Development of 7-Aminopyrazolo[1,5-a]pyrimidines as Anti-Candida and Antibacterial Agents

Full text of DOI:10.1080/00304948.2020.1833622

Organic Preparations and Procedures International
The New Journal for Organic Synthesis
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Development of 7-
Aminopyrazolo[1,5-a]pyrimidines as Anti-Candida
and Antibacterial Agents
Ranjana Aggarwal , Gulshan Singh , Virender Kumar , Eakta Masan & Pranay
Jain
To cite this article: Ranjana Aggarwal , Gulshan Singh , Virender Kumar , Eakta Masan &
Pranay Jain (2021) Development of 7-Aminopyrazolo[1,5-a]pyrimidines as Anti-Candida and
Antibacterial Agents, Organic Preparations and Procedures International, 53:1, 42-51, DOI:
10.1080/00304948.2020.1833622
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ORGANIC PREPARATIONS AND PROCEDURES INTERNATIONAL
2021, VOL. 53, NO. 1, 42–51
https://doi.org/10.1080/00304948.2020.1833622
EXPERIMENTAL PAPER
Development of 7-Aminopyrazolo[1,5-a]pyrimidines as
Anti-Candida and Antibacterial Agents
a
a
a
a
ꢀ
Ranjana Aggarwal , Gulshan Singh , Virender Kumar , Eakta Masan , and
Pranay Jainb
b
aDepartment of Chemistry, Kurukshetra University, Kurukshetra, Haryana, India; Department of
Biotechnology, University Institute of Engineering and Technology, Kurukshetra University, Kurukshetra,
Haryana, India
ARTICLE HISTORY Received 1 April 2020; Accepted 18 July 2020
We have previously developed1 a chemo and regioselective synthesis of pyrazol-1’-ylpyr-
azolo[1,5-a]pyrimidines 3a-h (Scheme 1). This was achieved by treatment of 5-amino-
3-hydrazinopyrazole dihydrochloride (1) with symmetrical and unsymmetrical b-diketones
2 in aqueous media. We have further found the pyrazol-1’-ylpyrazolo[1,5-a]pyrimidines
to be excellent antibacterial and antifungal agents2 for which structure-activity studies
showed that the antimicrobial potency was mainly influenced by the substituents at pos-
ition 7.
With these observations as background,3 we now describe the formation of the title
compounds by the reaction of 1 with 3(2)-aryl-3-oxoalkanenitriles. The novel com-
pounds were screened for their anti-Candida and antibacterial potency. Given the
advantages of green chemistry protocols,4–6 it was envisaged to condense 1 with com-
pounds 4 under solvent-free conditions. For example, one equivalent of 1 was thor-
oughly ground with two equiv of NaOAc and two equiv of 4a at room temperature.
Then the reaction mixture was heated on the water bath for 15 min to afford compound
5a (70%) as the exclusive product (Scheme 2). The presence of NaOAc was found to be
essential, as no product could be achieved without NaOAc. Following a similar protocol,
other pyrazolo[1,5-a]pyrimidines 5b-g were synthesized by condensing 1 with other
3(2)-aryl-3-oxoalkanenitriles 4b-g. All of the products were fully characterized (see
Experimental section).
The formation of compound 5a is attributed to the in situ generation of hydrazine by
C-N bond cleavage in 1 following the proposed mechanism depicted in Scheme 3.
To support the possible in situ generation of hydrazine, we undertook to synthesize
5a directly from hydrazine hydrate itself (Scheme 4). In exploring this possibility, the
reaction solvent was changed from toluene to a mixture of toluene and ethanol (9:1),
with a small amount of p-toluenesulfonic acid (PTSA). This indeed led to 5a (65%) in
10 hr, unambiguously determined from a comparison with an authentic sample.
CONTACT Ranjana Aggarwal
ranjana67in@yahoo.com
Department of Chemistry, Kurukshetra University,
Kurukshetra-136 119, Haryana, India
CSIR-National Institute of Science, Technology and Development Studies (NISTADS), Pusa Gate, K.S. Krishnan Marg,
New Delhi.
ß 2020 Taylor & Francis Group, LLC

