Synthesis and in vivo diuretic activity of some new benzothiazole sulfonamides containing quinoxaline ring system

Article abstract of DOI:10.3109/14756366.2015.1128425

A series of new 6-substituted-N-[3-{2-(substituted phenyl)-ethenyl} quinoxaline-2(1H)-ylidene]-1,3-benzothiazole-2-amine (4a–f) were designed and synthesized by condensing 2-amino-benzothiazole-6-sulfonic acid amide (1) with chalcones of quinoxaline-2-one (3a–f) in a hope to obtain promising and a new class of diuretic agents. Structures of all the newly synthesized compounds were characterized by spectral data and elemental analysis. The pharmacological studies in experimental rats indicates that compound 4c possesses excellent in vivo diuretic activity of 1.13 and appears to be a better diuretic agent than the reference drugs, acetazolamide (1.0) and urea (0.88). Insight of the binding mode of the synthesized compounds (ligand) into the binding sites of carbonic anhydrase enzyme (PDF code: 4KUV) was provided by docking studies, performed with the help of Maestro 9.0 docking software. Further pharmacokinetic and toxicological studies are needed to confirm the safety of compound 4c which emerged as a lead diuretic compound.

Full text of DOI:10.3109/14756366.2015.1128425

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and in vivo diuretic activity of some
new benzothiazole sulfonamides containing
quinoxaline ring system
Asif Husain, Diwakar Madhesia, Mohd Rashid, Aftab Ahmad & Shah Alam
Khan
To cite this article: Asif Husain, Diwakar Madhesia, Mohd Rashid, Aftab Ahmad & Shah Alam
Khan (2016): Synthesis and in vivo diuretic activity of some new benzothiazole sulfonamides
containing quinoxaline ring system, Journal of Enzyme Inhibition and Medicinal Chemistry,
DOI: 10.3109/14756366.2015.1128425
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1128425
Published online: 07 Jan 2016.
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J Enzyme Inhib Med Chem, Early Online: 1–8
2016 Taylor & Francis. DOI: 10.3109/14756366.2015.1128425
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SHORT COMMUNICATION
Synthesis and in vivo diuretic activity of some new benzothiazole
sulfonamides containing quinoxaline ring system
Asif Husain1, Diwakar Madhesia1, Mohd Rashid1,2, Aftab Ahmad3, and Shah Alam Khan4
2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi, India, Department of
Pharmaceutical Chemistry and Pharmacognosy, College of Pharmacy and Dentistry, Buraydah Private Colleges, Al-Qassim, Kingdom of Saudi
Arabia, 3Health Information Technology Department, Jeddah Community College, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia, and
4Department of Pharmacy, Oman Medical College, Muscat, Sultanate of Oman
Abstract
Keywords
A series of new 6-substituted-N-[3-{2-(substituted phenyl)-ethenyl} quinoxaline-2(1H)-ylidene]-
1,3-benzothiazole-2-amine (4a–f) were designed and synthesized by condensing 2-amino-
benzothiazole-6-sulfonic acid amide (1) with chalcones of quinoxaline-2-one (3a–f) in a hope to
obtain promising and a new class of diuretic agents. Structures of all the newly synthesized
compounds were characterized by spectral data and elemental analysis. The pharmacological
studies in experimental rats indicates that compound 4c possesses excellent in vivo diuretic
activity of 1.13 and appears to be a better diuretic agent than the reference drugs,
acetazolamide (1.0) and urea (0.88). Insight of the binding mode of the synthesized compounds
(ligand) into the binding sites of carbonic anhydrase enzyme (PDF code: 4KUV) was provided by
docking studies, performed with the help of Maestro 9.0 docking software. Further
pharmacokinetic and toxicological studies are needed to confirm the safety of compound 4c
which emerged as a lead diuretic compound.
Acetazolamide, benzothiazole, carbonic
anhydrase, Lipschitz value
History
Received 13 August 2015
Revised 24 November 2015
Accepted 24 November 2015
Published online 8 January 2016
Introduction
glaucoma, hyperkalemia, bromide intoxication, anginal syn-
drome, epilepsy, migraine and premenstrual depression. One of
the common adverse effects of diuretic therapy is ‘‘hyperurice-
mia’’ which is due to diminished extracellular and high degree of
urate re-absorption4.
