Synthesis, antihypertensive activity, and 3D-QSAR studies of some new p-hydroxybenzohydrazide derivatives

Article abstract of DOI:10.1002/ardp.201000008

p-Hydroxybenzohydrazide 2 on treatment with aromatic/aliphatic aldehyde followed by cyclization with carbon disulphide afforded compounds 4a-4n. Also, compound 2 by treatment of substituted isothiocyanate followed by the treatment of chloroacetic acid yields the corresponding compounds 6a-6i. All the test compounds were assayed for antihypertensive activity by non-invasive blood pressure measurement and invasive blood pressure measurement methods. The test compounds showed significant antihypertensive activity. The intact compounds were subjected to 3D-QSAR studies. The 3D-QSAR analysis was carried out by PHASE program and a statistically reliable model with good predictive power (r ²a=a0.98, q²a=a0.74) was achieved. The 3D-QSAR plots illustrated insights into the structure-activity relationship of these compounds which may aid in the design of potent p- hydroxybenzohydrazide derivatives as antihypertensive agents. One third of the world population is affected with cardiovascular diseases and the major part of it means hypertension. With the aim of obtaining new agents which might have a better or similar antihypertensive activity as the known standards, some novel p-hydroxybenzohydrazide derivatives were synthesized and tested. Copyright

Full text of DOI:10.1002/ardp.201000008

Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
119
Full Paper
Synthesis, Antihypertensive Activity, and 3D-QSAR Studies
of Some New p-Hydroxybenzohydrazide Derivatives
Ritesh P. Bhole and Kishore P. Bhusari
Department of Pharmaceutical Chemistry, Sharad Pawar College of Pharmacy, Nagpur, MS, India
p-Hydroxybenzohydrazide 2 on treatment with aromatic/aliphatic aldehyde followed by cyclization
with carbon disulphide afforded compounds 4a–4n. Also, compound 2 by treatment of substituted
isothiocyanate followed by the treatment of chloroacetic acid yields the corresponding compounds
6a–6i. All the test compounds were assayed for antihypertensive activity by non-invasive blood
pressure measurement and invasive blood pressure measurement methods. The test compounds
showed significant antihypertensive activity. The intact compounds were subjected to 3D-QSAR
studies. The 3D-QSAR analysis was carried out by PHASE program and a statistically reliable
model with good predictive power (r2 ¼ 0.98, q2 ¼ 0.74) was achieved. The 3D-QSAR plots
illustrated insights into the structure-activity relationship of these compounds which may aid in
the design of potent p-hydroxybenzohydrazide derivatives as antihypertensive agents.
Keywords: Antihypertensive activity / PHASE program / 3D-QSAR / Synthesis
Received: January 6, 2010; Accepted: February 9, 2010
DOI 10.1002/ardp.201000008
Introduction
of computational chemistry to drug discovery [16].
Quantitative drug design embraces two major activities,
the quantitative description of the structural differences
among a series of chemical compounds of biological interest,
and the formulation of ‘‘QSAR’’ (quantitative structure–
activity relationship) useful in the design of new and better
therapeutic agents [17]. A QSAR is a mathematical relation-
ship between a biological activity of a molecular system and
its geometric and chemical characteristics. QSAR attempts to
find a consistent relationship between biological activity and
molecular properties, so that these ‘‘rules’’ can be used to
evaluate the activity of new compounds: 3D models are
more easily interpretable than two-dimensional descriptors
or fingerprint-based QSAR models making it easier to suggest
new compounds for synthesis.
High blood pressure (BP) is one of the most potent risk factors
for the first and recurrent stroke [1]. One third of the world
population is affected with cardiovascular diseases and the
major part of it stems from hypertension [2–4]. Hypertension
is recognized as a major risk factor in a human patient with
cerebral hemorrhage and heart and renal disease; therefore,
diagnosis of hypertension is carried out by measuring blood
pressure on a regular basis [5–7]. p-Hydroxybenzohydrazide
analogs seem to be suitable parent compounds from which a
variety of biological activities are reported such as antitumor
[8, 9], anti-anginal [10], antitubercular [11, 12], antihyperten-
sive [13, 14], MAO (monoamine oxidase) enzyme inhibitor
[15], antibacterial [16], etc. Discovering three-dimensional
pharmacophores which can explain the activity of a series
of ligands is one of the most significant contributions
The chemistry of p-hydroxybenzohydrazide is of great
interest. This moiety has an immense importance for its
activity access against many diseases. In recent years, interest
has also been focused on thiadiazole derivatives and thiazo-
lidine derivatives of benzohydrazide [18–21].
Correspondence: Ritesh P. Bhole, Bhole, Department of Pharmaceutical
Chemistry, Sharad Pawar College of Pharmacy, Wanadongri, Hingna
Road, Nagpur-441 110, MS, India.
Results
E-mail: riteshb986@gmail.com, kpbhusari_spcp@rediffmail.com
Fax: þ91 7104 235-084
Synthesis
Some novel (4-hydroxyphenyl)-[(5-substituted alkyl/aryl)-2-thioxo-
1,3,4-thiadiazol-3-yl]methanones and N0-[(3-substituted alkyl/
Abbreviations: blood pressure (BP); invasive blood pressure measurement
(IBP); non-invasive blood pressure measurement (NIBP); partial least
square (PLS); Quantitative Structure-Activity Relationship (QSAR).
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120
R. P. Bhole and K. P. Bhusari
Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
aryl)-4-oxo-1,3-thiazolidin-2-ylidene]-4-hydroxybenzohydrazide
were synthesized with the aim of obtaining new agents
which might have more or a similar antihypertensive activity
than known ones. Thus, in the present investigation, 14
different derivatives of (4-hydroxyphenyl)-[5-substituted
alkyl/aryl)-2-thioxo-1,3,4-thiadiazol-3-yl]methanone and nine
derivatives of N’-[(3-substituted alkyl/aryl)-4-oxo-1,3-thiazoli-
din-2-ylidene]-4-hydroxybenzohydrazide were synthesized
and were evaluated for their antihypertensive activity. The
synthesis of the intermediate and target compounds was
performed by the reactions illustrated in Schemes 1 and 2.
Compound 2, 4-hydroxybenzohydrazide, was synthesized
by amination of compound 1 by hydrazine hydrate (80%;
Scheme 1). The physical and elemental analysis data con-
firmed the formation of the compound 2. To a solution of
Scheme 2. Synthesis of compounds 5a–i and 6a–i.
compound 2 in ethanol various aliphatic/aromatic aldehydes
were added. The mixture was refluxed and excess solvent was
distilled off to afforded N’-[(substituted alkyl/aryl)-methyli-
dene]-4-hydroxybenzohydrazide 3a–3n. To a solution of com-
pounds 3a–3n in ethanol, carbon disulphide in alc. KOH was
added to obtained cyclized (4-hydroxyphenyl)-[(5-substituted-
alkyl/aryl)-2-thioxo-1,3,4-thiadiazol-3-yl]-methanones 4a–4n.
The formation of various imines (Schiff’s bases) 3a–3n takes
place by the elimination of water from the compounds. The
excess of water was removed. Further, the nitrogen in the side
chain of N’-[(substituted alkyl/aryl) methylidene]-4-hydroxy-
benzohydrazide, with its lone pair of electrons, attacks the
carbon atom of carbon disulphide to give the intermediate,
which – on intermolecular rearrangement – afford the
cyclized products 4a–4n.
The IR spectra of compounds 3a–3n showed absorption
bands at 1500–1569 cmꢀ1 (C N stretching) and 1658–
–
–
1684 cmꢀ1 (C O stretching) due to the conversion of amine
–
–
to imine. Compound 3a showed the presence of character-
istic absorption peaks at 2986 and 2990 cmꢀ1 (CH(CH3)2
stretching); compound 3b showed absorption peaks at
2981, 2986, and 2990 cmꢀ1 (CH2CH2CH2CH3 stretching);
Scheme 1. Synthesis of compounds 3a–n and 4a–n.
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Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
New p-Hydroxybenzohydrazide Derivatives
121
compound 3d showed an absorption peak at 1498 cmꢀ1
(C-NO2 stretching); compounds 3e and 3g have absorption
peaks at 1080–1092 cmꢀ1 (Ar-Cl stretching), compound 3f at
1035 cmꢀ1 (Ar-F stretching). Absorption peaks of compound
3h are at 3010–3030 cmꢀ1 (Ar-CH stretching); compound 3i
showed an absorption band at 3537 cmꢀ1 (Ar-OH stretching)
and compounds 3j and 3l at 1050–1150 cmꢀ1 (O-CH3 stretch-
ing); finally, compound 3n showed an absorption band
at 1600 cmꢀ1 (furan), The compounds 4a–4n showed absorp-
tion bands at about 1189–1290 cmꢀ1 and 752–760 cmꢀ1
(C-S-C stretching) due to presence of the thioxo-thiadiazole
ring. The disappearance of the peak at 1500–1569 cmꢀ1
1555 cmꢀ1 (C-NO2 stretching). Compounds 5e and 5g showed
absorption peaks at 1045 cmꢀ1 (Ar-F stretching), compound
5f has absorption peaks at 700–750 cmꢀ1 due to C-Cl stretch-
ing, 5h showed a peak at 3010–3030 cmꢀ1 (Ar-CH stretching),
and compound 5i showed a peak at 1050–1150 cmꢀ1 (O-CH3
stretching). The IR spectra of the cyclized compounds 6a–6i
revealed the disappearance of the NH band at 3215–
3230 cmꢀ1
.
1
The H-NMR of compounds 5a–5i showed a downfield sig-
nal at d ¼ 10.80–11.81 ppm attributed to 3-substitued NH.
The signal due to aromatic protons appeared as a multiplet in
the range of d ¼ 7.2–8.1 ppm integrating for nine protons.
The cyclized compounds 6a–6i lacked the NH signals and
showed a new singlet signal at d ¼ 3.01–3.98 ppm attribu-
table to the thiazoline ring. Compounds 5a and 6a showed a
characteristic peak at d ¼ 1.12 ppm due to the presence of a
isopropyl group. Compound 5b and 6b showed a triplet at
d ¼ 0.95 and a multiplet at 1.28 ppm due to the presence of
the n-butyl group. Compounds 5h and 6h showed doublet d
value at 2.11 ppm (Ar-CH3), as well as the doublet at
d ¼ 3.83 ppm (Ar-OCH3) of compounds 5i and 6i.
Furthermore, compounds 5a–5i, and 6a–6i showed charac-
–
(C N) confirms the assigned structure.
–
In 1H-NMR spectra of compounds 3a–3n, the imine proton
appeared as a multiplet at d ¼ 2.1–2.3 ppm integrating for
one proton. The signal due to an aromatic proton appeared as
multiplet in the range of d values 7.2–8.1 ppm integrating for
four protons. The cyclized compounds 4a–4n showed the
absence of a signal at d ¼ 2.1–2.3 for imine. The compounds
showed a characteristic peak at d ¼ 5.33 (s, 1H, Ar-OH).
Compound 4a showed a peak at d ¼ 1.31–1.5 ppm (d, CH-
(CH3)2), while 4b showed peaks at d ¼ d 0.95 (t, 3H, butyl
CH3), 1.28 (m, 2H, butyl CH2), 1.51 (m, 2H, butyl CH2),
3.53 ppm (m, 2H, butyl CH2). Compound 4h had a peak at
d ¼ 2.2 ppm (Ar-CH3) and compounds 4j and 4l showed sin-
glets at d ¼ 1.2–1.31 ppm (Ar-OCH3); in compound 4m, a
singlet is seen at d ¼ 4.2–4.4 ppm (N(CH3)2), whereas com-
pound 4n showed a triplet at d ¼ 6.85–8.5 ppm (furan). Other
peaks, in addition, confirm the structures assigned.
