Microwave assisted one-pot synthetic route to imidazo[1,2-: A] pyrimidine derivatives of imidazo/triazole clubbed pyrazole and their pharmacological screening

Article abstract of DOI:10.1039/c8nj00670a

An efficient synthesis of imidazo[1,2-a]pyrimidine derivatives of pyrazole in excellent yield over a short reaction time based on a microwave-assisted, one-pot three-component condensation reaction of pyrazole aldehyde clubbed with imidazole 4 and triazole 5 nuclei, (substituted-phenyl/hetero-aryl)ethanones 6(a-g), and 2-amino benzimidazole 7 in the presence of the strong base KOH is described. All the compounds were screened for their preliminary in vitro antimicrobial, antituberculosis and antimalarial activities against a panel of pathogenic strains. The majority of the compounds exhibited excellent inhibitory action against S. typhi, S. pneumoniae, B. subtilis, and C. tetani. Some of the compounds showed good antifungal activity and moderate antituberculosis activity as compared to first line drugs. Two of the compounds 8b and 9b exhibited excellent antimalarial activity against P. falciparum strains.

Full text of DOI:10.1039/c8nj00670a

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Microwave assisted one-pot synthetic route
to imidazo[1,2-a]pyrimidine derivatives
of imidazo/triazole clubbed pyrazole
and their pharmacological screening†
Cite this:DOI: 10.1039/c8nj00670a
Pratibha Prasad, Anirudhdha G. Kalola and Manish P. Patel
*
An efficient synthesis of imidazo[1,2-a]pyrimidine derivatives of pyrazole in excellent yield over a short
reaction time based on a microwave-assisted, one-pot three-component condensation reaction of
pyrazole aldehyde clubbed with imidazole
4 and triazole 5 nuclei, (substituted-phenyl/hetero-
aryl)ethanones 6(a–g), and 2-amino benzimidazole 7 in the presence of the strong base KOH is
described. All the compounds were screened for their preliminary in vitro antimicrobial, antituberculosis
and antimalarial activities against a panel of pathogenic strains. The majority of the compounds exhibited
excellent inhibitory action against S. typhi, S. pneumoniae, B. subtilis, and C. tetani. Some of the
compounds showed good antifungal activity and moderate antituberculosis activity as compared to first
line drugs. Two of the compounds 8b and 9b exhibited excellent antimalarial activity against
P. falciparum strains.
Received 7th February 2018,
Accepted 18th June 2018
DOI: 10.1039/c8nj00670a
rsc.li/njc
1
. Introduction
The synthesis of nitrogen-containing fused heterocycles or
heterocyclic motifs containing multi-structures in one molecule
1
has attracted much attention in the last couple of decades.
Since small and simple heterocyclic scaffolds often exhibit
amazing multifaceted pharmacological properties, medicinal
chemists have been trying to constitute the most imperative
module of new active molecules for biological applications by
annulating different heterocycles such as indoles, pyrroles,
pyrimidines, imidazoles, pyrazoles and pyridines using various
2
synthetic strategies as it increases the range of potential
applications.
On the other hand, fused imidazole fragments and imidazo-
[1,2-a]-fused pyrimidine derivatives are important structural
moieties, which have been found to be used as core templates
for the synthesis of many drugs prescribed for a lot of pathologies
and natural products (Fig. 1). The synthesis of many imidazo-
Fig. 1 Alkaloids and drugs containing similar imidazo[1,2-a]pyrimidine/
pyridine derivatives.
[1,2-a] pyrimidines and pyridine fragments has attracted consider-
able attention from the pharmaceutical industry and these
compounds are widely recognized because of their important
5
biological activities and interesting therapeutic properties,
used as potent p38MAP kinase inhibitors, etc. Previously,
6
including anti-bacterial, anti-inflammatory, antifungal, anti- imidazo[1,2-a]pyrimidines have been prepared using different
7
microbial, antituberculosis, and anticancer activities, and also synthetic approaches.
Therefore, the development of efficient and practical synthetic
routes to generate imidazo[1,2-a]pyrimidine units for the synthesis
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar 388120,
of natural and biologically potent compounds is in great demand.
Gujarat, India. E-mail: patelmanish1069@yahoo.com
Electronic supplementary information (ESI) available. See DOI: 10.1039/c8nj00670a Typically, the synthesis of imidazo[1,2-a] heterocycles requires long
†
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8
reaction times and high temperatures via a conventional route, potent heterocycles, herein we present an efficient microwave-
hence limiting the accessibility of these biologically important induced one-pot route for the synthesis of imidazo[1,2-a]pyrimidine
structures. One of the ways to achieve this goal is the development derivatives of pyrazole fused to imidazole and triazole scaffolds
of cascade reactions in addition to replacing the oil bath with a triggered by eco-friendly base KOH with the belief that the
microwave reactor and opening a new window in microwave- amalgamation of separate pharmacophoric groups with analogous
assisted organic synthesis (MAOS), which allow the sequential activity into a single nucleus may produce novel heterocycles with
transformations of two or more reactants in the same reaction fascinating antimicrobial activity.
vessel, leading to performing the reactions in spectacularly
abbreviated time, minimizing the number of laboratory operations
and generation of waste chemicals, and increasing the yields
under much cleaner and easier reaction conditions. It has also
2
. Chemistry
gained the attention of chemists over the last decade as a very The required 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde
proficient tool. It has been applied to accelerate the course of many 2 was prepared via the Vilsmeier–Haack reaction according to a
9
organic reactions due to its unique advantages such as higher previously reported procedure. The substituted imidazo/1,2,4-
selectivity, lower quantities of side products and high yields and is triazole pyrazolyl aldehydes 4 and 5 were prepared by a nucleo-
4
recognized as a ‘‘green’’ technology in the field of organic synthesis.
philic displacement of the chloro group of starting material 2
Secondly, in support of this technology, multi-component with secondary amines 3a and b, respectively, using anhydrous
reactions (MCRs) offer excellent atom economy and are readily potassium carbonate in refluxing DMF solvent. Further, we
applicable for the preparation of extensive libraries of drug-like commenced this project by examining known reported synthetic
3
heterocycles. One-pot methods involving multi-component methods for the preparation of imidazo[1,2-a]pyrimidines
condensation using different reagents and catalysts are popular (Scheme 1). One such similar type of method involves a two-
in synthetic organic chemistry for the synthesis of heterocyclic step procedure using the conventional heating method beginning
compounds where speed is of the essence due to their reduced with chalcone followed by cyclocondensation with 1,2,4-triazol-
reaction times, simplicity of product isolation and higher yields. 3amine, which provides the dihydro-triazolo-pyrimidine analog,
1
0
Consequently, in view of the remarkable aforementioned which can be oxidized using either N-bromosuccinimide (NBS)
pharmacological activities of imidazo[1,2-a]pyrimidine derivatives or Br
11
2
to give 1,2,4-triazolo[1,5-a]pyrimidines. On the other hand,
and in continuation of our enduring research on the design and the other method involves condensation reactions employing
12
development of new methodologies for the synthesis of biologically high temperatures (e.g. 4220 1C in ethylene glycol, 190 1C in
Scheme 1 Synthesis of pyrazole based imidazo[1,2-a]pyrimidine derivatives 8(a–g) and 9(a–g). (i) DMF, POCl
h. (iii) Microwave method: A (one pot, one-step procedure) – EtOH : H O equimixture, KOH, 340 W/15–20 min; B and C (one pot, two-step procedure):
i) H O : EtOH/KOH equimixture, r.t., 10 min; B(ii) 340 W, 10–15 min; C(ii) 28 h reflux.
