Phenyl hydrazone bearing pyrazole and pyrimidine scaffolds: Design and discovery of a novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) against HIV-1 and their antibacterial properties

Article abstract of DOI:10.1039/c3ra41604f

A novel series of phenyl hydrazone bearing pyrazole and pyrimidine hybrid compounds has been designed using the molinspiration toolkit based on Lipinski's rule of five and developed via sequential reactions starting from the diazotization of different anilines and further active methylation with acetyl acetone, ethyl acetoacetate and ethyl cyanoacetate to generate hydrazono derivatives. The target hybrid compounds were synthesized on cyclisation of the resulting hydrazono derivatives with hydrazine, phenyl hydrazine and urea. These molecules have been subsequently tested for anti-HIV activity using TZM-bl cell lines. The MTT assay was also carried out for the cytotoxicity determination of the active compounds. Further, to exemplify the key structural features of the molecules, a molecular docking analysis of the most active compounds was performed at the NNIBP of the HIV-RT protein. The antibacterial activity of the target compounds was also determined against a panel of four Gram-positive and four Gram-negative human pathogens. All molecules showed a potent anti-HIV activity along with a prominent inhibition of bacterial organisms.

Full text of DOI:10.1039/c3ra41604f

View Article Online
View Journal
RSC Advances
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: U. P. Singh, H. R. Bhat, A. Verma, M. K.
Kumawat, R. Kaur, S. K. Gupta and R. K. Singh, RSC Adv., 2013, DOI: 10.1039/C3RA41604F.
This is an Accepted Manuscript, which has been through the RSC Publishing peer
review process and has been accepted for publication.
Accepted Manuscripts are published online shortly after acceptance, which is prior
to technical editing, formatting and proof reading. This free service from RSC
Publishing allows authors to make their results available to the community, in
citable form, before publication of the edited article. This Accepted Manuscript will
be replaced by the edited and formatted Advance Article as soon as this is available.
To cite this manuscript please use its permanent Digital Object Identifier (DOI®),
which is identical for all formats of publication.
More information about Accepted Manuscripts can be found in the
Information for Authors.
Please note that technical editing may introduce minor changes to the text and/or
graphics contained in the manuscript submitted by the author(s) which may alter
content, and that the standard Terms & Conditions and the ethical guidelines
that apply to the journal are still applicable. In no event shall the RSC be held
responsible for any errors or omissions in these Accepted Manuscript manuscripts or
any consequences arising from the use of any information contained in them.
www.rsc.org/advances
Registered Charity Number 207890

Page 1 of 37
RSC Advances
Dynamic Article Links ►
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
PAPER
Phenyl hydrazone bearing pyrazole and pyrimidine scaffolds: Design and
discovery of novel class of Non-Nucleoside Reverse Transcriptase Inhibitors
(NNRTIs) against HIV-1 and their antibacterial properties
Udaya Pratap Singh,*a,d Hans Raj Bhat,a Amita Verma,a Mukesh Kumar Kumawat,b Rajinder
Kaur, c S. K. Gupta c and Ramendra K. Singh*d
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Abstract
10 A novel series of phenyl hydrazone bearing pyrazole and pyrimidine hybrid compounds has been
designed using molinspiration toolkit on the basis of Lipinski’s rule of five and developed via
sequential reactions starting from diazotization of different anilines and further active methylation with
acetyl acetone, ethyl acetoacetate and ethyl cynoacetate to generate hydrazono derivatives. The target
hybrid compounds have been synthesized on cyclisation of resulting hydrazono derivatives with
15 hydrazine, phenyl hydrazine and urea. These molecules have been subsequently tested for antiꢀHIV
activity using TZMꢀbl cell lines. The MTT assay was also carried out for cytotoxicity determination of
active compounds. Further, to exemplify key structural features of the molecules, a molecular docking
analysis of most active compound was performed at NNIBP of HIVꢀRT protein. The antibacterial
activity of target compounds was also determined against a panel of four Gramꢀpositive and four
20 Gramꢀnegative human pathogens. All molecules showed potent antiꢀHIV activity along with
prominent inhibition of bacterial organisms.
25
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

CREATED USING THE RSC ARTICLE TEMPLATE (VER. 3.0) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS
RSC Advances
Page 2 of 37
View Article Online
ARTICLE TYPE
www.rsc.org/xxxxxx | XXXXXXXX
DOI: 10.1039/C3RA41604F
Introduction
Human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency
syndrome (AIDS), causes specific damage to the immune system of the humans both in industrialised
and resource poor countries [1]. Despite the decline in number of people newly infected worldwide,
HIV is still considered as one of the top three causes of morbidity and mortality. Globally, 34.0 million
people were living with HIV at the end of 2011 [2]. For the treatment of HIV, at some point between
clinical latency and progression to AIDS, antiretroviral (ARV) therapy is introduced. A highly active
antiretroviral therapy (HAART) or triple therapy, presently used for treatment, it includes two
5
10 nucleoside reverse transcriptase inhibitors (NRTIs) and one additional agent, such as protease inhibitor
(PI), nonꢀnucleoside reverse transcriptase inhibitor (NNRTI), or integrase strand transfer inhibitor, as
initial ARV regimens for treatmentꢀnaïve patients. This combination therapy is extremely effective at
lowering viral load and increasing CD4+ T cell levels. However, there has been some debate as to the
ideal point for introduction of HAART, and therefore World Health Organisation (WHO) had changed
15 their recommendation from commencement of ARV treatment at a CD4+ T cell count of 200 cells
mmꢀ3 or below to commencement at 350 cells mmꢀ3 in 2010 as this results in considerably better health
outcomes [3].
HIVꢀ1 Reverse transcriptase (RT), an enzyme encoded by the viral genome plays an essential
role in the replication of HIV catalysing the transcription of viral RNA into viral doubleꢀstranded DNA
20 [4]. It is a heterodimeric structure consisting of p66 and p51 subunits, where the p66 subunit is the
larger of the two and contains both NRTI and NNRTI binding sites. The p66 subunit consists of RNAꢀ
and DNAꢀdependent DNA polymerase and ribonuclease H (RNase H) domains, both of which are vital
for the process of converting singleꢀstranded RNA into doubleꢀstranded DNA. The p51 subunit, on the
other hand, plays a structural rather than catalytic role, although the protein sequence is identical to
25 that of p66 except that it excludes the RNase H domain [5]. There are two classes of reverse
2
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 3 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
transcriptase (RT) inhibitors used clinically, namely, nucleoside and nonꢀnucleoside reverse
transcriptase inhibitors, NRTIs and NNRTIs, respectively, each affecting the RT enzyme at a different
location [6]. NNRTIs serve as more advantageous than NRTIs owing to their unique antiviral activity,
high specificity, low toxicity and being independent of host cell metabolism to be converted into an
active form [7]. There are about 50 structurally different classes of small hydrophobic molecule
targeted at a specific allosteric binding site (an especially flexible pocket formed upon binding of the
inhibitor) situated about 10 Ǻ from the polymerase catalytic site and 60 Ǻ from the RNase H active
site within the HIVꢀ1 RT. As a result, stimulation of conformational changes at the substrate binding
site due to the binding of a nonꢀcompetitive inhibitor (NNRTI) in the allosteric site substantially
5
10 reduces the rate of incorporation of nucleotides, thereby halting DNA synthesis. Although they are
structurally quite diverse, all NNRTIs bind in the NNRTI binding pocket (NNIBP) in a similar
conformation and in identical fashion [8].
Consequently, the wide chemical opportunity due to flexibility of the NNIBP in RT and no
mammalian homology of this enzyme (HIVꢀ1 RT) exists, it is considered as a preferred option for the
15 discovery and development of selective newer small molecules. Yet, the main issue in NNRTI drug
design is incorporation of the inherent flexibility of the NNIBP, which is surrounded by five aromatic
(Tyrꢀ181, Tyrꢀ188, Pheꢀ227 and Trpꢀ229), six hydrophobic (Proꢀ59, Leuꢀ100, Valꢀ106, Valꢀ179, Leuꢀ
234 and Proꢀ236) and five hydrophilic (Lysꢀ101, Lysꢀ103, Serꢀ105, Aspꢀ132 and Gluꢀ224) amino
acids and thereby creating chances for π–π interactions and hydrogen bonding between the putative
20 inhibitor and the amino acid side chains, Fig. 1 [9]. Despite the innovative drug discovery initiatives,
the high cost associated with the HAART therapy also prevents availability of drug regimes in
resource poor countries, for instance in South Africa governmentꢀfunded antiretrovirals are made
available at a CD4+ T cell count of 200 cells mmꢀ3 or below, unless there is a tuberculosis coꢀinfection
or pregnancy [10].
25
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3