1
teristic H-NMR spectra confirming the assigned structure.
In the EI-MS spectra, molecular ion [Mþ] peaks, appeared
with different intensities and confirmed the molecular
weights of compounds 5a–5i and 6a–6i.
The molecular ion peaks were the base peaks for the com-
pounds; the appearance of an isotope peak [Mþ þ 2] as
intense as the molecular ion peak confirmed the presence
of a halogen atom in compounds 5e, 6e, 5f, 6f, 5g, and 6g. The
elemental analyses of the compounds 5a–5i and 6a–6i were
found within the limits of the theoretical values.
In the EI-MS spectra, the molecular ion peaks [Mþ], which
appeared at different intensities, confirmed the molecular
weight of compounds 3a–3n. Molecular ion peaks are the
base peaks for the compounds. The appearance of an isotopic
peak [M þ 2] along with the molecular ion peak confirmed
the presence of sulphur atoms in compounds 4a–4n.
Pharmacology
Antihypertensive activity of the synthesized compounds was
carried out by non-invasive blood pressure measurement
(NIBP). Their results were summarized in Table 1 and
Fig. 1. Percentage-inhibition data for non-invasive blood pres-
sure measurement are given in Table 2. Some of the com-
pounds, which show good antihypertensive activity, were
further assessed by invasive blood pressure (IBP) measure-
ment [22, 23]. The results for invasive blood pressure
measurement are given in Table 3 and Fig. 2. Percentage-
inhibition data for non-invasive blood pressure measurement
are given in Table 4. Both methods determining the changes
in blood pressure were performed by using Power Lab/4SP
with ML135 Dual Bio Amp computerized BP monitors auto-
matic cardiovascular system (AD instruments Pvt. Ltd.,
Australia). The reproducibility of the above experiment
was evaluated by using one way ANOVA technique and
was found to be statistically significant.
Addition of substituted isothiocyanates to the solution of
compound 2 in ethanol afforded the corresponding 2-[(4-
hydroxyphenyl) carbonyl]-N-substituted hydrazine carbo-
thioamide 5a–5i. In this reaction, compound 2 acts as a
nucleophile, having a lone pair of electrons on the nitrogen.
The reaction of 5a–5i with chloroacetic acid in boiling
ethanol containing fused sodium acetate undergoes a nucle-
ophilic addition–elimination reaction affording the corres-
ponding N’-[(3-substituted alkyl/aryl)-4-oxo-1,3-thiazolidin)-2-
ylidene]-4-hydroxybenzohydrazides 6a–6i.
The IR spectra of compounds 5a–5i showed NH and CS
stretching bands at 3215–3230 and 1309–1348 cmꢀ1, respect-
ively. Compound 5a showed the presence of characteristic
absorption peaks at 2986 and 2990 cmꢀ1 (CH(CH3)2stretching);
5b showed absorption peaks at 2984, 2989, and 2991 cmꢀ1
(CH2CH2CH2CH3 stretching), and 5d showed a band at
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122
R. P. Bhole and K. P. Bhusari
Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
Table 1. Antihypertensive activity data at a 10 mg/kg dose.
Compound
Average blood pressure (in mm Hg) at time (h)
0
1
2
3
4
5
6
7
8
9
10
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
6a
6b
6c
6d
6e
6f
6g
6h
6i
Control
Standard§
226 ꢁ 4
226 ꢁ 8
225 ꢁ 8
222 ꢁ 3
228 ꢁ 3
226 ꢁ 3
226 ꢁ 2
226 ꢁ 2
226 ꢁ 3
224 ꢁ 3
226 ꢁ 2
230 ꢁ 3
220 ꢁ 5
225 ꢁ 3
225 ꢁ 4
226 ꢁ 8
226 ꢁ 8
225 ꢁ 4
225 ꢁ 6
226 ꢁ 3
224 ꢁ 4
226 ꢁ 2
225 ꢁ 4
225 ꢁ 2
225 ꢁ 1
222 ꢁ 8
220 ꢁ 5
220 ꢁ 0
220 ꢁ 6
220 ꢁ 6
220 ꢁ 6
224 ꢁ 3
226 ꢁ 3
221 ꢁ 6
216 ꢁ 6
224 ꢁ 3
220 ꢁ 6
210 ꢁ 6
220 ꢁ 5
222 ꢁ 8
224 ꢁ 5
224 ꢁ 5
222 ꢁ 8
223 ꢁ 5
224 ꢁ 6
222 ꢁ 8
224 ꢁ 3
222 ꢁ 8
224 ꢁ 1
214 ꢁ 2
220 ꢁ 9
200 ꢁ 5
200 ꢁ 5
210 ꢁ 3
210 ꢁ 3
210 ꢁ 3
210 ꢁ 8
215 ꢁ 8
205 ꢁ 3
205 ꢁ 3
210 ꢁ 8
210 ꢁ 3
200 ꢁ 5
210 ꢁ 3
221 ꢁ 9
210 ꢁ 5
210 ꢁ 5
220 ꢁ 9
210 ꢁ 6
210 ꢁ 3
220 ꢁ 9
210 ꢁ 8
221 ꢁ 9
225 ꢁ 1
201 ꢁ 1
193 ꢁ 2
193 ꢁ 4
191 ꢁ 4
193 ꢁ 5
183 ꢁ 5
190 ꢁ 5
190 ꢁ 4
195 ꢁ 4
195 ꢁ 5
180 ꢁ 5
190 ꢁ 4
190 ꢁ 5
185 ꢁ 1
190 ꢁ 5
195 ꢁ 2
193 ꢁ 4
193 ꢁ 4
190 ꢁ 2
195 ꢁ 8
193 ꢁ 5
190 ꢁ 2
190 ꢁ 4
195 ꢁ 2
224 ꢁ 3
193 ꢁ 3
185 ꢁ 4
170 ꢁ 5
170 ꢁ 0
178 ꢁ 7
170 ꢁ 7
175 ꢁ 7
172 ꢁ 5
170 ꢁ 5
175 ꢁ 7
170 ꢁ 7
172 ꢁ 5
175 ꢁ 7
175 ꢁ 7
175 ꢁ 7
180 ꢁ 4
178 ꢁ 5
178 ꢁ 5
178 ꢁ 4
180 ꢁ 5
178 ꢁ 7
175 ꢁ 4
172 ꢁ 5
180 ꢁ 4
224 ꢁ 4
182 ꢁ 2
175 ꢁ 8
161 ꢁ 4
161 ꢁ 7
161 ꢁ 2
161 ꢁ 2
160 ꢁ 2
160 ꢁ 4
165 ꢁ 4
170 ꢁ 2
155 ꢁ 2
160 ꢁ 4
160 ꢁ 2
160 ꢁ 2
160 ꢁ 4
175 ꢁ 8
161 ꢁ 4
161 ꢁ 4
172 ꢁ 8
160 ꢁ 8
161 ꢁ 2
172 ꢁ 8
160 ꢁ 4
170 ꢁ 8
224 ꢁ 1
173 ꢁ 1
170 ꢁ 6
161 ꢁ 4
142 ꢁ 2
150 ꢁ 5
150 ꢁ 5
150 ꢁ 5
142 ꢁ 4
140 ꢁ 4
160 ꢁ 5
140 ꢁ 5
142 ꢁ 4
140 ꢁ 5
140 ꢁ 5
145 ꢁ 6
162 ꢁ 6
155 ꢁ 4
153 ꢁ 4
167 ꢁ 6
145 ꢁ 3
142 ꢁ 5
162 ꢁ 6
142 ꢁ 4
168 ꢁ 6
225 ꢁ 1
151 ꢁ 2
165 ꢁ 4
155 ꢁ 6
135 ꢁ 6
141 ꢁ 6
141 ꢁ 6
138 ꢁ 6
135 ꢁ 6
135 ꢁ 6
145 ꢁ 6
122 ꢁ 6
135 ꢁ 6
125 ꢁ 6
135 ꢁ 5
137 ꢁ 7
155 ꢁ 4
155 ꢁ 6
148 ꢁ 6
160 ꢁ 4
140 ꢁ 6
138 ꢁ 6
160 ꢁ 4
135 ꢁ 6
155 ꢁ 4
224 ꢁ 4
131 ꢁ 2
161 ꢁ 6
150 ꢁ 7
135 ꢁ 5
138 ꢁ 7
138 ꢁ 7
135 ꢁ 7
130 ꢁ 7
130 ꢁ 7
135 ꢁ 7
120 ꢁ 7
130 ꢁ 7
120 ꢁ 7
135 ꢁ 5
131 ꢁ 7
150 ꢁ 6
150 ꢁ 7
141 ꢁ 7
158 ꢁ 6
135 ꢁ 7
134 ꢁ 7
154 ꢁ 6
130 ꢁ 7
145 ꢁ 6
225 ꢁ 2
118 ꢁ 1
155 ꢁ 3
140 ꢁ 5
131 ꢁ 0
133 ꢁ 5
135 ꢁ 5
130 ꢁ 5
128 ꢁ 5
120 ꢁ 5
130 ꢁ 5
115 ꢁ 5
125 ꢁ 5
115 ꢁ 5
125 ꢁ 5
131 ꢁ 5
150 ꢁ 3
145 ꢁ 5
135 ꢁ 5
152 ꢁ 3
128 ꢁ 5
130 ꢁ 5
150 ꢁ 3
125 ꢁ 5
135 ꢁ 3
224 ꢁ 3
108 ꢁ 2
150 ꢁ 3
135 ꢁ 8
130 ꢁ 2
131 ꢁ 8
132 ꢁ 8
130 ꢁ 8
128 ꢁ 8
115 ꢁ 8
125 ꢁ 8
110 ꢁ 8
122 ꢁ 8
110 ꢁ 8
124 ꢁ 7
130 ꢁ 8
140 ꢁ 3
135 ꢁ 8
132 ꢁ 8
145 ꢁ 3
128 ꢁ 7
124 ꢁ 8
145 ꢁ 3
125 ꢁ 8
120 ꢁ 3
225 ꢁ 2
103 ꢁ 3
§
Standard: Valsertan.
250
200
150
100
50
4.a
4.b
4.c
4.d
4.e
4.f
4.g
4.h
4.i
4.j
4.k
4.l
4.m
4.n
6.a
6.b
6.c
6.d
6.e
6.f
Figure 1. Graph showing decrease in blood
pressure compared with standard by non-
invasive tail-cuff method.
6.g
6.h
6.i
Control
Standard
0
1
2
3
4
5
6
7
8
9
10
11
Time in h
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Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
New p-Hydroxybenzohydrazide Derivatives
123
Table 2. Comparative study of %-inhibition for antihypertensive activity.