3 2 3
, reflux 2 h. (ii) DMF, K CO , 80 1C, reflux
2
2
(
2
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Scheme 2 Plausible mechanistic pathway for the imidazo[1,2-a]pyrimidine derivative compounds.
1
3
14
1
-methyl-2-pyrrolidinone ), neat at 160 1C or refluxing in t-BuOH Michael adduct is accomplished according to the Claisen–Schmidt
15
in the presence of the strong base t-BuOK, or refluxing in DMF in condensation of 1-(4-substituted-phenyl/hetero-aryl)ethanones
the presence of triethylamine as a catalyst in an open vessel at 6(a–g) with substituted imidazo/1,2,4-triazole pyrazolyl aldehydes
20 1C, which is indeed the most common scenario, and none of 4/5 at room temperature and (ii) then the formed intermediate
16
1
the methods involve a one-pot method. By employing our own Michael adduct undergoes sequential [3+3] cycloaddition with
independent investigations in this area sequentially (one-pot three- 2-amino benzimidazole 7 followed by the removal of hydrogen to
component condensation, microwave irradiation protocol (340 W, furnish the final aromatized products (Scheme 2). The products
1
0–15 min, equimixture EtOH : H O, KOH)), the reaction can be obtained according to method A (Scheme 2 and Table 1) were in
2
efficiently tuned toward the exclusive formation of imidazo[1,2- the 68–78% range, and it appears that this unusual three-
a]pyrimidine derivatives 8(a–g) and 9(a–g) in good yield via component one-step reaction may lead to a significant number
method B. Transformations according to methods A, B and C of side products as all three components are added at once under
(Scheme 2 and Table 1) were in the 68–78%, 87–93% and 70–81% microwave exposure, thus increasing the possibility of side reactions.
ranges, respectively. In order to obtain a pure product, it required chromatographic
The formation of compounds 8(a–g) and 9(a–g) may proceed purification and thus did not lend itself a well high-throughput
in two steps: (i) the initial in situ formation of an intermediate methodology. On the basis of our mechanistic assumption, an
alternative one-pot, two-step procedure is elaborated (Table 1,
methods B and C).
Table 1 Synthesis and substituent patterns for the synthesized com-
pounds 8(a–g) and 9(a–g)
This protocol involved the initial in situ formation of Michael
adducts at room temperature, with the total consumption of
both starting material compounds 6(a–g) and 4/5 as evidenced
by TLC, followed by the addition of appropriate 2-aminobenimid-
azole, which resulted in quantitative yields in both experiments.
Although, in terms of reaction efficiency and facileness, efforts
have been made towards reducing the reaction time, both the
microwave-assisted heteroannulation experiments proved to be
very convenient in comparison to method C. The identities of
the synthesized compounds 8(a–g) and 9(a–g) were determined
Building blocks
Building blocks
Code
X
C
R
C
Yield (%) Code
X
R
C
Yield (%)
A
A
8
8
8
8
8
8
8
a
b
c
d
e
f
6
H
5
72
9
8
9a
9b
9c
9d
9e
9f
N
6
H
5
78
B
C
B
3
1
92
C
83
A
B
C
A
C
C
C
C
C
C
4-FC
6
H
4
70
7
1
N
N
N
N
N
N
4-FC
6
H
4
72
B
8
8
90
C
80
1
13
using H NMR, C NMR, and FT-IR spectral data and elemental
analysis, and the molecular weights of the compounds were
A
B
C
A
4-MeC
6
H
4
75
5
8
4-MeC
6
H
4
75
B
8
7
92
1
C
confirmed by mass spectrometry. In the H NMR spectra of
81
compounds 8(a–g) and 9(a–g), a singlet peak of the methyl proton
of pyrazole appeared at around d 2.258 ppm, while the aromatic
protons resonate as multiplets at d 6.76–8.12 ppm. There were no
amine proton peaks detected, which shows that the cyclisation
involved the aromatization of complete compounds. A peak was
observed at around d 13.07 ppm for the methane carbon of pyrazole
A
B
C
A
4-OMeC
6
H
4
75
2
0
4-OMeC
6
H
4
71
B
9
8
88
C
82
A
B
C
A
4-OHC
6
H
4
68
1
9
4-OHC
6
H
4
74
B
9
7
90
C
82
13
in the C NMR spectra of compounds 8(a–g) and 9(a–g). In FT-IR
spectra, compounds 8(a–g) and 9(a–g) exhibited an absorption band
A
B
C
A
SC
4
H
3
60
0
2
SC
4
H
3
67
B
8
7
82
À1
C
at around 1589–1651 cm for (–CQN) stretching.
75
A
B
C
A
g
NC
4
H
4
62
9g
NC
4
H
4
65
2.1. Optimization of the reaction conditions
B
7
6
8
8
81
C
To optimize the reaction conditions for the formation of the
target compound 8a, a mixture of respective 5-(1H-imidazol-1-
yl)-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 4, acetophe-
none 6a, and 2-aminobenzimidazole 7 in a 1 : 1 : 1.5 ratio was
73
Microwave assisted: method A – one pot, one-step procedure, method B –
one pot, two-step procedure. Conventional method: method C – one pot,
two-step procedure.
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Table 2 Optimization of the reaction conditions for 8a by employing the 4 and 2-aminobenzimidazole 7 as starting materials. Under the
most suitable method B
optimized conditions, a variety of structurally diverse 4-substituted
a
phenyl/heteroaryl ketones were investigated and a series of new
imidazo[1,2-a]pyrimidine derivatives of substituted pyrazole
with the 4-substituted phenyl/heteroaryl group residing in the
6-position of the fused imidazo-pyrimidine nucleus were obtained
in good yields. The results signified that aromatic ketones bearing
either electron-withdrawing or electron-donating functional groups,
such as fluoro, methyl, methoxy, or hydroxyl, gave consistently good
yields. Moreover, the heterocyclic aryl ketone gave compounds in
modest yields (Table 1).
Entry
Base (equiv.)
Solvent
Yield 8a (%)
1
2
3
4
5
6
7
8
9
No base
No base
No solvent
EtOH
EtOH
o05
o10
61
58
78
93
83
71
KOH, 1.0
KOH, 1.0
KOH, 1.0
KOH, 1.5
KOH, 2.0
NaOH, 1.5
H
2
O
EtOH : H
EtOH : H
EtOH : H
EtOH : H
EtOH : H
Glycol
t-BuOH
EtOH
t-BuOH
2
2
2
2
2
O (1 : 1)
O (1 : 1)
O (1 : 1)
O (1 : 1)
O (1 : 1)
K
2
CO
3
, 1.5
43
43
b
10
11
12
13
t-BuOK, 1.5
TEA, 1.5
DMAP, 1.5
Piperidine, 1.5
67
23
54
b
3. Pharmacology
3.1. In vitro antimicrobial activity
a
b
Isolated yield. TLC indicated a complicated reaction in which
significant amounts of side products were observed.
