Ureas/thioureas of benzo[d]isothiazole analog conjugated glutamic acid: Synthesis and biological evaluation

Article abstract of DOI:10.1002/ardp.201200470

A series of urea and thiourea derivatives of glutamic acid conjugated to 3-(1-piperazinyl)-1,2-benzisothiazole were synthesized, spectroscopically characterized, and evaluated for their in vitro antiglycation and urease inhibitory activities. Preliminary screening of the synthesized compounds 1-35 showed significant results. Amongst these, compounds 17-21 and 30-35 bearing fluoro and methoxy substituents, respectively, exhibited inhibitory potency greater than the reference standards. Hence, they may serve as new lead compounds for further development. Urea and thiourea derivatives of glutamic acid conjugated to 3-(1-piperazinyl)-1,2-benzisothiazole were evaluated for their in vitro antiglycation and urease inhibitory activities. Compounds 17-21 and 30-35 bearing fluoro and methoxy substituents, respectively, exhibited inhibitory potency greater than the reference compounds. Copyright

Full text of DOI:10.1002/ardp.201200470

Arch. Pharm. Chem. Life Sci. 2013, 346, 359–366
359
Full Paper
Ureas/Thioureas of Benzo[d]isothiazole Analog Conjugated
Glutamic Acid: Synthesis and Biological Evaluation
Anamika Sharma, Ramesh Suhas, and Dase Channe Gowda
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore, India
A series of urea and thiourea derivatives of glutamic acid conjugated to 3-(1-piperazinyl)-1,2-
benzisothiazole were synthesized, spectroscopically characterized, and evaluated for their in vitro
antiglycation and urease inhibitory activities. Preliminary screening of the synthesized compounds
1–35 showed significant results. Amongst these, compounds 17–21 and 30–35 bearing fluoro and
methoxy substituents, respectively, exhibited inhibitory potency greater than the reference
standards. Hence, they may serve as new lead compounds for further development.
Keywords: Antiglycation / Conjugation / Glutamic acid / Urease inhibition / Urea/thiourea
Received: December 16, 2012; Revised: March 2, 2013; Accepted: March 8, 2013
DOI 10.1002/ardp.201200470
Introduction
nephritis, ammonia, and urinary catheter encrustation [5, 6].
It is also a major cause of pathologies induced by Helicobacter
pylori as this allows bacteria to survive at the low pH of the
stomach and hence plays an important role in producing
peptic and gastric ulcers [5]. As a result, ureases have been
identified as important targets in research both for human
and animal health, as well as in agriculture.
Protein glycation, also known as the Maillard reaction is a
non-enzymatic reaction between reducing sugars and
proteins, produces Schiff bases which undergo Amadori re-
arrangement to form relatively stable compounds called
Amadori products. Under low pH or oxidative conditions,
Schiff bases or Amadori products degrade to generate reac-
tive carbonyls including methylglyoxal, which is a major
reactive carbonyl in vivo [1–3]. Its concentration in the blood
of diabetic patients was five folds of that in healthy individ-
uals [4]. Methylglyoxal glycates proteins in a much faster rate
than sugars, causing inter- and intramolecule crosslinks of
proteins, which causes aging.
Amino acids and their derivatives are versatile synthetic
building blocks for pharmaceutical applications; common
subunits in many bioactive compounds and natural products
([7] and references cited therein). Amongst, L-glutamate, a
neurotransmitter plays an important role in synaptic
plasticity [8], neurotoxicity and probably in some neurode-
generative disorders [9]. Glutamate binds to its receptor
protein via its three ionizable groups, namely a-amino, a-
and g-carboxylates, through hydrogen bonding or electro-
static interactions [10, 11].
The metalloenzyme urease (urea amidohydrolase; EC
3.5.1.5) catalyzes the hydrolysis of urea into ammonia and
carbon dioxide. It is present in a variety of plants, algae,
fungi, and bacteria [5]. Urease is involved in the pathogenesis
of hepatic encephalopathy, hepatic coma urolithiasis, pyelo-
It has been well established that urea and thiourea deriva-
tives have got a significant place in modern medicinal
chemistry. These have been reported in the literature to
possess antiglycating [12], antimicrobial [13] activity etc.
One of the important classes of heterocyclic compounds
is isothiazole and its derivatives which possess differential
pharmacological activities like anti-inflammatory [14], anti-
viral [15], analgesic, and antibacterial [16] and as inhibitors
for type II diabetes [17].
Correspondence: Dr. Dase Channe Gowda, Department of Studies in
Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India.
E-mail: dchannegowda@yahoo.co.in
Fax: þ91 821 2419361
Abbreviations: Boc, tert-butyloxycarbonyl; Boc-Glu(OBzl)-OH, 5-
(benzyloxy)-2-(tert-butoxycarbonylamino)-5-oxopentanoic acid; EDCI, 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt, N-hydroxybenzotriazole;
NMM, N-methyl morpholine; PBT, 3-(1-piperazinyl)-1,2-benzisothiazole; TCA,
trichloroacetic acid; TFA, trifluoroacetic acid.
Based on the above and the previous encouraging results
from our research group [18–22], interest was generated
to bridge urea/thiourea groups with heterocycle using an
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

360
A. Sharma et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 359–366
amino acid as the linker since the latter offers several
advantages in bioactivity [23].
carbonylic carbons in the range d 154.49–155.36 and 180.62–
180.86, respectively. The physical and analytical data of the
derivatives are presented in Tables 1 and 2, respectively.
Results and discussion
Biology
Chemistry
In the present investigation, we wish to report for the first
time urea/thiourea derivatives of double heterocyclic conju-
gated glutamic acid as effective antiglycating and urease
inhibitory agents. The title compounds were synthesized
1–35 and subjected to antiglycation and urease inhibitory
activities. The results obtained are presented in Table 3 and
the data represents average values from triplicate runs. Activity
of the test compounds was compared with rutin and thiourea,
which served as reference standards for antiglycation and
urease inhibitory activities, respectively. PBT was found to be
inactive. On conjugation with Boc-Glu(OBzl)-OH, compound 1
showed a slight enhancement in the activity. Further benzyl
group of 1 was deprotected to obtain 2, which showed decrease
in the activity. This difference in the activity revealed that free
acid has less effect on the activity. Compound 2 was further
conjugated with PBT to get 3, which showed an increase in the
activity compared to their counterparts.