Compound
Average blood pressure (in mm Hg) at time (h)
0
1
2
3
4
5
6
7
8
9
10
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
6a
6b
6c
6d
6e
6f
6g
6h
6i
Control
Standard§
ꢀ0.82
0
0.89
2.65
1.78
1.78
1.78
1.78
0
ꢀ0.89
0.88
3.57
0
1.78
6.25
1.78
0.89
0
2.2
11.5
11.1
6.6
6.6
6.6
6.6
4.4
7.07
8.8
6.6
6.6
11.1
6.6
1.7
6.6
6.6
2.2
6.6
6.6
2.2
6.6
1.7
–
13.8
14.6
14.7
13.8
18.3
15.1
15.1
12.9
25.9
19.6
15.1
15.1
17.4
15.1
12.9
13.8
13.8
15.1
12.9
13.8
15.1
15.1
12.9
–
17.4
24.7
24.1
20.5
24.1
21.8
23.2
24.1
23.8
24.1
23.2
21.8
21.8
21.8
19.6
20.5
20.5
20.5
19.6
20.5
21.8
23.2
19.6
–
21.8
28.7
28.1
28.1
28.1
28.5
28.5
26.3
29.2
30.8
28.5
28.5
28.5
28.5
21.8
28.1
28.1
23.2
28.5
28.1
23.2
28.5
24.1
–
24.4
28.7
36.8
37.7
37.7
37.7
36.8
37.7
37.1
37.7
36.8
37.7
38.0
35.8
28
31.4
36.8
25.7
35.5
36.8
28
36.8
25.3
–
26.3
31.4
39.7
41.5
41.5
42.8
39.7
39.7
40.2
45.5
39.7
44.1
40.2
39.3
31.4
31.4
38.3
28.5
37.5
40.1
28.5
39.7
32.1
–
28.4
33.6
40.2
43.1
43.1
44.4
42.2
42.2
42.4
46.6
42.2
46.6
40.2
42.0
33.6
33.6
41.7
29.7
40
30.8
38.0
42.0
45.0
49.5
46.4
44.1
46.4
44.6
50.8
44.1
50.8
44.6
42.0
33.6
35.8
44.1
32.1
44.1
44.1
33.0
44.1
48.6
–
33.3
40.2
42.4
46.2
46.0
48.8
45.7
48.8
46.0
51.3
45.7
51.3
45.5
42.4
38.8
40.1
45.6
35.5
44.4
46.2
35.5
44.1
48.8
–
ꢀ0.44
0.89
ꢀ1.78
ꢀ0.89
ꢀ0.89
ꢀ0.89
0
0
ꢀ0.89
ꢀ2.6
1.78
ꢀ0.44
ꢀ0.44
ꢀ0.89
ꢀ0.89
ꢀ0.44
ꢀ0.44
ꢀ0.89
0
0
0.89
0.44
0
0.89
0
0.89
–
5.75
42.2
31.5
42.2
37.7
–
ꢀ0.89
ꢀ0.44
–
3.57
14.5
18.6
28.3
31.0
33.7
45.3
47.8
52.9
54.7
§
Standard: Valsertan.
3D-QSAR study of the synthesized compounds
The synthesized compounds and their antihypertensive
activity data were used to generate 3D-QSAR models in order
to gain insights into the structural and molecular properties
of these compounds using the PHASE [24–26] program
(Schro¨dinger Computational Chemistry, USA). In general,
3D-QSAR is a useful tool in molecular design and medicinal
chemistry, which allows the prediction of the activity of
structurally diverse compounds, and also may assists in iden-
tifying new molecules with improved activity. The results are
summarized in Tables 5 to 7 and in Fig. 3.
Discussion
Synthesis
In the present investigation, different derivatives of (4-
hydroxyphenyl)-[5-substituted alkyl/aryl)-2-thioxo-1,3,4-thia-
diazol-3-yl]methanone and 4-hydroxy-N’-[-3-substituted alkyl/
aryl-4-oxo-1,3-thiazolidin-2-ylidene]benzohydrazide were syn-
thesized and their structures were confirmed from their
physical and analytical data. The selectivity of the substi-
tution as shown in Schemes 1 and 2 was believed to be
due to the electron density at N1 and N2. The latter nitrogen
Table 3. Antihypertensive activity of compounds by direct cannulation of carotid artery method (10 mg/kg dose).
Compound (10 mg/kg)
Average blood pressure (in mm Hg) at time (min)
0
15
30
60
120
180
240
4g
4j
4l
6f
6i
226 ꢁ 2
224 ꢁ 3
226 ꢁ 2
226 ꢁ 3
225 ꢁ 4
225 ꢁ 2
225 ꢁ 1
216 ꢁ 3
220 ꢁ 6
221 ꢁ 3
218 ꢁ 6
218 ꢁ 8
224 ꢁ 1
211 ꢁ 2
210 ꢁ 8
203 ꢁ 3
216 ꢁ 8
210 ꢁ 3
212 ꢁ 9
225 ꢁ 1
194 ꢁ 1
190 ꢁ 4
180 ꢁ 5
190 ꢁ 4
193 ꢁ 5
195 ꢁ 2
224 ꢁ 3
189 ꢁ 3
165 ꢁ 5
165 ꢁ 7
170 ꢁ 5
168 ꢁ 7
169 ꢁ 4
224 ꢁ 4
165 ꢁ 2
156 ꢁ 4
150 ꢁ 2
156 ꢁ 4
156 ꢁ 2
160 ꢁ 8
224 ꢁ 1
151 ꢁ 1
130 ꢁ 5
125 ꢁ 8
115 ꢁ 8
132 ꢁ 8
135 ꢁ 3
225 ꢁ 7
124 ꢁ 8
Control
Standard§
§
Standard: Valsertan.
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124
R. P. Bhole and K. P. Bhusari
Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
Figure 2. Graph showing decrease in blood
pressure compared with standard by direct can-
nulation of carotid artery.
Table 4. Comparative study of %-inhibition by the method of direct cannulation of the carotid artery (10 mg/kg dose).
Compound (10 mg/kg)
Average blood pressure (in mm Hg) at time (min)
0
15
30
60
120
180
240
4g
4j
4l
6f
6i
ꢀ0.4
0.4
ꢀ0.4
ꢀ0.4
0
3.5
1.7
1.3
2.6
2.6
–
6.6
9.7
4
6.6
5.7
–
15.1
19.6
15.1
13.8
12.9
–
26.3
26.3
24.1
25
24.5
–
30.3
33.0
30.3
30.3
28.5
–
42.2
44.4
48.8
41.3
40
Control
Standard§
–
0
–
50.5
3.5
14.5
18.9
26.5
33.2
§
Standard: Valsertan.
Table 5. Data set used for 3D QSAR analysis with corresponding actual and predicted pIC50 activity of compounds as antihypertensive
agents.
Compound
Antihypertensive activity
Compound
Antihypertensive activity
Actual pIC50
Predicted pIC50
Residuals
Actual pIC50
Predicted pIC50
Residuals
4a
4b
4c§
4d
4e§
4f
4g
4h
4i§
4j
0.30
0.07
0.07
0.06
0.01
0.02
0.07
0.02
0.17
0.05
0.05
0.07
N.D
N.D
005
004
001
002
005
001
010
004
001
002
N.D
N.D
ꢀ0.02
ꢀ0.02
0
4m§
4n
6a
6b
6c
6d
6e
6f
0.09
0.01
0.02
0.07
0.07
0.25
0.09
0.06
0.25
0.074
0.02
0.06
0.01
0.024
0.053
0.07
0.23
0.07
0.03
0.05
0.05
0.07
0.15
0
0.004
ꢀ0.017
0
ꢀ0.02
ꢀ0.02
ꢀ0.03
ꢀ0.2
ꢀ0.024
0.05
0
ꢀ0.02
ꢀ0.01
0.07
0.09
0.06
ꢀ0.05
6g
6h
6i§
§
Testset compounds; N.D: values not obtained.
Table 6. QSAR hypothesis score.
ID
Survival Survival -inactive Post-hoc Site Vector Volume Selectivity # Matches Energy Activity Inactive
ADHR.12H
AAHR.28
ADRR.32
AARR.40
38.835
39.791
29.208
32.206
32.097
34.164
27.696
32.702
3.72
0.95
0.931
0.885
0.804
0.849
0.847
2.202
2.362
2.42
37
37
33
33
0
4.638
4.42
4.638
4.602
1.737
1.627
1.512
1.504
3.516
3.675
3.67
0.85 0.862
0.91 0.92
0.91 0.909
0.01
5.191
5.169
2.422
H
: optimum model.
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Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
New p-Hydroxybenzohydrazide Derivatives
125
Table 7. PHASE 3D-QSAR statistical analysis.
Pharmacophore
Model 1
Model 2
Model 3
Model 4
Hypothesis
PLS & statistics for QSAR model
ADHRH
AAHR
ADRR
AARR
r2
0.98
0.05
54.1
1.41e12
3
0.94
0.03
27.1
4.22e13
3
0.84
0.02
43.2
2.56e12
3
0.92
0.04
28.1
3.22e13
3
SD §
F
P
No of PLS & factors
External test set for prediction
q2
rp
RMSE
0.74
0.91
0.1323
0.48
0.82
0.1321
0.54
0.81
0.1322
0.45
0.90
0.1222
§
H
SD: standard deviation; & PLS: partial least square; optimum model.
being richer in electron density is more reactive and provides
products of exclusive functionalization at N2 [11].
3D-QSAR study
A 3D-QSAR study was performed for compounds 4a–4n and
compounds 6a–6i to generate 3D-QSAR models. The statisti-
cally significant 3D-QSAR models were generated using 17
compounds as training set and were validated using a test
set with six compounds (Table 5) employing PHASE, the
pharmacophore modeling tool. A total of 101 five-point
hypotheses were obtained upon completion of the scoring
process and the top four high-ranking pharmacophore hy-
potheses were analyzed. The statistical and partial least
square (PLS) analyses results for four of the top ranking
pharmacophores (model 1–4) are summarized in Table 7.
Model 1 consisted of a hydrogen bond acceptor (A), one
hydrogen bond donor (D), a hydrophobic group (H), and one
aromatic ring (R). Model 2 was composed of two hydrogen-
bond acceptors (A), one hydrophobic group (H), and one
aromatic ring (R). Model 3 consisted of one hydrogen-bond
acceptor (A), one hydrogen bond donor (D), and two
aromatic rings (R). Model 4 possessed two hydrogen-bond
acceptor (A) and two aromatic rings (R). Model 1 exhibited
comparatively better PLS statistical qualities and excellent
prediction of the external test set compounds, and was,
therefore, considered for explaining the SAR of the com-
pounds. Model 1 showed a conventional correlation coef-
ficient (r2) of 0.98 with three components, test set
prediction (q2) of 0.74 and Pearson-R (rp) of 0.91. The high
value of variance ratio (F) observed for this model indicates
its statistical robustness which is further supported by the
significance level of the variance ratio (P). The value of
P < 0.05 indicates a greater degree of confidence and
means F is significant at the 95% level. The low standard
deviation (SD) and root-mean-squared error (RMSE)
contributes significantly to the model. The common
pharmacophore generated from the best PHASE hypothesis
with aligned compound 4j is shown in Fig. 3a. It
shows pharmacophoric features: black sphere for the
Antihypertensive activity
Antihypertensive activity testing for the compounds synthe-
sized according to Schemes 1 and 2 was carried out by the
non-invasive method (NIBP). In Scheme 1, 2,6 dichlorophenyl
compound 4g, 4-methoxyphenyl compound 4j, and 3,4-di-
substituted methoxyphenyl compound 4l had a remarkable
antihypertensive activity at 10 mg/kg. Although, 3-hydroxy-4-
methoxyphenyl compound 4k is slightly less potent than the
corresponding compounds 4g, 4j, and 4l. The other com-
pounds showed moderate activity. From Scheme 2, 2,4-
dichlorophenyl compound 6f and 2,4-dimethoxyphenyl com-
pound 6i showed remarkable antihypertensive activity. The
other compounds synthesized according to Scheme 2 showed
only moderate activity. It was observed that electron-with-
drawing or electron-donating substitutions may impart the
lipophilicity and show a better activity. The data are pre-
sented as means ꢁ S.E.M.; a repeated measures analysis of
variance was used to obtain the statistical significance
between and within the groups. Differences were considered
statistically significant at P < 0.05, and the F values for all
compounds in Schemes 1 and 2 are 80.33 ꢁ 0.5. The results of
%-inhibition are shown in Table 2. Few compounds showed
remarkable antihypertensive activity; these are compounds
4g, 4j, 4l, 6f, and 6i. They were further tested by the invasive
method (IBP; direct cannulation of carotid artery).
