Urea and thiourea derivatives of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine: Synthesis, characterization, antimicrobial activity and docking studies

Article abstract of DOI:10.1080/10426507.2019.1577845

An efficient and robust synthetic procedure was developed primarily for the synthesis of a precursor compound; 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine (11), from 2-chloropyrazine (7) through the chemical transformations such as hydrazine substitution, trifluoroacetyl group induction, cyclization and pyrazine ring reduction. A new series of urea derivatives 13a-e and thiourea derivatives 13f-j of compound 11 have been synthesized and the structures of all the compounds were confirmed using spectroscopic analyses such as IR, ¹H NMR, ¹³C NMR, LC-MS and HRMS. The newly synthesized compounds were screened for their in vitro antimicrobial activity against five bacteria and two fungi, in which compounds 13d, 13i and 13j displayed potential activity against bacterial strains and 13a, 13d, 13g and 13j against fungal strains with the MIC values in the range of 6.25–25.0 μg/mL. An overall comparison of the activity results revealed that thiourea derivatives contain better activity than that of urea compounds. Molecular docking studies on poly (ADP-ribose) polymerase 15 (ARTD7, BAL3) demonstrated that all the synthesized compounds possess significant binding energies (-8.1 to -9.8 kcal/mol) with no adverse effect in the active site of protein.

Full text of DOI:10.1080/10426507.2019.1577845

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: https://www.tandfonline.com/loi/gpss20
Urea and thiourea derivatives of 3-
(trifluoromethyl)-5,6,7,8-tetrahydro-[1,
2, 4]triazolo[4,3-a]pyrazine: Synthesis,
characterization, antimicrobial activity and
docking studies
Madhava Rao Mannam, Subba Rao Devineni, Chandra Mouli Pavuluri, Naga
Raju Chamarthi & Raja Sekhara P. Kottapalli
To cite this article: Madhava Rao Mannam, Subba Rao Devineni, Chandra Mouli Pavuluri,
Naga Raju Chamarthi & Raja Sekhara P. Kottapalli (2019): Urea and thiourea derivatives of 3-
(trifluoromethyl)-5,6,7,8-tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine: Synthesis, characterization,
antimicrobial activity and docking studies, Phosphorus, Sulfur, and Silicon and the Related
Elements, DOI: 10.1080/10426507.2019.1577845
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PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
https://doi.org/10.1080/10426507.2019.1577845
Urea and thiourea derivatives of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1, 2,
4]triazolo[4,3-a]pyrazine: Synthesis, characterization, antimicrobial activity and
docking studies
Madhava Rao Mannama, Subba Rao Devinenib , Chandra Mouli Pavulurib , Naga Raju Chamarthic , and
Raja Sekhara P. Kottapallia
b
aDepartment of Chemistry, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Andhra Pradesh, India; Institute of Surface-Earth
c
System Science, Tianjin University, Tianjin, China; Department of Chemistry, Sri Venkateswara University, Tirupathi, Andhra Pradesh, India
ABSTRACT
ARTICLE HISTORY
Received 16 May 2018
Accepted 30 January 2019
An efficient and robust synthetic procedure was developed primarily for the synthesis of a precur-
sor compound; 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine (11), from
2-chloropyrazine (7) through the chemical transformations such as hydrazine substitution, trifluor-
oacetyl group induction, cyclization and pyrazine ring reduction. A new series of urea derivatives
13a-e and thiourea derivatives 13f-j of compound 11 have been synthesized and the structures
KEYWORDS
Urea and thiourea
derivatives; sitagliptin
intermediate; structural
characterization; antimicro-
bial activity; molecular
docking; adenosine-5-
diphosphoribose enzyme
1
of all the compounds were confirmed using spectroscopic analyses such as IR, H NMR, 13C NMR,
LC-MS and HRMS. The newly synthesized compounds were screened for their in vitro antimicrobial
activity against five bacteria and two fungi, in which compounds 13d, 13i and 13j displayed
potential activity against bacterial strains and 13a, 13d, 13g and 13j against fungal strains with
the MIC values in the range of 6.25–25.0 mg/mL. An overall comparison of the activity results
revealed that thiourea derivatives contain better activity than that of urea compounds. Molecular
docking studies on poly (ADP-ribose) polymerase 15 (ARTD7, BAL3) demonstrated that all the syn-
thesized compounds possess significant binding energies (-8.1 to -9.8 kcal/mol) with no adverse
effect in the active site of protein.
GRAPHICAL ABSTRACT
Introduction
toxic and expensive,[5] and hence, it has emerged as one of
the principal public health problems throughout the
world.[6] Therefore, there is an urgent need to discover a
new class of antimicrobial agents with broad activity-spec-
trum against multi-resistant pathogens. Structural modifica-
tions of well-known existing antimicrobial agents and novel
classes of antimicrobials are two imperative strategies to
overcome the resistant problems.[7–8]
The increased consumption of antibiotics and inaccurate
diagnoses are major factors for antimicrobial resistance.[1]
Many antimicrobial resistant human pathogenic microbes
have been identified in past few decades such as penicillin-
resistant Streptococcus pneumonia, vancomycin-resistant
Enterococcus and methicillin-resistant Staphylococcus aur-
eus.[2–4] Treatment of such drug-resistant bacterial infections
is very difficult, especially in immunocompromised patients,
and requires a high dose of drugs that are possibly more
Heterocycles represent a prominent class of compounds
and have pharmaceutical importance due to their vital role
CONTACT Raja Sekhara P. Kottapalli
Rao Devineni subhaphd2010@yahoo.com
krsprasad_fed@kluniversity.in
Koneru Lakshmaiah Education Foundation, Vaddeswaram 522502, A.P., India; Subba
Tianjin University, ISESS, Tianjin 300072, China.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpss.
Supplementary material for this article can be accessed here.
ß 2019 Taylor & Francis Group, LLC