Diuretics, occasionally referred to as water pills, increase the
urine output. A diuretic primarily reduces the volume of
extracellular fluid, enhances the urinary excretion of sodium
chloride and secondarily increases the volume of urine excreted
by kidneys. The drugs of this class are frequently prescribed in
various heart and kidney related disorders. Diuretics are com-
monly indicated in the treatment of retention of fluid (edema),
hypertension, congestive heart failure, liver cirrhosis, epilepsy,
glaucoma and some kidney diseases. These drugs produce
diuresis by acting on distinct sites on nephron, however, the
primary mechanism involves inhibition of sodium ions
re-absorption in the renal tubules of the kidney1. The various
classes of therapeutic agents belonging to diuretics include
xanthine derivatives, mercurial agents, sugars or sugar deriva-
tives, thiazides, phenoxyacetic acids, aminomethylphenols, aro-
matic sulfonamides, steroids, pteridines and pyrazines. The most
common therapeutic class of diuretic includes loop diuretics (high
ceiling diuretic), thiazides, carbonic anhydrase (CA) inhibitors,
potassium-sparing diuretics and osmotic diuretics. Recently,
diuretics have been proposed to be safe and effective therapy in
the management of hypertension2. In addition, outcome of many
studies suggested them to be the first line therapy in management
of hypertension in elderly3. Some diuretics are also used in
CA or carbonate dehydratase is a family of enzymes that
occurs in various isoforms in kidney. CA derives its name due to
removal of a water molecule from carbonic acid. The mammalian
CA enzyme belongs to the alpha class and it catalyzes the rapid
interconversion of carbon dioxide and water to bicarbonate and
protons through a reversible reaction, which is otherwise slow in
its absence. In animals, the CA is primarily responsible for
maintaining acid base balance in blood and other body tissues,
and to help transport carbon dioxide out of tissues. Acetazolamide
(compound I) is a thiadiazole derivative which was introduced
into the clinical practice after World War II as a safer non
mercurial diuretic agent. It is a classical example of CA inhibitor
(CAI) which upon its administration alkalinizes and increases the
production of urine. Because of its alkalinizing property, it is
sometime administered along with methotrexate to speed up its
excretion in urine and thus prevents methotrexate induced
nephrotoxicity. Though, it is clinically used to lower blood
pressure, intraocular pressure, intracranial pressure, edema and
epilepsy, however, its clinical usefulness is limited. The inhibition
of CA by acetazolamide leads to accumulation of carbonic acid
and reduction in blood pH accompanied with inhibition of re-
absorption of bicarbonate, sodium and chloride ions from
proximal tubule of kidney. This led to increased excretion of
these ions along with water, leading to diuresis. It has been
Address for correspondence: Shah Alam Khan, Department of Pharmacy,
Oman Medical College, Muscat, Sultanate of Oman. Tel: +968
24504608, ext. 173. Mob: +968 97245361. E-mail: shahalamkhan@
yahoo.com

2
A. Husain et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
H
C
H
N
Figure 1. Structures of typical carbonic
anhydrase inhibitors (compounds I–VIII).
2
O
O
O
O
CH
H C
Cl
H N
3
3
O
S
2
S
S
H N
S
NH
2
NH
O
HN
SO NH
O
2
2
N
N
Cl
N
O
(I)
(II)
(III)
Cl
Cl
H
N
H C
H C
3
3
Cl
H
H
CH
N
3
N
N
SO NH
SO NH
2 2
2
2
N
OH
O
SO NH
O
2
2
O
(IV)
(VI)
(V)
NH
H
N
Cl
SO NH
O
O
HOOC
HOOC
SO NH
2 2
2
2
(VII)
(VIII)
reported that sulfonamide derivatives induce diuresis by inhibiting (Perkin–Elmer and LABINDIA, Applied Biosystem) model no.
CA II in cytosol as well as CA IV, XII and XIV isozymes bound API 3000 and FT/IR (Jasco, Japan), model no.410, respectively.
to membrane. Few of the benzothiadiazine derivatives such as The peaks in mass and IR spectra are presented as m/z and cm ꢀ 1
.
chlorothiazide (II), quinethazone, a quinazolinone derivative Elemental analysis (C, H and N) were performed on a Perkin–
(III), metolazone (IV), chlorthalidone, a pthalimindine diuretic Elmer 240 analyzer (Waltham, MA) and were found within the
(V), indapamide (VI), furosemide (VII) and bumetanide (VIII) acceptable range of 0.4% for each analyzed element.
act as CAIs with varying degree of therapeutic diuretic
efficiencies and different mechanisms (Figure 1). This could be
Synthesis
due to the presence of an unsubstituted primary sulfonamide
moiety in their structures which is responsible for effective zinc
binding in CAI activity5,6
Synthesis of 2-amino-benzothiazole-6-sulfonic acid amide (1)
.
Pre-cooled (5 ꢁC) glacial acetic acid (150 ml), KSCN (0.06 mol)
and p-amino sulfanilamide (0.06 mol) were added to form a
mixture7. This mixture was kept in ice bath and stirred
mechanically with gradual addition of bromine (0.02 mol) in
10 ml glacial acetic acid from a dropping funnel at such a rate that
temperature does not rise45 ꢁC. A continuous stirring was done
for an additional 3 h at temperature 0–10 ꢁC in an ice bath. The
separated hydrochloride salt was filtered, washed with acetic acid,
dried and then dissolved in hot water. The aqueous solution was
neutralized with aq. NH3 solution (25%) followed by filtration,
and washing with water. The pure compound (1) was obtained by
re-crystallization in benzene.
Diuretics constitute an important part of cardiovascular
therapy especially in controlling blood pressure in heterogeneous
population. These drugs are generally inexpensive, effective and
are well tolerated by the patients of all age groups at low doses.
However, the currently available diuretic therapy is associated
with many unpleasant side effects which warrant the design and
synthesis of a potent diuretic agent. Thereafter, we designed and
synthesized some new benzothiazole sulfonamides containing
quinoxaline ring and evaluated them for anticipated in vivo
diuretic activity.
Radish brown; m.p. 230 ꢁC; Yield 80%. Anal. Calcd. for
C7H7N3O2S2: C, 36.60; H, 3.18; N, 18.43. Found: C, 36.63; H,
4.61; N, 18.91. FTIR (KBr) ꢁmax (cm ꢀ 1): 3376 (N–Hstr), 3266
and 3244 (N–Hstr, –SO2NH2), 3015 (C–Hstr, Aromatic), 1694
Materials and methods
Chemicals and instrumentation
Chemicals (reagents and solvents) of high purity were purchased
from E. Merck Ltd (Mumbai, India), SD Fine Chemicals
(Mumbai, India)E. Merck Ltd (Mumbai, India), SD Fine
Chemicals (Mumbai, India) and used as such in all the experi-
ments. One end open capillary tubes were used to measure the
melting points on a liquid paraffin bath and are uncorrected.