RSC Advances
Page 4 of 37
View Article Online
DOI: 10.1039/C3RA41604F
Figure 1: HIVꢀ1 RT heterodimer showing Etravirine (TMCꢀ125) bound in the allosteric site (PDB
structures 3m8p).
The design and development of novel heterocyclic core scaffolds that fulfil the pharmacophoric
requirement of NNIBP and have cost effective synthesis could be an option worth considering.
Pyrazole, pyrimidine and hydrazone are notable heterocyclic scaffolds found to possess wide array of
pharmacological activities including antiꢀHIV [11] and antibacterial activity [12]. In continuation of
our ongoing efforts in the discovery of economic NNRTI agents, herein [13], we wish to report a novel
class of phenyl hydrazone bearing pyrazoles and pyrimidines in order to obtain more effective
5
10 candidates. These molecules were designed as per the pharmacophoric requirement to bind efficiently
with NNIBP, wherein pyrazole and pyrimidine rings act as pharmacophore and are able to create πꢀπ
interaction with amino acid residues present in NNIBP.
The people living with HIV/AIDS (PLWHA) also face serious health threats from
“opportunistic” infections (OIs). These infections are called “opportunistic” because they take
15 advantage of weakened immune system and cause devastating illnesses. According to Centre for
Disease Control (CDC), more than 20 OIs are considered as AIDSꢀdefining conditions, which include
4
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 5 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
various bacterial, protozoal and fungal infections [14]. Conversely, the infections caused by bacteria
are now recognized with increased prevalence and altered expression in patients with HIV infection. It
has been shown to cause substantially increased morbidity and mortality in both early and late stages
of HIV infection. As a result, antiꢀHIV agents possess antibacterial activity, can be able to cure the OIs
too and thereby radically improve the situation of PLWHA. Accordingly, designed molecules were
also tested for antibacterial activity against three Gramꢀpositive and five Gramꢀnegative human disease
causing pathogens.
5
Results and discussion
Design of compounds
10 The target compounds were designed using the Molinspiration java based webꢀapplet following
Lipinski’s rule of five (Ro5) [15]. It is web based software used to obtain parameters such as MiLogP,
TPSA and drug likeness. MiLogP (octanol/water partition coefficient) is calculated by the
methodology developed by Molinspiration as a sum of fragmentꢀbased contributions and correction
factors. The method is very robust and is able to process practically all organic and most
15 organometallic molecules. Topological Polar Surface Area (TPSA) is calculated based on the
methodology published by Ertl et al. as a sum of fragmentꢀbased contributions in which Oꢀ and Nꢀ
centered polar fragments are to be considered and calculated by surface areas that are occupied by
oxygen and nitrogen atoms and by hydrogen atoms attached to them [16]. TPSA has been shown to be
a very good descriptor characterizing drug absorption, including intestinal absorption, bioavailability,
20 Cacoꢀ2 permeability and blood brain barrier penetration. Thus, the TPSA is closely related to the
hydrogen bonding potential of a compound. The number of Rotatable Bonds – nrotb is a simple
topological parameter, which measures the molecular flexibility. It has been shown to be a very good
descriptor of oral bioavailability of drugs [17]. It is defined as any single nonꢀring bond, bound to
nonterminal heavy (i.e., nonꢀhydrogen) atom. However, amide CꢀN bonds are not considered because
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5

RSC Advances
Page 6 of 37
View Article Online
DOI: 10.1039/C3RA41604F
of their high rotational energy barrier. Predicted results of target compounds in terms of molecular
properties (molecular weight, MilogP and TPSA) are listed in Table 1. The entire set of designed
compounds, except 6b, showed no violations from the Lipinski’s Ro5, meaning hereby that all
molecules fulfilled the optimal requirement for drug absorption.
5
Table 1: Molinspiration data of the designed compounds
Compounds miLogP TPSA natoms
MW nON nOHNH nviolations nrotb volume
6a
6b
4.308
5.158
1.667
2.517
86.154
40.33
24.0 323.356
22.0 357.255
20.0 273.252
18.0 307.151
7
4
8
5
8
5
8
5
9
6
6
6
9
6
6
6
0
0
1
1
2
2
1
1
4
4
4
4
3
3
3
3
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
3
3
2
3
2
4
3
3
2
2
2
4
3
3
3
289.87
284.421
230.825
225.376
203.406
197.957
275.196
269.747
198.134
192.685
188.335
188.335
269.924
264.475
260.125
260.125
7a
113.07
67.246
7b
8a
1.338 115.969 18.0 247.214
8b
2.188
2.68
3.53
70.145
105.112 24.0 323.312
59.288 22.0 357.211
16.0 281.113
9a
9b
10a
10b
10c
10d
11a
11b
11c
11d
0.723 141.992 18.0 248.202
1.573
1.418
1.442
96.168
96.168
96.168
16.0 282.101
16.0
16.0
237.65
237.65
324.3
2.065 131.135 24.0
2.915
2.76
2.784
85.311
85.311
85.311
22.0 358.199
22.0 313.748
22.0 313.748
10
Scheme 1: Diazotization of anilines. Reagents and conditions: (i) NaNO2/HCl, stirring, 1h.
6
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 7 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
Scheme 2: Synthesis of hydrazono derivatives via active methylene compounds. Reagents and
conditions: Stirring 3h
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 7

RSC Advances
Page 8 of 37
View Article Online
DOI: 10.1039/C3RA41604F
Scheme 3: Synthesis of target compounds via cyclization of hydrazono derivatives using hydrazine,
urea and phenyl hydrazine
5
10
15
8
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 9 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
5
10
15
20
25
30
35
40
45
50
Figure 2: Proposed mechanism of pyrazole ring synthesis
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 9

RSC Advances
Page 10 of 37
View Article Online
DOI: 10.1039/C3RA41604F
5
10
15
20
25
Figure 3: Proposed mechanism of pyrimidine ring synthesis
Chemistry
30
The synthesis of target compounds viz. pyrazole and pyrimidine derivatives was achieved as
shown in Scheme 1ꢀ3 and the mechanism of pyrazole and pyrimidine ring generation in fig. 1 and 2.
The synthesis was initiated with the formation of diazonium chlorides of respective anilines 2(a-d)
upon diazotization of 1(aꢀd) using a mixture of sodium nitrite and concentrated HCl at 0ꢀ5 C.
Diazonium salts thus obtained were treated with calculated amounts of iceꢀcold active methylene
35 compounds, namely acetyl acetone, ethyl acetoacetate, ethyl cynoacetate in the presence of sodium
acetate to afford hydrazono derivatives 3, 4 and 5, respectively. These hydrazono derivatives further
underwent cyclization with hydrazine or phenyl hydrazine in boiling ethanol to the corresponding
10 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 11 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
pyrazolinꢀ5ꢀone derivatives of substituted anilines, compound 8(a,b) and 9(a,b). The structures of
1
13
these newly formed derivatives were ascertained by IR, H NMR, C NMR and elemental analysis.
The IR spectra showed the disappearance of the characteristic bands of acetyl carbonyl group and
carboxylic acid ester and appearance of strong bands in the 3107ꢀ3211 cmꢀ1 region, attributable to NH
stretching. The bands of pyrazolinone ring υ C=O groups appeared in the range of 1662ꢀ1676 cmꢀ1.
5
1
The HꢀNMR spectra of compounds 8(a,b) and 9(a,b) showed the presence of CH3 signal at δ 2.31ꢀ
2.51 ppm. The 13C NMR of 8b, for example, confirmed the absence of acetyl C=O and carboxylic acid
ester groups and appearance of two methyl signal in the high field region. The other carbon atoms of
pyrazolinone and phenyl moieties all appeared at the expected chemical shift. The compounds 10(aꢀd)
10 and 11(aꢀd) were synthesized via cyclization of 5(a,b) with hydrazine and phenyl hydrazine (Scheme
3). The IR spectra of 10(aꢀd) and 11(aꢀd), were characterized by the disappearance of υ CN band and
the appearance of band at 3217ꢀ3385 cmꢀ1 attributable to the stretching vibration of the υ NH2 group.
1
Diagnostically, a singlet at 5.61 ppm in the H NMR spectrum of 10b was attributable to the NH2
proton.
15
3,5ꢀDimethylꢀ4ꢀ((4ꢀnitrophenyl)diazenyl)ꢀ1ꢀphenylꢀ4,5ꢀdihydroꢀ1Hꢀpyrazole (6a) and its bromo
derivative 6b were obtained by thermal cyclization of 3(a,b) with phenyl hydarazine in glacial acetic
acid (Scheme 3). The IR spectra of 6(a,b) were characterised by disappearance of υ NH and acetyl
1
group C=O absorption bands and bands at 1455, 1445 cmꢀ1 indicated the presence of N=N. The H
NMR spectra of 6(a,b) showed the presence of two methyl groups and the aromatic protons all
20 appeared at expected chemical shift. The reaction of hydrazono derivative 3(a,b) with urea in ethanol
under reflux for 5h gave solid products 7(a,b). Compound 7a displayed bands in IR spectrum at 3118
13
(NH) and 1680 (pyrimidineꢀ2ꢀone C=O) cmꢀ1. Structure of 7a, was confirmed by CꢀNMR spectra,
which provided conclusive evidence for the formation of pyrimidinꢀ2ꢀone structure by showing singlet
at 196.60, 195.12, 166.16 and 139.65.
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 11