The in vitro anti-microbial activity was investigated against 24 h
old cultures of three Gram-positive, Gram-negative bacteria and
subjected to microwave irradiation by employing method B, three fungi by the disc diffusion method according to the
17
and the results of these comparative experiments are summarized National Committee for Clinical Laboratory Standards (NCCLS).
in Table 2. Initially, we sought to screen bases and their Mueller–Hinton broth was used as the nutrient medium to grow
equivalents including green solvents used in the reaction and dilute the compound suspension for the test bacteria and
(entries 1–13) to determine the one that can give the best results. Sabouraud Dextrose broth was used for fungal nutrition. The
It was found that with alkaline enhancement of the reaction compounds were tested at 1000 ppm in DMF solution. The strains
system the yield of product 8a improved (Table 2, entries 3–13). used for the activity were procured from MTCC (Microbial Type
No significant reaction was observed under either set of Culture Collection), Institute of Microbial Technology, Chandigarh.
conditions (entries 1 and 2). With this encouraging result, we The titled final 8(a–g) and 9(a–g) compounds were tested for
sought to screen inorganic (entries 3–11) and organic bases their anti-bacterial activity against Gram-negative bacteria such
(entries 12 and 13) used in the reaction, and found that the as Escherichia coli (MTCC 443), Salmonella typhi (MTCC 98) and
inorganic bases (entries 9–11) gave modest yields, and bases Vibrio cholerae (MTCC 3906) and Gram-positive bacteria such as
such as KOH (entry 6) and NaOH (entry 8) are more efficient Streptococcus pneumonia (MTCC 1936), Bacillus subtilis (MTCC
with almost quantitative yields. Similarly good yields were 441) and Clostridium tetani (MTCC 449), and their anti-fungal
detected when TEA was used as a base (entry 11).
activity against Candida albicans (MTCC 227), Trichophyton
In contrast, weaker bases such as K CO (entry 9) and organic rubrum (MTCC 297) and Aspergillus niger (MTCC 282). Ampicillin,
2
3
bases such as DMAP and piperidine (entries 12 and 13) gave ciprofloxacin, norfloxacin, nystatin, and griseofulvin were used as
decreased yields. We also studied the effect of the equiv. of the standard drugs for comparison of anti-bacterial and anti-fungal
base used, and found that 1 equiv. of KOH gave a comparatively activities, respectively. Discretion was evidenced by measuring
lower yield – i.e., the reaction was still incomplete (entries 3–5). the diameter of the inhibition zone at the end of 24 h for
When 1.5 equiv. of KOH was used (entry 6), it demonstrated bacteria at 35 1C and 48 h for fungi at 28 1C. The results are
superior activity and gave the best yield (93%). A further summarized in Table 3.
increase in the equiv. of KOH (entry 7) led to a decrease in the
reaction yield due to the multifarious reaction mixture. To find
3.2. In vitro antituberculosis activity
the most appropriate solvent for the reaction, various green The motivating results obtained by antimicrobial screening
solvents (glycol, t-BuOH, EtOH, H O) and an EtOH + H O prompted us to execute the preliminary screening of the title
2
2
mixture were used. Among the different reaction media, the synthesized compounds 8(a–g) and 9(a–g) for their in vitro
use of glycol and t-BuOH solvents in contrast to EtOH led to a antituberculosis activity against the Mycobacterium tuberculosis
significant decrease in the yield. However, it was in fact pleasing H37Rv strain. Primary screening of all the synthesized compounds
À1
to note that there was a significant increase in the yield of 8a was conducted at a concentration of 250 mg mL
using
18
with the choice of solvent as water (entry 4) and ethanol (entry 3) L o¨ wenstein–Jensen medium as described by Rattan. Rifampicin
as well, with a negative aspect that the desired product obtained and isoniazid were used as the standard drugs for comparison, and
was a sticky solid in the former and a lower ratio than the expected the results are summarized in Table 4.
one in the latter case. Hence, the appropriate reaction conditions
3.3. In vitro antimalarial activity
were restrained to the selection of an eco-friendly and safe solvent
medium – EtOH : H
2
O (1 : 1) (entries 5–8). Thus, during these All the synthesized compounds were screened for their in vitro
studies, we ascertained that the best results were obtained antimalarial activity against chloroquine and quinine sensitive
using this system. Employing the optimized conditions, we strains of P. falciparum. All experiments were performed in
next studied the scope of the reaction using the same aldehyde duplicate, and the IC50 mean values are given in Table 5.
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Table 3 % Yields of the synthesized compounds 8(a–g) and 9(a–g) and their in vitro antimicrobial activity (MIC, mg mL
)
Gram-negative bacteria
Gram-positive bacteria
Fungi
Entry
E.C. MTCC
43
S.T. MTCC
98
V.C. MTCC
3906
S.P. MTCC
1936
B.S. MTCC
441
C.T. MTCC
449
C.A. MTCC
227
T.R. MTCC
297
A.N. MTCC
282
4
4
5
200
250
100
100
250
250
100
200
100
200
62.5
100
250
250
125
100
100
62.5
250
100
62.5
100
250
100
25
200
250
100
100
200
200
100
100
62.5
125
100
250
200
100
125
200
100
25
100
62.5
62.5
100
200
100
62.5
100
100
100
62.5
200
200
100
125
200
100
50
200
100
100
100
125
250
100
62.5
100
250
125
250
62.5
200
200
100
250
50
62.5
200
250
62.5
100
250
200
250
100
250
62.5
250
250
200
100
100
250
100
50
1000
500
41000
500
41000
1000
1000
500
1000
41000
1000
100
1000
500
1000
500
41000
1000
500
41000
41000
100
1000
41000
—
8
8
8
8
8
8
8
9
9
9
9
9
9
9
a
b
c
d
e
f
g
a
b
c
d
e
f
500
41000
41000
41000
250
1000
500
500
500
62.5
41000
41000
1000
500
41000
1000
500
500
41000
—
—
250
200
100
200
100
250
100
250
100
25
100
1000
1000
41000
500
1000
—
g
Ampicillin
Ciprofloxacin
Norfloxacin
Nystatin
—
—
—
10
—
10
—
10
—
10
—
100
—
—
—
—
100
500
100
Greseofulvin
—
—
—
—
—
—
500
500
100
Boldfaced values indicate the active compounds; E.C., Escherichia coli; S.T., Salmonella typhi; V.C., Vibrio cholerae; S.P., Streptococcus pneumoniae;
B.S., Bacillus subtilis; C.T., Clostridium tetani; C.A., Candida albicans; T.R., Trichophyton rubrum; A.F.: Aspergillus niger; MTCC, microbial-type culture
collection; ‘—’ indicates not tested.