Silica gel G-coated TLC plates (Merck, Darmstadt, Germany)
with the solvent systems (toluene: ethyl acetate: formic acid
(5:4:1, v/v/v) or benzene: acetone (9:1, v/v)] were used to check
the purity and completion of reactions. Iodine vapors or UV light
were used to locate the spots on TLC chromatogram. Bruker
spectrospin DPX-400 MHz (Rheinstetten, Germany) was used to
record the 1H NMR and 13C NMR spectra of compounds in
DMSO-d6/CDCl3 using tetramethylsilane (TMS) as an internal
reference. The chemical shift (ꢀ) values are reported in parts per
million (ppm) while coupling constants (J) in Hz. The exchange-
able protons of (OH and NH) were ascertained by the D2O
shaking test. Mass and IR spectra were recorded on LCMS/MS
1
(C¼Nstr); H NMR (400 MHz, DMSO-d6, TMS): ꢀ/ppm: 5.81(s,
2H, –NH2), 6.58 (d, 1H), 7.45 (d, 1H), 6.89 (s, 1H); 13C-NMR
(DMSO-d6): ꢀ/ppm: 154.63 (C¼N), 141.74, 138.49, 129.13,
128.17, 127.53, 125.71(Ar-C); ESI-MS (m/z): 230 (M+).
Synthesis of 3-methylquinoxaline-2(1H)-one (2)
3-Methylquinoxaline-2(1H)-one was synthesized from pyruvic acid
and o-phenylenediamine by the following procedure8: equimolar
quantities of pyruvic acid (0.01) and o-phenylenediamine (0.01)
were refluxed in ethanol for 3 h. Upon completion of the reaction,
the contents were cooled down to separate out the solid. The
compound was filtered out and re-crystallized with ethanol.
Dark gray; m.p. 206 ꢁC; Yield 80%. Anal. Calcd. for
C9H8N2O: C, 67.49; H, 5.03; N, 17.49. Found: C, 67.51; H,
5.21; N, 17.45. FTIR (KBr) ꢁmax (cm ꢀ 1) 3322 (N–Hstr), 3051 (C–
Hstr, Aromatic), 2955 (C–Hstr, Aliphatic), 1694 (C¼Ostr); 1H

DOI: 10.3109/14756366.2015.1128425
Sulfonamide endowed quinoxaline and benzothiazole rings as diuretics
3
NMR (400 MHz, CDCl3, TMS): ꢀ/ppm: 2.5 (s, 3H, CH3), 6.70– 6.93 (d, 1H, ¼CH-Ar), 7.13–8.42 (m, 11H, Ar-H), 8.98 (s, 1H,
7.81 (m, 4H, Ar-H), 8.39 (s, 1H, NH); 13C-NMR (DMSO-d6): NH); 13C-NMR (DMSO-d6): ꢀ/ppm: 170.53, 166.45, 157.28
ꢀ/ppm:
128.52, 124.45, 123.72, 121.17 (Ar-C), 23.42(CH3); ESI-MS 131.57, 129.03, 128.56, 127.63, 125.53, 124.74, 123.01, 122.87,
167.51(C¼O),
157.63(C¼N),
135.27,
133.21, (C¼N), 138.47, 136.49 (CH¼CH), 136.78, 135.21, 134.92,
(m/z): 168 (M+).
121.36, 120.45, 119.31, 116.82, 113.69, 112.56, 110.64 (Ar-C),
30.75 (CH3, OCH3); ESI-MS (m/z): 489 (M+).
Synthesis of 3-[2-substituted phenylethenyl]quinoxalin-2(1H)-
one (3a–f)
2-{3-[2–(3,4-Dimethoxyphenyl)-ethenyl]-1H-quinoxalin-2-ylide-
neamino}-1,3 benzothiazole-6-sulfonic acid amide (4d)
A mixture of compound 2 (0.01 mol) and different aromatic
aldehydes (0.01 mol) were fused at 165 ꢁC for 3 h in presence of Light Blue; m.p. 210–211 ꢁC; Yield 82%. Anal. Calcd. for
piperidine (0.5 ml). The obtained product was re-crystallized C25H21N5O4S2: C, 57.79; H, 4.07; N, 13.48. Found: C, 58.15; H,
using ethanol to obtain compound 3a–f.
4.13; N, 13.45. FTIR (KBr) ꢁmax (cm ꢀ 1): 3330 (N–Hstr), 3266
and 3244 (N–Hstr, –SO2NH2), 3058 (C–Hstr, Aromatic), 2850 (C–
General procedure for the synthesis of 6-substituted-N-[3-{2–(4- Hstr, –O–CH3), 1632 (C¼Nstr), 1097 (C–Ostr, Ar–O–CH3), 628
1
substitutedphenyl)-ethenyl} quinoxaline-2(1H)-ylidene]
-1,3-benzothiazole-2-amine (4a–f)
(C–S–Cstr); H NMR (400 MHz, CDCl3, TMS): ꢀ/ppm: 3.73 (s,
6H, –OCH3), 7.27 (d,1H, ¼CH–C¼),7.67 (d, 1H, ¼CH-Ar), 7.13–
8.42 (m, 10H, Ar-H), 8.88 (s, 1H, NH); 13C-NMR (DMSO-d6):
ꢀ/ppm: 169.34, 165.21, 155.46 (C¼N), 138.56, 133.27 (CH¼CH),
136.71, 136.13, 135.05, 132.45, 130.29, 127.15, 126.89, 125.32,
124.63, 123.69, 121.81, 121.17, 120.45, 118.58, 117.37, 115.61,
111.89, 111.13 (Ar-C), 34.19, 34.15 (CH3, OCH3); ESI-MS (m/z):
519 (M+).