In the invasive method, the 4-methoxyphenyl compound 4j
showed a remarkable antihypertensive activity and the 3,4-
disubstituted methoxyphenyl compound 4l had good anti-
hypertensive activity.
From the ANOVA analysis and %-inhibition studies, it was
concluded that the substitution (R) with phenyl/substituted-
phenyl showed a better activity than substitution (R) with an
aliphatic group/furan.
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126
R. P. Bhole and K. P. Bhusari
Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
Figure 3. Visual representation of atom-based PHASE QSAR. PHASE-3D plots of the crucial pharmacophore regions
based on the model displayed with compound 4j. Positive coefficient-favored areas (contributing for increase in activity)
are represented by gray cubes. Negative coefficient-favored areas (contributing for decrease in activity) are represented by
black cubes.
hydrogen-bond acceptor (A) with the arrows pointing in the
direction of lone pair, gray sphere for the donor group (D), and
the torus for aromatic rings (R). In addition, the 3D-QSAR
results are also visualized as 3D plots of crucial pharmacophore
regions in which the gray cubes (regions) indicate increase –
and black cubes (regions) indicate decrease – in activity for
specific feature in the ligand regions.
the substructure fragments with hydrogen-bond donor in
this area may reduce the activity (compounds 4a, 4b, 6d, and
6g). In the negative ionic plots (Fig. 3d), gray regions at the 40-
position of the 5-phenyl ring suggest that increased activity
may be anticipated by moderate negatively charged sub-
stituents at the 40-position (compounds 4h and 4j), whereas
black region surrounding the 6-position of the benzohydra-
zide ring disfavor the activity (compound 6h). The hydro-
phobic (nonpolar) plot indicates desired/undesired
hydrophobic regions around two phenyl rings (Fig. 3b).
The gray region observed in the vicinity of the OCH3 group
and the 40-position of the benzohydrazide ring signifies that
substitution with a hydrophobic group may result in
enhanced activity (compounds 4j and 4i), whereas a slight
variation in the phenyl ring results in decreased activity
(compounds 6a and 6b). The disfavored black region in the
vicinity of the 40-position of the 5-phenyl ring suggests that a
hydrophobic group in this area results in decreased activity
(compounds 6d and 6h).
Figures 3b to 3d show the 3D-QSAR plots with specific
features relating to the most active conformer overlaid with
the best-generated pharmacophore hypothesis (compound
4j) and their relation with the biological activity of the com-
pounds as antihypertensive agents by generating volume-
occupied maps.
In the hydrogen-bond donor plots (Fig. 3c), the gray
regions at the 4-position of the benzohydrazide ring show
the significance of protons in the hydrogen-bond formation
with the receptor surface (compounds 4j, 4i, and 6i). The
black regions in the vicinity of 30- and 40-position of the
phenyl ring of p-hydroxybenzohydrazide suggested that
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Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
New p-Hydroxybenzohydrazide Derivatives
127
diethyl ether, and recrystallized from methanol affording the
desired white product 3a. Yield: 1.17 g (78%); m. p.: 123–1248C
(rectified spirit); Rf: 0.59 (acetonitrile/methanol 1:1); IR (KBr), in
This study demonstrates that the activity may be increased
by placing electron-releasing group(s) such as methoxy-
phenyl/dimethoxyphenyl on the p-hydroxybenzohydrazide
ring. Moreover, aromatic substitution should be retaining
for a better activity. Also, activity may be increase by placing
the hydrophobic groups at a particular position of the phenyl
ring of the p-hydroxybenzohydrazide.
cmꢀ1: 3090 (Ar-OH, NH), 1672–1682 (hydrazide –C O), 1500–
–
–
1569 (–C N), 2986, 2990 (CH(CH ) stretching); 1H-NMR (DMSO-
–
–
3 2
d6) d: 2.09 (s, 1H, CH3), 4.00 (m, 1H, isopropyl CH), 4.27 (s, 1H,
NCH2), 4.32 (s, 1H, NHNH2), 5.24 (s, 2H, Ar-OH), 8.42 (s, 1H, CH-N),
–
12.13 (d, 2H, CONHN CH); EI-MS (m/z, 100%): 206 [M þ 2] (100).
–
Anal. calcd. for C11H14N2O2: C, 64.08; H, 6.84; N, 13.57. Found: C,
64.06; H, 6.84; N, 13.58.
Experimental
Compound 3c
Chemistry
A solution of the corresponding compound 2 (1.5 g, 10 mmol) in
ethanol (40 mL) was refluxed with benzaldehyde (1.06 g,
10 mmol) affording the white product 3c by following the pro-
cedure as described for 3a (refluxing for 4 h). Yield: 1.14 g
(76%); m. p.: 172–1738C (rectified spirit); Rf: 0.7 (acetonitrile/
methanol 1:1); IR (KBr), in cmꢀ1: 3120–3221 (Ar-OH, NH),
Chemicals were obtained from Fluka Chemical Co. (Germany).
Melting points (m. p.) were detected with open capillaries using
Thermonik Precision Melting point cum Boiling point apparatus
(C-PMB-2, Mumbai, India) and are uncorrected. IR spectra (KBr)
were recorded on FTIR-8400s spectrophotometer (Shimadzu,
Japan). 1H-NMR spectra were obtained using a Varian EM 390
spectrophotometer (Varian, USA) using CDCl3. All chemical-shift
values were recorded as d (ppm). The purity of compounds was
–
1668–1682 (hydrazide –C O), 1569 (C-Ar stretching), 1510–
–
1582 (–C N); 1H-NMR (DMSO-d6) d: 2.09 (s, 1H, CH3), 4.27 (s,
1H, NCH2), 4.32 (s, 1H, NHNH2), 5.31 (s, 1H, Ar-OH), 8.42 (s, 1H,
–
–
controlled by thin layer chromatography (silica gel, HF254-361
,
–
CH-N),/11.95 (d, 2H, CONHN CH), 6.1–7.5 (m, 4H, Ar-H); EI-MS
–
type 60, 0.25 mm, Merck, Darmstadt, Germany). Elementary
analysis was performed at RTM Nagpur University, India.
Elementary analyses for C. H, N were within ꢁ0.4% of theoretical
values.
Specific examples presented below illustrate the general syn-
thetic procedures.
(m/z, 100%): 240 [M þ 2] (100). Anal. calcd. for C14H12N2O2: C,
69.96; H, 5.02; N, 11.65. Found: C, 69.99; H, 5.03; N, 11.66.
Compound 3d
A solution of the corresponding compound 2 (1.5 g, 10 mmol) in
ethanol (40 mL) was refluxed with 4-nitrobenzaldehyde (1.6 g,
10 mmol) afforded the yellowish product 3d by following the
procedure as described for 3a (refluxing for 5 h). Yield: 1.0 g
(67%); m. p.: 189–1908C (rectified spirit); Rf: 0.7 (acetonitrile/
methanol 1:1); IR (KBr), in cmꢀ1: 3180–3230 (Ar-/OH, NH),
Synthesis of 2
A mixture of compound 1 (1.5 g, 0.02 mol) and hydrazine
hydrate (80%) (4.12 mL, 0.08 mol) was refluxed for 12 h. The
excess solvent was removed under reduced pressure and the
reaction mixture was cooled to 4–58C. The solid crystals which
separated were filtered, washed with cold water, dried, and
–
1668–1682 (hydrazide –C O), 1487–1555 (C-NO stretching),
2
–
1578 (C-Ar stretching), 1520–1582 (–C N); 1H-NMR (DMSO-d6)
–
–
d: 2.09 (s, 3H, CH3), 4.27 (s, 1H, NCH2), 5.29 (s, 1H, Ar-OH), 8.22
recrystallized from ethanol to obtain
a white product 2
–
(s, 1H, CH-N), 11.78 (d, 2H, CONHN CH); EI-MS (m/z, 100%): 285
–
(1.4 g, m. p.: 172–1738C). Yield: 1.2 g (80%); m. p.: 170–1718C
(ethanol/water); Rf: 0.62 (acetonitrile/methanol 1:1); IR (KBr), in
cmꢀ1: 3351 (alcohol O-H & C-O stretching), 3013 (Ar-H stretching),
1622 (C-O stretching), 1185, 1034 (alcohol O-H stretching), 832
(benzene 1,4 disubstituted); 1H-NMR (300 MHz, DMSO-d6) d: 9.5 (s,
1H, CONH), 5.32 (s, 2H, NH2); EI-MS (m/z, 100%): 152 [M þ 2] (100).
Anal. calcd. for C7H8N2O2: C, 55.26; H, 5.31; N, 18.40. Found: C,
55.26; H, 5.30; N, 18.46.
[M þ 2] (100). Anal. calcd. for C14H11N3O4: C, 58.96; H, 3.89; N,
14.70. Found: C, 59.96; H, 3.85; N, 14.73.
Compound 3e
A solution of the corresponding compound 2 (1.5 g, 10 mmol) in
ethanol (40 mL) was refluxed with 4-chlorobenzaldehyde (1.4 g,
10 mmol) affording the desired white product 3e by following
the procedure as described for 3a (refluxing for 5 h). Yield: 1.2 g
(81%); m. p.: 165–1668C (rectified spirit); Rf: 0.56 (acetonitrile/
methanol, 1:1); IR (KBr), in cmꢀ1: 3200–3280 (Ar-OH, NH), 1658–
General procedure for synthesis of 3a–3n
A solution of the corresponding compound 2 (1.5 g 10 mmol) in
ethanol (40 mL) was refluxed with various aliphatic/aromatic
aldehydes (10 mmol) for 3 h. The excess of solvent was removed
under reduce pressure. After cooling to room temperature, a
white solid appeared. This crude product was filtered, washed
with diethyl ether, dried, and recrystallized from rectified spirit.
The physical and analytical data of some selected compounds
are given below
–
1684 (hydrazide –C O), 1080–1092 (Ar-Cl stretching), 1578 (C-Ar
–
1
–
stretching), 1520–1582 (–C N); H-NMR (DMSO-d ) d: 4.27 (s, 2H,
–
NCH2), 5.29 (s, 1H, Ar-OH), 8.22 (s, 1H, CH-N), 11.78 (s, 2H,
CONHN CH), 7.35–8.01 (m, 8H, ArH); EI-MS (m/z, 100%): 274.29
6
–
–
[M þ 2] (100). Anal. calcd. for C14H11ClN2O2: C, 61.16; H, 4.09; N,
10.22. Found: C, 61.21; H, 4.04; N, 10.20.
Compound 3f
A solution of the corresponding compound 2 (1.5 g, 10 mmol) in
ethanol (40 mL) was refluxed with 4-fluorobenzaldehyde (1.24 g,
10 mmol) affording the desired product 3f (1.3 g, m. p.: 173–
1748C) by following the procedure as described fort 3a (refluxing
for 6 h). Yield: 1.1 g (76%); m. p.: 173–1748C (rectified sprit); Rf:
0.7 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 3280–3320
–
Compound 3a
A solution of the corresponding compound 2 (1.5 g, 10 mmol) in
ethanol (40 mL) was refluxed with propenaldehyde (0.4 g,
10 mmol) for 3 h. The excess of solvent was removed under
reduce pressure. After cooling to room temperature, a white
solid appeared. This crude product was filtered, washed with
(Ar-OH, NH), 1658–1684 (hydrazide –C O), 3010–3030
–
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128
R. P. Bhole and K. P. Bhusari
Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
1
–
(Ar-CH stretching), 1578 (C-Ar stretching), 1621–1633 (–C N); H-
–
The reaction mixture was heated under reflux for 6 h. It was
concentrated and poured into crush ice. The resultant solid was
filtered, dried, and recrystallized to give the white product 4a.