À1
Table 4 In vitro antituberculosis activity (% inhibition) of compounds drug ampicillin (MIC 100 mg mL ) against Gram-negative
À1
against M. tuberculosis H37Rv (at a concentration of 250 mg mL
)
bacteria, against E. coli compound 8f, against S. typhi compounds
b, 9b, and 9e and against V. cholera compound 8g were found to
have outstanding activity (MIC 62.5 mg mL ); whereas compounds
a, 8b, 8e, 9b, 9d, and 9f against E. coli, compounds 5, 8c, 9a, 9d,
and 9f against S. typhi and compounds 8a, 8b, 8e, 8f, 9b, and
e against V. cholerae showed significant equipotency (MIC
00 mg mL ) to the standard drug ampicillin (MIC 100 mg mL ).
In inhibiting Gram-positive bacteria, against S. pneumoniae
compounds 5, 8a, 8e, and 9b (MIC 62.5 mg mL ) showed
excellent potential and compounds 4, 8b, 8d, 8f, 8g, 9a, and
8
Entry
% Inhibition
Entry
% Inhibition
À1
4
5
62
69
61
91
78
64
83
65
61
9a
9b
9c
9d
9e
9f
9g
70
94
62
85
90
74
73
98
99
8
8
8
8
8
8
8
8
a
b
c
d
e
f
9
1
À1
À1
Rifampicin
Isoniazid
À1
g
À1
9
e (MIC 100 mg mL ) showed equipotency to the standard drug
ampicillin (MIC 100 mg mL ); against B. subtilis compounds 8f
and 9d (MIC 62.5 mg mL ) and 8c and 9b (MIC 125 mg mL )
À1
À1
Table 5 In vitro antimalarial activity of the compounds
À1
À1
À1
Entry
IC50 (mg mL
)
Entry
IC50 (mg mL )
showed outstanding activity in comparison to the standard
4
5
1.24
1.32
0.051
0.030
1.84
1.52
1.19
1.75
0.079
9a
9b
9c
9d
9e
9f
9g
1.45
0.041
1.50
1.45
0.054
0.83
0.092
0.020
0.268
À1
drugs ampicillin (MIC 250 mg mL ) and norfloxacin (MIC
À1
1
00 mg mL ), while compounds 5, 8a, 8b, 8e, 8g, and 9g
(MIC 100 mg mL ) showed equivalent results to norfloxacin
MIC 100 mg mL ) and excellent potency compared to ampicillin
MIC 250 mg mL ). Similarly, compounds 4, 8b, and 9d (MIC
62.5 mg mL ) against C. tetani showed excellent activity compared
to the standard drugs ampicillin (MIC 2500 mg mL ) and
ciprofloxacin (MIC 100 mg mL ). Compounds 8c, 8g, 9f, and 9g
MIC 100 mg mL ) exhibited greater potency than ampicillin (MIC
8
8
8
8
8
8
8
a
b
c
d
e
f
À1
À1
À1
(
(
À1
Chloroquine
Quinine
g
À1
À1
À1
(
2
4
. Results and discussion
À1
À1
50 mg mL ) and equipotency to ciprofloxacin (MIC 100 mg mL ).
4.1. Biological results
4
.1.2. In vitro antifungal activity. In vitro antifungal screening
4
.1.1. In vitro antibacterial activity. Upon reviewing the data (Table 3) demonstrated that, against C. albicans, com-
À1
antimicrobial screening data (Table 3), it has been observed pound 9b (MIC 100 mg mL ) exhibited equivalent activity
that a majority of the compounds showed excellent potential to nystatin (MIC 100 mg mL ), and along with it, 8f (MIC
against S. typhi, S. pneumonia, B. subtilis and C. tetani compared 250 mg mL ) exhibited fabulous activity compared to griseo-
to the standard drug ampicillin. In comparison to the standard fulvin (MIC 500 mg mL ), whereas compounds 5, 8b, 8c, 9a,
À1
À1
À1
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À1
and 9f (MIC 500 mg mL ) were of comparable potential to containing rings demonstrated increased activity against all
À1
griseofulvin (MIC 500 mg mL ). Against T. rubrum, compounds pathogens in comparison to triazole. In combination com-
À1
8
b, 8d, 8e, 9b, 9e, and 9f (MIC 500 mg mL ) were found to be pounds with benzylic substitutions (–F, –OH) were found highly
À1
equally active as nystatin and griseofulvin (MIC 500 mg mL ). active against S. pneumoniae, S. typhi and C. tetani (8b and 8e)
À1
Compounds 8b and 9e (MIC 100 mg mL ) were equivalent to and showed excellent antifungal activity against fungal strains
À1
both antifungal standard drugs (MIC 100 mg mL ).
T. rubrum (8b and 8e) and A. niger (8b), these substitutions also
4.1.3. In vitro antitubercular activity. Encouraged by the enhanced antimalarial and antitubercular activities. Secondly,
antimicrobial screening results, we screened for the antitubercular compounds with –OMe and –Me substituents were found to be
activity of the target compounds 8(a–g) and 9(a–g). Compounds 8b, reluctant towards all the strains in the former case; 8c was only
9
9
b and 9e were found to possess the highest potency with 91, 94 and active against B. subtilis in the former case, while the one
0% inhibition, respectively. On the other hand, two compounds 8e without any substituent showed inhibition against S. pneumoniae
and 9d are moderately active against M. tuberculosis H37Rv. All other and malarial strains. Further, in combination with heteroatom-
compounds showed poor inhibition of M. tuberculosis growth. containing motifs 6f and g increased antibacterial activity
.1.4. In vitro antimalarial activity. Compounds 8a, 8b, 8g, against E. coli, B. subtilis (8f), and V. cholerae (8g) was shown;
b, 9e and 9g showed principal activity against the P. falciparum increased antifungal activity against C. albicans and T. rubrum (8f);
4
9
strain in comparison to quinine (IC50 0.268), as their IC50 and antimalarial activities against P. falciparum strain (8g).
values were in the 0.030–0.092 range. While compound 8b The triazole ring increases the antibacterial activity against
was found to possess moderate activity (i.e. IC50 0.030) aligned S. pneumoniae (5), but in combination with the phenyl ring
with chloroquine, the remaining compounds showed less anti- having an electron withdrawing group (–F) exhibits increased
malarial activity against the P. falciparum strain in comparison antimicrobial potency against C. tetani, S. pneumoniae, and
to chloroquine and quinine drugs. From the above results, it C. albicans, and also amplifies the antitubercular and antimalarial
can be concluded that in this set of synthesized heterocyclics, activities (9b). Further analysis shows that electron releasing
compounds 8b, 9b and 9e may become a promising new class groups (–Me, –OMe) decrease the potency towards fungal strains:
of antimicrobial, antitubercular and antimalarial agents in the the latter showed higher inhibition against B. subtilis (9d) and also
future.
increased antitubercular activity, but the compound with the
4.1.5. Structure–activity relationship (SAR). The structural former substituent was poorly active against all pathogens. The
activity relationship analysis (Fig. 2) demonstrated that the compound with the –OH group replacement (9e) exhibited
imidazole and triazole nuclei at the fifth position of the pyrazole excellent pronounced antimalarial, antitubercular, and anti-
ring in connotation with the different natures of groups at the para microbial activities against P. falciparum, Mycobacterium tuberculosis
position of the phenyl ring and various heterocyclic motifs 6(a–g) at H37Rv, S. typhi and T. rubrum strains, respectively; while the
the sixth position of the pyrimidine ring are prone to incongruity compounds without any phenyl ring substitution were dubious
in biological activities. The imidazole nucleus is responsible for against any strains. On the other hand, in association with sulphur
improved antibacterial activity against C. tetani (4) but in and nitrogen containing heterocycles, compound 5 demonstrated
combination with various substituted phenyl and heterocycle enhanced antifungal activity against T. rubrum and A. niger (9f) in
Fig. 2 Structure–activity relationships for the antimicrobial, antimalarial and antituberculosis activities of the synthesized compounds 8(a–g) and 9(a–g).