6-Substituted-N-[3-{2–(4-substitutedphenyl)-ethenyl} quinoxa-
line-2(1H)-ylidene]-1,3 benzothia zole-2-amine (4a–f) were
synthesized from compounds 1 and 3a–f. Compounds 1 and
3a–f were refluxed in an equimolar ratio in ethanol for 13–16 h.
The reaction mixture was heated to obtain a concentrated mixture
which upon cooling afforded a solid product which was filtered
and purified by recrystallization in ethanol.
2-{3-[2–(4-Nitrophenyl)-ethenyl]-1H-quinoxalin-2-ylidenea-
mino}-1,3 benzothiazole-6-sulfonic acid amide (4e)
2-{3-[2-Phenylethenyl]-1H-quinoxalin-2-ylideneamino}-1,3
benzothiazole-6-sulfonic acid amide (4a)
Yellowish brown; m.p. 238–240 ꢁC; Yield 75%. Anal. Calcd. for
C23H16N6O4S2: C, 54.75; H, 3.20; N, 16.66. Found: C, 54.81; H,
3.27; N, 16.71. FTIR (KBr) ꢁmax (cm ꢀ 1): 3300 (N–Hstr), 3360
and 3270 (N–Hstr, –SO2NH2), 3058 (C–Hstr, Aromatic), 1629
(C¼Nstr), 1570 (N¼Ostr., Ar-NO2), 1338 (C–Nstr), 857 (C–Hdef, p-
disubst. Bz), 628 (C-S-Cstr); 1H NMR (400 MHz, DMSO-d6,
TMS): ꢀ/ppm: 6.9 (d, 1H, ¼CH-C¼),7.2 (d, 1H, ¼CH-Ar),
7.13–8.42 (m, 11H, Ar-H), 8.8 (s, 1H, NH); 13C-NMR
(DMSO-d6): ꢀ/ppm: 170.85, 167.34, 156.89 (C¼N), 139.72,
137.19 (CH¼CH), 138.56, 137.45, 134.19, 133.59, 131.78,
129.36, 127.81, 126.67, 125.39, 120.93, 120.15, 119.27,
118.34, 117.13, 116.58, 115.25, 113.42, 112.74 (Ar-C); ESI-MS
(m/z): 504 (M+).
Brown solid; m.p. 230–231 ꢁC; Yield 62%. Anal. Calcd. for
C23H17N5O2S2: C, 60.11; H, 3.73; N, 15.24. Found: C, 60.15; H,
3.75; N, 16.45. FTIR (KBr) ꢁmax (cm ꢀ 1): 3330 (N–Hstr), 3266
and 3244 (N–Hstr, –SO2NH2), 1629 (C¼Nstr), 1338 (C–Nstr), 711
1
(C–Hdef, monosubst. Bz), 628 (C–S–C str.); H NMR (400 MHz,
CDCl3, TMS): ꢀ/ppm: 6.9 (d,1H, ¼CH–C¼), 7.2 (d, 1H, ¼CH-
Ar), 6.5–8.79 (m, 12H, Ar-H), 8.8 (s, 1H, NH); 13C-NMR
(DMSO-d6): ꢀ/ppm: 164.15, 158.27, 154.63 (C¼N), 140.32,
139.73 (CH¼CH), 135.91, 134.28, 132.64, 131.46, 130.83,
129.25, 129.13, 128.37, 126.41, 124.16, 123.14, 122.92, 120.34,
118.72, 116.85, 115.70, 114.79, 110.51 (Ar-C); ESI-MS (m/z):
459 (M+).
2-{3-[2–(4-Dimethylaminophenyl)-ethenyl]-1H-quinoxalin-2-yli-
deneamino}-1,3 benzothiazole -6-sulfonic acid amide (4f)
2-{3-[2–(4-Chlorophenyl)-ethenyl]-1H-quinoxalin-2-ylidenea-
mino}-benzothiazole-6-sulfonic acid amide (4b)
Brownish gray; m.p. 228–230 ꢁC; Yield 72%. Anal. Calcd. for
C25H22N6O2S2: C, 59.74; H, 4.41; N, 16.72. Found: C, 59.78; H,
3.91; N, 16.75. FTIR (KBr) ꢁmax (cm ꢀ 1): 3395 (N–Hstr), 3366
and 3244 (N–Hstr, –SO2NH2), 3150 (C–Hstr, N–Me2), 3058
Blackish brown; m.p. 235–237 ꢁC; Yield 67%. Anal. Calcd. for
C23H16ClN5O2S2: C, 55.92; H, 3.26; N, 14.18. Found: C, 55.95;
H, 3.75; N, 14.35. FTIR (KBr) ꢁmax (cm ꢀ 1): 3376 (N–Hstr), 3266
& 3244 (N–Hstr, –SO2NH2), 1629 (C¼Nstr), 1338 (C–Nstr), 813
(C–Hdef, p-disubst. Bz), 748 (Ar C–Clstr), 628 (C–S–Cstr); 1H
NMR (400 MHz, CDCl3, TMS): ꢀ/ppm: 6.9 (d, 1H, ¼CH–C¼),
7.2 (d, 1H, ¼CH-Ar), 6.5–8.9 (m, 11H, Ar-H), 8.8 (s, 1H, NH);
13C-NMR (DMSO-d6): ꢀ/ppm: 165.76, 156.23, 155.74 (C¼N),
141.56, 138.27 (CH¼CH), 134.87, 133.21, 132.17, 131.65,
131.17, 130.59, 129.38, 128.49, 125.53, 124.63, 123.01, 121.36,
120.45, 119.31, 116.82, 113.69, 112.56, 110.64 (Ar-C); ESI-MS
(m/z): 493 (M+).
