effect at CNS. Present study explored that substitution of 1,3,4-thiadiazoles at third position and sulphonamido moiety at fifth position leads to the development of new chemical entities with potent anticonvulsant activity as compared to diuretic activity.
Seizures are caused by abnormal stimulation of nerves in the brain by other nerves. Generally, anticonvulsants reduce the excitability of the neurons (nerve cells) of the brain. When neuron excitability is decreased, seizures are theoretically reduced in intensity and frequency of occurrence or, in some instances, are virtually eliminated.[1] CA isozymes, which are strongly inhibited by aromatic and heterocyclic sulphonamides. hCA I and hCA II isoenzyme inhibitors involved in the anticonvulsant activity, shown by many sulphonamide drugs with CA inhibitory properties.[4-7]
The synthesized compounds showed antiepileptic effect, may be due to their inhibitory effect on brain carbonic anhydrase; specifically inhibition of hCA I and hCA II isoenzyme, leads to an increased transneuronal chloride gradient, increased chloride current, and increased inhibition.
Experimental Protocols
QSAR Study
a) The dataset and parameters. The CA inhibition data of sulphonamides and 1,3,4-thiadiazole data have been reported in terms of inhibitory concentration of 50% of enzyme inhibition (IC50 in micromoles). The inhibition data were converted to negative logarithmic values (concentration in moles). These values were used for subsequent QSAR analyses as response variable. The models for CA-IV inhibition were constructed based on the training set and the generated models were then validated: internally (using the leave one out technique) and externally (predicting the activities of the test set).[28"30,33] Molecular structure were generated and optimised with CS Chem Draw Ultra 6.0 and Chem 3D Ultra (Cambridge Soft.) respectively)34 first by molecular mechanics (MM2) and re-optimised by MOPAC-AM1 until the root mean square (RMS) gradient value becomes smaller than 0.0001 kcal/molA.
b) Statistical computation. The relationship between response variable (as a dependent variable) and various physicochemical as well as structural descriptors (as independent variables), were established by step-wise linear multiple regression analysis using SYSTAT 10.2 and VALSTAT running on a Pentium 4 processor (CPU 3.0 GHz HT).[35-36] Significant descriptors were chosen on the basis of statistical data of analysis.[37-38]
Model I
pIC=[5.3164(±0.4683)]+HLC[0.3005(±0.1034)]+LUMO[-0.7018(±0.3847)]+VDWE [0.0835(±0.0522)]
N=30, R=0.9186, R2=0.8439, variance=0.1040, SD=0.3225, F=46.8575, Q2=0.7669, SPRESS=0.3941, Sdep=0.3669
This model has an outlier (compound Im) because their residual values exceeded twice the standard error of estimate. When this outlier was removed from the dataset, a significant Model II has been found which is able to explain 0.0829 of variance of CA-IV inhibition. This model has a high internal predictivity as shown by the good Q2 value of 0.8066.
Model II
pIC=[5.3104(±0.4189)]+HLC[0.2875(±0.0930)]+LUMD[-0.7033(±0.3441)]+VDWE [0.0864(±0.0467)]
N=29, R=0.9310, R2=0.8668, variance=0.0829, std=0.2879, F=54.219, Q2=0.8066, SPRESS=0.3633, SDEP=0.3374
The parameters used in the model were almost independent, which can be seen from the Pearson correlation matrix (Table
4, Figure 2). Two best models were selected from series, out of which Model II is selected as the best. Since, this model has better statistically significant value, minimum standard deviation, low variance, and statistically significant F value.