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the former case and increased antimalarial activity (9g) in the latter plates precoated with silica gel, 60F, 0.25 mm thickness) (Merck,
case and was poorly active against bacterial strains. Thus, appraising Darmstadt, Germany) was used for monitoring the progress of all
the activity data, it is noteworthy that the various pyrimidine target reactions, purity, and homogeneity of the synthesized compounds
compounds with the imidazole nucleus were found to be much (eluent ethyl acetate:hexane 7:3). The melting points of all the
more pharmacologically active than those with the triazole nucleus. titled compounds were determined by an open-tube capillary
The overall activity of the target compounds depends not only on method and are uncorrected. UV radiation and/or iodine were
the nature of the nucleus attached at the fifth position of the used as the visualizing agents. Mass spectra were recorded on a
pyrazole ring but also on the peripheral substituents appended Shimadzu LCMS 2010 spectrometer (Shimadzu, Tokyo, Japan)
through the phenyl ring and heteroaromatic moieties, and purchased under the PURSE program of DST at Sardar Patel
positional changes.
University, Vallabh Vidyanagar. The IR spectra were recorded
using KBr on a Perkin-Elmer Spectrum GX FT-IR Spectrophot-
ometer (Perkin-Elmer, USA) and only the characteristic peaks are
reported at 254 cm . Elemental analysis (% C, H, and N) was
carried out using a Perkin-Elmer 2400 series-II elemental analyzer
5. Conclusion
À1
In summary, we have reported a proficient, facile, mild green
environmentally benign synthetic protocol for the synthesis of
imidazo[1,2-a]pyrimidine derivatives of pyrazole clubbed with
the imidazole and triazole nuclei in good yield based on a
microwave-assisted, one-pot three-component condensation
reaction for the first time. The key step in the suggested
mechanistic pathway involves the base-mediated formation of
a,b-unsaturated ketone generated in situ followed by cyclization
with 2-amino benzimidazole to afford the final targeted product
through an easy way. All the synthesized compounds were
examined for their various biological activities with the hope
of discovering new structural classes of agents with antimicrobial,
antimalarial and antitubercular activities, which can be of use
for further meticulous pre-clinical explorations. A majority of
compounds have emerged as effective inhibitory agents against
S. typhi, S. pneumoniae, B. subtilis, and C. tetani. Compounds 8f
and 9b showed outstanding activity against C. albicans, while
compounds 8b and 9e showed against A. niger as compared
to griseofulvin and nystatin. Compounds 8b, 9b, 9e and 9d
exhibited the best antimalarial and antituberculosis activities.
The fluoro, hydroxyl, and thiophene groups and the imidazole
nucleus in comparison to that of the triazole nucleus to some extent
in the imidazopyrimidine derivative of pyrazole compounds are
considered to be responsible for the biological activity.
(
Perkin-Elmer, USA) and all the compounds are within Æ0.4% of
1 13
theory specified. H NMR and C NMR spectra were recorded in
CDCl solvent on a Bruker Avance 400F (MHz) spectrometer
Bruker Scientific Corporation Ltd, Switzerland) using the residual
3
(
solvent signal as an internal standard at 400 MHz and 100 MHz
respectively. Chemical shifts are reported in parts per million (ppm).
6
.1.1. General procedure for the synthesis of substituted
-(heteroaryl)-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehydes
4 and 5). 5-Chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde
(1 mmol), substituted heteroaryl compound imidazole 2/1,2,4-
5
(
1
triazole 3 (1 mmol) and anhydrous potassium carbonate (2 mmol)
in dimethylformamide (5 mL) were charged into a 100 mL round
bottom flask equipped with a mechanical stirrer and condenser.
The reaction mixture was refluxed and heated at 80 1C for 2 h and
the progress of the reaction was monitored by TLC. After the
completion of the reaction (as evidenced by TLC), the reaction
mixture was cooled to room temperature and then poured into
ice cold water (50 mL) with continuous stirring followed by
neutralization with dilute protonic acid. The separated precipitates
of 5-(1H-imidazol/1,2,4-triazol-1-yl)-3-methyl-1-phenyl-1H-pyrazole-4-
carbaldehydes 4 and 5 were filtered, thoroughly washed with water,
dried, and recrystallized from ethanol.
6
.1.2. General procedure for the synthesis of 4-(5-(1H-imidazol/
,2,4-triazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-(4-substituted-
phenyl/hetero-aryl)benzo[4,5]imidazo[1,2-a]pyrimidine derivatives
8a–g and 9a–g)
1
(
6
. Experimental
6.1.2.1. Microwave irradiation method
6.1. Chemistry
Method A (one pot, one-step). In a 50 mL round bottom
All reactions were performed with commercially available flask, equimolar amounts of the corresponding 5-(1H-imidazol/
reagents, which were used without further purification. Phenylhy- 1,2,4-triazol-1-yl)-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde
drazine, potassium hydroxide and acetophenones were purchased 4/5 (1.0 mmol), 1-(4-substituted-phenyl/hetero-aryl)ethanones
from S.D. Fine Chem Ltd, Vadodara, Gujarat, India. 2-Amino- 6a–g (1.0 mmol) and 2-aminobenzimidazole 7 (1.5 mmol) in
benzimidazole was purchased from Sigma-Aldrich. Solvents were
2
H O : EtOH (1 : 1) containing powdered potassium hydroxide
purchased from Spectrochem and were purified and dried before (1.5 mmol) were mixed thoroughly and subjected to microwave
being used. The microwave-assisted reactions are conducted in a irradiation at 340 W for 15–20 min. After the completion of the
‘
‘RAGA’s Modified Electromagnetic Microwave System,’’ wherein reaction (evidenced by TLC, ethyl acetate : hexane (7 : 3)), the
microwaves are generated by a magnetron at a frequency of resulting solution was cooled to room temperature. Then, an
450 MHz having adjustable output power levels, i.e., 10 levels EtOH–H O equimixture was added to it and heated with
2
2
from 140 to 700 W and with an individual sensor (a fiber-optic vigorous stirring. The solid mass that separated upon cooling
sensor) for temperature control attached to a reflux condenser was filtered, washed well with a cold water : ethanol equimixture,
with constant stirring (thus avoiding the risk of high pressure dried and purified by chromatography using ethyl acetate : hexane
development). Thin-layer chromatography (TLC, on aluminum (7 : 3) as eluent to obtain a pure solid sample.