RSC Advances
Page 12 of 37
View Article Online
DOI: 10.1039/C3RA41604F
Anti-HIV activity
Initially ten compounds from the target dataset, viz. 8a, 8b, 9a, 9b, 10a, 10b, 10c, 11a, 11b and
11c were selected on the basis of in silico study to determine the possible antiꢀHIV activity against the
TZMꢀbl cells at the concentration of 50 ꢁg mLꢀ1. The results are presented in Table 2. It was observed
that compounds 8a, 8b, 10a and 10c showed more than 85 % inhibition of HIV at a test dose with
maximum 98 % in the case of compound 10b. Out of these, six compounds showed more than 40 %
inhibition were subsequently subjected to cytotoxicity determination using MTT assay (Table 3). It has
been observed that the increase in concentration of compounds 8a, 8b, 10(a-c) from 25 ꢁg mLꢀ1 to 125
ꢁg mLꢀ1 (fiveꢀfold increase) does not appreciably lower the percentage of cell viability. Hence, it was
5
10 inferred that, molecules showed potent antiꢀHIV activity, while presenting no significant toxicity at
test dosages under in vitro conditions.
Table 2 AntiꢀHIV activity of the selected compounds
Compound
8a
8b
9a
9b
10a
10b
10c
11a
11b
11c
Concentration (in ꢁg mLꢀ1) Percentage of Inhibition
50
50
50
50
50
50
50
50
50
50
85
91
0
10
91
98
93
41
20
0
15
Table 3 Cytotoxicity assay of the compounds†
Compound
8a
8b
10a
10b
125 ꢁg mLꢀ1
25 ꢁg mLꢀ1
5 ꢁg mLꢀ1
1 ꢁg mLꢀ1
0.2 ꢁg mLꢀ1
31
27
33
27
20
40
34
26
32
28
25
49
87
32
81
44
40
84
82
96
80
40
52
86
90
102
89
70
67
90
10c
11a
20 †: Results presented as percentage of cell viability
12 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 13 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
Molecular docking
It is a significant computational method used to forecast the binding of the ligand to the receptor
binding site. The molecules 8b and 10b were docked within NNIBP of HIVꢀRT to assess their mode of
interaction and structural requirement for HIVꢀ1 RT inhibition (Table 4). The formation of one
hydrogen bond was revealed between HIVꢀ1 RT Leu234 and pyrazole N on inspecting the postꢀdock
pose of compound 8b at NNIBP inside the HIVꢀ1 RT protein (Fig. 5). It was fashioned via Leu234
with the involvement of hydrogen of pyrazole nitrogen as shown in Fig. 4, Table 4. Further, formation
one of πꢀ π and σꢀ π interactions with Tyr318 and Val106, respectively via phramacophoric pyrazole
5
10 nuclei was also observed. In another highly active molecule, 10b, the pyrazole ring was oriented as
faceꢀtoꢀface with HIVꢀ1 RT Tyr181 creating πꢀπ stacking interaction, while showing the formation of
one hydrogen bond via hydrazone with Tyr188.
A close inspection of best docked pose of compound 10b, clearly establish that it attained a
“horseshoeꢀlike conformation as that of second generation NNRTIs and similar interaction with
15 Tyr181 and Tyr188 of p66 subunit in the NNIBP, Fig 5 [18]. The compound 8b, however, did no show
similar orientation. Therefore, on the basis of conformational analysis of postꢀdocked poses of ligands
8b and 10b, it was inferred that higher activity of compound 10b was attributable to its conformational
flexibility within the NNIBP.
Further, these molecules were evaluated by a scoring function to distinguish between good (near
20 native) and bad (decoy) docking solutions. Scoring functions for molecular docking are traditionally
either physicsꢀbased or knowledgeꢀbased and differ mainly in the derivation of the mathematical
models. It is very vital as it used to compute the strength of the nonꢀcovalent interaction, called as
binding affinity, between two molecules after they have been docked. To define binding affinity of
ligands with receptor in a better way, docked compounds (8b and 10b) were rigorously analysed
25 through scoring function as shown in Table 5.
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 13

RSC Advances
Page 14 of 37
View Article Online
DOI: 10.1039/C3RA41604F
The affinity of the molecules against the receptor was initially determined by LUDI3 score. It is a de
novo ligand design program that reckons interaction sites with a receptor and suggests small molecular
fragments that are able to interact with the receptor. This program analyses a geometrical fit of given
chemicals into the binding site and calculates other determinants of good binding, such as hydrogen
bonds, lipophilic interactions, ionic interactions, and acylic interactions [19]. In general, a higher Ludi
score represents higher affinity and stronger binding of a ligand to the receptor, as is shown in the case
of 10b (510, most active) than 8b (460, less active).
5
Piecewise Linear Potential is a fast and simple docking function that has been shown to elucidate
the protein ligand binding affinities in an efficient way. The PLP scores are measured in arbitrary units
10 and higher PLP scores indicate stronger receptorꢀligand binding. Two versions of the PLP function are
available and used in the present case: PLP1 [20] and PLP2 [21]. In the PLP1 function, each nonꢀ
hydrogen ligand or nonꢀhydrogen receptor atom is assigned a PLP atom type. Hydrogen atoms are
excluded from consideration. Whereas, in PLP2 function, PLP atom typing remains the same as in
PLP1. In addition, an atomic radius is assigned to each atom except for hydrogen. It is worthwhile to
15 mention that the most active compound (10b) was also found in conformity of PLP scoring functions,
showing higher values than corresponding less active molecule, 8b.
The PMF scoring functions were developed on the basis of statistical analysis of the 3D structures
of proteinꢀligand complexes. These were found to correlate well with proteinꢀligand binding free
energies. The scores are calculated by summing pairwise interaction terms over all interatomic pairs of
20 the receptorꢀligand complex [22]. Again, compound 10b being highly active, prevailed over 8b in
terms of PMF scoring, proving the experimental results.
The internal nonꢀbonded ligand energy is calculated for each new conformation that is generated.
Bad conformations with high internal ligand energies (typically resulting from internal close contacts)
are discarded. The internal ligand energy consists of a van der Waals (vdW) term and an optional
25 electrostatic term. The nonꢀbonded vdW energy is computed using a standard 9ꢀ6 (unsoftened)
14 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 15 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
potential using forcefield parameters consistent with the forcefield employed. Readings revealed 10b
as an efficient ligand owing to its high negative ligand internal energy than compound 8b.
Additionally, these molecules were analysed using Lig score and dock score, which classified as
empirical and knowledge based scoring functions, respectively [23]. The most active molecule, 10b,
presented higher values than corresponding less active molecule 8b, which in generalized manner, held
true for highly active ligands.
5
10
15
20
Figure 4 Postꢀdocked conformation of ligand 8b in complex with the HIVꢀ1 RT at NNIBP
Figure 5 Postꢀdocked conformation of ligand 10b in complex with the HIVꢀ1 RT at NNIBP
25
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 15