Synthetic Study
Melting point was determined in one end open capillary tubes on a Thermonik Precision melting point apparatus (C-PMP-2, Mumbai, India) and are uncorrected. Infrared (IR) spectra were recorded for the compounds on Shimadzu FT-IR 8400s spectrophotometer in KBr. 1H nuclear magnetic resonance (1H NMR) spectra were recorded for the compounds on Varian EM-390 apparatus. Chemical shifts are reported in parts per million (ppm) using tetramethylsilane (TMS) as an internal standard. Elemental analysis (C, H, N and S) were undertaken with Elemental Vario EL III Carlo Erba 1106 analyzer. The purity of the compounds was confirmed by thin layer chromatography using silica gel glass plates and a solvent system of benzene: ethanol (8:2). The spots were developed in iodine chamber and visualized under ultra violet lamp. The log p values were determined using ChemDraw software
General procedure. 5-(4-Amino)benzenesulphonamido-1,3,4-thiadiazol-2-sulphonamide was synthesized in following sequence:
5-[(4-Acetamido)benzenesulphonamido]-1,3,4-thiadiazol-2-(N-benzoyl)-sulphonamide (8a). [5-Amino-1,3,4-thiadiazol-2-[N-benzoylsulphonamide] (4a) was suspended in a 1:1 mixture of acetone-water, and the stoichometric amount of 4-acetamidobenzenesulphonyl chloride (7a) and base was added concomitantly. The reaction mixture was magnetically stirred for several hours, the solvent was evaporated, then the pH was adjusted to 2 with 5N hydrochloric acid and the recrystallised from aqueous ethanol.
5-[(4-Amino)benzenesulphonamido]-1,3,4-thiadiazol-2-(N-benzoyl)sulphonamide (9a). The synthesized compound, (8a) (2.0 g, 0.0041 mol) was dissolved in 20 % aqueous sodium hydroxide and refluxed for 45 minutes, allowed to cool and acidified with dilute hydrochloric acid, to precipitate out the hydrolyzed product (9a), which was separated by filtration at vacuum, and recrystallised.
5-(4-Amino)benzenesulphonamido-1,3,4-thiadiazol-2-sulphonamide (10a). The compound (8a) (2.0 g, 0.0042 mol) was refluxed with 10-15 mL of 70 per cent sulphuric acid (3:4 by volume) for 30 minutes, allowed to cool, filtered off and rendered the filtrate alkaline with 10-20 per cent sodium hydroxide solution. The precipitate thus formed was filtered at vacuum and recrystallised to obtained compound (10a).
Adopting the above procedures, other compounds (8b-g), (9b-g) and (10b) were synthesized and characterized. The characterized data of these compounds are as follows:
N-[(5-amino-1,3,4-thiadiazol-2-yl)sulfonyl]benzamide (4a). Yield 76 %, m.p. 2340C, Rf 0.61. m/z (GC-MS): 268 (100%, Base peak, M+). Found: C 38.04, H 2.82, N 19.72, S 22.57 %. C9H8N4O3S2 (284.31) requires C 38.02, H 2.84, N 19.71, S 22.56 %. FT-IR (KBr) vmax cm-1: 3446-3368 (N-H), 3193-3002 (Ar C-H), 1685 (C=O), 6833-588 (C-S), 1072 -1026 (S=O), 1326, 1178 (sulphonamide), 3070-3002 (heteroaromatic C-H), 1685-1629 (heteroaromatic C=N). 1H NMR (DMSO-d6) SH ppm: 3.320 (s, 1H, CONH), 7.0187.853 (m, 5H, Ar-H), 8.025 (s, 1H, thiadiazole, C-H), 8.611 (s, 1H, acetamido-H).
N-{[5-({[4-(acetylamino)phenyl]sulfonyl}amino)-1,3,4-thiadiazol-2-yl]sulfonyl}benzamide (8a). Yield 32.54 %, m.p. 2160C, Rf 0.54, LogP 2.159. Found: C 42.44, H 3.12, N 14.55, S 19.87 %. C17H15N5O6S3 (481.52) requies C 42.40, H 3.14, N 14.54, S 19.98 %. m/z (GC-MS): 458 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3520-3400 (N-H), 3110-3012 (Ar C-H), 17001680 (amide C=O), 700-600 (C-S), 1070-1030 (S=O), 1370-1335, 1170-1155 (sulphonamide), 3265 (sulphonamide N-H), 3077-3003
(heteroaromatic C-H), 3500-3220 (heteroaromatic N-H), 1640 (heteroaromatic C=N). A.max (chloroform) (lgs)/nm 235. 1H NMR (DMSO-d6) SH ppm: 1.l0T(s, 1H, Ar-SO2H), 2.378 (s, 3H, CH3), 3.320 (s, 6H, CONH), 4.740 (s, 1H, N-H), 7.281- 7.785 (m, 6H, Ar-H), 8.025 (s, 1H, thiadiazole, C-H), 8.611 (s, 1H, acetamido-H). 13C NMR 5c ppm: 169.7, 168.9, 141.6, 135.5, 134.3, 128.9, 128.7, 128.7, 127.5, 127.5, 127.4, 122.0, 121.9, 121.8, 23.1.