Boc-Glu(OBzl)-OH was conjugated to PBT using EDCI/HOBt as
coupling agent and NMM as base to get 1. The side chain
benzyl group of 1 was deprotected with 1 N NaOH/MeOH to
get 2 which was conjugated with PBT to get 3. Boc group of 1
and 3 was deblocked using TFA and further reacted with
isocyanates and isothiocyanates to get urea and thiourea
derivatives 4–35, respectively (Scheme 1). All the derivatives
4–35 were obtained in high yields. Synthesis was confirmed
by IR, 1H NMR, 13C NMR, mass and elemental analysis. The IR
spectra displayed absorption bands at 3304–3399, 2029–
2048, and 1629–1642 cmꢀ1 for –NH–, –C S, and –C O
–
–
–
–
groups, respectively. 1H NMR spectra exhibited multiplets
for the aromatic protons at d 7.21–7.93 and urea/thiourea
NH protons at d 8.03–8.25 and 8.23–8.77, respectively.
13C NMR spectral analysis of the ureas and thioureas showed
the typical absorptions bands for the carbonylic and thio-
O
O
O
H
O
H
H
H
N
.
TFA H2N
N
N
S
N
N
Boc
Boc
OH
O
N
O
+
N
O
.
HCl HN
N
N
O
(i)
(iii)
N
N
(iv)
X
N
N
N
S
S
R
O
O
O
N
S
PBT
O
1
_
4
14
(ii)
O
N
H
N
Boc
O
H
(i)
N
N
Boc
N
N
N
S
+
.
HN
N
N
HCl
S
O
N
N
N
S
O
OH
PBT
2
N
S
3
(iii)
O
H
H
N
O
N
N
.
H2N
TFA
N
N
X
N
N
R
(iv)
N
S
N
O
S
O
N
N
N
N
N
S
S
_
15 35
_
_
H4
Reagents and conditions: (i) EDCI/HOBt, NMM, 0°C to rt, (ii) 1 N NaOH/MeOH, 2 h, 0°C to rt, (iii) TFA, 45 min, rt, (iv) NMM, DMF, R C6
N=C=X, 0°C to rt, where R = H, F, Cl, Br, OCH3 and X = O, S
Scheme 1. Urea/thiourea derivatives of PBT conjugated glutamic acid.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 359–366
Derivatives of Benzoisothiazole Analog Conjugated Glu
361
Table 1. Physical data of the synthesized compounds.
Entry
Rf
Yield Yield m.p.
(g) (%) (8C)
Mol. Fr.
Mol. wt.
Elemental analysisa)
Rfa
Rfb
Calculated
Observed
C
H
N
S
C
H
N
S
1
2
3
4
5
6
7
8
0.42 0.50 4.579 85.00 Gum
C28H34N4O5S
C21H28N4O5S
538.66
448.54
649.83
575.65
591.72
575.65
575.65
591.72
587.69
603.75
587.69
603.75
587.69
603.75
668.83
684.90
686.82
702.89
686.82
686.82
702.89
703.28
719.34
703.28
719.34
763.79
747.73
763.79
747.73
698.86
714.92
698.86
714.92
698.86
714.92
62.43 6.36 10.40
56.23 6.29 12.49
59.15 6.05 15.09
62.59 5.25 12.17
60.89 5.11 11.84 10.84 60.88 5.14 11.86 10.83
62.59 5.25 12.17
62.59 5.25 12.17
60.89 5.11 11.84 10.84 60.88 5.13 11.85 10.86
63.36 5.66 11.92 5.46 63.35 5.65 11.91 5.47
61.67 5.51 11.60 10.62 61.68 5.53 11.63 10.61
63.36 5.66 11.92 5.46 63.35 5.65 11.93 5.45
61.67 5.51 11.60 10.62 61.68 5.53 11.63 10.65
63.36 5.66 11.92 5.46 63.35 5.67 11.91 5.43
61.67 5.51 11.60 10.62 61.68 5.50 11.62 10.61
61.06 5.43 16.75 9.59 61.10 5.42 16.74 9.58
59.62 5.30 16.36 14.05 59.60 5.31 16.37 14.09
59.46 5.14 16.31 9.34 59.50 5.15 16.32 9.35
58.10 5.02 15.94 13.69 58.10 5.03 15.95 13.70
59.46 5.14 16.31
59.46 5.14 16.31
58.10 5.02 15.94 13.69 58.10 5.03 15.95 13.70
58.07 5.02 15.93 9.12 58.10 5.03 15.92 9.14
56.77 4.90 15.58 13.37 56.80 4.88 15.59 13.40
58.07 5.02 15.93 9.12 58.10 5.01 15.92 9.13
56.77 4.90 15.58 13.37 56.80 4.91 15.59 13.40
53.47 4.62 14.67 12.59 53.50 4.63 14.68 12.60
54.61 4.72 14.99
53.47 4.62 14.67 12.59 53.50 4.63 14.68 12.60
54.61 4.72 14.99
60.15 5.48 16.03
58.80 5.36 15.67 13.46 58.80 5.35 15.68 13.45
60.15 5.48 16.03 9.18 60.11 5.49 16.04 9.19
58.80 5.36 15.67 13.46 58.80 5.35 15.66 13.50
60.15 5.48 16.03 9.18 60.12 5.47 16.04 9.17
58.80 5.36 15.67 13.46 58.80 5.34 15.65 13.50
5.95 62.40 6.35 10.41
7.15 56.21 6.28 12.48
9.87 59.13 6.06 15.10
5.57 62.59 5.24 12.16
5.96
7.14
9.88
5.58
0.35 0.41 3.559 93.70
0.37 0.44 4.201 81.53
0.45 0.50 0.180 84.22
0.49 0.56 0.178 81.02
0.43 0.48 0.176 82.35
0.50 0.58 0.175 81.88
0.56 0.62 0.169 76.92
0.42 0.48 0.184 84.32
0.41 0.46 0.170 75.84
0.43 0.49 0.182 83.41
0.42 0.48 0.181 80.74
0.45 0.52 0.179 82.03
0.38 0.42 0.181 80.74
0.43 0.52 0.178 86.47
0.41 0.48 0.180 90.90