RSC Advances
Page 16 of 37
View Article Online
DOI: 10.1039/C3RA41604F
Table 4 Interaction of compound 8b and 10b in the NNIBP of HIVꢀ1 RT protein structure
Compound 3m8p.pdb
Hydrogen bond
Piꢀpi
PiꢀSigma
8b
Amine NH…..Leu234
Hydrazone, N…Tyr188
PyrazoleꢀTyr318
PyrazoleꢀTyr181
Pyrazole—Val106
NO
10b
Table 5 Docking scores of compound 8b and 10b
S. No
1
2
3
4
Scoring parameter
Ludi3
ꢀPLP1
ꢀPLP2
ꢀPMF
8b
460
59.41
58.21
14.67
1.52
10b
510
68.51
63.78
59.62
2.85
5
Jain
6
7
8
9
Lig Internal energy
Lig Score 1 Dreiding
Lig score2 Dreiding
Dock Score
ꢀ1.79
2.16
4.34
38.98
ꢀ51.54
ꢀ1.54
2.61
4.86
39.24
ꢀ33.53
10
Binding Energy (in kcal molꢀ1)
5
Antibacterial activity
All synthesized compounds were screened for their minimum inhibitory concentration (MIC,
ꢁg/mL) against a panel of four Gramꢀpositive, viz. Staphylococcus aureus (NCIMꢀ2079), Bacillus
subtilis (NCIMꢀ2063), Bacillus cereus (NCIMꢀ2156), Staphylococcus Epidermidis and four Gramꢀ
10 negative bacterial strains viz. Pseudomonas aeruginosa (NCIMꢀ2036), Escherichia coli (NCIMꢀ2065),
Proteus Mirabilis and Proteus vulgaris for determination of their antibacterial efficacy using cefixime
as a standard drug and results were presented in Table 6. It is noteworthy to mention here that the
entire set of test compounds showed marked to considerable inhibition pattern against the bacterial
strains and even some cases showed more potent activity than the reference standard. From table 6, it
15 was inferred that compound 6a showed more potent activity against B. cereus and B. subtilis, equiꢀ
16 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 17 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
potent to S. aureus, P. aeruginosa and E. coli in comparison to cefixime and considerable to least
activity against rest of the strains. Introduction of halogen atom (6b, bromo) in the place of nitro group
causes drastic reduction in activity against P. aeruginosa and E. coli. On the contrary, in comparison to
the standard, it showed marked increase in activity against S. epidermidis and potent in the case of rest
of the strains. On substituting the pyrazole with diꢀmethyl pyrimidine, 7a, having nitro group on
phenyl hydrazone showed moderate to less activity against B. cereus, P. aeruginosa, E. coli, S.
epidermidis and B. subtilis, it showed considerably potent activity against S. aureus, P. mirabilis, and
equipotent to P. vulgaris in comparison to standard. A significant shift in antibacterial profile was
observed on replacing nitro group with bromo resulting in potent to significant activity against entire
5
10 range of bacterial strains. Notably, mild to no activity was observed against all the three Gram positive
strains by compound 8a except S. epidermidis. Additionally, this compound showed more potent
activity against P. mirabilis and P. vulgaris in comparison to standard. However, not very significant
difference in activity was witnessed on insertion of bromo in the place of nitro group, while a presence
of extra phenyl on the pyrazole revealed prominent inhibition pattern ranging from potent to
15 considerable activity on the entire set of tested microorganisms. The compound 10a showed potent
activity against P. mirabilis and considerable to less activity against rest of the test strains. However,
equiꢀpotent activity was disclosed in the case of Gramꢀpositive microorganisms and P. aeruginosa and
showed much significant activity against rest of the strains on substituting nitro with bromo group. A
substantial loss in activity was observed on introduction of 3ꢀchloro at the phenyl of hydrazone, 10c,
20 against entire set of the bacterial strains except significantly potent activity against S. aureus, E. coli
and P. mirabilis. To our surprise, drastic upsurge in activity of 10d was reported against entire
bacterial strains and showed the compound potent in majority of cases except P. vulgaris and E.
epidermidis on isomeric replacement of chloro from second to fourth position. Moderate to least
activity was disclosed by compound 11a against S. aureus, P aeruginosa and S. epidermidis. However,
25 it also showed equipotent activity against B. subtilis, B. cereus and P. vulgaris; and significant to more
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 17

RSC Advances
Page 18 of 37
View Article Online
DOI: 10.1039/C3RA41604F
potent activity against rest of the microorganisms. Conversely, significant increase in potency and
inhibitory activity of compound 11b was reported against entire panel of tested bacterial strains except
S. epidermidis on introduction of bromo into the core structure. The drastic decline in activity was
reported by compound 11d against majority of strains except S. epidermidis, which showed better
inhibition pattern. Additionally, no major noticeable change in activity profile was reported by 11d, an
isomeric counterpart of 11c, compound 11d.
5
On the basis of antibacterial spectrum of the tested compounds, structureꢀactivity relationship
(SAR) studies suggested that activity was radically improved by the presence of halogen atom and was
further substantiated by presence of additional phenyl ring on the heterocyclic core. The ease of
10 penetration into the bacteria via altering the integrity of cell wall due to the presence of electron
withdrawing group may be the plausible factor to switch the potency of the compound, which is
further found in agreement with the corresponding physicoꢀchemical parameter discussed in Table 1.
Moreover, presence of additional phenyl ring makes the compound more lipophilic and enhances
antibacterial activity via increasing the permeability of the compounds across the cell wall, for
15 instance, 6a, 6b, 9a, 9b and 11(aꢀd). It was also revealed that presence of methyl group at R1 as shown
in Fig. 6, made the molecules more active towards both the tested strains, while activity abolishes on
introduction of basic NH2 group. The presence of CH3 along with other CH3 of pyrazole enhances the
activity, suggesting the role of diꢀmethyl pyrazole, while drastic reduction in activity was observed on
introduction of >C=O group at R2 position against Gramꢀpositive strains along with no significant
20 change against Gramꢀnegative organisms, Fig. 6. Moreover, replacement of pyrazole with pyrimidine
nuclei resulted in marginal loss of activity. On closely inspecting the Table 1, the factors like TPSA,
molecular weight and volume of the entire compounds were found in optimal range and contributed
towards the better absorption and considerable bioꢀactivity. A significant change in activity was
reported by the compounds having paraꢀchloro group rather than its isomeric meta configuration, viz.
25 10c
and
10d;
11c
and
11d.
18 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 19 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
Figure 6: Structure–activity relationship (SAR) of designed target derivatives as antibacterial agent.
Table 6: Antibacterial data of target compounds (presented as MIC in ꢁg mLꢀ1).
5
Compounds S. aureus B. subtilisB. cereus S.
P. aeruginosa E. coliP. mirabilis P. vulgaris
Epidermidis
6a
12.5
3.125
6.25
3.125
50
3.125
1.56
100
3.125
100
50
3.125
6.25
12.5
12.5
25
1.56
50
100
6.25
50
3.125
50
3.125 50
50
6b
25
50
12.5
1.56
1.56
12.5
12.5
6.25
3.125
3.125
1.56
3.125
1.56
50
7a
6.25
6.25
100
100
3.125
12.5
12.5
6.25
25
50
7b
12.5
12.5
12.5
12.5
1.56
12.5
6.25
100
50
1.56
6.25
3.125
1.56
3.125
6.25
3.125
25
3.125 1.56
12.5 3.125
12.5 6.25
3.125 25
8a
8b
25
9a
1.56
3.125
12.5
12.5
6.25
3.125
50
9b
50
12.5
10a
10b
10c
10d
11a
11b
11c
11d
Cefixime
12.5 3.125
6.25 1.56
3.125 6.25
3.125 3.125
6.25 6. 25
6.25 1.56
1.56
1.56
1.56
50
25
1.56
50
25
12.5
50
6.25
6.25
12.5
12.5
6.25
100
50
3.125
50
3.125
100
6.25
3.125
6.25
50
100
50
12.5
12.5
50
50
12.5
50
12.5 100
3.125 12.5
12.5
3.125
Experimental
Design of the compounds
10 Design of the target compounds was done in accordance with Lipinski’s rule of 5 (Ro5) using
Molinspiration web applet.
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 19