N-{[5-({[4-(acetylamino)phenyl]sulfonyl}amino)-1,3,4-thiadiazol-2-yl]sulfonyl}-4-chlorobenzamide (8b). Yield 40.12 %, m.p. 2240C, Rf 0.62, LogP 2.717. Found: C 39.56, H 2.74, N 13.57, S 18.63 %. C17H14N5O6S3Cl (515.97) requires C 39.57, H 2.73, N 13.57, S 18.641 %. m/z (G3C-MS): 462 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3567-3385 (N-H), 3108-3045 (Ar C-H), 16611525 (amide C = O), 760-636 (C-S), 1097-1026 (S=O), 1383, 1330, 1177, 1141 (sulphonamide), 3385 (sulphonamide N-H), 2920 (CH3-O), 1531-1482 (Ar C-NO2), 852 (Ar C-N) 543 (C-Cl). Xmax (chloroform) (lgs)/nm 238. 1H NMR (DMSO-d6) 5H ppm: 1.217 (s 1H, Ar-SO2H), 2.332 (s, 3H, CH3), 3.496 (s, 1H, CONH), 4.740 (s, 1H, N-H), 7.242- 7.453 (m, 3H, Ar-H), 8.009 (s, 1H, thiadiazole, C-H), 8.036 (s, 1H, Acetamido-H). 13C NMR Sc ppm: 170.1, 169.1, 141.0, 137.3, 135.4, 132.2, 129.5, 129.1, 128.8,128.7, 128.2, 127.6, 122.0, 121.8, 22.5.
N-{[5-({[4-(acetylamino)phenyl]sulfonyl}amino)-1,3,4-thiadiazol-2-yl]sulfonyl}-4-nitrobenzamide (8c). Yield 28.13 %, m.p. 2420C, Rf 0.58, LogP 2.156. Found: C 38.79, H 2.69, N 15.94, S 18.27 %. C18H17N6O8S3 (526.52) requires C 38.78, H 2.68, N 15.96, S 18.27 %. m/z (GC-MS): 531 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3367-3110 (N-H), 3110-3024 (Ar C-H), 1445 (amide C=O), 711-655 (C-S), 1128-1080 (S=O), 1348, 1313, 1184 (sulphonamide), 3238 (sulphonamide N-H), 3077-3003 (heteroaromatic C-H), 2360-2341 (heteroaromatic N-H), 1720 (heteroaromatic C=N), 1515-1452 (Ar C-NO2), 896-852 (Ar C-N). Xmax (chloroform) (lgs)/nm 248. 1H NMR (DMSO-d6) 5H ppm: 2T32 (s, 3H, CH3), 3.541 (s, 1H, CON(-H) Ar), 7.241- 7.483 (m, 4H, Ar-H), 8.017-8.259 (m, 4H, Ar-H), 8.110 (s, 1H, acetamido N-H), 8.129 (s, 1H, Sulphonamido N-H). 13C NMR Sc ppm: 170.1, 168.9, 141.7, 140.3, 135.2, 128.7, 128.5, 127.4, 121.8,121.7, 127.5, 121.3, 23.0.