0.36 0.43 0.102 96.50
0.34 0.41 0.100 92.40
0.39 0.47 0.098 92.70
0.44 0.53 0.175 83.00
0.49 0.58 0.185 86.00
0.45 0.54 0.129 79.40
0.47 0.56 0.134 80.60
0.39 0.47 0.179 83.00
0.36 0.44 0.186 84.00
0.50 0.60 0.190 81.00
0.42 0.50 0.180 78.00
0.49 0.59 0.200 85.00
0.45 0.53 0.180 79.00
0.44 0.53 0.185 86.10
0.45 0.54 0.180 81.80
0.47 0.56 0.168 78.10
0.49 0.59 0.178 80.90
0.40 0.48 0.187 87.00
0.41 0.49 0.191 87.00
196
69
91
121
130
153
129
89
73
103
93
128
98
85
C
32H39N7O4S2
C30H30FN5O4S
C30H30FN5O3S2
C30H30FN5O4S
5.57 62.56 5.25 12.17
5.57 62.58 5.24 12.14
5.58
5.56
C30H30FN5O4S
C30H30FN5O3S2
C31H33N5O5S
C31H33N5O4S2
C31H33N5O5S
C31H33N5O4S2
C31H33N5O5S
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
C
31H33N5O4S2
C34H36N8O3S2
C34H36N8O2S3
C34H35FN8O3S2
C34H35FN8O2S3
C34H35FN8O3S2
C34H35FN8O3S2
C34H35FN8O2S3
C34H35ClN8O3S2
C34H35ClN8O2S3
C34H35ClN8O3S2
C34H35ClN8O2S3
C34H35BrN8O2S3
C34H35BrN8O3S2
C34H35BrN8O2S3
C34H35BrN8O3S2
C35H38N8O4S2
C35H38N8O3S3
C35H38N8O4S2
C35H38N8O3S3
C35H38N8O4S2
C35H38N8O3S3
110
82
114
129
180
109
89
9.34 59.50 5.15 16.33
9.34 59.50 5.13 16.32
9.33
9.36
118
98
119
123
148
123
148
92
79
119
92
8.58 54.60 4.71 14.98
8.59
8.58 54.60 4.74 14.97
9.18 60.18 5.49 16.01
8.56
9.17
139
118
a)
Values obtained are within ꢁ0.4% of the calculated ones.
With a view to further increase the potency of the com-
pounds, Boc groups of 1 and 3 were removed and converted to
urea/thiourea derivatives, which exhibited a wide spectrum
of activity. Single heterocyclic conjugated derivatives 4–14
showed less potency towards antiglycation and urease inhibi-
tory activity when compared to double heterocyclic conju-
gated derivatives 15–35. This clearly explained that double
conjugation has a pronounced effect on biological activity. In
the series, compounds 15–21 and 30–35 showed effective
activity towards antiglycation and urease inhibition when
compared to standard rutin and thiourea, respectively.
Results appeared that compounds containing –NH–CS–NH–
are more active than –NH–CO–NH–, which could be due
to bioisosteric effects [24]. Divalent sulfur being a highly
versatile atom is characterized by the ability to interact both
with electron-poor and with electron-rich functional groups,
which would help the molecule to bind to receptors more
efficiently [25].
Particularly, compounds containing methoxy group 30–35
exhibited excellent activity, which is attributed to the electron
donating nature of –OCH3. Additionally, presence of methoxy
in para position has shown enhanced activity than ortho which
in turn has exhibited more activity than meta. This revealed
that positioning of the substituent on the aryl group played an
important role in inhibition studies. Thus, the preferential
order of the substituent for the activity was found to be p>o>m.
On the other hand, it seems to be interesting to point out
that among the halogen substituted derivatives (F, Cl, Br),
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362
A. Sharma et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 359–366
Table 2. Characterization data of the synthesized compounds by IR, 1H NMR, 13C NMR, and mass.
Entry
1
IR (cmS1
nmaxnujol
)
1H NMR (d) ppm
13C NMR (d) ppm
Mass
–
1.40 (9H,s,Boc), 1.82–2.80 (4H,m,Glu-CH2),
3.50–3.85 (8H,m,CH2), 4.71–4.83 (1H,t,Glu-CH),
5.12 (2H,s,benzyl-CH2), 7.27–7.91 (9H,m,ArH),
8.01–8.23 (1H,m,NH)
173.28, 170.90, 163.12, 155.23, 152.06, 136.28,
129.81, 127.89, 127.48, 127.32, 124.39, 124.05,
80.24, 69.80, 49.42, 44.56, 41.16, 30.26, 29.61,
27.48
539 (Mþ1)
2
3
4
3332 (NH)
1641 (CO)
1.40 (9H,s,Boc), 1.77–2.56 (4H,m,Glu-CH2), 3.40–3.90
(8H,m,CH2), 4.69–4.90 (1H,t,Glu-CH), 7.26–7.91
(4H,m,ArH), 8.00–8.10 (1H,m,NH), COOH (exchanged)
1.40 (9H,s,Boc), 1.82–2.86 (4H,m Glu-CH2), 3.43–3.92
(16H,m,CH2), 4.57–4.89 (1H,t,Glu-CH), 7.25–7.92
(8H,m,ArH), 8.02–8.21 (1H,m,NH)