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Method B (one-pot, two-step). 5-(1H-Imidazol/1,2,4-triazol-1- m.p. 208 1C; IR (KBr, n, cm ): 2967 (aromatic ring –CH), 1589
1
yl)-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 4/5 (1.0 mmol), (–CQN), 1371 (Ar-CH
3 6
); H NMR (400 MHz, DMSO-d ): d 2.25 (s,
1
3
1
-(4-substituted-phenyl/hetero-aryl)ethanones 6a–g (1.0 mmol) and 3H, CH ), 2.43 (s, 3H, ArCH ), 6.77–8.12 (m, 17H, ArH) ppm;
potassium hydroxide (1.5 mmol) in a H O : EtOH equimixture were NMR (100 MHz, DMSO-d ): d 13.1, 21.6, 109.8, 112.6, 116.0,
C
À
3
À
3
À
2
6
mixed and stirred at room temperature for 10–15 min until the 116.3, 119.9, 120.9, 122.5, 123.0, 126.6, 127.3, 129.0, 129.8, 129.9,
formation of a precipitate. After the completion of the reaction (as 130.1, 131.2, 132.4, 134.1, 136.9, 137.2, 138.7, 145.5, 148.8, 151.7,
evidenced by TLC), 2-aminobenzimidazole 7 (1.5 mmol) was added 159.8; anal. calc. for C30H N (481.55): C, 74.83; H, 4.81; N,
23 7
to the mixture, which was then subjected to microwave irradiation 20.36; found: C, 75.06; H, 5.12; N, 20.62%.
at 340 W for 10–15 min in a 50 mL round bottom flask. An EtOH–
6
.1.2.6. 4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-
2
H O mixture (1 : 1) was added to the reaction mixture and heated
with vigorous stirring. Then the solid mass that separated upon
cooling was removed by filtration, washed well with cold EtOH–
4
-yl)-2-(4-methoxyphenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (8d).
Yellow solid; m.p. 222 1C; IR (KBr, n, cm ): 3035 (aromatic
ring –CH), 1610 (–CQN), 1368 (Ar-CH
DMSO-d ): d 2.26 (s, 3H, CH ), 3.90 (s, 3H, –OCH
3
6
4H, ArH), ppm; C NMR (100 MHz, DMSO-d ): d 13.1, 55.5,
À1
1
3
); H NMR (400 MHz,
H O (1 : 1) and dried in air and purified by leaching in an equal
2
6
À
3
À
), 6.75–8.20 (m,
volume ratio of chloroform and methanol, affording pure solid
compounds.
1
3
1
1
1
À
06.7, 109.9, 112.5, 114.4, 119.9, 120.7, 122.2, 123.1, 125.2,
26.3, 127.4, 128.7, 129.0, 129.3, 129.6, 129.8, 131.2, 134.1,
6.1.2.2. Conventional method
Method C (one pot, two-step). 5-(1H-Imidazol/1,2,4-triazol-1- 136.9, 138.2, 145.5, 148.8, 152.0, 160.6; MS (m/z) calc.: 497.55,
yl)-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 4/5 (1.0 mmol), found: 498.23 [M + 1]; anal. calc. for C30 O: C, 72.42; H,
-(4-substituted-phenyl/hetero-aryl)ethanones 6a–g (1.0 mmol) 4.66; N, 19.71; found: C, 72.73; H, 4.45; N, 20.14%.
23 7
H N
1
and potassium hydroxide (1.5 mmol) in a H O : EtOH equimixture
2
6
.1.2.7. 4-(4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-
were mixed and stirred at room temperature for 10–15 min until the
formation of a precipitate. Subsequently, after the completion of the
reaction (as evidenced by TLC) in a 50 mL round bottom flask,
4
-yl)benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)phenol (8e). Yellow
solid; m.p. 218 1C; IR (KBr, n, cm ): 2989 (aromatic ring
À1
1
–
(
CH), 1591 (–CQN), 1373 (Ar-CH
400 MHz, DMSO-d ): d 2.26 (s, 3H, CH
ArH), 13.52 (s, 1H, ArOH) ppm; C NMR (100 MHz, DMSO-d
3
) 3350 (–OH); H NMR
2-aminobenzimidazole 7 (1.5 mmol) was added and then the
6
À
3
), 6.75–8.08 (m, 17H,
reaction mixture was allowed to stir for 28 h until chalcone was
completely consumed. Then to the resulting reaction mass,
water (10 mL) was added. The mixture was ultrasonically agitated
for 30 min and then filtered. The solid mass that separated upon
cooling was filtered, washed well with a cold water : ethanol equi-
mixture, dried and purified by leaching in an equal volume ratio of
chloroform and methanol, affording pure solid compounds.
1
3
À
À
6
):
d 13.2, 106.2, 112.6, 117.1, 119.4, 120.6, 120.6, 122.7, 123.6,
1
1
26.7, 127.6, 127.8, 129.5, 129.7, 129.9, 134.3, 136.9, 138.9,
45.1, 145.2, 146.4, 148.2, 148.9, 153.6, 155.9; anal. calc. for
C H N O (483.52): C, 72.04; H, 4.38; N, 20.28; found: C, 73.97;
2
9
21 7
H, 4.63; N, 19.86%.
6
.1.2.8. 4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-
6
.1.2.3. 4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-
4
-yl)-2-(thiophen-2-yl)benzo[4,5]imidazo[1,2-a]pyrimidine (8f). Yellow
solid; m.p. 234 1C; IR (KBr, n, cm ): 3054 (aromatic ring –CH),
575 (–CQN), 1378 (Ar-CH
s, 3H, CH ), 6.76–8.13 (m, 16H, Ar-H), ppm; C NMR (100 MHz,
yl)-2-phenylbenzo[4,5]imidazo[1,2-a]pyrimidine (8a). Yellow solid;
m.p. 215 1C; IR (KBr, n, cm ): 3011 (aromatic ring –CH), 1651
–CQN), 1370 (Ar-CH
H, –CH
À1
À1
1
1
(
3 6
); H NMR (400 MHz, DMSO-d ): d 2.26
1
(
3
3
); H NMR (400 MHz, DMSO-d
6
): d 2.26 (s,
13
3
À
1
3
À
3
), 6.75–8.24 (m, 18H, Ar-H), ppm; C NMR (100 MHz,
): d 13.0, 100.0, 107.0, 109.9, 112.6, 119.9, 120.9, 122.4,
À
À
DMSO-d
26.1, 126.4, 127.3, 128.5, 129.0, 129.5, 129.9, 132.1, 133.4, 136.9,
37.9, 142.5, 145.2, 148.9, 151.5, 153.6, 155.9; anal. calc. for
S: C, 68.48; H, 4.04; N, 20.70; found: C, 68.51; H, 4.25;
N, 19.98%.
6
): d 13.2, 106.7, 110.9, 112.6, 119.8, 120.7, 122.5, 123.2,
DMSO-d
6
1
1
1
1
4
23.1, 126.5, 127.9, 129.0, 129.8, 131.2, 131.6, 134.1, 136.2, 137.0,
37.2, 138.5, 145.6, 148.8, 151.8, 156.0, 156.4; MS (m/z) calc.:
67.52, found: 468.14 [M + 1]; anal. calc. for C H N : C, 74.50;
H, 4.53; N, 20.97; found: C, 75.02; H, 4.13; N, 21.05%.