RSC Advances
Page 20 of 37
View Article Online
DOI: 10.1039/C3RA41604F
Structural Investigation
All chemicals were of analytical grade and used directly. Melting points determined in open capillary
tubes with electrothermal melting point apparatus (MPꢀ1) are uncorrected. The completion of reaction
was checked by thin layer chromatography (TLC) using silica gelꢀG coated Alꢀplates (0.5 mm
thickness; Merck) and the plates were illuminated under UV (254 nm) and evaluated in iodine. FTꢀIR
(in 2.0 cmꢀ1, flat, smooth, abex, KBr) spectra were recorded on Perkin ElmerꢀSpectrum RXꢀI
5
1
13
spectrometer. H NMR spectra were recorded on Bruker Avance II 400 NMR Spectrometer and C
NMR spectra on Bruker Avance II 100 NMR spectrometer in DMSOꢀd6 using TMS as the internal
standard. Chemical shift is reported in parts per million (ppm, δ), and signals are described as s
10 (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Solvents were purified prior to use
according to standard protocols.
Diazotization of substituted anilines
A solution of substituted anilines 1(aꢀd) in concentrated HCl (3ꢀ4 mL) was cooled to 0ꢀ5 C under
15 ice, and a cold sodium nitrite solution (1.5 g in 10 mL of water) added to it dropꢀwise during 10 min.
The reaction mixture was then stirred for 1h which afforded the desired diazonium salt of respective
aniline.
General procedure for the preparation of hydrazono derivatives
To an iceꢀcold mixture of appropriate active methylene compound [acetyl acetone (i), ethyl
20 acetoacetate (ii), ethyl cyanoacetate (iii)] (0.01 mol) and sodium acetate (0.05 mol) in ethanol (50 mL)
was added dropꢀwise with stirring a solution of diazonium salt compound (0.01 mol) over 20 min. The
stirring was continued for 1 h and the reaction mixture was then left for 3 h at room temperature. The
solid product was collected and recrystallized from ethanol to give the corresponding hydrazono
derivative (3, 4 and 5).
25
20 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 21 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
General procedure for cyclisation reactions
Method A: With Hydrazine hydrate
A mixture of appropriate hydrazone (4 a,b and 5 aꢀd) and hydrazine hydrate (0.01 mol) in ethanol (30
mL) was heated under reflux for 6ꢀ8 h. The solvent was concentrated and the reaction product was
allowed to cool. The separated product was filtered off, washed with water, dried and recrystallized
from ethanol.
5
3ꢀMethylꢀ4ꢀ(2ꢀ(4ꢀnitrophenyl)hydrazono)ꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 8a
Paleꢀyellow crystals; Yield: 85 %; M.P.: 240ꢀ242 C; FTIR υmax (in cmꢀ1): 3197.98 (Secondary ꢀNH
10 stretching), 1676.14 (ꢀC=O stretching), 1566.20 (ꢀNH bending), 1527.62 (Asymmetrical, ꢀNO2
stretching), 1433.11 (Symmetrical, ꢀNO2 stretching), 1350.17 (ꢀC=Cꢀ stretching, Ar), 1242.16ꢀ1203.58
1
(ꢀCꢀN stretching), 1070.49ꢀ572.86 (ꢀCꢀH bending, Ar); H NMR (400MHz, CDCl3ꢀd6, TMS) δ ppm:
1.18ꢀ1.19 (d, 3H J=8 Hz, CH3), 2.16 (s, 1H, NH, ArꢀH), 2.50ꢀ2.51 (m, 1H, CH, aliphatic), 7.80ꢀ7.83
(d, 1H J=12 Hz, ArꢀH), 7.88ꢀ7.90 (d, 1H J=8 Hz, ArꢀH), 7.98 (s, 1H, amide); 13C NMR (100MHz,
15 CDCl3) δ ppm: 159.91 (Cꢀamide), 148.63 (CꢀNH, Ar), 142 (CꢀNO2, Ar), 121.23 (CH, Ar), 109.72
(CH, Ar), 78.71 (CH, aliphatic), 40.24 (CH, aliphatic), 11.49 (CH3). Elemental Analysis for
C10H9N5O3: C, 48.58, H, 3.67, N, 28.33 (calculated): C, 48.60, H, 3.69, N, 28.30 (found).
4ꢀ(2ꢀ(4ꢀBromophenyl)hydrazono)ꢀ3ꢀmethylꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 8b
20 Darkꢀpale yellow crystals; Yield: 81%; M.P.: 220ꢀ222 C; FTIR υmax (in cmꢀ1): 3211.48 (Secondary ꢀ
NH stretching), 1666.50 (ꢀC=O stretching), 1587.42ꢀ1566.20 (ꢀNH bending), 1483.26ꢀ1375.25 (ꢀC=Cꢀ
stretching, Ar), 1249.87 (ꢀCꢀN stretching), 1070.49 (ꢀCꢀBr stretching), 968.27ꢀ594.08 (ꢀCꢀH bending,
1
Ar); H NMR (400MHz, CDCl3ꢀd6, TMS) δ ppm: 1.19 (d, 3H J=4 Hz, CH3), 2.13 (s, 1H, NH,
aromatic), 2.50 (m, 1H, CH, aliphatic alicyclic), 7.34ꢀ7.37 (d, 1H J=12 Hz, ArꢀH), 7.44ꢀ7.48 (d, 1H
13
25 J=16 Hz, ArꢀH), 8.01(s, 1H, CꢀNH, amide); C NMR (100MHz, CDCl3) δ ppm: 160.23 (Cꢀamide),
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 21

RSC Advances
Page 22 of 37
View Article Online
DOI: 10.1039/C3RA41604F
146.64 (CꢀNH, Ar), 140.58 (CH, Ar), 132.0 (CH, Ar), 128.71 (CꢀBr, Ar), 78.12 (CH, aliphatic), 38.98
(CH, aliphatic), 11.43 (CH3, aliphatic); Elemental Analysis for C10H9BrN4O: C, 42.73, H, 3.23, N,
19.93 (calculated): C, 42.70, H, 3.22, N, 19.93 (found).
5
3ꢀAminoꢀ4ꢀ(2ꢀ(4ꢀnitrophenyl)hydrazono)ꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 10a
Brown crystals; Yeild: 78%; M.P.: 291ꢀ293 C ; FTIR υmax (in cmꢀ1): 3685.07-3462.22 (Primary ꢀNH
stretching), 3197.98 (Secondary ꢀNH stretching), 1674.21 (ꢀC=O stretching), 1624.06 (Primary ꢀNH
bending), 1581.63 (Asymmetrical, ꢀNO2 stretching), 1533.41 (Symmetrical, ꢀNO2 stretching), 1354.03
(Secondary ꢀNH bending), 1249.87ꢀ1209.37 (ꢀC=Cꢀ stretching, Ar), 1109.07 (ꢀCꢀN stretching),
1
10 989.48ꢀ682.80 (ꢀCꢀH bending, Ar); H NMR (400MHz, CDCl3ꢀd6, TMS) δ ppm: 1.05ꢀ1.08 (m, 1H,
CH, aliphatic), 1.19 (s, 2H, NH2), 2.50ꢀ2.51 (m, 1H, CH, aliphatic), 3.28 (s, 1H, NH, Ar), 7.51ꢀ7.55 (t,
1H J=16 Hz, ArꢀH), 7.77ꢀ7.79 (d, 1H J=8 Hz, ArꢀH), 7.84ꢀ7.86 (d, 1H J=8 Hz, ArꢀH), 7.97 (s, 1H,
13
amide); C NMR (100MHz, CDCl3) δ ppm: 158.36 (Cꢀamide), 148.75 (CꢀNH, Ar), 130.12 (CꢀNO2,
Ar), 120.95 (CH, Ar), 117.72 (CH, Ar), 78.69 (CH, aliphatic alicyclic), 40.24 (NH2ꢀCH, aliphatic
15 alicyclic); Elemental Analysis for C9H8N6O3: C, 43.55; H, 3.25; N, 33.86 (calculated): C, 43.58; H,
3.22; N, 33.80 (found).
3ꢀAminoꢀ4ꢀ(2ꢀ(4ꢀbromophenyl)hydrazono)ꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 10b
Darkꢀbrown crystals; Yeild: 89 %; M.P.: 265ꢀ267 C; FTIR υmax (in cmꢀ1): 3355.78ꢀ3300.20 (Primary
20 ꢀNH stretching), 3188.82 (Secondary ꢀNH stretching), 1641.42 (ꢀC=O stretching), 1598.99 (Primary ꢀ
NH bending), 1521.48 (Secondary ꢀNH bending), 1485.19ꢀ1363.67 (ꢀC=Cꢀ stretching, Ar), 1247.94 (ꢀ
1
CꢀN stretching), 1112.93 (ꢀCꢀBr stretching), 1082.49ꢀ572.86 (ꢀCꢀH bending, Ar); H NMR (400MHz,
CDCl3ꢀd6, TMS) δ ppm: 2.50ꢀ2.51 (d, 2H J=4 Hz, NH2), 3.49 (m, 1H, CH, aliphatic alicyclic), 4.31
(m, 1H, CH, aliphatic alicyclic), 5.61(s, 1H, NH, ArꢀH), 7.40ꢀ7.50 (m, 1H, ArꢀH), 8.09 (s, 1H, amide);
25 13C NMR (100MHz, CDCl3) δ ppm: 149.62 (Cꢀamide), 141.10 (CH, Ar), 131.86 (CH, Ar), 116.74
22 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 23 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
(CH, Ar), 115.87 (CꢀBr, Ar), 78.92 (CH, aliphatic), 40.21 (CH, aliphatic); Elemental Analysis
C9H8BrN5O: C, 38.32; H, 2.86; N, 24.83 (calculated): C, 38.35; H, 2.89; N, 24.86 (found).
3ꢀAminoꢀ4ꢀ(2ꢀ(3ꢀchlorophenyl)hydrazono)ꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 10c
5
Darkꢀbrown crystals: Yeild: 87 %; M.P.: 160ꢀ162
C; FTIR υmax (in cmꢀ1): 3385.07 ꢀ3325.28
(Primary ꢀNH stretching), 3186.40 (Secondary ꢀNH stretching), 1683.86 (ꢀC=O stretching), 1637.56ꢀ
1600.92 (Primary ꢀNH bending), 1481.33 (Secondary ꢀNH bending), 1404.40ꢀ1253.73 (ꢀC=Cꢀ
stretching, Ar), 1207.44(ꢀCꢀN stretching), 1114.06 (ꢀCꢀCl stretching), 999.13ꢀ588.57 (ꢀCꢀH bending,
Ar); 1H NMR (400MHz, CDCl3ꢀd6, TMS) δ ppm: 2.07 (s, 2H, NH2, aliphatic alicyclic), 2.50ꢀ2.51 (m,
10 1H, CH, aliphatic alicyclic), 3.46ꢀ3.50 (m, 1H, CH, aliphatic alicyclic), 5.34 (s, 1H, NHꢀAr), 6.99ꢀ7.01
(t, 1H J=8 Hz, ArꢀH ), 7.17ꢀ7.19 (d, 1H J=8 Hz, ArꢀH), 7.23ꢀ7.27 (t, 1H J=4 Hz, ArꢀH), 7.50ꢀ7.51 (d,
2H J=4 Hz, ArꢀH), 7.82 (s, 1H, amide); 13C NMR (100MHz, CDCl3) δ ppm: 158.56 (Cꢀamide), 143.20
(CꢀNH, Ar), 134.60 ( ClꢀC, Ar), 130.25 (CH, Ar), 123.51 (CH, Ar), 114.30 (CH, Ar), 78.35 ( CH,
aliphatic alicyclic), 40.25 ( CꢀNH2, aliphatic alicyclic); Elemental Analysis for C9H8ClN5O: C, 45.49;
15 H, 3.39; N, 29.47 (calculated): C, 45.50; H, 3.35; N, 29.45 (found).
3ꢀAminoꢀ4ꢀ(2ꢀ(4ꢀchlorophenyl)hydrazono)ꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 10d
Brown crystals: Yeild: 79 %; M.P.: 225ꢀ227
C; FTIR υmax (in cmꢀ1): 3313.71 (Primary ꢀNH
stretching), 3172.90 (Secondary ꢀNH stretching), 1649.14 (ꢀC=O stretching),1587.42 (Primary ꢀNH
20 bending), 1489.05ꢀ1444.68 (Secondary ꢀNH bending), 1396.46ꢀ1265.30 (ꢀC=Cꢀ stretching, Ar),
1
1109.07 (ꢀCꢀN stretching), 1008.77 (ꢀCꢀCl stretching), 871.82ꢀ594.08 (ꢀCꢀH bending, Ar); H NMR
(400MHz, CDCl3ꢀd6, TMS) δ ppm: 1.05ꢀ1.09 (m, 1H, CH, aliphatic alicyclic), 1.20 (s, 2H, NH2,
aliphatic), 2.50ꢀ2.51 (m, 1H, CH, aliphatic alicyclic), 3.30ꢀ3.49 (m 1H, CH, aliphatic alicyclic), 5.51
(s, 1H, NH, Ar), 7.04ꢀ7.08 (d, 1H J=16 Hz, ArꢀH), 7.24 (s, 1H, ArꢀH), 7.30ꢀ7.34 (t, 1H J=16 Hz, Arꢀ
13
25 H), 7.40ꢀ7.42 (d, 1H J=8 Hz, ArꢀH), 8.05 (s, 1H, amide); C NMR (100MHz, CDCl3) δ ppm: 158.86
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 23