Yield: 1.15 g (61%); m. p.: 177–1788C (DMF/water); Rf: 0.69 (aceto-
NMR (DMSO-d6) d: 4.12 (s, 2H, NCH2), 4.31 (s, 1H, NHNH2), 5.40 (s,1H,
NH ), 11.04 (s, 2H, CONHN CH), 7.55–8.01 (m, 8H, ArH); EI-MS
(m/z, 100%): 254.31 [M þ 2] (100). Anal. calcd. for C15H14N2O2: C,
–
–
2
70.80; H, 5.51; N, 11.01. Found: C, 70.83; H, 5.52; N, 11.02.
nitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 1189-1290 (C S stretch-
–
–
ing), 1689–1720 (–C O), 1568 (C-Ar stretching); 1H-NMR (DMSO-
–
–
d6) d: 1.31–1.53 (m, 4H, CH-(CH3)2), 2.97 (s, 3H, CH3–N), 5.23 (s, 1H,
Ar-OH), 8.10 (s, 1H, CH-N); EI-MS (m/z, 100%): 280 [M þ 2] (100).
Anal. calcd. for C12H12N2O2S2: C, 50.41; H, 4.21; N, 9.92. Found: C,
51.41; H, 4.31; N, 9.99.
Compound 3h
A solution of the corresponding compound 2 (1.5 g, 10 mmol) in
ethanol (40 mL) was refluxed with 4-methylbenzaldehyde (1.2 g,
10 mmol) affording the desired white product 3h by following
the procedure as described for 3a (refluxing for 7 h). Yield: 1.03 g
(69%); m. p.: 181–1828C (rectified spirit); Rf: 0.68 (acetonitrile/
methanol, 1:1); IR (KBr), in cmꢀ1: 3266–3380 (Ar-OH, NH), 1658–
Compound 4c
To a solution of compound 3c (2.4 g, 0.01 mol) in ethanol
(50 mL), a mixture of potassium hydroxide (0.54 g, 0.01 mol)
in ethanol (10 mL) and carbon disulphide (20 mL) was added.
The reaction mixture was heated under reflux for 8 h. It was
concentrated. Following the procedure described for 4a gave the
white product 4c. Yield: 1.7 g (71%); m. p.: 191–1928C (DMF/
–
1684 (hydrazide –C O), 1578 (C-Ar stretching), 1621–1633
–
(–C N); 1H-NMR (DMSO-d6) d: 4.02 (s, 2H, NCH2), 4.17 (s, 1H,
–
–
NHNH ), 5.45 (s, 1H, Ar-OH), 11.24 (s, 2H, CONHN CH), 7.55–
–
–
2
8.01 (m, 8H, ArH); EI-MS (m/z, 100%): 267.34 [M þ 2] (100). Anal.
calcd. for C14H12N2O3: C, 51.99; H, 3.45; N, 13.04. Found: C, 52.01;
H, 3.43; N, 13.00.
water); Rf: 0.56 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1
:
–
–
1191–1240 (C S stretching), 1680–1710 (–C O), 1577 (C-Ar
–
–
stretching); 1H-NMR (DMSO-d6) d: 5.23 (s, 1H, Ar-OH), 7.0–8.5
(m, 9H, Ar-H); EI-MS (m/z, 100%): 314 [M þ 2] (100). Anal. calcd.
for C15H10N2O2S2: C, 57.28; H, 3.20; N, 8.90. Found: C, 57.31; H,
3.21; N, 8.91.
Compound 3i
A solution of the corresponding compound 2 (1.5 g, 10 mmol) in
ethanol (40 mL) was refluxed with 4-hydroxybenzaldehyde
(1.25 g, 10 mmol) affording the desired whitish product 3i by
following the procedure as described for 3a (refluxing for 6 h).
Yield: 1.09 g (73%); m. p.: 156–1578C (rectified spirit); Rf: 0.65
(acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 3276–3390 (Ar-OH,
NH), 1050–1150 (O-CH3 stretching), 1658–1682 (hydrazide
Compound 4d
To a solution of compound 3d (2.8 g, 0.01 mol) in ethanol
(50 mL), a mixture of potassium hydroxide (0.54 g, 0.01 mol)
in ethanol (10 mL) and carbon disulphide (20 mL) was added.
The reaction mixture was heated under reflux for 7 h. It was
concentrated. Following the procedure described for 4a afforded
the yellowish product 4d. Yield: 1.7 g (62%); m. p.: 237–2388C
(DMF/water); Rf: 0.66 (acetonitrile/methanol, 1:1); IR (KBr), in
–C O), 1568 (C-Ar stretching), 1610 (–C N); 1H-NMR (DMSO-d6)
–
–
–
–
d: 1.21 (t, 3H, OCH3-C6H4), 5.27 (s, 1H, Ar-OH), 8.22 (s, 1H,
–
CH-N), 11.78 (2H, CONHN CH); EI-MS (m/z, 100%): 270 [M þ 2]
–
(100). Anal. calcd. for C15H14N2O3: C, 66.61; H, 5.22; N, 10.30.
Found: C, 66.66; H, 5.22; N, 10.36.
cmꢀ1: 1191–1240 (C S stretching), 1680–1720 (–C O), 1487
–
–
–
–
(Ar-NO2 stretching), 1577 (C-Ar stretching); 1H-NMR (DMSO-d6)
d: 5.33 (s, 1H, Ar-OH), 6.88–7.86 (m, 4H, Ar-H1) 8.09–8.33 (m,
4H, Ar-H2); EI-MS (m/z, 100%): 359 [M þ 2] (100). Anal. calcd.
for C15H9N3O4S2: C, 50.11; H, 2.41; N, 11.57. Found: C, 50.13;
H, 2.52; N, 11.69.
Compound 3j
A solution of the corresponding compound 2 (1.5 g, 10 mmol) in
ethanol (40 mL) was refluxed with 4-methoxybenzaldehyde
(1.36 g, 10 mmol) affording the desired white product 3j by
following the procedure as described for 3a (refluxing for 6 h).
Yield: 0.96 g (64%); m. p.: 165–1668C (rectified spirit); Rf: 0.65
(acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 3276–3390 (Ar-OH,
Compound 4e
–
NH), 1654–1684 (hydrazide –C O), 1568 (C-Ar stretching), 1610
–
To a solution of compound 3e (2.7 g, 0.01 mol) in ethanol
(50 mL), a mixture of potassium hydroxide (0.54 g, 0.01 mol)
in ethanol (10 mL) and carbon disulphide (20 mL) was added.
The reaction mixture was heated under reflux for 8 h. It was
concentrated. Following the procedure described for 4a afforded
the white product 4e. Yield: 2.2 g (80%); m. p.: 237–2388C (DMF/
1
–
(–C N); H-NMR (DMSO-d ) d: 2.35 (d, 6H, CH ), 4.22 (s, N-CH), 8.10
–
(s, 1H, CH-N), 11.97 (2H, CONHN CH); EI-MS (m/z, 100%): 283
6
3
–
–
[M þ 2] (100). Anal. calcd. for C16H17N3O2: C, 67.81; H, 6.02; N,
14.81. Found: C, 67.83; H, 6.05; N, 14.83.
water); Rf: 0.59 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1
:
1191–1240 (C S stretching), 1092–1100 (Ar-Cl stretching), 1680–
General procedure for synthesis of compounds 4a–4n
To a mixture of the corresponding compounds 3a–3n (0.01 mol)
in ethanol (50 mL), a solution of potassium hydroxide (0.01 mol)
in ethanol (10 mL) was added followed by carbon disulphide
(20 mL). The reaction mixture was heated under refluxed for
6 h. It was concentrated and poured into crush ice. The resultant
solid was filtered, dried, and recrystallized from a mixture of
DMF and water (1:1).
–
–
1
–
–
1720 (–C O), 1577 (C-Ar stretching); H-NMR (DMSO-d ) d: 5.22 (s,
6
1H, Ar-OH), 6.88–7.78 (m, 4H, Ar-H1), 7.52–7.77 (m, 4H, Ar-H2); EI-
MS (m/z, 100%): 348 [M þ 2] (100). Anal. calcd. for C15H9ClN2O2S2:
C, 51.61; H, 2.53; N, 9.96. Found: C, 51.65; H, 2.60; N, 10.16.
Compound 4h
To a solution of compound 3h (2.5 g, 0.01 mol) in ethanol
(50 mL), a mixture of potassium hydroxide (0.54 g, 0.01 mol)
in ethanol (10 mL) and carbon disulphide (20 mL) was added.
The reaction mixture was heated under reflux for 8 h. It was
concentrated. Following the procedure described for 4a afforded
Compound 4a
To a solution of compound 3a (1.9 g, 0.01 mol) in ethanol
(50 mL), a mixture of potassium hydroxide (0.54 g, 0.01 mol)
in ethanol (10 mL) and carbon disulphide (20 mL) was added.
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Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
New p-Hydroxybenzohydrazide Derivatives
129
the whitish product 4h. Yield: 2.1 g (79%); m. p.: 212–2138C (DMF/
Compound 5a
water); Rf: 0.67 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1
:
To a solution of compound 2 (1.5 g 0.01 mol) in ethanol (50 mL),
isopropyl isothiocyanate (1.01 g, 0.01 mol) was added. The reac-
tion mixture was refluxed for 12 h. Excess solvent was removed
under vacuum. The residue was washed with diethyl ether and
recrystallized to obtain the white product 5a. Yield: 1.0 g
(70%); m. p.: 225–2268C (methanol); Rf: 0.67 (acetonitrile/meth-
anol, 1:1); IR (KBr), in cmꢀ1: 3213, 3229 (NH stretching), 2986,
–
–
1191–1240 (C S stretching), 1670–1730 (–C O), 1577 (C-Ar
–
–
stretching); 1H-NMR (DMSO-d6) d: 2.2 (s, 3H, CH3), 5.12 (s, 1H,
Ar-OH), 6.88–7.78 (m, 4H, Ar-H1), 7.21–7.71 (m, 4-H, Ar-H); EI-MS
(m/z, 100%): 328 [M þ 2] (100). Anal. calcd. for C16H12N2O2S2: C,
58.51; H, 3.50; N, 8.49. Found: C, 58.52; H, 3.68; N, 8.53.
–
–
2990 (CH(CH3)2 stretching), 1732 (C O stretching), 1315 (C S
Compound 4i
–
–
stretching); 1H-NMR (CDCl3) d: 1.2 (d, 6H, isopropyl CH3), 4.00
(m, 1H, isopropyl CH), 7.74 (s, 1H, CONH), 7.2–7.78 (m, 4H, Ar-H),
5.21 (s,1H, -OH); EI-MS (m/z, 100%): 253 [M þ 2] (100). Anal. calcd.
for C11H15N3O2S: C, 52.13; H, 5.98; N, 16.59. Found: C, 52.15; H,
5.97; N, 16.59.
To a solution of compound 3i (2.5 g, 0.01 mol) in ethanol
(50 mL), a mixture of potassium hydroxide (0.54 g, 0.01 mol)
in ethanol (10 mL) and carbon disulphide (20 mL) was added.