1.83–2.85 (4H,m,Glu-CH2), 3.47–3.87 (8H,m,CH2),
4.76–4.92 (1H,t,Glu-CH), 5.15 (2H,s,benzyl-CH2),
7.22–7.99 (13H,m,ArH), 8.05–8.24 (2H,m,NH)
179.98, 170.86, 163.16, 155.31, 152.18, 127.45,
124.36, 124.06, 80.29, 49.43, 44.55, 41.18,
30.08, 29.59, 27.45
171.18, 170.76, 163.18, 155.29, 152.02, 127.49,
124.39, 124.08, 80.23, 49.42, 44.57, 41.19,
29.61, 27.85, 27.56
447 (Mꢀ1)
650 (Mþ1)
577 (Mþ1)
3287 (NH)
1631 (CO)
3220 (NH)
1686 (CO)
173.28, 170.76, 163.28, 155.29, 153.16, 152.08,
136.38, 130.89, 130.28, 129.86, 127.86, 127.49,
127.39, 124.39, 124.20, 124.03, 120.06, 115.60,
69.79, 49.53, 44.58, 41.18, 30.32, 27.43
180.69, 173.22, 170.72, 163.25, 160.21, 152.02,
136.31, 130.92, 130.32, 129.89, 129.38, 129.32,
127.42, 127.35, 124.33, 124.02, 121.41, 116.08,
69.82, 52.84, 44.55, 41.19, 30.34, 27.51
173.28, 170.83, 163.19, 156.85, 154.96, 152.02,
136.33, 134.17, 130.85, 129.86, 127.82, 127.42,
127.38, 124.27, 124.03, 118.34, 117.85, 117.15,
69.81, 49.62, 44.56, 41.17, 30.33, 27.52
173.25, 170.84, 163.16, 155.65, 154.86, 152.03,
136.39, 136.25, 129.85, 127.84, 127.43, 127.34,
124.28, 124.02, 119.97, 115.14, 69.82, 49.62,
44.54, 41.12, 30.35, 27.83
5
3327 (NH)
1636 (CO)
2038 (CS)
1.81–2.84 (4H,m,Glu-CH2), 3.45–3.85 (8H,m,CH2),
4.75–4.91 (1H,t,Glu-CH), 5.14 (2H,s,benzyl-CH2),
7.23–7.98 (13H,m,ArH), 8.25–8.75 (2H,m,NH)
593 (Mþ1)
577 (Mþ1)
577 (Mþ1)
593 (Mþ1)
588 (Mþ1)
604 (Mþ1)
588 (Mþ1)
604 (Mþ1)
588 (Mþ1)
604 (Mþ1)
6
3325 (NH)
1631 (CO)
1.82–2.84 (4H,m,Glu-CH2), 3.45–3.87 (8H,m,CH2),
4.75–4.92 (1H,t,Glu-CH), 5.12 (2H,s,benzyl-CH2),
7.23–7.97 (13H,m,ArH), 8.04–8.23 (2H,m,NH)
7
3396 (NH)
1638 (CO)
1.83–2.84 (4H,m,Glu-CH2), 3.47–3.86 (8H,m,CH2),
4.76–4.91 (1H,t,Glu-CH), 5.13 (2H,s,benzyl-CH2),
7.23–7.99 (13H,m,ArH), 8.03–8.23 (2H,m,NH)
8
3319 (NH)
1638 (CO)
2034 (CS)
1.82–2.83 (4H,m,Glu-CH2), 3.43–3.83 (8H,m,CH2),
4.77–4.93 (1H,t,Glu-CH), 5.13 (2H,s,benzyl-CH2),
7.22–7.97 (13H,m,ArH), 8.24–8.73 (2H,m,NH)
180.78, 173.26, 170.75, 163.26, 156.03, 152.03,
136.33, 135.58, 129.86, 127.85, 127.43, 127.38,
125.28, 124.32, 124.03, 116.83, 69.83, 52.82,
44.56, 41.17, 30.26, 27.84
9
3345 (NH)
1634 (CO)
1.82–2.84 (4H,m,Glu-CH2), 3.43–3.95 (8H,m,CH2),
4.70–4.81 (1H,t,Glu-CH), 3.94 (3H,s,OCH3),
5.12 (2H,s,benzyl-CH2), 7.24–7.96 (13H,m,ArH),
8.03–8.25 (2H,m,NH)
1.81–2.84 (4H,m,Glu-CH2), 3.42–3.94 (8H,m,CH2),
4.75–4.89 (1H,t,Glu-CH), 3.96 (3H,s,OCH3),
5.13 (2H,s,benzyl-CH2), 7.22–7.97 (13H,m,ArH),
8.25–8.76 (2H,m,NH)
1.81–2.84 (4H,m,Glu-CH2), 3.46–3.91 (8H,m,CH2),
4.73–4.82 (1H,t,Glu-CH), 3.96 (3H,s,OCH3),
5.14 (2H,s,benzyl-CH2), 7.23–7.96 (12H,m,ArH),
8.03–8.24 (2H,m,NH)
1.82–2.85 (4H,m,Glu-CH2), 3.41–3.98 (8H,m,CH2),
4.77–4.89 (1H,t,Glu-CH), 3.94 (3H,s,OCH3),
5.15 (2H,s,benzyl-CH2), 7.22–7.96 (13H,m,ArH),
8.26–8.76 (2H,m,NH)
1.82–2.86 (4H,m,Glu-CH2), 3.42–3.92 (8H,m,CH2),
4.79–4.85 (1H,t,Glu-CH), 3.93 (3H,s,OCH3),
5.12 (2H,s,benzyl-CH2), 7.23–7.95 (12H,m,ArH),
8.02–8.21 (2H,m,NH)
1.81–2.84 (4H,m,Glu-CH2), 3.42–3.93 (8H,m,CH2),
4.77–4.85 (1H,t,Glu-CH), 3.94 (3H,s,OCH3),
5.14 (2H,s,benzyl-CH2), 7.23–7.95 (13H,m,ArH),
8.26–8.75 (2H,m,NH)
173.26, 169.83, 163.16, 154.46, 152.02, 149.83,
136.32, 129.84, 128.35, 127.83, 127.41, 127.39,
124.38, 124.26, 124.03, 121.08, 120.16, 112.64,
69.82, 49.82, 49.63, 44.53, 41.18, 30.28, 27.82
180.63, 173.28, 169.93, 163.14, 154.94, 152.04,
136.33, 129.86, 128.88, 127.83, 127.43, 127.35,
126.32, 125.33, 124.24, 124.02, 121.46, 112.84,
69.84, 52.93, 49.67, 44.55, 41.16, 30.26, 27.81
173.26, 169.93, 163.14, 161.38, 154.63, 152.01,
137.16, 136.34, 130.03, 129.88, 127.84, 127.44,
127.38, 124.28, 124.05, 116.74, 113.86, 110.84,
69.85, 49.84, 49.65, 44.53, 41.18, 30.28, 27.83
180.58, 173.24, 169.98, 163.18, 161.38, 152.02,
139.08, 136.38, 130.16, 129.88, 127.86, 127.42,
127.38, 124.28, 124.03, 119.16, 116.93, 111.02,
69.75, 57.81, 49.72, 44.53, 41.18, 30.28, 27.81