ORGANIC PREPARATIONS AND PROCEDURES INTERNATIONAL
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Scheme 1. General reaction of 5-amino-3-hydrazinopyrazole dihydrochloride (1) and b-diketones (2).
Scheme 2. Reaction of 5-amino-3-hydrazinopyrazole dihydrochloride 1 with 3(2)-aryl-3-oxoalkaneni-
triles 4.
Scheme 3. Generation of hydrazine in situ.
We surmise that formation of 5a directly from hydrazine hydrate follows the mech-
anistic pathway shown in Scheme 5. Thus hydrazine hydrate 6 condenses with one
mole of 4a to give the intermediate 5-amino-1H-pyrazole 7. The exocyclic amino group
of 7 reacts with the carbonyl group of a second molecule of 4a to give the intermediate
8. Finally, intramolecular nucleophilic addition gives 5a.
The robust character of the protocol used in this mechanistic exploration encouraged
us to use it to prepare more 7-aminopyrazolo[1,5-a]pyrimidines (5h-5o). These com-
pounds had further structural variations at positions 2, 3, 5 and 6 of the pyrazolo[1,5-
a]pyrimidine moiety (Scheme 6). We anticipated these would be useful for estimating

44
R. AGGARWAL ET AL.
Scheme 4. Formation of 5a directly from hydrazine hydrate.
Scheme 5. Proposed mechanism for formation of 5a from hydrazine.
structure-activity relationships in the microorganisms we wished to assay. We also used
this method to again prepare compounds 5a-5g. The results of our synthesis efforts are
summarized in Table 3. Following our synthetic methods, no purification was needed.
Compounds 5a-o were screened for their in vitro anti-Candida activity against four
pathogenic Candida strains, namely Candida albicans (MTCC 3017), Candida glabrata
(MTCC 3019), Candida tropicalis (MTCC 184) and Candida parapsilosis (MTCC 1965)
obtained from Microbial Type Culture Collection, Institute of Microbial Technology
(IMTECH), Chandigarh. Compounds showing zones of inhibition of 12 mm or more
were further screened for their minimum inhibitory concentration (MIC) through the
agar-well diffusion assay technique. The results are compiled in Table 1. For evaluating
anti-Candida activity the known antifungal fluconazole was used as standard drug. It is
worth mentioning that most of these compounds showed anti-Candida activity against
C. albicans. Interestingly, compounds 5i, 5l, and 5o showed activity against C. glabrata,
an organism which was resistant to fluconazole. The anti-Candida activity against C.
tropicalis increases to a significant extent when the hydrogen of the phenyl residue of
compound 5a is replaced with a chlorine in 5b, bromine in 5d and fluorine in 5c.
Compounds 5a-o were assayed in vitro for their antibacterial activity against two
Gram-positive bacteria {Staphylococcus aureus (MTCC 890), Streptococcus mutans
(MTCC 3160)} and two Gram-negative bacteria {Pseudomonas aeruginosa (MTCC
2295), Escherichia coli (MTCC 43)} obtained from MTCC, IMTECH, Chandigarh. For
evaluating antibacterial activity, ciprofloxacin was used as the standard drug. The results
of antibacterial activity are presented in Table 2. In general, the results reveal that many
of the tested compounds showed antibacterial potency towards S. mutans and P.

ORGANIC PREPARATIONS AND PROCEDURES INTERNATIONAL
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Scheme 6. Preparation of compounds 5a-o.
aeruginosa. The data in Table 2 suggest that the movement of an aryl residue from pos-
ition-2 to position-3 increases the antibacterial potency of the compounds. Replacement
of hydrogen (5a) and chlorine (5b) with a methyl group (5c) causes a huge loss of anti-
bacterial activity. The presence of a methyl group on positions 2 and 5 decreases the
potency drastically (5f).
In summary, we have disclosed convenient methods for the preparation of novel 7-
aminopyrazolo[1,5a]pyrimidines in good to very good yields and high purity. We have
examined the activities of our compounds in eight micro-organisms and discovered use-
ful activities. Some of the activities appear to be related to substitution patterns on the
pyrimidines, suggesting that further work will be warranted in forming wider structure-
activity relationships. Finally, our work demonstrates the productive interaction of
mechanistic organic chemistry with organic synthesis.
Experimental section
Melting points were determined in open capillaries and are uncorrected. The IR spectra
of the compounds were recorded on a Buck Scientific IR M-500 spectrophotometer