(C–Hstr, Aromatic), 1629 (C¼Nstr), 1338 (C–Nstr), 837 (C–Hdef
,
p-disubst. Bz), 687 (C–S–Cstr); 1H NMR (400 MHz, CDCl3,
TMS): ꢀ/ppm: 3.07 (s, 6H, NMe2), 6.73 (d, 1H, ¼CH-Ar), 7.41
(d,1H, ¼CH–C¼), 8.7 (s, 1H, NH), 7.44–8.64 (m, 11H, Ar-H);
13C-NMR (DMSO-d6): ꢀ/ppm: 169.85, 165.71, 155.35 (C¼N),
138.83, 138.13 (CH¼CH), 139.01, 137.15, 135.26, 132.75,
132.13, 130.72, 128.54, 126.35, 125.71, 124.36, 123.17, 120.68,
119.92, 115.23, 115.12, 112.63, 111.28, 110.81 (Ar-C), 27.81,
27.76 (CH3); ESI-MS (m/z): 502 (M+).
2-{3-[2–(4-Methoxyphenyl)-ethenyl]-1H-quinoxalin-2-ylidenea-
mino}-1,3 benzothiazole-6-sulfonic acid amide (4c)
Pharmacology
Diuretic activity
Greenish dark gray; m.p. 234–237 ꢁC; Yield 60%. Anal. Calcd. for
C24H19N5O3S2: C, 58.88; H, 3.91; N, 14.31. Found: C, 58.91; H, The diuretic activity was performed by adopting the Lipschitz
3.87; N, 14.56. FTIR (KBr) ꢁmax (cm ꢀ 1): 3376 (N–Hstr), 3266 method9. An approval of experimental protocol was obtained from
and 3244 (N–Hstr, –SO2NH2), 3058 (C–Hstr, Aromatic), 2853 (C– the Institutional Animal Ethical Committee (173 CPCSEA 28
Hstr, –O–CH3), 1628 (C¼Nstr), 1097 (C–Ostr, Ar–O–CH3), 813 January 2010 – Proposal No. 647), Central Animal House
(C–Hdef, p-disubst. Bz), 628 (C–S–Cstr); 1H NMR (400 MHz, Facility, Jamia Hamdard University, New Delhi, India. Fifty-
CDCl3, TMS): ꢀ/ppm: 3.74 (s, 3H, –OCH3), 6.26 (d, ¼CH–C¼), four healthy adult Wistar albino rats (180–200 g) were selected

4
A. Husain et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
and divided in to nine groups having six animals in each (n¼6) were made in order to establish whether the scale is to be set at
group. The rats were acclimatized in standard environmental 100 ppm or higher13.
conditions for one week. The standard diet and water ad libitum
were given to all rats during the protocol of the study which was Molecular docking studies
withdrawn from all animals 15 h prior to the experiment. The rats
Molecular docking studies help in understanding and predicting
were kept at room temperature 25 5 ꢁC throughout the experi-
the enzyme–ligand interactions in vivo. Docking studies of the
ment. The total excreted urine (from 0 to 5 h) of rats kept in
newly synthesized compounds against CA enzyme were carried
metabolic cage was collected to measure the diuretic activity of
out to rationalize the results obtained in this study. The newly
the compounds. In the beginning of each experiment, the animal’s
synthesized compounds were evaluated through molecular
cages, funnel and measuring cylinder were pre-coated with liquid
modeling and docking studies. The molecular docking studies
paraffin to facilitate the better collection with a minimum loss of
of all compounds were performed on 3D structure of CA enzyme
urine. Each animal was placed in a separate metabolic cage which
with the help of Maestro 9.0 program (Schrodinger Inc., NY) by
was fitted with a wire mesh at the bottom and a funnel to collect
64 bit operating system under window 7 with an HCl computer
the urine. Stainless steel sieves were placed in the funnel to retain
[Intel (R) Core (TM) i5–2400 CPU at 3.10 GHz, 8 GB memory].
and separate feces from urine and allow the urine alone to pass
The 3D structure of the enzyme for this study was downloaded
through the sieves. The urine was collected for a total duration of
from the Protein Data Bank (code; PDB ID: 4KUV). This enzyme
5 h. The test compounds were administered orally (p.o.) at a dose
is known to have 96% similarity with the human cell enzyme and
of 45 mg/kg body weight in 5 ml of 0.9% NaCl solution. Control
it is also reported that all active site residues in the vicinity of
group received 5 ml of 0.9% NaCl solution only. The activity of
cofactor of this enzyme have exactly the same counterparts14. The
test compounds was compared with standard diuretic acetazola-
enzyme structure was refined and checked for any missing atoms,
mide (45 mg/kg body weight in 5 ml of 0.9% NaCl solution).
bonds and contacts. All residues excluding ligand molecules and
Diuretic activity was calculated by using following formula9–13
.
water molecules were deleted manually. The ligand molecules
were build using the builder molecule and then their energy was
minimized. The grid box tools were used to generate an active
site. The conformer having the lowest energy was chosen and its
energy was minimized. The best binding modes of compound 4c
in the active site of 4KUV is shown in Figures 2 and 3.