173.26, 169.94, 163.16, 155.96, 154.58, 152.04,
136.32, 131.98, 129.86, 127.83, 127.49, 127.33,
124.26, 124.03, 119.96, 113.24, 69.79, 49.79,
49.63, 44.54, 41.16, 30.31, 27.88
10
11
12
13
14
3313 (NH)
1632 (CO)
2033 (CS)
3306 (NH)
1625 (CO)
3328 (NH)
1635 (CO)
2038 (CS)
3374 (NH)
1637 (CO)
3343 (NH)
1634 (CO)
2043 (CS)
180.59, 173.28, 169.98, 163.18, 155.98, 152.02,
136.33, 131.92, 129.88, 127.86, 127.39, 127.32,
124.26, 124.04, 119.93, 113.26, 69.71, 52.86,
49.65, 44.56, 41.18, 30.39, 27.85
15
16
3219 (NH)
1683 (CO)
1.98–2.86 (4H,m,Glu-CH2), 3.48–4.98 (16H,m,CH2),
4.71–4.87 (1H,t,Glu-CH), 7.25–7.92 (13H,m,ArH),
8.05–8.23 (2H,m,NH)
1.82–2.88 (4H,m,Glu-CH2), 3.40–4.98 (16H,m,CH2),
4.78–4.85 (1H,t,Glu-CH), 7.22–7.91 (13H,m,ArH),
8.25–8.75 (2H,m,NH)
171.28, 170.98, 163.18, 155.18, 152.08, 138.06,
129.86, 129.08, 127.58, 124.36, 124.08, 120.18,
49.44, 44.50, 41.18, 27.85, 27.58
180.86, 171.24, 170.24, 163.16, 152.09, 135.08,
129.39, 127.86, 127.56, 125.18, 124.32, 124.09,
52.75, 44.54, 41.19, 27.86, 27.59
669 (Mþ1)
685 (Mþ1)
3325 (NH)
1630 (CO)
2031 (CS)
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 359–366
Table 2. (continued )
Derivatives of Benzoisothiazole Analog Conjugated Glu
363
Entry
17
IR (cmꢀ1
nmaxnujol
)
1H NMR (d) ppm
13C NMR (d) ppm
Mass
3319 (NH)
1631 (CO)
1.81–2.82 (4H,m,Glu-CH2), 3.48–3.86 (16H,m,CH2),
4.75–4.91 (1H,t,Glu-CH), 7.21–7.99 (12H,m,ArH),
8.04–8.23 (2H,m,NH)
171.22, 170.78, 163.18, 155.36, 153.88, 152.02,
130.86, 130.20, 127.42, 124.32, 124.19, 124.08,
120.02, 115.89, 49.62, 44.56, 41.17, 27.82,
27.56
180.66, 171.31, 170.88, 163.12, 160.20, 152.01,
130.89, 130.28, 129.72, 127.44, 124.33, 124.08,
121.32, 116.01, 52.82, 44.55, 41.12, 27.81,
27.53
171.32, 170.82, 163.18, 156.86, 154.92, 152.01,
134.18, 130.86, 127.43, 124.23, 124.01, 118.36,
117.88, 117.18, 49.65, 44.57, 41.18, 28.82,
27.51
688 (Mþ1)
704 (Mþ1)
688 (Mþ1)
18
19
3339 (NH)
1629 (CO)
2040 (CS)
1.82–2.89 (4H,m,Glu-CH2), 3.46–3.80 (16H,m,CH2),
4.74–4.89 (1H,t,Glu-CH), 7.23–7.98 (12H,m,ArH),
8.24–8.73 (2H,m,NH)
3320 (NH)
1638 (CO)
1.81–2.80 (4H,m,Glu-CH2), 3.45–3.90 (16H,m,CH2),
4.73–4.88 (1H,t,Glu-CH), 7.21–7.99 (12H,m,ArH),
8.03–8.22 (2H,m,NH)
20
21
22
3398 (NH)
1634 (CO)
1.89–2.83 (4H,m,Glu-CH2), 3.46–3.89 (16H,m,CH2),
4.78–4.90 (1H,t,Glu-CH), 7.21–7.97 (12H,m,ArH),
8.05–8.23 (2H,m,NH)
1.82–2.89 (4H,m,Glu-CH2), 3.45–3.92 (16H,m,CH2),
4.70–4.91 (1H,t,Glu-CH), 7.24–7.99 (12H,m,ArH),
8.25–8.76 (2H,m,NH)
1.83–2.87 (4H,m,Glu-CH2), 3.46–3.92 (16H,m,CH2),
4.72–4.87 (1H,t,Glu-CH), 7.21–7.98 (12H,m,ArH),
8.06–8.24 (2H,m,NH)
170.70, 170.23, 163.12, 155.68, 154.88, 152.00,
136.28, 127.57, 124.44, 124.06, 119.96, 115.13,
49.60, 44.55, 41.16, 28.39, 27.85
180.77, 170.42, 170.15, 163.19, 156.01, 152.01,
135.56, 127.56, 125.29, 124.43, 124.08, 116.85,
52.75, 44.60, 41.19, 28.19, 27.83
171.23, 170.89, 163.12, 154.86, 152.02, 139.46,
135.21, 132.14, 127.48, 125.86, 124.24, 124.03,
120.10, 119.56, 49.63, 44.56, 41.18, 27.82,
27.38
688 (Mþ1)
704 (Mþ1)
3316 (NH)
1632 (CO)
2039 (CS)
3341 (NH)
1637 (CO)
704 (Mþ1)
706 (Mþ2)
23
3298 (NH)
1629 (CO)
2029 (CS)
1.85–2.81 (4H,m,Glu-CH2), 3.41–3.89 (16H,m,CH2),
4.71–4.84 (1H,t,Glu-CH), 7.26–7.99 (12H,m,ArH),
8.23–8.75 (2H,m,NH)
180.62, 171.42, 170.79, 163.14, 152.08, 139.96,
135.41, 132.46, 127.41, 126.01, 124.28, 124.09,
122.46, 121.28, 52.83, 44.53, 41.12, 27.92,
27.39
720 (Mþ1)
722 (Mþ2)
24
25
26
3365 (NH)
1638 (CO)
1.82–2.80 (4H,m,Glu-CH2), 3.43–3.91 (16H,m,CH2),
4.72–4.86 (1H,t,Glu-CH), 7.24–7.98 (12H,m,ArH),
8.06–8.24 (2H,m,NH)
1.82–2.86 (4H,m,Glu-CH2), 3.41–3.89 (16H,m,CH2),
4.76–4.89 (1H,t,Glu-CH), 7.21–7.99 (12H,m,ArH),
8.23–8.77 (2H,m,NH)
1.81–2.86 (4H,m,Glu-CH2), 3.45–3.93 (16H,m,CH2),
4.77–4.90 (1H,t,Glu-CH), 7.22–7.99 (12H,m,ArH),
8.25–8.73 (2H,m,NH)
171.40, 170.81, 163.13, 155.01, 152.01, 136.96,