RSC Advances
Page 24 of 37
View Article Online
DOI: 10.1039/C3RA41604F
(C, amide), 149.65 (CꢀNH, Ar), 141.55 (CꢀCl, Ar), 129.12 (CH, Ar), 123.98 (CH, Ar), 123.40 (CH,
Ar), 114.75 (CH, Ar), 78.85 (C, aliphatic, alicyclic), 40.21 (C, aliphatic alicyclic): Elemental Analysis
C9H8ClN5O: C, 45.49; H, 3.39; N, 29.47 (calculated): C, 45.47; H, 3.36; N, 29.50 (found).
5
Method B: With Phenyl hydrazine
To a solution of appropriate hydrazone (3a,b; 4a,b and 5a-d) in glacial acetic acid (40 mL), phenyl
hydrazine (0.012 mol) and anhydrous sodium acetate (0.01 mol) were added. The reaction mixture was
then heated under reflux for 4 h. The resulting mixture was then poured into iceꢀcold water and stored
in refrigerator overnight. The crude product, was washed with water, dried and recrystallized from
10 ethanol.
3,5ꢀDimethylꢀ4ꢀ((4ꢀnitrophenyl)diazenyl)ꢀ1ꢀphenylꢀ4,5ꢀdihydroꢀ1Hꢀpyrazole 6a
Orange crystals; Yeild: 84 %; M.P.: 102ꢀ104
C; FTIR υmax (in cmꢀ1): 3100.64 (Secondary ꢀNH
stretching), 2924.09 (ꢀCꢀH stretching, ꢀCH3), 1670.35 (ꢀC=O stretching), 1597.08(Asymmetrical, ꢀNO2
15 stretching), 1548.84ꢀ1386.82 (ꢀC=Cꢀ stretching, Ar), 1455, 1445 (N=N) 1330.88 (Symmetrical, ꢀNO2
stretching), 1235.96 (Secondary ꢀNH bending), 1193.94 (ꢀCꢀN stretching), 1022.27ꢀ682.80 (ꢀCꢀH
1
bending, Ar); H NMR (400MHz, CDCl3ꢀd6, TMS) δ ppm: 1.21 (s, 3H, CH3), 2.48ꢀ2.49 (d, 1H J=4
Hz, CH, aliphatic alicyclic), 2.50ꢀ2.51 (d, 1H J=4 Hz, CH, aliphatic alicyclic), 2.63ꢀ2.64 (d, 3H J=4
Hz, CH3), 7.41ꢀ7.44 (m, 1H, ArꢀH), 7.50ꢀ7.52 (d, 1H J=8 Hz, ArꢀH), 7.85ꢀ7.86 (d, 1H J=4 Hz, ArꢀH),
20 7.88ꢀ7.89 (d, 1H J=4 Hz, ArꢀH), 8.02 (s, 1H, ArꢀH), 8.26ꢀ8.27 (d, 1H J=4 Hz, ArꢀH); 13C NMR
(100MHz, CDCl3) δ ppm: 156.38 (C, imine), 147.11 (C, imine, imidazole), 142.83 (CꢀNO2, Ar),
141.10 (Cꢀimidazole, Ar), 138.30 (CH, Ar), 136.25 (CH, Ar), 128.98 (CH, Ar), 128 (CH, Ar), 124.39
(CH, Ar), 122.10 (CH, Ar), 78.78 ((CH3)2ꢀN, imidazole), 40.25 (CꢀCH3, imidazole), 13.95 (CH3),
10.95 (CH3); Elemental Analysis for C17H17N5O2: C, 63.15; H, 5.30; N, 21.66 (calculated): C, 63.10;
25 H, 5.32; N, 21.68 (found).
24 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 25 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
4ꢀ((4ꢀBromophenyl)diazenyl)ꢀ3,5ꢀdimethylꢀ1ꢀphenylꢀ4,5ꢀdihydroꢀ1Hꢀpyrazole 6b
C; FTIR υmax (in cmꢀ1): 3045.60ꢀ2958.80 (ꢀCꢀH
Yellowish crystals; Yeild: 80 %; M.P.: 110ꢀ112
stretching, ꢀCH3), 1662.64 (ꢀC=O stretching), 1598.99ꢀ1506.41 (ꢀC=Cꢀ stretching, Ar), 1455, 1445
1
5
(N=N), 1114.86 (ꢀCꢀBr stretching), 1064.71ꢀ522.71 (ꢀCꢀH bending, Ar); H NMR (400MHz, CDCl3ꢀ
d6, TMS) δ ppm: 0.92 (s, 3H, CH3), 1.24ꢀ1.25 (d 3H J=4 Hz, CH3), 2.60 (m 1H, CH, aliphatic
alicyclic), 2.64ꢀ2.65 (d, 1H J=4 Hz, CH, aliphatic alicyclic), 7.51ꢀ7.52 (t, 1H J=4 Hz, ArꢀH), 7.59ꢀ7.60
(d, 1H J=4 Hz, ArꢀH), 7.61ꢀ7.62 (t, 1H J=4 Hz, ArꢀH), 7.70 (m, 1H, ArꢀH), 7.71ꢀ7.72 (d, 1H J=4 Hz,
13
ArꢀH); C NMR (100MHz, CDCl3) δ ppm: 152.36 (N=CꢀCH3, imine, aliphatic alicyclic), 143.92 (Cꢀ
10 N, Ar), 139.49 ((CH3)2ꢀN, Ar), 136.20 (CH, Ar), 132.11 (CH, Ar), 129.28 (CH, Ar), 128.17 (CH, Ar),
124.88 (CH, Ar), 123.54 (CꢀBr, Ar), 77.36 (Cꢀ(CH3)2, aliphatic alicyclic ), 76.78 (CH3)2ꢀCH3, aliphatic
alicyclic), 14.16 (CH3), 11.39 (CH3); Elemental Analysis for C17H17BrN4: C, 57.15; H, 4.80; N, 15.68
(calculated); C, 57.13; H, 4.83; N, 15.65 (found).
15 3ꢀMethylꢀ4ꢀ(2ꢀ(4ꢀnitrophenyl)hydrazono)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 9a
Reddishꢀorange crystals; Yeild: 88 %; M.P.: 181ꢀ183 C; FTIR υmax (in cmꢀ1): 3107.32 (Secondary ꢀ
NH stretching), 1662.64 (ꢀC=O stretching), 1560.41 (ꢀNH bending), 1527.62 (Asymmetrical, ꢀNO2
stretching), 1490.97 (Symmetrical, ꢀNO2 stretching), 1330.88 (ꢀC=Cꢀ stretching, Ar), 1236.37 (ꢀCꢀN
1
stretching), 1147.65ꢀ570.93 (ꢀCꢀH bending, Ar); H NMR (400MHz, CDCl3ꢀd6, TMS) δ ppm: 1.88ꢀ
20 1.89 (d, 3H J=4 Hz, CH3), 2.31 (m, 1H, CH, aliphatic), 2.50ꢀ2.51 (m, 1H, CH, aliphatic), 3.29 (s, 1H,
NH, Ar), 7.13ꢀ7.17 (t, 1H J=16 Hz, ArꢀH), 7.35ꢀ7.40 (m, 1H, ArꢀH), 7.59ꢀ7.63(t, 1H J=16 Hz, ArꢀH),
13
7.87ꢀ7.90 (d, 1H J=12 Hz, ArꢀH), 7.93ꢀ7.96 (d, 1H J=12 Hz, ArꢀH); C NMR (100MHz, CDCl3) δ
ppm: 156.18 (Cꢀamide), 148.58 (CꢀNH, Ar), 142 (CꢀN(CH3)2, Ar), 128.57 (CH, Ar), 124.64 (CH, Ar),
121.82(CH, Ar), 119 (CH, Ar), 117.65 (CH, Ar),110.50 (CH, Ar), 78.79 ( (CH3)2CꢀN, aliphatic),
25 11.55 (CH3); Elemental Analysis for C16H13N5O3: C, 59.44; H, 4.05; N, 21.66 (calculated); C, 59.42;
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 25