The reaction mixture was heated under reflux for 7 h. It was
concentrated. Following the procedure described for 4a, afforded
the whitish product 4i. Yield: 1.9 g (79%); m. p.: 218–2198C (DMF/
water); Rf: 0.77 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1
:
1191–1240 (C S stretching), 1670–1730 (–C O), 3600–3400 (Ar-
Compound 5b
–
–
–
–
To a solution of compound 2 (1.5 g 0.01 mol) in ethanol (50 mL),
n-butyl isothiocyanate (1.15 g, 0.01 mol) was added. The reaction
mixture was refluxed for 12 h. Following the procedure as
described for 5a afforded the white product 5b. Yield: 1.1 g
(74%); m. p.: 168–1698C (methanol); Rf: 0.66 (acetonitrile/meth-
1
OH), 1577 (C-Ar stretching); H-NMR (DMSO-d6) d: 5.35 (s, 1H, Ar-
OH), 6.88–7.78 (m, 4H, Ar-H), 7.01–7.85 (m, 4H, Ar-H);
EI-MS (m/z, 100%): 330 [M þ 2] (100). Anal. calcd. for
C15H10N2O3S2: C, 54.51; H, 3.02; N, 8.39. Found: C, 54.53; H,
3.05; N, 8.48.
anol, 1:1); IR (KBr) [cmꢀ1]: 3213, 3224 (NH stretching), 1731 (C O
–
–
1
–
–
stretching), 1316 (C S stretching); H-NMR (CDCl ) d: 0.95 (t, 3H,
3
butyl CH3), 1.28 (m, 2H, butyl CH2), 1.51 (m, 2H, butyl CH2), 3.53
(m, 2H, butyl CH2), 7.73 (s, 1H, CONH), 6.8–7.78 (m, 4H, Ar-H), 5.3
(s, 1H, -OH); EI-MS (m/z, 100%): 267 [M þ 2] (100). Anal. calcd.
for C12H17N3O2S: C, 53.83; H, 6.41; N, 15.69. Found: C, 53.91; H,
6.41; N, 15.72.
Compound 4j
To a solution of compound 3j (2.7 g, 0.01 mol) in ethanol
(50 mL), a mixture of potassium hydroxide (0.54 g, 0.01 mol)
in ethanol (10 mL) and carbon disulphide (20 mL) was added.
The reaction mixture was heated under reflux for 6 h. It was
concentrated. Following the procedure described for 4a afforded
the whitish product 4j. Yield: 1.9 g (74%); m. p.: 202–2038C (DMF/
water); Rf: 0.67 (acetonitrile/methanol, 1:1); IR (KBr) [cmꢀ1]: 1191–
Compound 5c
To a solution of compound 2 (1.5 g, 0.01 mol) in ethanol (50 mL),
phenyl isothiocyanate (1.35 g, 0.01 mol) was added. The reaction
mixture was refluxed for 12 h. Following the procedure as
described for 5a afforded the white product 5c. Yield: 1.2 g
(82%); m. p.: 175–1768C (methanol); Rf: 0.66 (acetonitrile/meth-
–
–
–
1240 (C S stretching), 1670–1730 (–C O), 3600–3400 (Ar-OH),
–
1577 (C-Ar stretching); 1H-NMR (DMSO-d6) d: 1.2–1.31 (s, 3H,
OCH3), 5.26 (s, 1H, Ar-OH), 6.88–7.78 (m, 4H, Ar-H1), 7.10–7.71
(m, 4H, Ar-H2); EI-MS (m/z, 100%): 343 [M þ 2] (100). Anal. calcd.
for C16H12N2O3S2: C, 55.72; H, 3.50; N, 8.10. Found: C, 55.80; H,
3.51; N, 8.13.
anol, 1:1); IR (KBr), in cmꢀ1: 3217, 3232 (NH), 1738 (C O stretch-
–
–
1
–
–
ing), 1313 (C S stretching); H-NMR (CDCl ) d: 5.35 (s, 1H, Ar-OH),
3
6.8–7.62 (m, 9H, Ar-H), 7.75 (s, 1H, CONH); EI-MS (m/z, 100%): 287
[M þ 2] (100). Anal. calcd. for C14H13N3O2S: C, 58.53; H, 4.51; N,
14.59. Found: C, 58.20; H, 4.56; N, 14.62.
Compound 4m
To a solution of compound 3m (2.8 g, 0.01 mol) in ethanol
(50 mL), a mixture of potassium hydroxide (0.54 g, 0.01 mol)
in ethanol (10 mL) and carbon disulphide (20 mL) was added.
The reaction mixture was heated under reflux for 10 h. It was
concentrated. Following the procedure described for 4a afforded
the white product 4m. Yield: 2.2 g (74%); m. p.: 209–2108C (DMF/
Compound 5d
To a solution of compound 2 (1.5 g, 0.01 mol) in ethanol (50 mL),
4-nitrophenyl isothiocyanate (1.8 g, 0.01 mol) was added. The
reaction mixture was refluxed for 10 h. Following the procedure
as described for 5a afforded the yellowish product 5d. Yield: 0.8 g
(56%); m. p.: 189–1908C (methanol); Rf: 0.66 (acetonitrile/meth-
water); Rf: 0.56 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1
:
1181–1230 (C S stretching), 1680–1740 (–C O), 3600–3400 (Ar-
–
–
–
–
1
anol, 1:1); IR (KBr), in cmꢀ1: 3215, 3230 (NH), 1731 (C O stretch-
–
OH), 1577 (C-Ar stretching); H-NMR (DMSO-d6) d: 4.2–4.4 (s, 6H,
N(CH3)2), 7.2–7.78 (m, 4H, Ar-H1), 7.65–7.8 (m, 4H, Ar-H2), 5.21 (s,
1H, -OH); EI-MS (m/z, 100%): 357 [M þ 2] (100). Anal. calcd.
for C17H15N3O2S2: C, 57.11; H, 4.20; N, 11.74. Found: C, 57.12;
H, 4.23; N, 11.76.
–
1
–
–
ing), 1315 (C S stretching); H-NMR (CDCl ) d: 5.33 (s, 1H, Ar-OH),
3
6.88–7.82 (m, 8H, ArH), 7.78 (s, 1H, CONH), 10.40 (s, 1H, NH), 11.81
(s, 1H, NHAr); EI-MS (m/z, 100%): 322 [M þ 2] (100). Anal. calcd.
for C14H12N4O4S: C, 50.59; H, 3.64; N, 16.83. Found: C, 50.60; H,
3.64; N, 16.86.
General procedure for synthesis of 5a–5i
To a solution of 2 (0.01 mol) in ethanol (50 mL), various aliphatic/
aromatic isothiocyanates (0.01 mol) were added. The reaction
mixture was refluxed for 12 h. Excess solvent was removed under
vacuum. The residue was washed with diethyl ether and recrys-
tallized using methanol.
Compound 5e
To a solution of compound 2 (1.5 g, 0.01 mol) in ethanol (50 mL),
4-fluorophenyl isothiocyanate (1.5 g, 0.01 mol) was added. The
reaction mixture was refluxed for 11 h. Following the procedure
as described in 5a afforded the white product 5e. Yield: 1.2 g
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130
R. P. Bhole and K. P. Bhusari
Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
(82%), m. p.: 192–1938C (methanol); Rf: 0.7 (acetonitrile/metha-
General procedure for synthesis of compound 6a–6i
A mixture of the thiosemicarbazide (0.01 mol), chloroacetic acid
(0.01 mol), and sodium acetate (0.2 mol) in ethanol (60 mL) was
refluxed for 10 h. The mixture was cooled, diluted with water till
turbidity, and left overnight to obtain the product. The product
was filtered, dried, and recrystallized using aqueous ethanol.
nol, 1:1); IR (KBr), in cmꢀ1: 3217, 3232 (NH), 1738 (C O stretch-
–
–
1
–
–
ing), 1313 (C S stretching); H-NMR (CDCl ) d: 5.35(s, 1H, Ar-OH),
3
6.78–7.82 (m, 8H, ArH), 7.78 (s, 1H, CONH), 10.40 (s, 1H, NH), 11.81
(s, 1H, NHAr); EI-MS (m/z, 100%): 305 [M þ 2] (100). Anal. calcd.
for C14H12FN3O2S: C, 55.07; H, 3.94; N, 13.74. Found: C, 55.07; H,
3.96; N, 13.76.
Compound 6a
Compound 5f
A mixture of compound 5a (2.57 g, 0.01 mol), chloroacetic acid
(0.01 mol), and sodium acetate (0.2 mol) in ethanol (60 mL) was
heated under reflux for 10 h. The mixture was cooled and diluted
with enough water to develop turbidity. The mixture was left
overnight for complete separation of the product. The separated
product was filtered and dried to give the corresponding whitish
product 6a. Yield: 2.21 g (86%); m. p.: 225–2268C (methanol); Rf:
0.55 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 3212 (NH
To a solution of compound 2 (1.5 g, 0.01 mol) in ethanol (50 mL),
2,4-dichlorophenyl isothiocyanate (2.0 g, 0.01 mol) was added.
The reaction mixture was refluxed for 12 h. Following the pro-
cedure as described in 5a, afforded the white product 5f. Yield:
1.1 g (79%); m. p.: 202–2038C (methanol); Rf: 0.62 (acetonitrile/
methanol, 1:1); IR (KBr), in cmꢀ1: 3212, 3235 (NH), 1734 (C O
–
–
1
–
stretching), 700–750 (C-Cl stretching), 1317 (C S stretching); H-
–
NMR (CDCl3) d: 5.22 (s, 1H, Ar-OH), 6.71–7.54 (m, 7H, ArH), 7.74 (s,
1H, CONH), 0.43 (s, 1H, NH), 11.82 (s, 1H, NHAr); EI-MS (m/z, 100%):
356 [M þ 2] (100). Anal. calcd. for C14H11Cl2N3O2S: C, 48.50; H,
2.81; N, 10.59. Found: C, 48.50; H, 2.80; N, 10.60.
–
stretching), 2986, 2990 (CH(CH ) stretching), 1732 (C O stretch-
3 2
–
ing), 1315 (C S stretching); 1H-NMR (CDCl3) d: 1.12 (d, 6H, iso-
–
–
propyl CH3), 3.96 (m, 1H, isopropyl CH), 5.31 (s, 1H, Ar-OH), 3.01
(s, 2H, thiazolidine CH2), 7.74 (s, 1H, CONH); EI-MS (m/z, 100%):
293 [M þ 2] (100). Anal. calcd. for C13H15N3O3S: C, 53.21; H, 5.15;
N, 14.30. Found: C, 53.23; H, 5.15; N, 14.32.
Compound 5g
To a solution of compound 2 (1.5 g, 0.01 mol) in ethanol (50 mL),
2,6-diflurophenyl isothiocyanate (1.7 g, 0.01 mol) was added. The
reaction mixture was refluxed for 12 h. Following the procedure
as described for 5a, afforded the white product 5g. Yield: 1.3 g
(87%); m. p.: 212–2138C (methanol); Rf: 0.56 (acetonitrile/meth-
Compound 6b
A mixture of compound 5b (2.67 g, 0.01 mol), chloroacetic acid
(0.01 mol) and sodium acetate (0.2 mol) in ethanol (60 mL) was
heated under reflux for 10 h. The mixture was cooled and diluted
with enough water to develop turbidity. The mixture was left
overnight for complete separation of the product. The separated
product was filtered and dried to give the corresponding whitish
product 6b. Yield: 2.2 g (85%); m. p.: 206–2078C (methanol); Rf:
anol, 1:1); IR (KBr), in cmꢀ1: 3217, 3232 (NH), 1738 (C O stretch-
–
–
1
–
–
ing), 1313 (C S stretching); H-NMR (CDCl ) d: 5.35 (s, 1H, Ar-OH),
3
6.78–7.82 (m, 8H, ArH), 7.68 (s, 1H, CONH), 10.40 (s, 1H, NH), 11.81
(s, 1H, NHAr); EI-MS (m/z, 100%): 323 [M þ 2] (100). Anal. calcd.
for C14H11F2N3O2S: C, 52.01; H, 3.39; N, 13.00. Found: C, 52.01; H,
3.43; N, 13.00.