132.96, 127.86, 127.42, 124.31, 124.08, 119.98,
49.82, 44.53, 41.13, 27.96, 27.41
180.69, 171.32, 170.86, 163.18, 152.01, 136.13,
133.06, 127.91, 127.39, 125.36, 124.36, 124.11,
52.83, 44.52, 41.18, 27.81, 27.52
180.81, 171.31, 170.71, 163.18, 152.01, 136.92,
132.92, 132.06, 130.48, 127.91, 127.31, 124.31,
124.08, 122.42, 52.91, 44.51, 41.12, 27.83,
27.51
704 (Mþ1)
706 (Mþ2)
3342 (NH)
1630 (CO)
2041 (CS)
3389 (NH)
1632 (CO)
2029 (CS)
720 (Mþ1)
722 (Mþ2)
764 (Mþ1)
766 (Mþ2)
27
28
3319 (NH)
1637 (CO)
1.83–2.89 (4H,m,Glu-CH2), 3.41–3.89 (16H,m,CH2),
4.74–4.78 (1H,t,Glu-CH), 7.22–7.99 (12H,m,ArH),
8.06–8.25 (2H,m,NH)
171.36, 170.73, 163.13, 154.86, 152.08, 138.01,
131.42, 127.33, 127.18, 124.32, 124.08, 123.31,
120.46, 120.18, 49.61, 44.58, 41.13, 27.89,
27.54
180.69, 171.32, 170.79, 163.12, 152.02, 137.24,
131.08, 127.82, 127.34, 125.46, 125.42, 124.33,
124.01, 123.42, 52.89, 44.53, 41.19, 27.63,
27.42
748 (Mþ1)
750 (Mþ2)
3310 (NH)
1635 (CO)
2019 (CS)
1.82–2.84 (4H,m,Glu-CH2), 3.46–3.90 (16H,m,CH2),
4.75–4.88 (1H,t,Glu-CH), 7.22–7.98 (12H,m,ArH),
8.24–8.76 (2H,m,NH)
764 (Mþ1)
766 (Mþ2)
29
30
3319 (NH)
1639 (CO)
1.81–2.85 (4H,m,Glu-CH2), 3.41–3.91 (16H,m,CH2),
4.73–4.89 (1H,t,Glu-CH), 7.23–7.99 (12H,m,ArH),
8.04–8.24 (2H,m,NH)
1.81–2.84 (4H,m,Glu-CH2), 3.43–3.94 (16H,m,CH2),
4.71–4.82 (1H,t,Glu-CH), 3.93 (3H,s,OCH3),
7.21–7.95 (12H,m,ArH), 8.03–8.24 (2H,m,NH)
171.33, 170.81, 163.19, 155.01, 152.01, 138.21,
132.08, 127.38, 124.36, 124.08, 122.36, 120.82,
49.63, 44.56, 41.13, 27.73, 27.49
170.32, 169.92, 163.19, 154.49, 152.02, 149.86,
128.39, 127.36, 124.39, 124.36, 124.08, 121.09,
120.18, 112.61, 49.81, 49.62, 44.53, 41.18,
27.86, 27.34
180.62, 170.21, 169.93, 163.12, 154.92, 152.01,
128.86, 127.31, 126.30, 125.34, 124.36, 124.01,
121.42, 112.82, 52.92, 49.68, 44.51, 41.19,
27.91, 27.42
170.36, 169.92, 163.18, 161.39, 154.62, 152.06,
137.18, 130.01, 127.33, 124.31, 124.01, 116.72,
113.86, 110.86, 49.82, 49.63, 44.55, 41.17,
27.81, 27.39
748 (Mþ1)
750 (Mþ2)
3341 (NH)
1632 (CO)
699 (Mþ1)
715 (Mþ1)
699 (Mþ1)
31
32
3319 (NH)
1634 (CO)
2039 (CS)
1.82–2.83 (4H,m,Glu-CH2), 3.41–3.93 (16H,m,CH2),
4.75–4.89 (1H,t,Glu-CH), 3.97 (3H,s,OCH3),
7.22–7.96 (12H,m,ArH), 8.26–8.76 (2H,m,NH)
3304 (NH)
1629 (CO)
1.81–2.81 (4H,m,Glu-CH2), 3.45–3.92 (16H,m,CH2),
4.73–4.81 (1H,t,Glu-CH), 3.95 (3H,s,OCH3),
7.22–7.96 (12H,m,ArH), 8.04–8.25 (2H,m,NH)
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

364
A. Sharma et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 359–366
Table 2. (continued )
Entry
33
IR (cmꢀ1
nmaxnujol
)
1H NMR (d) ppm
13C NMR (d) ppm
Mass
3324 (NH)
1639 (CO)
2031 (CS)
1.82–2.85 (4H,m,Glu-CH2), 3.41–3.98 (16H,m,CH2),
4.78–4.86 (1H,t,Glu-CH), 3.95 (3H,s,OCH3),
7.21–7.95 (12H,m,ArH), 8.26–8.75 (2H,m,NH)
180.59, 170.33, 169.96, 163.16, 161.86, 152.01,
139.02, 130.18, 127.39, 124.32, 124.03, 119.18,
116.92, 111.01, 52.88, 49.71, 44.52, 41.18,
27.81, 27.39
715 (Mþ1)
34
35
3371 (NH)
1634 (CO)
1.83–2.81 (4H,m,Glu-CH2), 3.43–3.91 (16H,m,CH2),
4.79–4.85 (1H,t,Glu-CH), 3.93 (3H,s,OCH3),
7.24–7.96 (12H,m,ArH), 8.03–8.22 (2H,m,NH)
1.81–2.88 (4H,m,Glu-CH2), 3.42–3.9 (16H,m,CH2),
4.75–4.86 (1H,t,Glu-CH), 3.95 (3H,s,OCH3),
7.23–7.95 (12H,m,ArH), 8.25–8.73 (2H,m,NH)
170.18, 169.87, 163.18, 155.98, 154.52, 152.03,
131.96, 127.32, 124.38, 124.06, 119.92, 113.21,
49.71, 49.62, 44.51, 41.18, 27.82, 27.47
180.69, 170.16, 169.73, 163.13, 156.43, 152.01,
131.74, 127.91, 127.26, 124.43, 124.09, 113.81,
52.79, 49.62, 44.55, 41.19, 27.88, 27.47
699 (Mþ1)
715 (Mþ1)
3340 (NH)
1630 (CO)
2047 (CS)
Table 3. Antiglycation and urease inhibitory activities of
synthesized compounds.
compounds containing fluoro proved to be an activity
enhancer which is in good agreement with earlier reports
[13, 19]. This may be due to the more electron withdrawing
nature of the fluoro compared to other halogens.