Total urine output
Urinary excretion ¼
ꢂ 100
Total liquid intake
Urinary excretion of treated group
Urinary excretion of control group
Diuretic action ¼
ꢂ 100
ꢂ 100
Results and discussion
Diuretic action of treated group
The synthesis of the quinoxaline derivatized benzothiazole
sulfonamides were started from chalcones (3a–f) and 6-sulfony-
lamino-1,3-benzothiazole-2-amine (1). 6-sulfonylamino-1,3-ben-
zothiazole-2-amine was synthesized by the reaction of KSCN
(0.06 mol) and p-sulfanilamide (0.06 mol) in glacial acetic acid
(150 ml) pre-cooled to 5 ꢁC using bromine as a catalyst. The series
(4a–f) was synthesized in a single step from different chalcones
(3a–f) and 6-sulfonylamino-1,3-benzothiazole-2-amine (1) by
refluxing them in absolute alcohol for 12–13 h. The 3-[2-
(substituted phenyl)-ethenyl] quinoxalin-2(1H)-one (3a–f) were
prepared from 3-methylquinoxaline-2(1H)-one (2) and substituted
aryl amines. 3-methylquinoxaline-2(1H)-one was synthesized by
refluxing o-phenylenediamine with pyruvic acid in the presence
of ethanol as outlined in synthetic scheme (Figure 4) and space fill
model of compounds (4a–f) in Figure 5.
Diuretic activity ¼
Diuretic action of control group
Effect on electrolyte concentration
The concentration of Na+ and K+ ions was measured by calibra-
tion method using flame photometry. The primary standard
solutions of sodium and potassium of 1000 ppm concentration by
weight was prepared for flame photometric analysis.
These solutions were diluted before use to the proper
concentrations in the following manner. The collected urine
(1–3 ml) was diluted with distilled water up to 100 ml and the
resultant diluted solution was used directly for the measurement
of both Na+ and K+ ions. The calibration charts used for these
determinations depend entirely concentration of urine. Trial runs
Figure 2. Binding mode of compound 4c into
the binding sites of carbonic anhydrase
enzyme (PDB code: 4KUV) showing hydro-
gen bond (dotted lines) with ASN62 and pi–
pi interaction with HIE64 and HIS94.

DOI: 10.3109/14756366.2015.1128425
Sulfonamide endowed quinoxaline and benzothiazole rings as diuretics
5
In the IR spectral analysis, compounds, showed absorp- bonds through its amino and carbonyl group. ASN62 residue is
tion bands in the range of 3390–3318 cm ꢀ 1 due to trans- said to be the same residue at which the natural inhibitor is known
ene C–H stretching bonds, 2600–2550 cm ꢀ 1 due to S–H, to bind14.
1600–1550 cm ꢀ 1 due to aromatic C–H groups, 1540–
A hydrogen bond interaction was observed between the
1500 cm ꢀ 1 due to imine C¼N stretching and 800–720 cm ꢀ 1 active hydrogen of amino group (–NH2) of thiazole ring of
1
due to aromatic C–H deformation. The H NMR spectra further compound 4c which acts as a hydrogen bond donor with the
confirmed the chemical structure and the peaks found in the range carboxyl group (C¼O) of the side chain residue of ASN62
˚
of 6.5–8.00 ppm were due to presence of different aromatic (2.12 A) that acts as hydrogen bond acceptor is illustrated in
protons, signals at 8–10 ppm was characteristic of imino proton Figure 2. The sulfonamide group of compound 4c appears to
and the peaks in the region of 10–13.1 ppm was ascribed to play an important role in formation of strong hydrogen bond
sulfonamide-proton. The peaks for the methylene protons were because the lone pair electrons on nitrogen atom of the amide
observed in the range of 3.8–4.9 ppm. Two doublets in the range group delocalized into the carbonyl group (C¼O) of com-
of 7.5–8 ppm having coupling constant of 15.6 Hz were very pound. Pi–Pi interaction of the compound with the target also
characteristic of a trans-proton of the ethylene group. seems to play an important role in inhibitory activity, as
The results of docking studies of compound 4c into the active phenyl ring of benzothiazole nucleus appears to be properly
˚
sites of enzyme showed hydrogen bond, ꢂ interaction and oriented toward the more lipophilic area of HIE64 (4.30 A) and
hydrophobic molecular interactions which are believed to be formed CH–ꢂ interaction. Another Pi–Pi interaction as
˚
responsible for the observed affinity of the tested molecule. quinoxaline ring properly oriented toward HIS94 (5.43 A and
Interestingly, compound 4c does not have a zwitter ion, however, 4.78) and formed CH–ꢂ interaction is illustrated in Lig plot
it attaches to the ASN62 residue of enzyme by making hydrogen (Figure 3). The other hydrophobic interactions formed between
Figure 3. Lig plot of compound 4c showing
interaction into the binding sites of carbonic
anhydrase enzyme (PDB code: 4KUV),
hydrogen bond (pink dotted line on top left)
˚
with ASN62 (2.12 A) and pi–pi interaction
(green solid line shown as three arrows on
˚
right) with HIE64 (4.30 A) and HIS94
˚
(5.43 A and 4.78).
H
C
Figure 4. Synthetic protocol of compounds
(4a–f).