RSC Advances
Page 26 of 37
View Article Online
DOI: 10.1039/C3RA41604F
H, 4.09; N, 21.70 (found).
4ꢀ(2ꢀ(4ꢀBromophenyl)hydrazono)ꢀ3ꢀmethylꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 9b
Brightꢀorange crystals; Yeild: 76 %; M.P.: 145ꢀ147 C; FTIR υmax (in cmꢀ1): 3211.48 (Secondary ꢀ
NH stretching), 1666.50 (ꢀC=O stretching), 1587.42ꢀ1564.27 (ꢀNH bending), 1483.26ꢀ1375.25 (ꢀC=Cꢀ
stretching, Ar), 1249.87 (ꢀCꢀN stretching), 1055.06 (ꢀCꢀBr stretching), 968.27ꢀ594.08 (ꢀCꢀH bending,
Ar); 1H NMR (400MHz, CDCl3ꢀd6, TMS) δ ppm: 1.18 (d, 3H J=4 Hz, CH3), 2.23 (s, 1H, NH, Ar),
2.51 (m, 1H, CH, aliphatic alicyclic), 7.08ꢀ7.12 (t, 1H J=16 Hz, ArꢀH), 7.27ꢀ7.34 (m, 1H, ArꢀH), 7.41ꢀ
5
13
7.44(d, 1H, J=9 Hz, ArꢀH), 7.68 (s, 1H, ArꢀH), 7.84ꢀ7.86 (d, 1H J=8 Hz, ArꢀH); C NMR (100MHz,
10 CDCl3) δ ppm: 156.83 (Cꢀamide), 147.91 (CꢀNH, Ar), 139.99 (CH, Ar), 137.56 (CH, Ar), 132.01 (CH,
Ar), 128.37 (CH, Ar), 124.51 ( CH, Ar), 117.73 ( CH, Ar), 117.04 (CH, Ar), 78.06 (CH, aliphatic),
40.26 (CH, aliphatic), 11.32 (CH3, aliphatic); Elemental Analysis for C16H13BrN4O: C, 53.80; H, 3.67;
N, 15.68 (calculated); C, 53.83; H, 3.65; N, 15.66 (found).
15 3ꢀAminoꢀ4ꢀ(2ꢀ(4ꢀnitrophenyl)hydrazono)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 11a
Paleꢀyellow crystals; Yeild: 72%; M.P.: 85ꢀ87 C; FTIR υmax (in cmꢀ1): 3385.07-3325.28 (Primary ꢀ
NH stretching), 3186.40 (Secondary ꢀNH stretching), 1683.86 (ꢀC=O stretching), 1637.56 (Primary ꢀ
NH bending), 1600.92 (Secondary ꢀNH bending), 1481.33(Asymmetrical, ꢀNO2 stretching), 1404.18
(Symmetrical, ꢀNO2 stretching), 1253.73 1249.87ꢀ1207.44 (ꢀC=Cꢀ stretching, Ar), 1114.86 (ꢀCꢀN
20 stretching), 999.13ꢀ580.57 (ꢀCꢀH bending, Ar); 1H NMR (400MHz, CDCl3ꢀd6, TMS) δ ppm: 1.29ꢀ1.36
(m, 1H, CH, aliphatic alicyclic), 1.89 (s, 2H, NH2, aliphatic alicyclic), 2.50ꢀ2.51 (m, 1H, CH, aliphatic
alicyclic), 3.11 (s, 1H, NH, Ar), 7.45ꢀ7.54 (m, 1H, ArꢀH), 7.75ꢀ7.77 (d, 2H J=8 Hz, ArꢀH), 7.78ꢀ7.80
(d, 2H J=8 Hz, ArꢀH), 7.82ꢀ7.83 (d, 1H J=4 Hz, ArꢀH), 7.84ꢀ7.85 (d, 1H J=4 Hz, ArꢀH), 7.89ꢀ7.92 (d,
1H J=12 Hz, ArꢀH); 13C NMR (100MHz, CDCl3) δ ppm: 160.45 (Cꢀamide), 148.47 (CꢀNH, Ar),
25 143.18 ((CH3)2ꢀNꢀC, Ar), 142.06 (NO2ꢀC, Ar), 130.14 (CH, Ar), 129.83 (CH, Ar), 121.58 (CH, Ar),
26 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 27 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
119.22 (CH, Ar), 118.29 (CH, Ar), 114.42 (CH, Ar), 110.83 (CH, Ar), 78.07 ( CH, aliphatic alicyclic),
62.11 ( CH, aliphatic alicyclic); Elemental Analysis for C15H12N6O3: C, 55.55; H, 3.73; N, 25.91
(calculated); C, 55.53; H, 3.70; N, 25.94 (found).
5
3ꢀAminoꢀ4ꢀ(2ꢀ(4ꢀbromophenyl)hydrazono)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 11b
Paleꢀyellow crystals; Yeild: 82%; M.P.: 130ꢀ132
C; FTIR υmax (in cmꢀ1): 3217.27 (Primary ꢀNH
stretching), 3198.02 (Secondary ꢀNH stretching), 1687.71 (ꢀC=O stretching), 1600.92 (Primary ꢀNH
bending), 1535.43 (Secondary ꢀNH bending), 1479.40ꢀ1377.17 (ꢀC=Cꢀ stretching, Ar), 1328.95ꢀ
1
1263.37(ꢀCꢀN stretching), 1132.21 (ꢀCꢀBr stretching), 1068.56ꢀ576.72 (ꢀCꢀH bending, Ar); H NMR
10 (400MHz, CDCl3ꢀd6, TMS) δ ppm: 1.89ꢀ1.91 (d, 3H J=8 Hz, CH3), 2.50ꢀ2.52 (m, 1H, CH, aliphatic),
3.26 (s, 1H, NH, ArꢀH), 4.24ꢀ4.33 (m, 1H, CH, aliphatic), 7.32ꢀ7.46 (m, 1H, ArꢀH), 7.93 (s, 1H,
13
amide); C NMR (100MHz, CDCl3) δ ppm: 160.83 (Cꢀamide), 141.46 (CꢀNH, Ar), 131.67 (CꢀNO2,
Ar), 117.62 (CH, Ar), 111.20 (CH, Ar), 78.61 (CH, aliphatic), 39.60 (CH, aliphatic), 13.96 (CH3);
Elemental Analysis for C15H12BrN5O: C, 50.30; H, 3.38; N, 19.55 (calculated); C, 50.33; H, 3.35; N,
15 19.50
3ꢀAminoꢀ4ꢀ(2ꢀ(3ꢀChlorophenyl)hydrazono)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 11c
Lightꢀyellow crystals; Yeild: 86%; M.P.: 160ꢀ162 C; FTIR υmax (in cmꢀ1): 3228.84 (Primary ꢀNH
stretching), 3082.25 (Secondary ꢀNH stretching), 1699.29 (ꢀC=O stretching), 1602.85ꢀ1558.48
20 (Primary ꢀNH bending), 1498.69ꢀ1460.11 (Secondary ꢀNH bending), 1371.39ꢀ1328.95 (ꢀC=Cꢀ
stretching, Ar), 1251.80 (ꢀCꢀN stretching), 1112.93 (ꢀCꢀCl stretching), 1018.41ꢀ586.36 (ꢀCꢀH bending,
Ar); 1H NMR (400MHz, CDCl3ꢀd6, TMS) δ ppm: 1.29ꢀ1.35 (m, 1H, CH, aliphatic alicyclic), 1.92 (s,
2H, NH2, aliphatic alicyclic), 2.51 (m, 1H, CH, aliphatic alicyclic), 3.20 (s, 1H, CꢀNH, Ar), 4.25ꢀ4.33
(m, 1H, CH, aliphatic alicyclic), 7.00ꢀ7.02 (d, 1H J=8 Hz, ArꢀH), 7.06ꢀ7.09 (m , 1H, ArꢀH), 7.20ꢀ7.30
25 (m, 1H, ArꢀH), 7.36ꢀ7.38 (d, 1H J=8 Hz, ArꢀH), 7.41 (s, 1H, ArꢀH), 7.46 (s, 1H, ArꢀH); 13C NMR
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 27