27 19 7
C H N
29
21 7
6
.1.2.9. 4-4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-
6
.1.2.4. 4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-
4
-yl)-2-(1H-pyrrol-2-yl)benzo[4,5]imidazo[1,2-a]pyrimidine (8g). Orange
solid; m.p. 215 1C; IR (KBr, n, cm ): 3071 (aromatic ring –CH), 1643
–CQN), 1370 (Ar-CH ), H NMR (400 MHz, DMSO-d ): d 2.26 (s, 3H,
CH ), 6.75–8.21 (m, 16H, Ar-H), 9.73 (s, 1H, –NH) ppm; C NMR
3
(100 MHz, DMSO-d
23.1, 126.4, 127.3, 127.9, 128.9, 129.4, 129.9, 131.1, 133.4, 134.1,
36.9, 137.3, 138.4, 142.3, 145.5, 148.6, 151.9, 161.1; MS (m/z) calc.:
56.50, found: 457.13 [M + 1]; anal. calc. for C27 : C, 71.04; H,
.42; N, 24.55; found: C, 69.97; H, 4.13; N, 24.74%.
À1
4
-yl)-2-(4-fluorophenyl)benzo[4,5]imidazo[1,2-a]pyrimidine (8b). Orange
solid; m.p. 210 1C; IR (KBr, n, cm ): 3021 (aromatic ring –CH), 1591
–CQN), 1367 (Ar-CH ); H NMR (400 MHz, DMSO-d ): d 2.26 (s, 3H,
À1
1
(
3
6
1
1
3
(
–
d
6
1
1
(
3
6
À
À
À
13
3
CH ), 6.75–8.23 (m, 17H, Ar-H), ppm; C NMR (100 MHz, DMSO-
À
À
6
): d 13.0, 106.9, 109.9, 112.5, 119.8, 120.9, 122.4,
): d 13.1, 106.9, 109.9, 112.5, 119.9, 120.7, 122.3, 123.1, 126.4,
27.4, 127.8, 128.9, 129.8, 131.1, 133.4, 134.1, 136.9, 137.2, 138.3,
42.3, 145.5, 148.8, 151.9, 161.0, 162.5; anal. calc. for C29
485.51): C, 71.74; H, 4.15; F, 3.91; N, 20.19; found: C, 71.47; H, 4.91;
N, 20.24%.
1
1
4
4
H20FN
7
20 8
H N
6.1.2.10. 4-(3-Methyl-1-phenyl-5-(1H-1,2,4-triazol-1-yl)-1H-pyrazol-
6
.1.2.5. 4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4- 4-yl)-2-phenylbenzo[4,5]imidazo[1,2-a]pyrimidine (9a). Yellow solid;
À1
yl)-2-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyrimidine (8c). Yellow solid; m.p. 212 1C; IR (KBr, n, cm ): 2984 (aromatic ring –CH),
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1
1
2
(
623 (–CQN), 1379 (Ar-CH
.28 (s, 3H, CH
3
); H NMR (400 MHz, DMSO-d
6
): d (100 MHz, DMSO-d
6
): d 13.2, 106.7, 110.9, 112.6, 120.7, 122.3,
13
À
3
), 7.19–8.27 (m, 17H, ArH) ppm; C NMR 123.6, 126.2, 126.3, 127.7, 128.5, 129.1, 129.3, 129.8, 132.1,
100 MHz, DMSO-d ): d 13.3, 104.4, 106.9, 110.0, 112.8, 116.3, 133.4, 136.9, 137.9, 142.6, 145.3, 148.9, 151.4, 153.6, 155.9;
À
6
120.8, 122.3, 123.5, 126.5, 129.3, 129.9, 130.1, 132.4, 133.4, 135.3, MS (m/z) calc.: 474.54, found: 475.02 [M + 1]; anal. calc. for
1
36.9, 138.2, 145.6, 148.9, 151.7, 153.4, 159.8, 163.3; MS (m/z) C H N S: C, 65.81; H, 3.82; N, 23.61; found: C, 65.45; H, 4.21;
2
6
18 8
20 8
calc.: 468.51, found: 469.02 [M + 1]; anal. calc. for C28H N : C, N, 23.14%.
7
1.78; H, 4.30; N, 23.92; found: C, 72.01; H, 4.46; N, 23.24%.
6
.1.2.16. 4-(3-Methyl-1-phenyl-5-(1H-1,2,4-triazol-1-yl)-1H-pyrazol-
6.1.2.11. 2-(4-Fluorophenyl)-4-(3-methyl-1-phenyl-5-(1H-1,2,4-
4
-yl)-2-(1H-pyrrol-2-yl)benzo[4,5]imidazo[1,2-a]pyrimidine (9g). Orange
solid; m.p. 235 1C; IR (KBr, n, cm ): 3068 (aromatic ring –CH), 1627
–CQN), 1378 (Ar-CH ); H NMR (400 MHz, DMSO-d ): d 2.28 (s, 3H,
triazol-1-yl)-1H-pyrazol-4-yl)benzo[4,5]imidazo[1,2-a]pyrimidine (9b).
Orange solid; m.p. 206 1C; IR (KBr, n, cm ): 2993 (aromatic ring
À1
À1
1
(
3
6
1
–
CH), 1642 (–CQN), 1372 (Ar-CH
d 2.28 (s, 3H, CH
3
); H NMR (400 MHz, DMSO-d
6
):
13
CH ), 6.92–8.17 (m, 15H, ArH), 10.24 (s, 1H, NH) ppm; C NMR
À
3
À
À
13
3
), 7.19–8.24 (m, 16H, ArH), ppm; C NMR
): d 13.2, 104.4, 107.0, 111.1, 112.8, 116.3,
À
À
6
(100 MHz, DMSO-d ): d 13.2, 106.5, 110.8, 112.6, 120.6, 122.2, 123.4,
25.9, 126.4, 127.5, 128.4, 129.2, 129.3, 129.9, 132.1, 133.4,
36.9, 137.8, 142.6, 145.3, 148.9, 151.4, 153.6, 155.9; anal. calc.
for C26
7.89; H, 4.31; N, 27.54%.
(100 MHz, DMSO-d
6
1
1
1
1
20.8, 122.4, 123.6, 126.5, 127.4, 129.9, 130.0, 132.5, 133.4, 135.4,
36.9, 138.3, 145.5, 148.9, 151.7, 153.6, 159.7, 163.3; anal. calc. for
19 9
H N : (457.49) C, 68.26; H, 4.19; N, 27.55; found: C,
C H FN (486.50): C, 69.13; H, 3.94; N, 23.03; found: C, 68.87; H,
28
19
8
6
4.04; N, 22.85%.
6
.2. Biological evaluation
.2.1. In vitro evaluation of antimicrobial activity. The
MICs of the synthesized compounds 8(a–g) and 9(a–g) were
6
.1.2.12. 4-(3-Methyl-1-phenyl-5-(1H-1,2,4-triazol-1-yl)-1H-pyrazol-
6
4
-yl)-2-(p-tolyl)benzo[4,5]imidazo[1,2-a]pyrimidine (9c). Yellow solid;
m.p. 219 1C; IR (KBr, n, cm ): 3087 (aromatic ring –CH), 1651
–CQN), 1369 (Ar-CH ); H NMR (400 MHz, DMSO-d ): d 2.27 (s,
3 3
H, CH ), 2.44 (s, 3H, CH ), 7.18–8.16 (m, 16H, ArH), ppm;
C NMR (100 MHz DMSO-d
12.8, 120.7, 122.1, 123.6, 126.3, 127.8, 129.3, 129.8, 133.5, 136.9,
37.9, 142.2, 144.0, 145.3, 145.5, 153.6, 157.9, 158.9, 159.4, 160.9; MS
m/z) calc.: 482.54, found: 483.13 [M + 1]; anal. calc. for C29 : C,
2.18; H, 4.60; N, 23.22; found: C, 71.95; H, 4.46; N, 23.43%.