R'
R
O
H
N
H
N
O
NH
NH
O
O
2
O
ethanol
OH
+
C C
H C
piperidine
N
CH
3
N
2
3
R'
R
o-Phenylenediamine Pyruvic acid 3-Methyl quinoxaline-2-one (2)
Chalcones(3a-f)
NH
N
S
ethanol
Br
2
2
NH
+
KSCN
2
H NO S
H NO S
2
2
2
2
R'
R
p-Amino sulfanilamide
2-Amino-benzothiazole-6-sulfonic
acidamide(1)
N
N
S
H NO S
2
2
R = H, Cl, OCH , NO , N(CH )
3 2
HN
N
3
2
R' = H, OCH
3
(4a-f)

6
A. Husain et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Figure 5. Space-filling models of 4a–f series compounds.
NO
−
H+
E-Zn2+ OH2 + HCO3 (2)
(1)
2
+
+
CO2
H2O
HCO3
H O
2
−
−
E-Zn2+ OH−
E-Zn2+ HCO3
N
S
E-Zn2+ OH2
E-Zn2+ OH− + H+
−OH
(3)
Tetrahedral adduct
(Sulfonamide)
N
O
S
−
−OH
NH
O
HN
N
O
+2
His119
O
Zn
+2
Zn
+2
Zn
94His
His119
His119
His94
His94
His96
His96
His96
(a)
(b)
+2
+2
H O
+
E-Zn
OH + I
E-Zn
I
2
2
(Substitution reaction)
- BH+
B
Figure 7. Proposed mechanism of inhabitation of carbonic anhydrase: by
binding to the ZN2+ ion of the enzyme by substituting non-protein zinc
ligand, t generate a tetrahedral adduct.
O
H
O
OH2
+2
+ H2O
O−
+2
Zn
Zn
−
His119
His119
- HCO3
and His10 were involved in the proton-shuttling processes
between the active site and the environment. Further, it was
also observed that amino-acid residues 92 and 131 are found to be
involved in the binding of quite a few sulfonamide/sulfamate
inhibitors. The zinc-bound water is also involved in formation of
hydrogen bond interactions with the –OH group of Thr199 amino
acid, which in turn form a bridge to the carboxylate moiety of
His94
His94
His96
His96
(d)
(c)
Figure 6. Zinc binding in carbonic anhydrase inhabitation activity.
the phenyl ring and aliphatic portion with the remains of Glu106. These hydrogen ion interactions proved to be quite
amino acids residues are shown in Figures 2 and 3. Compound helpful in enhancing the nucleophilicity of the zinc-bound water
molecule, and to orient the substrate (CO2) in a favorable position
4c showed a glide score value of ꢀ5.94 which clearly indicates
for a nucleophilic attack. The enzyme with hydroxide bound to
Zn2+ (a) is an active form of the enzyme which is a basic in
nature. This strong nucleophile attacks carbon dioxide (CO2)
molecule, bound in a hydrophobic pocket in its neighborhood.
that the synthesized molecule interacts in a befitting manner
with the enzyme.
Heterocyclic sulfonamides act by inhibiting enzyme CAs. It
has been known that Zn2+ metal ions are essential for catalysis in
all a-CA enzymes catalyzed reactions investigated till date. The This elusive substrate-binding site contains residues Val121,
˚
ion is shown to be situated at the base of a 15-A deep active-site
cleft by X-ray crystallographic data and is synchronized by three
Val143 and Leu198 in the case of the human isozyme CA (II) (b)
which leads to the formation of bicarbonate coordinated to
histidine residues viz His94, His96 and His119 and a hydroxide Zn2+ (c). The water molecules then displaces bicarbonate ion
which is liberated into the solution, forming a catalytically
inactive acid form of the enzyme, with water coordinated to
ion/water molecule. It was also evidenced that the cluster of
histidine residues comprising of His64, His4, His3, His17, His15

DOI: 10.3109/14756366.2015.1128425
Sulfonamide endowed quinoxaline and benzothiazole rings as diuretics
7
Table 1. In vivo diuretic activity of synthesized compounds (4a–f) using a rat animal model.
Diuresis
Dose
(mg/kg
body wt.)
Total urinary
output (ml)
Normal saline
intake (ml)
Urinary
excretion (%)
Diuretic
action
Diuretic
activity
Lipschitz
value
Treatment
1
4a
4b
4c
4d
4e
4f
Cont
Urea
Acet
45
45
45
45
45
45
45
4.4 0.357**
3.5 0.320*
3.4 0.217*
5.3 0.413**
2.8 0.262
3.4 0.346*
3.2 0.365
2.2 0.250
3.0 0.12
3.1 0.11
2.9 0.19
3.0 0.12
3.2 0.10
2.8 0.16
3.1 0.15
3.0 0.13
3.0 0.18
2.9 0.16
146.6
112.9
117.24
176.66
87.5
121.42
103.22
73.33
136.6
155.17
2.0
1.07
0.75
0.75
1.13
0.56
0.78
0.66
0.47
.88
1.21
0.85
0.85
1.28
0.63
0.88
0.75
–
1.53
1.59
2.4
1.2
1.65
1.4
1.0
2.11
1.86
–
1000
45
4.1 0.450**
4.5 0.452**
1.0
1.13
1.0
Each value in total urinary output column represents the mean SEM (n ¼ 6).
Cont, control; Acet, acetazolamide.
*p50.05.