RSC Advances
Page 28 of 37
View Article Online
DOI: 10.1039/C3RA41604F
(100MHz, CDCl3) δ ppm: 160.82 (Cꢀamide), 143.08 (CꢀNH, Ar), 141.95 ( (CH3)2ꢀN, Ar), 134.54 (Clꢀ
C, Ar), 130.35 (CH, Ar), 124.04 (CH, Ar), 115.85 (CH, Ar), 114.67 (CH, Ar ), 110.82 (CH, Ar),
104.97 (CH, Ar), 104.53 (CH, Ar), 78.31 ( CH, aliphatic alicyclic), 61.93 ( CH, aliphatic alicyclic);
Elemental Analysis for : C, 57.42; H, 3.86; Cl, 11.30; N, 22.32; Elemental Analysis for C15H12ClN5O:
C, 57.42; H, 3.86; N, 22.32 (calculated); C, 57.45; H, 3.87; N, 22.34 (found)
5
3ꢀAminoꢀ4ꢀ(2ꢀ(3ꢀchlorophenyl)hydrazono)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ5(4H)ꢀone 11d
Darkꢀgreen crystals; Yeild: 82%; M.P.: 210ꢀ212
C; FTIR υmax (in cmꢀ1): 3211.48 (Primary ꢀNH
stretching), 3163.26 (Secondary ꢀNH stretching), 2980.02 (ꢀCꢀH stretching, ꢀCH3), 1741.72 (ꢀC=O
10 stretching), 1687.71ꢀ1593.20 (Primary ꢀNH bending), 1498.69 (Secondary ꢀNH bending), 1373.32ꢀ
1311.59 (ꢀC=Cꢀ stretching, Ar), 1241.70 (ꢀCꢀN stretching), 1109.93 (ꢀCꢀCl stretching), 1022.27ꢀ
1
599.86 (ꢀCꢀH bending, Ar); H NMR (400MHz, CDCl3ꢀd6, TMS) δ ppm: 1.30ꢀ135 (m, 1H, CH,
aliphatic alicyclic), 1.92 (s, 2H, NH2, aliphatic alicyclic), 2.50ꢀ2.51 (m, 1H, CH, aliphatic alicyclic),
3.29 (s 1H, NH, Ar), 4.25ꢀ4.33 (m, 1H, CH, aliphatic alicyclic), 7.06ꢀ7.10 (t, 1H J=16 Hz, ArꢀH),
15 7.12ꢀ7.16 (t, 1H J=16 Hz, ArꢀH), 7.28ꢀ7.41 (m, 1H, ArꢀH), 7.46ꢀ7.48 (d, 1H J=8 Hz, ArꢀH), 7.99 (s,
13
1H, ArꢀH); C NMR (100MHz, CDCl3) δ ppm: 161.11 (C, amide), 141.80 (CꢀNH, Ar), 140.75 (Cꢀ
NH, Ar), 129.18 (CꢀCl, Ar), 128.89 (CH, Ar), 125.45 (CH, Ar), 124.53 (CH, Ar), 116.04 (CH, Ar),
115.76 (CH, Ar), 115.13 (CH, Ar), 111.15 (CH, Ar), 103.88 (CH, Ar), 103.40 (CH, Ar), 78.72 (C,
aliphatic, alicyclic), 61.72 (C, aliphatic alicyclic); Elemental Analysis for : C, 57.42; H, 3.86; N, 22.32
20 (calculated); C, 57.44; H, 3.85; N, 22.35 (found).
Method C: With Urea
A mixture of hydrazono derivative (3a,b; 0.005 mol) and urea (0.01 mol) in ethanol (50 mL) was
heated under reflux for 5 h. After cooling to room temperature, crushed ice was added and the mixture
25 was stirred for 1 h. The separated product was collected by filtration and recrystallized from ethanol.
28 | Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 29 of 37
RSC Advances
View Article Online
DOI: 10.1039/C3RA41604F
4,6ꢀdimethylꢀ5ꢀ(2ꢀ(4ꢀnitrophenyl)hydrazono)pyrimidinꢀ2(5H)ꢀone 7a
Lightꢀyellow crystals; Yeild: 78%; M.P.: 165ꢀ166 C; FTIR υmax (in cmꢀ1): 3118.90 (Secondary ꢀNH
stretching), 1680.00 (ꢀC=O stretching), 1597.06(Asymmetrical, ꢀNO2 stretching), 1496.76
(Symmetrical, ꢀNO2 stretching), 1355.96 (Secondary ꢀNH bending), 1311.12ꢀ1163.80 (ꢀC=Cꢀ
5
1
stretching, Ar), 1116.78 (ꢀCꢀN stretching), 1049.28ꢀ611.43 (ꢀCꢀH bending, Ar); H NMR (400MHz,
CDCl3ꢀd6, TMS) δ ppm: 1.72 (s, 3H, CH3), 1.91 (s, 3H, CH3), 7.68ꢀ7.70 (d, 1H J=16 Hz, ArꢀH), 8.11
(s, 1H, ArꢀNH), 8.20ꢀ8.23 (d, 1H J=12 Hz, ArꢀH); 13C NMR (100MHz, CDCl3) δ ppm: 196.80 (C,
carbonyl), 195.12 (C, imine), 166.16 (C, imine), 139.65 (CꢀNH, Ar), 132.42 (CꢀNO2, Ar), 131.42 (CH,
10 Ar), 118.28 (CH, Ar), 116.95 (CH, Ar), 30.21 (CH3), 12.12 (CH3); Elemental Analysis for
C12H11N5O3: C, 52.75; H, 4.06; N, 25.63 (calculated); C, 52.76; H, 4.02; N, 25.60 (found).
5ꢀ(2ꢀ(4ꢀBromophenyl)hydrazono)ꢀ4,6ꢀdimethylpyrimidinꢀ2(5H)ꢀone 7b
Darkꢀyellow crystals; Yeild: 88%; M.P.: 90ꢀ92
C; FTIR υmax (in cmꢀ1): 3072.60 (Secondary ꢀNH
15 stretching), 1666.50 (ꢀC=O stretching), 1624.06ꢀ1413.82 (ꢀC=Cꢀ stretching, Ar), 1375.25ꢀ1317.38 (ꢀ
NH bending), 1187.79 (ꢀCꢀN stretching), 1070.49 (ꢀCꢀBr stretching), 929.69ꢀ507.39 (ꢀCꢀH bending,
Ar); 1H NMR (400MHz, CDCl3ꢀd6, TMS) δ ppm: 2.49 (s, 3H, CH3), 2.61 (s, 3H, CH3), 7.28 (s, 1H, Nꢀ
NH, Ar), 7.29ꢀ7.31 (d, 1H J=8 Hz, ArꢀH), 7.51ꢀ7.53 (d, 1H J=8 Hz, ArꢀH); 13C NMR (100MHz,
CDCl3) δ ppm: 198.20 (Cꢀcarbonyl), 196.90(Cꢀimine), 166.10 (Cꢀimine), 140.67(NHꢀC, Ar), 133.53
20 (CH, Ar), 132.69 (CH, Ar), 118.64 (CH, Ar), 117.65 (CH, Ar ), 77.36 (CꢀBr, Ar), 31.69 (CH3), 26.61
(CH3); Elemental Analysis for C12H11BrN4O: C, 46.93; H, 3.61; N, 18.24 (calculated); C, 46.93; H,
3.61; N, 18.24 (found).
Biological Activity
Anti-HIV activity
25 For antiꢀHIV screening of the compounds, TZMꢀbl cells (4×104/ well) were seeded in 24 well plates
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 29