À1
1
7
1
determined by a broth microdilution method. Serial dilutions
were prepared in primary and secondary screening. DMSO was
used as a diluent to achieve the preferred concentration of
compounds to test upon standard bacterial strains. The tubes
were then incubated overnight. The control tube without any
antibiotic was instantly subcultured (before inoculation) by
dispersing a loopful evenly over a quarter of plate of medium
appropriate for the growth of the test organism and put for
incubation at 37 1C overnight. The MIC is defined as the lowest
concentration of an antibiotic or a test sample allowing no
visible growth. The MIC of the control organism was read to
check the accuracy of the compound concentrations. The
amount of growth from the control tube before incubation
(which represents the original inoculum) was compared. All the
tubes not showing visible growth (in the same manner as the
control tube described above) were subcultured and incubated
overnight at 37 1C. The subcultures might show similar numbers
of colonies (indicating bacteriostatic activity), a reduced number
of colonies (indicating a partial or slow bactericidal activity) and
no growth (if the whole inoculum was killed). Each synthesized
(
3
6
3
À
À
À
1
3
6
): d 13.2, 21.5, 101.3, 107.3, 111.3,
1
1
(
7
22 8
H N
6.1.2.13. 2-(4-Methoxyphenyl)-4-(3-methyl-1-phenyl-5-(1H-1,2,4-
triazol-1-yl)-1H-pyrazol-4-yl)benzo[4,5]imidazo[1,2-a]pyrimidine (9d).
Yellow solid; m.p. 223 1C; IR (KBr, n, cm ): 3064 (aromatic ring
CH), 1610 (–CQN), 1379 (Ar-CH
d 2.28 (s, 3H, CH ), 3.904 (s, 3H, OCH ), 7.03–8.24 (m, 16H, ArH),
À1
1
–
3
); H NMR (400 MHz, DMSO-d
6
):
À
3
À
6
3
À
13
ppm; C NMR (100 MHz, DMSO-d ): d 13.2, 21.5, 101.3, 107.3,
1
1
1
2
11.3, 112.8, 120.7, 122.1, 123.6, 126.3, 127.8, 129.3, 129.8, 133.5,
36.9, 137.9, 142.2, 144.0, 145.3, 145.5, 153.6, 157.9, 158.9, 159.4,
60.9; anal. calc. for C29 O (498.54): C, 69.87; H, 4.45; N,
2.48; found: C, 69.72; H, 4.83; N, 21.97%.
22 8
H N
6
.1.2.14. 4-(4-(3-Methyl-1-phenyl-5-(1H-1,2,4-triazol-1-yl)-1H-
pyrazol-4-yl)benzo[4,5]imidazo[1,2-a]pyrimidin-2-yl)phenol (9e).
Yellow solid; m.p. 247 1C; IR (KBr, n, cm ): 3017 (aromatic
À1
À1
compound was diluted to obtain 2000 mg mL concentration as
a stock solution. The test must include a second set of the same
dilutions inoculated with an organism of known sensitivity. In
1
ring –CH), 1611 (–CQN), 1371 (Ar-CH
400 MHz, DMSO-d ): d 2.29 (s, 3H, CH
ArH), 13.54 (s, 1H, OH) ppm; C NMR (100 MHz, DMSO-d
3
), 3340 (–OH); H NMR
(
6
3
), 7.11–8.01 (m, 16H,
À
À1
1
3
primary screening 500, 250 and 200 mg mL concentrations of
À
À
6
):
d 13.2, 106.3, 112.7, 117.1, 119.4, 119.3, 120.6, 122.7, 123.6,
the synthesized compounds were taken. The active synthesized
compounds found in this primary screening were further tested
in a second set of dilutions against all microorganisms. The
compounds that were found to be active in primary screening
were similarly diluted to obtain 100, 62.5, 50, 25 and 12.5 mg mL
concentrations. The highest dilution showing at least 99%
1
1
(
2
26.7, 127.7, 127.8, 129.4, 129.9, 134.3, 136.8, 138.8, 145.1, 145.2,
46.4, 148.3, 148.9, 153.7, 155.9; anal. calc. for C H N O
484.51): C, 69.41; H, 4.16; N, 23.13; found: C, 68.85; H, 4.24; N,
2
8 20 8
À1
3.31%.
6.1.2.15. 4-(3-Methyl-1-phenyl-5-(1H-1,2,4-triazol-1-yl)-1H-pyrazol- inhibition is taken as the MIC.
4
-yl)-2-(thiophen-2-yl)benzo[4,5]imidazo[1,2-a]pyrimidine (9f). Yellow
6.2.2. In vitro evaluation of antituberculosis activity. The
À1
solid; m.p. 227 1C; IR (KBr, n, cm ): 2986 (aromatic ring –CH), drug susceptibility and the antituberculosis activity of the test
1
d 2.28 (s, 3H, CH
1
643 (–CQN), 1376 (Ar-CH
3 6
); H NMR (400 MHz, DMSO-d ): compounds against M. tuberculosis H37Rv were investigated by
1
3
17
), 7.13–8.15 (m, 15H, ArH) ppm; C NMR the L ¨o wenstein–Jensen slope method with a slight modification,
À
3
À
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À1
where 250 mg mL dilution of each test compound was added to SICART, Vallabh Vidyanagar, for elemental analysis and Microcare
liquid L o¨ wenstein–Jensen medium, and then the media were Laboratory, Surat, for the biological screening of the compounds
sterilized by an inspissation method. The title compounds solution reported herein. One of the authors (P. Prasad) is grateful to the
À1
of 250 mg mL concentration was prepared in DMSO. A culture of UGC, New Delhi, for a BSR Research Fellowship.
M. tuberculosis H37Rv growing in L o¨ wenstein–Jensen medium was
harvested in 0.85% saline in bijou bottles. These tubes were then
incubated at 37 1C for 24 h followed by streaking of M. tuberculosis
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incubated at 37 1C. Growth of bacilli was seen after 12 days,
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1
9
method described by Trager and Jensen. The P. falciparum
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20
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21
with 5% D-sorbitol when the parasites were in the ring stage. The
stock solutions were additionally diluted in whole medium (RPMI
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was adjusted to 1–2% parasitaemia and 2.5% haematocrit in
hypoxanthine-free RPMI-1640 culture medium with 10% human
plasma and was exposed to 7 concentrations of each compound for
a single cycle of parasite growth of 48 h at 37 1C. A positive control
with reference to antimalarial drugs at standard concentrations was
used in each experiment. The standard drugs chloroquine and
quinine were used as the reference antimalarial agents, blood
smears were read blind and each duplicate experiment was
repeated three times.
7
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3
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