N-{[5-({[4-(acetylamino)phenyl]sulfonyl}amino)-1,3,4-thiadiazol-2-yl]sulfonyl}-3-nitrobenzamide (8d). Yield 21.87 %, m.p. 2370C, Rf 0.61, LogP 1.481. Found: C 38.76, H 2.69, N 15.96, S 18.29 %. C18H17N6O8S3 (526.52) requires C 38.78, H 2.68, N 15.96, S 18.27 %. m/z (GC-MS): 534 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3527-3415 (N-H), 3100-3000 (Ar C-H), 1531 (amide C=O), 713-619 (C-S), 1087 (S=O), 1352, 1311, 1176 (sulphonamide), 3265 (sulphonamide N-H), 3077-3003 (heteroaromatic C-H), 2341-2331 (heteroaromatic N-H), 1720 (heteroaromatic C=N), 1456-1531 (Ar C-NO2), 827 (Ar C-N). Xmax (chloroform) (lgs)/nm 252. 1H NMR (DMSO-d6) 5H ppm: 2.214 (s, 3H, CH3), 3.452 (s, 1H, CON(-H) Ar), 7.447- 7.881 (m, 2H, Ar-H), 8.219 (s, 1H, acetamido N-H), 8.437 (s, 1H, Sulphonamido N-H), 7.904-8.724 (m, 4H, Ar-H). 13C NMR Sc ppm: 169.9, 169.1, 148.3,
141.3, 135.3, 135.1, 133.2, 129.6, 127.4,c 127.2, 124.5, 122.4, 121.4,
121.4, 22.8.
N-{[5-({[4-(acetylamino)-5-chloro-2-methylphenyl]sulfonyl} amino)-1,3,4-thiadiazol-2-yl] sulfonyl} benzamide (8e). Yield 26.60 %, m.p. 2490C, Rf 0.64, LogP 3.204. Found: C 40.79, H 3.05, N 13.20, S 18.17 %. C18H16N5O6S3Cl (529.99) requires C 40.79, H 3.04, N 13.21, S 18.15 %. m/z (G3C-MS): 519 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3398-3137-(N-H), 3107-3010-(Ar C-H), 1708-1685 (amide C=O), 692-621 (C-S), 1081-1054 (S=O), 1365, 1311, 1187 (sulphonamide), 3319 (sulphonamide N-H), 23602341 (heteroaromatic N-H), 1708 (heteroaromatic C=N), 592-532 (C-Cl), 972-846 (C-N), 2894 (C-H). Xmax (chloroform) (lgs)/nm 252. 1H NMR (DMSO-d6) 5H ppm: 2.054m(s, 1H, C(=O)CH3), 2.452 (s, 1H, Ar C-CH3), 3.365 (s, 1H, CON(-H)Ar), 7.404-7.753 (m, 3H, Ar-H), 7.647- 7.818 (m, 2H, Ar-H), 8.124 (s, 1H, acetamido N-H), 8.548 (s, 1H, Sulphonamido N-H). 13C NMR Sc ppm: 170.1, 168.8,
140.3, 136.2, 134.2, 134.2, 132.4, 128.9, 128.8, 127.7, 127.6, 127.3,
126.3, 123.0, 22.6, 16.2.
N-{[5-({[4-(acetylamino)-5-chloro-2-methylphenyl]sulfonyl}
amino)-1,3,4-thiadiazol-2-yl] sulfonyl}-4-chlorobenzamide (8f). Yield 39.44 %, m.p. 2570C, Rf 0.61, LogP 3.763. Found: C 38.29, H 2.65, N 12.42, S 17.07 %. C18H15N5O6S3Cl2 (564.44) requires C 38.30, H 2.68, N 12.41, S 17.04 %. m/z (GC-MS): 581 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3359-3338 (N-H), 3082-3045 (Ar C-H), 1691-1629 (amide C=O), 678-669 (C-S), 1035 (S=O), 1342, 1319, 1157 (sulphonamide), 3338 (sulphonamide N-H), 23812341 (heteroaromatic N-H), 1785-1735 (heteroaromatic C=N), 559 (C-Cl), 960-827 (C-N), 2943 (C-H). Xmax (chloroform) (lgs)/nm 248. 1H NMR (DMSO-d6) SH ppm: 2.062"s, 1H, C(=O)CH3), 2.564 (s, 1H, Ar C-CH3), 3.447 (s, 1H, CON(-H) Ar), 7.173-7.392 (m, 2H, Ar-H), 7.423- 7.588 (m, 2H, Ar-H), 7.973 (s, 1H, acetamido N-H), 8.213 (s, 1H, Sulphonamido N-H). 13C NMR Sc ppm: 169.8, 169.0, 140.2, 137.4, 135.9, 135.1, 132.4, 129.1, 129.1,128.9, 128.8, 127.7,
126.4, 122.9, 23.0, 16.5.