46
R. AGGARWAL ET AL.
Table 1. In vitro anti-Candida activity and minimum inhibitory concentration (MIC) of 5a-o.
Diameter of zone of inhibition (mm)a (MIC (mg/ml))
Compound
C. glabrata
C. albicans
C. tropicalis
C. parapsilosis
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
–
–
–
–
–
–
10
–
–
–
–
11
–
–
10
–
28 (125)
12 (210)
14 (300)
12 (260)
26 (250)
–
12
10
–
–
11
12 (210)
24 (250)
14 (320)
26 (500)
18 (400)
–
–
–
–
–
12 (250)
–
–
–
–
–
–
–
–
–
–
–
11
13 (625)
12 (1250)
10
12
13 (1250)
–
–
10
34 (50)
12 (1250)
10
–
10
–
Fluconazole
aMean of three replicates.
1
using KBr pellets (ꢀmax in cmꢁ1); H and 13C NMR spectra were determined on a
Bruker instrument at 300 and 75 MHz, respectively. Chemical shifts are expressed on
the d-scale downfield from TMS as an internal standard. Elemental analyses were per-
formed at NIPER (Mohali), Chandigarh, India. All the compounds gave C, H and N
analysis within 0.40% of the theoretical values. Mass spectra were measured in EI
mode on a Shimadzu QP-5000 spectrometer at NIPER (Mohali), Chandigarh, India.
Thin layer chromatography (TLC) was performed on silica gel (100-200), using ethyl
acetate/petroleum ether (20% v/v) as the eluting solvent. 3(2)-Aryl-3-oxoalkanenitriles
4a-e were prepared from a-bromoacetophenones and potassium cyanide following the
literature procedure.7 Similarly, 2-aryl-3-oxoalkanenitriles 4f-g were also prepared fol-
lowing the literature protocol.8,9 5-Amino-3-hydrazinopyrazole dihydrochloride 1 was
prepared by condensing hydrazine hydrate with malononitrile.10
Representative solvent-free preparation. 7-Amino-2,5-diphenylpyrazolo[1,5-
a]pyrimidine (5a)
5-Amino-3-hydrazinopyrazole dihydrochloride (0.378 g, 2 mmol) 1 was ground with
sodium acetate (0.328 g, 4 mmol) with a dry pestle and mortar at room temperature
under solvent-free conditions. To the resulting mixture was added 3-phenyl-3-oxopro-
panenitrile (0.580 g, 4 mmol) 4a and the mixture was ground thoroughly for 5 minutes
at room temperature, then heated on a water bath for 15 minutes until TLC showed
complete disappearance of starting materials. The addition of aqueous ethanol (50%) to
the crude mixture led to the separation of the solid product which was filtered, dried
and recrystallized from aqueous ethanol (50%) to give pure 5a (70%). Following the
same synthetic protocol, compounds 5b-g were synthesized, with mean yields of 73%.
Direct Reactions with Hydrazine Hydrate. 7-Aminopyrazolo[1,5-a]pyrimidines 5a-
5g, 5h-5o
To hydrazine hydrate 6 (0.05 g, 1 mmol) was added the appropriate 3(2)-aryl-3-oxoalka-
nenitrile (2 mmol) 4a-g in toluene/EtOH (9:1) followed by a catalytic amount of p-