Entry
X
R
Antiglycationa)
Urease inhibitory
activitya)
In summary, the present study reports the synthesis and
evaluation of antiglycation and urease inhibitory activities
of a series of urea/thiourea derivatives of glutamic acid
conjugated to 3-(1-piperazinyl)-1,2-benzisothiazole. From
the biological activity studies, several conclusions can be
deduced: (i) Urea/thiourea derivatives of 1 and 3 enhance
the activity. (ii) Compounds carrying thiourea moiety
proved to be more potent when compared with urea
analogues. (iii) Among halogen-substituted derivatives,
fluoro-containing compounds exhibited more activity.
(iv) Methoxy substituent bearing compounds exhibited
excellent activity. Hence, this study supports that compounds
17–21 and 30–35 are highly potent with good antiglycation
and urease inhibitory activities. The results further encour-
age us for the synthesis and evaluation of new derivatives
bearing different amino acids.
IC50 (mM)
IC50 (mM)
1
2
3
4
5
6
7
8
–
–
–
O
S
O
O
S
O
S
O
S
O
S
O
S
O
S
O
O
S
O
S
O
S
S
O
S
O
O
S
O
S
–
–
–
2F
2F
3F
4F
4F
2OCH3
2OCH3
3OCH3
3OCH3
4OCH3
4OCH3
H
H
2F
2F
3F
4F
4F
3Cl
3Cl
4Cl
4Cl
2Br
3Br
95 ꢁ 0.7
160 ꢁ 0.8
80 ꢁ 0.2
56 ꢁ 0.3
49 ꢁ 0.2
67 ꢁ 0.3
45 ꢁ 0.1
39 ꢁ 0.3
33 ꢁ 0.4
48 ꢁ 0.3
52 ꢁ 0.2
39 ꢁ 0.2
43 ꢁ 0.4
30 ꢁ 0.2
30 ꢁ 0.6
25 ꢁ 0.5
22 ꢁ 0.4
21 ꢁ 0.1
23 ꢁ 0.3
22 ꢁ 0.1
21 ꢁ 0.2
115 ꢁ 0.9
95 ꢁ 0.6
57 ꢁ 0.1
48 ꢁ 0.3
135 ꢁ 0.8
180 ꢁ 0.9
145 ꢁ 0.4
150 ꢁ 0.5
14 ꢁ 0.5
11 ꢁ 0.4
22 ꢁ 0.1
19 ꢁ 0.2
12 ꢁ 0.3
10 ꢁ 0.1
41.9 ꢁ 0.2
–
20.5 ꢁ 0.2
29.0 ꢁ 0.3
19.0 ꢁ 0.2
29.0 ꢁ 0.3
20.5 ꢁ 0.1
30.5 ꢁ 0.4
26.5 ꢁ 0.2
18.0 ꢁ 0.3
20.5 ꢁ 0.1
17.5 ꢁ 0.1
29.5 ꢁ 0.3
24.5 ꢁ 0.2
17.0 ꢁ 0.1
15.0 ꢁ 0.2
14.5 ꢁ 0.1
13.0 ꢁ 0.1
14.5 ꢁ 0.4
10.0 ꢁ 0.1
15.0 ꢁ 0.2
13.0 ꢁ 0.1
9.0 ꢁ 0.1
39.0 ꢁ 0.5
36.0 ꢁ 0.6
37.5 ꢁ 0.4
32.5 ꢁ 0.1
37.5 ꢁ 0.4
44.5 ꢁ 0.2
39.0 ꢁ 0.8
33.0 ꢁ 0.3
8.8 ꢁ 0.5
5.6 ꢁ 0.1
13.0 ꢁ 0.4
12.4 ꢁ 0.4
6.6 ꢁ 0.2
4.6 ꢁ 0.1
–
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Rutin
Thiourea
Experimental
Boc-Glu(OBzl)-OH, EDCI, HOBt and TFA were purchased from
Advanced Chem. Tech. (Louisville, Kentucky, USA). NMM and
phenylisocyanates/isothiocyanates were purchased from Sigma
Chemical Co. (St. Louis, MO). All chemicals and reagents were
obtained from Aldrich (USA), Spectrochem Pvt. Ltd. (India) and
Rankem Pvt. Ltd. (India) and were used without further purifi-
cation. Melting points were determined on a Superfit melting
point apparatus (India) and are uncorrected. FT-IR was performed
using a Jasco spectrometer (Japan) using nujol media. 1H NMR
(300 MHz) and 13C NMR (100 MHz) spectra were recorded on a
Bruker multinuclear FT-NMR spectrometer (Japan) using CDCl3
or DMSO(d6) as solvent. The chemical shifts are reported as parts
per million (ppm) using TMS as an internal standard. Mass
spectra were obtained on Agilent LCMS (Ireland). Progress of
the reaction was monitored by TLC using silica gel coated on
glass plates with the solvent system comprising chloroform/
methanol/acetic acid in the ratio 98:02:03 (Raf )/95:05:03 (Rfb)
3Br
4Br
2OCH3
2OCH3
3OCH3
3OCH3
4OCH3
4OCH3
–
O
S
–
–
–
21.5 ꢁ 0.1
a)
Values are mean of three determinations, the ranges of which
are <5% of the mean in all case.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 359–366
Derivatives of Benzoisothiazole Analog Conjugated Glu
365
and the compounds on the TLC plates were detected by iodine
vapors. All the chemicals and reagents used for biological studies
were purchased from Aldrich (USA).
mixture was stirred for 8 h slowly warming to room tempera-
ture. DMF was evaporated under high vacuum and the residue
was poured into about 20 mL ice-cold 90% saturated KHCO3
solution and stirred for 15 min. The precipitated compound
was extracted into chloroform and washed sequentially with
5% NaHCO3 solution (2 ꢂ 20 mL), water (2 ꢂ 20 mL), 0.1 N
HCl (2 ꢂ 20 mL) followed by brine. The organic layer was dried
over anhydrous sodium sulfate. The solvent was removed under
reduced pressure, triturated with n-hexane, filtered and dried.