N-{[5-({[4-(acetylamino)-5-methoxy-2-nitrophenyl]sulfonyl}
amino)-1,3,4-thiadiazol-2-yl]sulfonyl}benzamide (8g). Yield 25.30 %, m.p. 2640C, Rf 0.52 LogP 0.694.. Found: C 38.88, H 2.88, N 15.12, S,, 17.27 %. C18H15N6O9S3 (590.99) requires C 38.85, H 2.90, N 15.10, S 17.28 %. m/z (GC-MS): 541 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3537-3427 (N-H), 3124-3028 (Ar C-H), 1691-1528 (amide C=O), 717-609 (C-S), 1022-1078 (S=O), 1367, 1333, 1181, 1157 (sulphonamide), 3265 (sulphonamide N-H), 2847 (CH3-O), 1528-1487 (Ar C-NO2), 823 (Ar C-N stretching). Xmax (chloroform) (lgs)/nm 261. 1H NMR (DMSO-d6) 5H ppm: 2.054 (s, 1H, C(=O)CH3), 3.714 (s, 1H, CON(-H) Ar), 4.245 (s, 1H, Ar O-CH3), 7.204-7.938 (m, 3H, Ar-H), 8.12- 8.417 (m, 2H, Ar-H), 8. 241 (s, 1H, acetamido N-H), 8.548 (s, 1H, Sulphonamido N-H). 13C NMR Sc ppm: 169.7, 169.1, 159.3, 138.2, 137.7, 134.5, 132.3, 131.1, 131.1, 130.8, 129.1, 128.9, 127.7, 127.6, 116.9, 116.5, 112.4, 55.9, 22.8.
N-[(5-{[(4-aminophenyl)sulfonyl]amino}-1,3,4-thiadiazol-2-yl) sulfonyl]benzamide (9a). Yield 60.27 %, m.p. 25 90C, Rf 0.57, LogP 2.448. Found: C 40.98, H 2.97, N 15.95, S 21.89 % C15H13N5O5S3 (439.49) requires C 40.99, H 2.98, N 15.94, S 21.89 %. m/z (GC-MS): 429 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3573-3295 (N-H), 3075 (Ar C-H), 1687-1513 (amide C=O), 712662 (C-S), 1128-1026 (S=O), 1330, 1177, 1125 (sulphonamide), 3385 (sulphonamide N-H). Xmax (chloroform) (lgs)/nm 238. 1H NMR (DMSO-d6) SH ppm: 1.24T(s, 1H, Ar-SO2NH), 3.526 (s, 2H, Ar-NH2), 6.895-6.923 (s, 2H, Ar-unsymmetrical pattern), 7.2368.392 (m, 3H, Ar-H), 8.143 (s, 1H, Sulphonamido N-H). 13C NMR Sc ppm: 170.2, 152.0, 134.4, 132.3, 129.8, 128.8, 128.7, 128.2, 128.1, 127.6, 127.6, 116.5, 116.3.
N-[(5-{[(4-aminophenyl)sulfonyl]amino}-1,3,4-thiadiazol-2-yl)sulfonyl]-4-chloro benzamide (9b). Yield 70.64 %, m.p. 2670C, Rf 0.52, LogP 3.007. Found: C 38.01, H 2.57, N 14.75, S 20.29 %. C15H12N5O5S3Cl (473.93) requires C 38.01, H 2.55, N 14.78, S 20.30 %. m/z (GC-MS): 481 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3561-3372 (N-H), 3097-3015 (Ar C-H), 1698 (amide C=O), 763-553 (C-S), 1087-1056 (S=O), 1341, 1330, 1170, 1139 (sulphonamide), 3324-3197 (sulphonamide N-H), 782 (C-Cl). Xmax (chloroform) (lgs)/nm 242. 1H NMR (DMSO-d6) 5H ppm: 1.329 (s, 1H, Ar-SO2NH), 3.225 (s, 2H, Ar-NH2), 6.985-7.014 (s, 2H, Ar-unsymmetrical pattern), 7.623-8.432 (m, 2H, Ar-H), 8. 344 (s, 1H, Sulphonamido N-H). 13C NMR Sc ppm: 170.1, 151.5, 137.5, 132.2, 130.1, 129.2, 129.1, 129.0, 128.9, 128.2, 128.1, 116.3, 116.2.