ORGANIC PREPARATIONS AND PROCEDURES INTERNATIONAL
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Table 2. In vitro antibacterial activity and minimum inhibitory concentrations (MIC) of 5a-o.
Diameter of zone of inhibition (mm)a (MIC (mg/ml))
Compound
E. Coli
S. aureus
S. mutans
P. aeruginosa
5a
5b
5c
5d
5e
5f
13 (125)
14 (250)
Not determined
–
12 (125)
13 (125)
12 (500)
13 (500)
14 (250)
–
13 (125)
12 (250)
12 (250)
–
–
–
–
–
–
–
11 (500)
11 (250)
–
10
5g
5h
5i
5j
5k
5l
5m
5n
–
–
–
10
12
12 (78)
11
10
19 (313)
15 (1250)
13 (78)
13 (78)
17 (380)
12
–
12
–
–
–
12
–
21 (313)
12
12
19 (20)
16 (1250)
15 (313)
11
11
14 (1250)
13 (78)
12
–
–
–
5o
10
40 (4)
Ciprofloxacin
37 (5)
31
32
aMean of three replicates.
toluenesulfonic acid. The mixture was refluxed until the completion of reaction (4 hr),
as determined by TLC. On completion, the excess solvent was distilled off, and the solid
thus obtained was filtered off. The solid was dissolved in water and neutralized with
aqueous sodium bicarbonate solution. Finally, the product was filtered and dried in air
to give 5a-g. No further purification was needed.
A mixture of hydrazine hydrate 6 (0.05 g, 1 mmol) and 3(2)-aryl-3-oxoalkanenitrile
(1 mmol) 4a-g in toluene/EtOH (9:1) was refluxed for 30 min. Then, to this reaction
mixture another 1 mmol of 3(2)-aryl-3-oxoalkanenitrile 4a-e was added along with a
catalytic amount of p-toluenesulfonic acid. The mixture was again refluxed until com-
pletion of reaction (4 hr), as determined by TLC. On completion of reaction as moni-
tored on TLC, the excess solvent was removed by distillation and the solid obtained was
filtered off. The solid was dissolved in water and treated with aqueous sodium bicarbon-
ate solution. The product was filtered off and dried in air to give 5h-o. No further puri-
fication was needed.
Evaluation of anti-Candida and antibacterial activity: preliminary screening
The approximate number of microorganisms in a liquid suspension is determined by
comparing the turbidity of the test organism suspension with a McFarland standard. It
is a mixture of two chemical constituents {9.95 ml barium chloride (1.175%) þ 0.05 mL
sulfuric acid (1.0%)}. The reaction between the two chemicals results in production of a
fine precipitate of barium sulfate. When shaken well, the turbidity of 0.5 McFarland
standard is visually comparable to a bacterial suspension of approximately 15x108 cells/
mL. The inocula of different test pathogens were adjusted according to the above
McFarland standard. Anti-Candida and antibacterial activities of the compounds were
evaluated by the agar-well diffusion method. Using a sterilized cork borer, well plates of
6 mm diameter were punched in solidified malt extract agar plates containing the test
pathogens adjusted as above at 0.5 McFarland standard. Then 250 lL of the chemical
compound (1 mg/mL) was added into the well with the help of a sterilized micropipette.

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The inoculated plates were kept in an upright position in an incubator at 37 ꢂC for
24 hrs. The plates were observed for the diameter of the zone of growth inhibition
around the well which was measured in mm on a Himedia zone scale.11
Determination of minimum inhibitory concentration (MIC)
A twofold serial dilution of compound (1 mg/mL) in 20% dimethylsulfoxide (DMSO)
was made. Dilution was done in sterile distilled water (1:1) to achieve a decreasing con-
centration range. A 250 lL volume of each dilution was introduced into each well (in
triplicate) in the specific media agar plates, already seeded with 100 lL of standardized
microbial inocula of the test microbial strain. All test plates were incubated aerobically
at 37 ꢂC for 24 hrs and observed for the inhibition zones. The lowest concentration of
each extract showing a clear zone of inhibition (>8 mm) was considered as the MIC.12
Acknowledgments
We are thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India,
for providing fellowships to Eakta and Gulshan Singh. We acknowledge the co-operation of Dr.
Dionisia Sanz and Dr. Rosa M. Claramunt, Departamento de Quimica Organica y Bio-organica,
Facultad de Ciencias, UNED, Senda del Rey 9, E-28040 Madrid, Spain, for NMR studies.
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