0.59 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 1732 (C O
–
–
1
stretching), 3212 (NH stretching); H-NMR (CDCl3) d: 0.95 (t, 3H,
butyl CH3), 1.28 (m, 2H, butyl CH2), 1.51 (m, 2H, butyl CH2), 3.53
(m, 2H, butyl CH2), 3.01 (s, 2H, thiazolidine CH2), 4.72 (s, 1H, NH),
5.35 (s, 1H, Ar-OH), 7.73 (s, 1H, CONH); EI-MS (m/z, 100%): 307
[M þ 2] (100). Anal. calcd. for C14H17N3O3S: C, 53.91; H, 6.35; N,
15.62. Found: C, 53.91; H, 6.41; N, 15.72.
Compound 5h
To a solution of compound 2 (1.5 g, 0.01 mol) in ethanol (50 mL),
2,6-dimethylphenyl isothiocyanate (1.6 g, 0.01 mol) was added.
The reaction mixture was refluxed for 13 h. Following the pro-
cedure as described in 5a afforded the pale white product 5h.
Yield: 1.0 g (69%); m. p.: 198–1998C (methanol); Rf: 0.66 (aceto-
Compound 6c
nitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 3010–3030 (Ar-CH), 3217,
A mixture of compound 5c (2.8 g, 0.01 mol), chloroacetic acid
(0.01 mol), and sodium acetate (0.2 mol) in ethanol (60 mL) was
heated under reflux for 10 h. The mixture was cooled and diluted
with enough water to develop turbidity. The mixture was left
overnight for complete separation of the product. The separated
product was filtered and dried to give the corresponding whitish
product 6c. Yield: 2.1 g (84%); m. p.: 256–2578C (methanol); Rf:
1
3232 (NH), 1738 (C O stretching), 1313 (C S stretching); H-NMR
–
–
–
–
(CDCl3) d: 5.30 (s, 1H, Ar-OH), 2.11 (d, 2H, CH3), 6.78–7.82 (m, 7H,
ArH), 7.66 (s, 1H, CONH), 10.44 (s, 1H, NH), 11.80 (s, 1H, NHAr); EI-
MS (m/z, 100%): 316 [M þ 2] (100%). Anal. C16H17N3O2S; C, 60.91/
60.93; H, 5.39/5.43; N, 13.32/13.32.
0.66 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 1732 (C O
–
–
Compound 5i
1
stretching), 3218 (NH stretching); H-NMR (CDCl3) d: 3.04 (s, 2H,
To a solution of compound 2 (1.5 g, 0.01 mol) in ethanol (50 mL),
2,6-dimethylphenyl isothiocyanate (1.9 g, 0.01 mol) was added.
The reaction mixture was refluxed for 12 h. Following the
procedure as described for 5a afforded the yellowish product
5i. Yield: 1.12 g (75%); m. p.: 203–2048C (methanol); Rf: 0.57
(acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 3219, 3230 (NH),
thiazolidine CH2), 4.72 (s, 1H, NH), 5.35 (s, 1H, Ar-OH), 7.73 (s, 1H,
CONH), 6.38–7.85 (m, 9H, Ar-H); EI-MS (m/z, 100%): 327 [M þ 2]
(100). Anal. calcd. for C16H13N3O3S: C, 58.51; H, 4.55; N, 14.52.
Found: C, 58.52; H, 4.56; N, 14.62.
1732 (C O stretching), 1310 (C S stretching); 1H-NMR
–
–
–
–
Compound 6d
(CDCl3) d: 5.31 (s, 1H, Ar-OH), 3.83 (d, 2H, OCH3), 6.30–7.82 (m,
7H, ArH), 7.69 (s, 1H, CONH), 10.40 (s, 1H, NH), 11.80 (s, 1H, NHAr);
EI-MS (m/z, 100%): 347 [M þ 2] (100). Anal. calcd. for
C16H17N3O4S: C, 55.31; H, 4.90; N, 12.08. Found: C, 55.32; H,
4.93; N, 12.10.
A mixture of compound 5d (3.2 g, 0.01 mol), chloroacetic acid
(0.01 mol), and sodium acetate (0.2 mol) in ethanol (60 mL) was
heated under reflux for 10 h. The mixture was cooled and diluted
with enough water to develop turbidity. The mixture was left
overnight for complete separation of the product. The separated
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Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
New p-Hydroxybenzohydrazide Derivatives
131
product was filtered and dried to give the corresponding yellow-
ish product 6d. Yield: 1.6 g (52%); m. p.: 241–2428C (methanol); Rf:
0.56 (acetonitrile/methanol, 1:1), IR (KBr), in cmꢀ1: 3214 (NH
overnight for complete separation of the product. The separated
product was filtered and dried to give the corresponding whitish
product 6h. Yield: 2.6 g (87%); m. p.: 217–2188C (methanol); Rf:
0.61 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 3280 (NH
–
stretching), 1731 (C O stretching), 1555 (NO2 stretching);
–
1H -NMR (CDCl3) d: 3.98 (s, 2H, thiazolidine CH2), 4.60 (s, 1H,
NH), 5.23 (s, 1H, Ar-OH), 7.70 (s, 1H, CONH), 6.38–7.85 (m, 8H, Ar-
H); EI-MS (m/z, 100%): 372 [M þ 2] (100). Anal. calcd.
for C16H12N4O5S: C, 50.51; H, 3.55; N, 16.82. Found: C, 50.60;
H, 3.64; N, 16.86.
stretching), 1773 (C O stretching); 1H-NMR (CDCl3) d: 1.47 (d,
–
–
3H, CH3), 3.16 (s, 2H, thiazolidine CH2), 4.26 (s, 1H, NH), 5.34 (s,
1H, Ar-OH), 7.62 (s, 1H, CONH), 6.48–7.85 (m, 7H, Ar-H); EI-MS (m/z,
100%): 355 [M þ 2] (100). Anal. calcd. for C18H17N3O3S: C, 60.91;
H, 5.43; N, 13.30. Found: C, 60.93; H, 5.43; N, 13.32.
Compound 6e
Compound 6i
A mixture of compound 5e (3.0 g, 0.01 mol), chloroacetic acid
(0.01 mol), and sodium acetate (0.2 mol) in ethanol (60 mL) was
heated under reflux for 10 h. The mixture was cooled and diluted
with enough water to develop turbidity. The mixture was left
overnight for complete separation of the product. The separated
product was filtered and dried to give the corresponding whitish
product 6e. Yield: 2.7 g (89%); m. p.: 223–2248C (methanol); Rf: 0.7
A mixture of compound 5i (3.4 g, 0.01 mol), chloroacetic acid
(0.01 mol), and sodium acetate (0.2 mol) in ethanol (60 mL) was
heated under reflux for 10 h. The mixture was cooled and diluted
with enough water to develop turbidity. The mixture was left
overnight for complete separation of the product. The separated
product was filtered and dried to give the corresponding whitish
product 6i. Yield: 2.6 g (84%); m. p.: 203–2048C (methanol); Rf:
0.61 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 3280 (NH
(acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 3220 (NH stretch-
1
ing), 1792 (C O stretching), 1045 (C-F stretching); H-NMR (CDCl )
stretching), 1773 (C O stretching); 1H-NMR (CDCl3) d: 3.73 (s,
–
–
–
–
3
d: 3.76 (s, 2H, thiazolidine CH2), 4.36 (s, 1H, NH), 5.3 (s, 1H, Ar-OH),
8.04 (s, 1H, CONH), 6.38–7.85 (m, 8H, Ar-H); EI-MS (m/z, 100%): 372
[M þ 2] (100). Anal. calcd. for C16H12FN3O3S: C, 55.05; H, 3.95; N,
13.78. Found: C, 55.07; H, 3.96; N, 13.76.
6H, OCH3), 3.76 (s, 2H, thiazolidine CH2), 4.16 (s, 1H, NH), 5.41
(s, 1H, Ar-OH), 7.82 (s, 1H, CONH), 6.48–7.85 (m, 7H, Ar-H); EI-MS
(m/z, 100%): 387 [M þ 2] (100). Anal. calcd. for C18H17N3O5S: C,
55.31; H, 4.90; N, 12.06. Found: C, 55.32; H, 4.93; N, 12.10.
Compound 6f
Pharmacological studies
A mixture of compound 5f (3.5 g, 0.01 mol), chloroacetic acid
(0.01 mol), and sodium acetate (0.2 mol) in ethanol (60 mL) was
heated under reflux for 10 h. The mixture was cooled and diluted
with enough water to develop turbidity. The mixture was left
overnight for complete separation of the product. The separated
product was filtered and dried to give the corresponding whitish
product 6f. Yield: 2.6 g (75%); m. p.: 215–2168C (methanol); Rf:
0.56 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 3220 (NH
Antihypertensive activity by non-invasive tail-cuff method
The antihypertensive drugs were evaluated by studying their
response on elevated blood pressure. A drug that reduces the
elevated BP (blood pressure) to normal is said to be antihyper-
tensive drug.
The newly synthesized compounds 4a–4n (see Scheme 1) and
6a–6i (Scheme 2) were tested for their antihypertensive activity.
A Norwegian strain of inbreed albino rats weighing 200–250 g
was used. (The animals were purchased from the National
Toxicological Centre, Pune, India.) All rats were housed in a
temperature- (258C) and humidity-controlled room (75 ꢁ 5%)
with a 12-h light/dark cycle. Valsertan was used as standard drug.
stretching), 1743 (C O stretching); 1H-NMR (CDCl3) d: 3.76 (s,
–
–
2H, thiazolidine CH2), 4.30 (s, 1H, NH), 5.28 (s, 1H, Ar-OH),
7.82 (s, 1H, CONH), 6.38–7.85 (m, 7H, Ar-H); EI-MS (m/z, 100%):
396 [M þ 2] (100). Anal. calcd. for C16H11Cl2N3O3S: C, 47.21; H,
3.11; N, 11.76. Found: C, 47.20; H, 3.11; N, 11.80.
Induction of hypertension: fructose-induced hypertension
Groups of eight male Wistar rats weighing 210–250 g were used
(purchased from National Toxicological Centre, Pune, India).
Rats were housed two per cage on a 12-h light and 12-h dark
cycle and were allowed free access to standard laboratory diet
(Purina rat chow) and drinking fluid. Drinking fluid consisted of
a fructose solution (10%). Body weight, food and fluid intake of
each rat were measured every week during the treatment. By
using the tail-cuff method, systolic blood pressure and pulse rate
was measured before and every week during treatment.
Compound 6g
A mixture of compound 5g (3.2 g, 0.01 mol), chloroacetic acid
(0.01 mol), and sodium acetate (0.2 mol) in ethanol (60 mL) was
heated under reflux for 10 h. The mixture was cooled and diluted
with enough water to develop turbidity. The mixture was left
overnight for complete separation of the product. The seprated
product was filtered and dried to give the corresponding whitish
product 6g. Yield: 2.4 g (78%); m. p.: 219–2208C (methanol); Rf:
0.56 (acetonitrile/methanol, 1:1); IR (KBr), in cmꢀ1: 3280 (NH
stretching), 1773 (C O stretching); 1H-NMR (CDCl3) d: 3.06 (s,
–
–
2H, thiazolidine CH2), 4.36 (s, 1H, NH), 5.23 (s, 1H, Ar-OH),
7.82 (s, 1H, CONH), 6.38–7.85 (m, 7H, Ar-H); EI-MS (m/z, 100%):
363 [M þ 2] (100). Anal. calcd. for C16H11F2N3O3S: C, 52.01; H,
3.41; N, 13.00. Found: C, 52.01; H, 3.43; N, 13.00.