N-[(5-{[(4-aminophenyl)sulfonyl]amino}-1,3,4-thiadiazol-2-yl)sulfonyl]-4-nitro benzamide (9c). Yield 52.06 %, m.p. 2830C, Rf 0.44, LogP 1.263. Found: C 37.20, H 2.51, N 17.34, S 19.86 % C15H12N6O7S3 (484.49) requires C 37.19, H 2.50, N 17.35, S 19.86 %. m/z (GC-MS): 512 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3407-3126 (N-H), 3107-3010 (Ar C-H), 1708-1685 (amide C=O), 692-621 (C-S), 1515-1452 (Ar C-NO2), 1054-1081 (S=O), 1365, 1311, 1187 (sulphonamide), 3319 (sulphonamide N-H),
2360-2341 (heteroaromatic N-H), 1708 (heteroaromatic C=N), 972-846 (C-N), 2894 (C-H). Xmax (chloroform) (lge)/nm 273. 1H NMR (DMSO-d6) SH ppm: 2.42 f(s, 1H, Ar-SO2NH), 4.032 (s, 2H, Ar-NH2), 6.985-7.014 (s, 2H, Ar-unsymmetrical pattern), 6.6347.327 (m, 2H, Ar-H), 8.611 (s, 1H, Sulphonamido N-H). 13C NMR Sc ppm: 169.9, 151.9, 151.4, 140.2, 129.2, 128.6, 128.5, 128.4, 128.1, 121.3, 121.3, 116.4, 116.2.
N-[(5-{[(4-aminophenyl)sulfonyl]amino}-1,3,4-thiadiazol-2-yl)sulfonyl]-3-nitro benzamide (9d). Yield 46.41 %, m.p. 2740C, Rf 0.47, LogP 1.151. Found: C 37.19, H 2.51, N 17.36, S 19.87 %. C15H12N6O7S3 (484.49) requires C 37.19, H 2.50, N 17.35, S 19.86 %. m/z (GC-MS): 519 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3412-3197 (N-H), 3107-2925 (Ar C-H), 1712-1654 (amide C=O), 723-681 (C-S), 1487-1411 (Ar C-NO2), 1109-1020 (S=O), 1368, 1333, 1173 (sulphonamide), 3320 (sulphonamide N-h), 2378-2331 (heteroaromatic N-H), 1712 (heteroaromatic C=n), 2862 (C-H). Xmax (chloroform) (lge)/nm 265. 1H NMR (DMSO-d6) SH ppm: 1. 495 (s, 1H, Ar-SO2NH), 3. 503 (s, 2H, Ar-NH2), 6. 985-7.014 (s, 2H, Ar-unsymmetrical pattern), 6. 436-7. 273 (m, 2H, Ar-H), 7.932 (s, 1H, Sulphonamido N-H). 13C NMR Sc ppm: 169.8, 151.4, 148.3, 135.2, 133.4, 129.9, 129.6, 128.4, 128.1, 124.3, 122.3,
116.6, 116.3.
N-[(5-{[(4-amino-5-chloro-2-methylphenyl)sulfonyl]amino}-1,3,4-thiadiazol-2-yl) sulfonyl] benzamide (9e). Yield 68.62 %, m.p. 2630C, Rf 0.48, LogP 3.494. Found: C 39.40, H 2.88, N 14.34, S 19.70 %. C16H14N5O5S3Cl (487.96) requires C 39.38, H 2.89, N 14.35, S 19.71 %. m/z (G3C-MS): 495 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3467-3178 (N-H), 3102-2936 (Ar C-H), 17341686 (C^X 713-690 (C-S), 559-522 (C-Cl), 1134-1047(S=O), 1387, 1324, 1157 (Sulphonamide), 3370 (sulphonamide N-H), 2395 (heteroaromatic N-H), 1784 (heteroaromatic C=N), 2912 (C-H stretching). Xmax (chloroform) (lge)/nm 272. 1H NMR (DMSO-d6) SH ppm: 1.249%, 1H, Ar-SO2NH), 3.990 (s, 3H, Ar-CH3), 3.526 (s, 2H, Ar-NH2), 6.895-6.923 (s, 2H, Ar-unsymmetrical pattern), 7.236-7.581 (m, 6H, Ar-H), 7.604-7.735 (s, 2H, NH2), 8.367-8.392 (s, 1H, thiadiazole, C-H). 13C NMR Sc ppm: 170.1 147.6, 135.6, 134.3, 132.2, 130.6, 128.5, 128.5, 127i, 127.7, 127.3, 122.6, 117.9, 16.5.