**p50.01 (Dunnett’s multiple comparison test with Control group).
Table 2. Effect of synthesized compounds (4a–f) on electrolyte
concentration in excreted urine of rats.
benzothiazole-6-sulfonic acid amide (4c) was highly significant,
i.e. 5.30 0.413 (p50.01), which is increased by4200% as
compared to the control group. Urinary excretion of compound 1
was found to be in between urea (136.6%) and standard
acetazolamide (155.17%). Diuretic action of compound 4c was
observed to be 2.4 in comparison with urea (2.11) and
acetazolamide (1.86). Diuretic activity of compound 4c was
found to be 1.13 times more potent than acetazolamide, while 1,
4a, 4b and 4e were calculated as 1.07, 0.75, 0.75 and 0.78,
respectively (Table 1). The Lipschitz value (the ratio T/U, in
which T is response of the test compound, and U, that of urea
treatment, indices of 1.0 and higher are regarded as a positive
effect in terms of diuretic activity) shows that 4c is 1.28 times as
potent as urea, as far as urinary output is concerned, and
compound 1 is also more potent (1.21 times) than urea.
Compound 1 showed a significant increase in Na+ excretion,
2.41 0.102 (p50.01) which was slightly higher than the
standards, urea (1.72 0.073) and acetazolamide (1.80 0.041)
(Table 2). It is evident from the obtained results that compound 1
possess significant kaliuretic property (p50.01), 1.08 0.036,
however, 4e emerged as a stronger kaliuretic (1.75) with regards
to the Na+/K+ ratio (Table 2).
Electrolyte concentration (mEq/l)
Dose
(mg/kg
body wt.)
Treatment
Na+
K+
Na+/K+
1
4a
4b
4c
4d
4e
4f
Cont
Urea
Acet
45
45
45
45
45
45
45
–
2.41 0.102**
1.64 0.124
1.68 0.076
2.14 0.095*
1.30 0.076**
1.40 0.075**
1.50 0.070
0.84 0.045
1.72 0.073
1.80 0.041
1.08 0.036**
0.91 0.040
0.88 0.038
0.71 0.046
0.60 0.044**
0.81 0.035
0.70 0.041
0.41 0.11
2.23
1.8
1.9
3.01
2.16
1.75
2.14
2.04
2.86
2.22
1000
45
0.61 0.043*
0.81 0.043
Each value represents the mean SEM (n ¼ 6).
*p50.05.
**p50.01 (Dunnett’s multiple comparison test with Standard group,
acetazolamide).
Cont., control; Acet, acetazolamide.
Zn2+ (d). To regenerate the active basic form, i.e. hydroxide
bound to Zn2+ (a), a proton transfer reaction from the active site to
the environment may be assisted either by active-site residues
(such as His64, the proton shuttle in isozymes I, II, IV, VI, VII, IX
and XII–XIV among others) or by the buffers present in the
medium (Figure 6). The schematical representation of the process
may be shown by reactions 2 and 3. In reaction 3, the proton
transfer regenerating the zinc-hydroxide species of the enzyme is
The structure activity relationship study illustrated that the
substitution with halogens at 6th position of benzothiazole ring
decreases the activity, but shows a good promising effect as
compared to other standard diuretics. From the analysis of results,
compound 4c (2-{3-[2–(4-methoxyphenyl)-ethenyl]-1H-quinoxa-
lin-2-ylideneamino}-benzothiazole-6-sulfonic
acid
amide)
appeared to be the most promising and the lead compound as
diuretic among all the newly synthesized aromatic sulfonamide
compounds.
a rate-limiting step in the catalysis (Figure 7)15–18
.
The rats were kept in metabolic cages for the measurement of
the diuretic activity of the synthesized compounds. The diuretic
activity was measured by collecting the total excreted urine of rats
after administration of the test compounds and was compared
Conclusions
Six differently substituted quinoxaline derivatives of benzothiazole
with standard and control groups of the rats. The diuretic activity (4a–f) were synthesized with an aim to obtain diuretic compounds
was evaluated as per the reported method14. Compounds 1 and with a better spectrum. These compounds were evaluated for their
diuretic activity in experimental rat animal model. The data
obtained from pharmacological studies indicates that compound 4c
4a–f were evaluated for diuretic activity using acetazolamide as
reference drug.
The results of the in vivo diuretic activity of the newly showed excellent diuretic activity of 1.13 in comparison to 1.0 for
synthesized compounds are presented in Table 1. Compounds 1,
4a, 4b, 4c and 4e demonstrated excellent cumulative urine output,
acetazolamide and 0.88 for Urea. Compound 1 also showed very
good diuretic activity (0.81). Additionally, molecular docking
among these compound, the urinary output of 2-{3-[2–(4- studies were also carried out with the help of Maestro 9.0 program
(Schrodinger Inc) to ascertain the binding patterns of compound 4c
methoxyphenyl)-ethenyl]-1H-quinoxalin-2-ylideneamino}-1,3

8
A. Husain et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
containing ethoxyphthalimide moiety via key intermediate
6-chloro 1,3 benzothiazole 2-amine. Indian J Chem 2010;49B:
818–25.
into the binding sites of CA enzyme (PDB code: 4KUV). In light of
the current results, the compound 4c may be worth to explore and is
a subject of further detailed studies in search of prospective new
diuretics.
9. Lipschitz WL, Hadidian Z, Kerpcsar A. Bioassay of diuretics.
J Pharmacol Exp Ther 1943;79:97–110.
10. Majeed J, Shaharyar M. Synthesis and in-vivo diuretic activity of
some novel pyrimidine derivatives. J Enzyme Inhib Med Chem
2011;26:819–26.
11. Meera R, Devi P, Muthumani P, et al. Evaluation of diuretic activity
from Tylophora indica leaves extracts. J Pharm Sci Res 2009;1:
112–16.
Declaration of interest
The authors report no declarations of interest.
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