Chemistry
Synthesis of benzyl 5-(4-(benzo[d]isothiazol-3-yl)-
piperazin-1-yl)-4-(tert-butoxycarbonylamino)-
5-oxopentanoate (1)
PBT was synthesized as reported in the literature method [26]. To
Boc-Glu(OBzl)-OH (4.050 g, 0.012 mol) dissolved in acetonitrile
(40 mL) and cooled to 08C was added NMM (1.13 mL, 0.012 mol).
To this solution EDCI (2.295 g, 0.012 mol) was added and stirred
while maintaining the temperature at 08C. After stirring the
reaction mixture for 10 min at this temperature, HOBt
(1.837 g, 0.012 mol) in DMF (18 mL) was added. The reaction
mixture was stirred for an additional 10 min and a pre-cooled
solution of PBT (3.066 g, 0.012 mol) in DMF (30 mL) was added
slowly. After 20 min, pH of the solution was adjusted to 8 by
the addition of NMM and the reaction mixture was stirred over-
night at room temperature. Acetonitrile was removed under
reduced pressure and the residual DMF was poured into about
500 mL ice-cold 90% saturated KHCO3 solution and stirred
for 30 min. The precipitated product was extracted into chloro-
form. The organic layer was washed with 5% NaHCO3 solution
(3 ꢂ 60 mL), water (1 ꢂ 60 mL), 0.1 N cold HCl solution
(2 ꢂ 60 mL) and brine (1 ꢂ 60 mL). The chloroform layer was
dried over anhydrous sodium sulfate. The solvent was removed
under reduced pressure. The obtained product was triturated
with n-hexane, filtered and dried.
Biology
Sodium phosphate buffer (pH 7.4) was prepared by mixing
Na2HPO4 (67 mM) containing sodium azide (3 mM) and phos-
phate buffer saline (PBS) which was prepared by mixing NaCl
(137 mM) þ Na2HPO4 (8.1 mM) þ KCl (2.68 mM) þ KH2PO4
(1.47 mM) and pH 10 was adjusted with NaOH (0.25 mM). BSA
(10 mg/mL) and anhydrous glucose (50 mg/mL) solutions were
prepared in sodium phosphate buffer.
In vitro antiglycation assay [27]
In 96-well plate assays, each well contained 60 mL of reaction
mixtures {20 mL bovine serum albumin (BSA) (10 mg/
mL) þ 20 mL of anhydrous glucose (50 mg/mL) þ 20 mL test
sample}. Glycated control contained 20 mL BSA þ 20 mL
glucose þ 20 mL sodium phosphate buffer, while blank control
contains 20 mL BSA and 40 mL sodium phosphate buffer.
Reaction mixture was incubated at 378C for 7 days. After incu-
bation, 6 mL (100%) of TCA was added in each well and centri-
fuged (15,000 rpm) for 4 min at 48C. After centrifugation, the
pellets were rewashed with 60 mL (10%) of TCA. The supernatant
containing glucose, inhibitor and interfering substance was
removed and the pellet containing the advanced glycated end
product (AGE)-BSA was dissolved in 60 mL PBS. Assessment of
fluorescence spectrum (excitation 370 nm), and changes in fluor-
escence intensity (excitation 370 nm to emission 440 nm), based
on AGEs were monitored by using spectrofluorimeter Varioskan
(Germany) and % inhibition was calculated using:
Synthesis of 5-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)-
4-(tert-butoxycarbonylamino)-5-oxopentanoic acid (2)
1 (5.0 g, 0.009 mol) was hydrolyzed in methanol (50 mL) using
cold solution of 1 N NaOH (14 mL) for 2 h. Completion of the
reaction was monitored by TLC and the solvent was evaporated,
cooled, neutralized with cold 1N HCl, extracted with chloroform,
washed with 1 N HCl followed by water and dried over anhy-
drous sodium sulfate. The solvent was removed under pressure,
triturated with ether, filtered and dried to obtain debenzylated
compound 2.
ꢀ
ꢁ
Fluorescence of sample
% Inhibition ¼ 1 ꢀ
ꢂ 100
Fluorescence of glycated sample
Rutin was used as the standard antiglycating agent.
Synthesis of tert-butyl 1,5-bis(4-(benzo[d]isothiazol-3-yl)-
piperazin-1-yl)-1,5-dioxopentan-2-ylcarbamate (3)
Urease inhibitory assay [28]
The title compound 3 was synthesized using Boc-Glu-PBT (5.382 g,
0.01 mol), PBT (2.555 g, 0.01 mol), EDCI/HOBt as coupling agent
and NMM as base and following the procedure as described for 1.
Reaction mixtures comprising 25 mL of enzyme (jack bean ure-
ase) solution and 55 mL of buffers containing 100 mM urea were
incubated with 5 mL of the test compounds (0.5 mM concen-
tration) at 308C for 15 min in 96-well plates. Urease activity
was determined by measuring ammonia production using the
indophenol method. Briefly, 45 mL each of phenol reagent
(1% w/v phenol and 0.005% w/v sodium nitroprusside) and
70 mL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride
NaOCl) were added to each well. The increasing absorbance at
630 nm was measured after 50 min, using a microplate reader.
The entire assays were performed at pH 6.8. Percentage inhi-
bitions were calculated from the formula:
General procedure for the synthesis of urea/thiourea
derivatives (4–35)
The Boc group of 1 and 3 (0.0003 mol) was removed by stirring with
2.0 mL of TFA for 45 min at room temperature. After completion
of the reaction monitored by TLC, reaction mixture was concen-
trated at high vacuum to get TFA.H-Glu(OBzl)-PBT/TFA.H-Glu(PBT)-
PBT which was then triturated with dry ether, filtered and dried.
To
a solution of TFA.H-Glu(OBzl)-PBT/TFA.H-Glu(PBT)-PBT
ꢀ
Thiourea was used as the standard inhibitor of urease.
ꢁ
(0.001 mol) in DMF (1 mL), cooled to 08C was added NMM
(0.219 mL, 0.002 mol). To this solution respective substituted
phenyl isocyanates/isothiocyanates (0.0012 mol) was added
drop-wise maintaining the temperature at 08C. The reaction
OD test well
OD control
% Inhibition ¼ 1 ꢀ
ꢂ 100
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

366
A. Sharma et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 359–366
The authors have declared no conflict of interest.
[13] R. Suhas, S. Chandrashekar, D. C. Gowda, Eur. J. Med. Chem.
2012, 48, 179–191.
´
[14] L. Kuczynski, M. Kuriata, B. Ciupka, Pol. J. Pharmacol. Pharm.
1984, 36, 485–491.
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