N-[(5-{[(4-amino-5-chloro-2-methylphenyl)sulfonyl]amino}-1,3,4-thiadiazol-2-yl) sulfonyl]-4-chlorobenzamide (9f). Yield 53.29 %, m.p. 2680C, Rf 0.58, LogP 4.052. Found: C 39.80, H 2.51, N 14.43, S 18.42 %. C16H13N5O5S3Cl2 (522.41) requires C 39.79, H 2.51, N 13.41, S 18.41 %. m/z (GC-MS): 516 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3407-3126 (N-H), 3107-3010 (Ar C-H), 1708-1685 (amide C=O), 692-621 (C-S), 1054-1081 (S=O), 1365, 1311, 1187 (sulphonamide), 3319 (sulphonamide N-H), 23602341 (heteroaromatic N-H), 1708 (heteroaromatic C=N), 972-846 (C-N), 2894 (C-H), 592-532 (C-Cl). Xmax (chloroform) (lge)/nm 285. 1H NMR (DMSO-d6) 5H ppm: 2.071"s, 1H, Ar-SO2NH), 4.139 (s, 3H, Ar-CH3), 4. 652 (s, 2H, Ar-NH2), 6. 539-7.214 (s, 2H, Ar-unsymmetrical pattern), 7.623-7.851 (m, 6H, Ar-H), 7.046-7.973 (s, 2H, NH2), 8.534-8.743 (s, 1H, thiadiazole, C-H). 13C NMR Sc ppm: 169.8, 147.5, 137.7, 135.6, 132.2, 130.5, 129.0, 129.0, 128.8, 128.8,
127.7, 122.4, 117.8, 16.9.
N-[(5-{[ (4-amino-5-methoxy-2-nitrophenyl)sulfonyl]amino}-1,3,4-thiadiazol-2-yl) sufonyl]benzamide (9g). Yield 21.24 %, m.p. 2520C, Rf 0.47, LogP 4.006. Found: C 37.37, H 2.71, N 16.33, S 18.72 %. C16H13N6O8S3Cl (514.51) requires C 37.35, H 2.74, N 16.33, S 1870 %. m/z (GC-MS): 523 (100%, Base peak, M+). FT-IR (KBr) vmax cm-1: 3359-3338 (N-H), 3082-3045 (Ar C-H), 1691-1629 (amide C=O), 678-669 (C-S), 1035 (S=O), 1342, 1319, 1157 (sulphonamide), 3338 (sulphonamide N-H), 23812341 (heteroaromatic N-H), 1515-1452 (Ar C-NO2), 1785-1735 (heteroaromatic C=N), 559 (C-Cl), 960-827 (C-N), 2943 (C-H). Xmax (chloroform) (lge)/nm 288. 1H NMR (DMSO-d6) SH ppm: 2л46 (s, 1H, Ar-SO2NH), 3.927 (s, 3H, Ar-OCH3), 4.201 (s, 2H, Ar-NH2), 6.539-7.412 (s, 2H, Ar-unsymmetrical pattern), 7.0237.851 (m, 5H, Ar-H), 6.957-7.077 (s, 2H, NH2). 13C NMR Sc ppm:
170.0, 154.1, 140.8, 138.4, 134.3, 132.2, 128.6, 128.4, 127.6, 127.3,
125.3, 115.6, 112.8, 56.1. 5-{[(4-Aminophenyl)sulfonyl]amino}-1,3,4-thiadiazole-
2-sulfonamide (lúa). Yield 42.04 %, m.p. 194nC, Rf 0.60, LogP 0.840. Found: C 28.67, H 2.71, N 20.89, S 28.68 %. C8H9N5O4S3 (335.38) requires C 28.65, H 2.70, N 20.88, S 28.68 %. m/z (GC-MS): 319 (100%, Base peak, M+). FT-lR (KBr) vmax cm-1: 33983378 (N-H), 3120-2793 (Ar C-H), 706 (C-S), 1080-1026 (S=O), 1358, 1293, 1080 (sulphonamide), 3320 (sulphonamide N-h), 2378-2351 (heteroaromatic N-H), 1686 (heteroaromatic C=N). A.max (chloroform) (lg8)/nm 260. 1H NMR (DMSO-d6) SH ppm: 1.254 (s, 1H, Ar-SO2NH), 7.228-8.123 (m, 6H, Ar-H), 8.611 (s, 1H, sulphonamido-H). 13C NMR Sc ppm: 151.4, 129.9, 128.2, 128.1,
116.4, 116.3.