Evaluation of hypertension
Since maximum effects on the chosen parameters were achieved
after 4–6 weeks, the duration of the treatment could get limited
to this time. Statistical analysis was performed using a one-way
analysis of variance, followed by the Newman–Keuls test.
The experimental work was carried out in accordance with the
guidelines provided by the Committee for the Purpose of Control
and Supervision of Experiments in Animal (CPCSEA)
The newly synthesized compounds 4a–4n (Scheme 1) and
6a–6i (Scheme 2) were used to study antihypertensive activity.
Compound 6h
A mixture of compound 5h (3.1 g, 0.01 mol), chloroacetic acid
(0.01 mol), and sodium acetate (0.2 mol) in ethanol (60 mL) was
heated under reflux for 10 h. The mixture was cooled and diluted
with enough water to develop turbidity. The mixture was left
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132
R. P. Bhole and K. P. Bhusari
Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
25 groups for NIBP and 7 groups for IBP, consisting of six rats
each of Norwegian strain of inbreed albino rats (male), weighing
200–250 g, were used. Valsartan was used as standard drug. All
rats were housed in a temperature- and humidity-controlled
room with a 12-h light/dark cycle. Antihypertensive activity
was tested via two methods viz. non-invasive blood pressure
measurement (NIBP), which measures the systolic blood pres-
sure, and invasive blood pressure measurement (IBP), which
measures the diastolic blood pressure. Only those compounds
which had demonstrated a significant activity in the NIBP were
further tested by IBP method.
Five compounds, i. e. 4g, 4j, 4l, 6f, and 6i, had shown significant
activity by non-invasive blood pressure measurement (tail-cuff
method); they were further evaluated for their antihypertensive
activity by invasive blood pressure measurement (IBP, direct
cannulation of the carotid artery). Antihypertensive activity,
carried out by the cannulation method gave the diastolic blood
pressure (DBP). The data are presented as means ꢁ S.E.M. A
repeated measures analysis of variance was used to obtain the
statistical significance between and within groups. The differ-
ences were considered statistically significant at P < 0.05, and
the F value for all compounds was F: ꢁ0.5.
Non-invasive blood pressure (NIBP) measurement
Indirect (non-invasive) blood pressure was determined with
a Power Lab/4SP with ML135 Dual Bio Amp and computerized
BP monitor (AD instruments Pvt. Ltd., Australia). This system
measures the systolic blood pressure (SBP) by recording the
cuff pressure at which the interrupted blood flow returns to
the tail.
Hypertension was induced by an increase of fructose in the
animals’ diet. Rats were trained for the experiment. On the first
day of the experiment, the test compounds, i. e. 4a–4n and 6a–6i,
were administered by oral feeding using an oral feeding needle.
The test compounds and standard drug valsertan were prepared
in carboxymethyl cellulose solution (5%) and given orally (dose
10 mg/kg). One group of animals was treated with standard drug
valsartan. One out of the twenty-five groups was treated with
vehicle.
Invasive blood pressure (IBP) measurements
Blood pressure measurements were performed by direct cannu-
lation of the carotid artery. The mean arterial blood pressure
(MAP) was measured by cannulation technique employing a
Power Lab/4SP with ML135 Dual Bio Amp and computerized
BP monitor (AD instruments Pvt. Ltd., Australia).
Animals were anesthetized with sodium pentobarbital
(35 mg/kg, i.p.), and the left carotid artery was exposed, cannu-
lated, and exteriorized between the scapulas. Blood pressure
was measured directly from cannula using transducers attach-
ments of the above instrument. After the animals recovered
from surgery, a base-line blood pressure was established.
They were dosed orally with the test compounds via a feeding
needle. Acute effects were determined by monitoring the blood
pressure at 15, 30, 60, 120, 180, and 240 min after oral dosing.
Average readings were calculated by employing the ANOVA
method.
The tail pulse was detected by passage of the tail through a
narrow tail-cuff sensor attached to the amplifier. BP measure-
ments were started by automatic inflation of the tail-cuff to
greater than 200 mmHg and then release of pressure. The results
were recorded in form of a graph. The computer was provided
with two tracings: as soon as one had started, the other was
stopped at the same time. The lower trace channel plotted cuff
pressure, which was calibrated at 500 mmHg at full scale. The
tracing was sharply raised when applied to the tail cuff and fell
off gradually during 15 to 20 s during the test. The upper trace
channel monitors the pulse, with fluctuations about the centre
line suddenly appearing at the onset of pulsations. The first onset
of the pulse was taken as the systolic blood pressure. Initiation of
pulse pressure was determined when the baseline amplitude
increased in accordance with the set maximal inflated cuff
pressures. The maximal inflation was set at 200 mmHg.
Blood pressure recording were considered to be successful
when the animal did not move and a clear initial pulse could
be seen. Ten tail-cuff measurements were made in a session. The
BP for the session was accepted as the average of four BP readings
that were within 5 mmHg or the average of ten readings that
were within 8 mmHg. BP measurements were done thrice per
week for two weeks.
3D-QSAR study
Data set for analysis
A data set comprising 23 compounds, i. e. 4a–4n and 6a–6i, were
used in the present study. The in-vivo biological activity data
was reported as IC50. The IC50 values were converted to
pIC50 ¼ –log(IC50).
IC50 ¼ pIC50 ¼ ꢀlogC þ logðitÞ
where
(1)
C ¼ molar concentration ¼ ½concentration ðmg=mLÞ
ꢂ 0:001= molecular weightꢃ
logðitÞ ¼ log½%inhibition=100 ꢀ %inhibitionꢃ:
The data set consists of some highly active and inactive com-
pounds, with few compounds in between them. Table 5
represents pIC50 values with its actual pIC50 values and
PHASE-predicted pIC50 values. A total of 23 compounds was
available with pIC50 values, of which 17 compounds were chosen
for the training set and six compounds were selected for the test
sets based on the Tanimoto similarity coefficient [27].
Prior to dosing the animals, the initial graph reading was
taken to record the BP before administration of the drug.
After 1 h of dosing, recordings were taken as mentioned above.
Each compound was evaluated three times in one week, and
average of the recordings was noted, accordingly.
3D-QSAR
The experiment was repeated at two different dose levels (5
and 2.5 mg/kg) for those compounds that showed significant
statistical differences between test and control groups.
Average readings were calculated by employing the one-way
ANOVA method.
3D-QSAR studies were undertaken by PHASE 3D-QSAR (a high-
performance program for ligand-based drug design [25]) model
workflow using 23 compounds. The following five steps were
performed.
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Arch. Pharm. Chem. Life Sci. 2011, 2, 119–134
New p-Hydroxybenzohydrazide Derivatives
133
pharmacophore provides a hypothesis to explain how the active
molecules bind to the receptor. The scoring procedure provides a
ranking of the different hypotheses, which allows making
rational choices about which hypotheses are most appropriate
for further investigation. Scoring with respect to actives was
conducted using default parameters for site, vector, and volume
terms. Ligand activity, expressed as –log10(IC50), was incorpo-
rated into the score with a weight of 1.0, and relative confor-
mational energy (kJ/mol) was included with a weight of 0.01.
Hypotheses that emerged from this process were subsequently
scored with respect to inactives, using a weight of 1.0. The
inactive molecules were scored to observe the alignment of these
molecules with respect to the pharmacophore hypothesis to
enable making a decision on the selection of the hypothesis.
Larger is the difference between the scores of active and inac-
tives, better is the hypothesis at distinguishing the actives from
inactives. Their results are summarized in Table 6.
Step I: Preparing ligands
The 3D conversion and minimization was performed using
LigPrep (MMFF force field) incorporated in PHASE. A maximum
of 100 conformers were generated per structure using a pre-
process minimization of 100 steps and post-process minimi-
zation of 50 steps. Each minimized conformer was filtered
through a relative energy window of 11.4 kcal/mol (50 kJ/mol)
´
˚
and a minimum atom deviation of 2.00 A.
Step II: Creating pharmacophore sites
The second step in developing a pharmacophore model is to use a
set of pharmacophore features to create sites for all the ligands.
Each ligand structure is represented by a set of points in 3D
space, which coincide with various chemical features that may
facilitate noncovalent binding between the ligand and its target
receptor. PHASE provides a built-in set of six pharmacophore
features, hydrogen bond acceptor (A), hydrogen bond donor (D),
hydrophobic group (H), negatively ionizable (N), positively ion-
izable (P), and aromatic ring (R). The rules that are applied to map
the positions of pharmacophore sites are known as feature
definitions, and they are represented internally by a set of
SMARTS (Smiles ARbitory Target Specification) patterns. Each
pharmacophore feature is defined by a set of chemical structure
patterns. All user-defined patterns are specified as SMARTS
queries and assigned one of the three possible geometries, which
define physical characteristic of the site (i) Point: the site is
located on a single atom in the SMARTS query; (ii) Vector: the
site is located on a single atom in the SMARTS query, and it will
be assigned directionality according to one or more vectors
originating from the atom; (iii) Group: the site is located at
the centroid of a group of atoms in the SMARTS query. For
aromatic rings, the site is assigned directionality defined by a
vector that is normal to the plane of the ring. A default setting
having acceptor (A), donor (D), hydrophobic (H), negative (N),
positive (P), and aromatic ring (R) was used for the creation of
pharmacophore sites. No user-defined feature was employed for
the present study.
Step V: Building QSAR model
Atom-based 3D-QSAR models were generated for the hypotheses
using the 17 compound in the training set with three partial
˚
least square (PLS) factors and a grid spacing of 1 A. The 3D-QSAR
results were visualized using 3D plots of crucial pharmacophore
regions (Fig. 3). As compound 4j showed good activity, the com-
mon pharmacophores were generated for the best PHASE hy-
pothesis with this compound (Fig. 3a). PHASE 3D-plots of crucial
pharmacophore regions based on the hypothesis generated were
displayed with compound 4j. Positive coefficient-favored areas
(contributing for increase in activity) are represented by gray
cubes. Negative coefficient-favored areas (contributing for
decrease in activity) are represented by black cubes. The visual
representations are covered in Figs. 3b to 3d. The summary of
PHASE 3D-QSAR statistical analysis is given in Table 7.
PHASE-QSAR statistics and model validation
The PHASE descriptors served as independent variables and
activity values as dependent variables in deducing 3D-QSAR
models by PLS regression analysis method. PHASE-QSAR models
do not use internal cross-validation techniques but rather uses
distinct training and test sets. The true external test sets assists
in the validation of the 3D-QSAR model and helps in the gener-
ation of validation parameters like q2 and rp values. The predic-
tive ability of each analysis was determined from a test of six
ligands that were not included in the training set. The test set
molecules were aligned and their activities were predicted by
each PLS analysis. The statistical results are summarized in
Table 7.
The second step in developing a pharmacophore model was to
use a set of pharmacophore features and to create sites for all the
ligands. The different sites for pharmacophore were created.
Step III: Finding a common pharmacophore
Active and inactive thresholds of pIC50 were 0.2 and 0.3, respect-
ively. The thresholds were applied to the training set for devel-
oping the common pharmacophore hypotheses. After applying,
default feature definitions to each ligand and common pharma-
cophores containing four sites were generated using a terminal
´
˚
box size of 1 A, and all active compounds match. Any single
Authors wish to thanks Dr. P. Trivedi, Dean, R. G. P. V., University,
Bhopal and Dr. C. Kartikeyan, Asst. Prof. R. G. P. V., University Bhopal,
India for their valuable help in QSAR studies.
pharmacophore in the group ultimately becomes a common
pharmacophore hypothesis which gives an explanation how
ligands bind to the receptor. Common pharmacophores are
identified using a tree-based partitioning technique that groups
together similar pharmacophores according to their intersite
distances, i. e., the distances between pairs of sites in the
pharmacophore.
The authors have declared no conflict of interest.
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