5-{[(4-Amino-5-methoxy-2-nitrophenyl)sulfonyl]amino}-1,3,4-thiadiazole-2-sulfonamide (lúb). Yield 34.58 %, m.p. 222nC, Rf 0.65, LogP -1.174. Found: C 26.36, H 2.47, N 20.49, S 23.43 %. C9H10N6O7S3 (410.41) requires C 26.34, H 2.46, N 20.48, S 23.44 o^. m/z (GC-MS): 396 (100%, Base peak, M+). FT-lR (KBr) vmax cm-1: 3358-3298 (N-H), 3127-2761 (Ar C-H), 726 (C-S), 1078-1014 (S=O), 1532-1476 (Ar C-NO2), 1358, 1293, 1080 (sulphonamide), 3320 (sulphonamide N-H), 2398-2371 (heteroaromatic N-h), 1677 (heteroaromatic C=N), 569-527 (C-Cl), 2873 (C-H). Xmax (chloroform) (lg8)/nm 278. 1H NMR (DMSO-d6) 5H ppm: 1. 425 (s, 1H, Ar-SO2NH), 3.108(s, 3H, OCH3), 3.527 (s, 2H, Ar-NH2), 7.3348.312 (m, 2H, Ar-H), 8.611 (s, 1H, sulphonamido-H). 13C NMR Sc ppm: 153.1, 140.2, 138.7, 125.3, 115.2, 112.9, 56.1.
Pharmacology
The anticonvulsant evaluations were undertaken using reported procedure.[28-3033] Male albino mice (CF-1 strain or swiss, 25-35 g) and rats (Sprague-Dawley or Wistar, 100-150 g) were used as experimental animals. The tested compounds were suspended in polyethylene glycol 400.
Anticonvulsant screening. lnitially all the compounds were administered orally in a volume of 100 mg/kg body weight of mice, 60 min prior to the electroshock. The electroshock induced in animal by passing a current of 45 mA for 0.2 sec duration through electroconvulsiometer (Techno lndia) using corneal electrodes. The following phases in sequence and time in each phase (in seconds) was noted.[39-41]
1. Tonic flexon : Contraction of muscle throughout the body
and forelimbs.
2. Tonic extension : Extension of extremities.
3. Clonus : Stage of relaxation after extension.
4. Stupor : Stage of unconsciousness before recovery
(Generally more than one minute).
While in scPTZ test, all the drugs were administered orally 30 min prior to the administration of pentylenetetrazole (6 mg/kg) by subcutaneous injection. The animals were observed for 1 and 3 hour by placing in a separate cage. The duration of seizures (tonic-clonic convulsions) were recorded.[42] Activity was established using the MES and scPTZ test and these data are presented in Table 5.
Diuretic activity. The Lipschitz method was employed for the assessment of diuretic activity. [43] The rats were deprived of food for 18 h and tap water ad libitum was allowed. The urine was collected quantitatively for a total period of four hours and the urine collected of initially of 20 min. was discarded.[44] The effect of the compounds was compared with standard and the control group. The ratio (T/C, T/S) of urine volume of treated and control group was determined. [45-46] The results are shown in Table 6.
Acknowledgements. The authors are highly thankful to Director, Sophisticated Analytical Instrument Facility
(SAIF), Chandigarh, Pune University, Pune for providing the necessary spectral analysis, and Head, Department of Pharmacy, SGVU, Jaipur for providing necessary laboratory facilities to carry out this research work.
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Received 31.01.2013 Accepted 23.02.2013