2
M. R. MANNAM ET AL.
NH2
known in the field of drug discovery that the structural
modification of either drug intermediates or target com-
pounds with assimilation of functional groups like urea and
thiourea can lead new drug-like compounds. Therefore, con-
sidering the biological importance of 1,2,4-triazole fused
molecules as well as urea and thiourea derivatives, we
expected that the structural modifications of fused molecule
11 with incorporation of urea and thiourea functionalities
may produce promising chemotherapeutic agents.
Based on the above considerations and as extension of
our discovery for potential antimicrobial agents, we
attempted to synthesize a new class of urea and thiourea
derivatives of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1, 2,
4]triazolo[4,3-a]pyrazine (11). All the compounds synthe-
sized in this study were evaluated for their in vitro anti-
microbial activity. Their molecular docking studies were also
performed to know the binding orientation of the title prod-
ucts into the enzyme, poly (ADP-ribose) polymerase 15
(ARTD7, BAL3), macro domain 2 complex with adenosine-
5-diphosphoribose enzyme (3V2B).
Cl
F
OH
N
O
N
OH
N
N
O
N
N
N
N
N
F
N
N
N
N
HO
OH
Alprazolam (3)
Fluconazole (1)
N
Ribavirin (2)
NH
N
N
N
N
O
N
Cl
N
H
Rizatriptan (4)
Amoxapine (5)
N
CF3
N
N
R1
F
F
N
N
N
N
F3C
N
X = O,S
N
NH2
O
F
Sitagliptin (6)
Title products 13a-j
Figure 1. Biologically active 1,2,4-triazole and piperazine derivatives and
title products.
in metabolism. They can be used as biomimetics as well as
active pharmacophore in a wide variety of drugs, vitamins
and many natural products.[9] Particularly, 1,2,4-triazole
derivatives are recognized as one of the most promising che-
motherapeutic agents in medicinal chemistry.[10] For
example, fluconazole (1) is used in the treatment of fungal
infections,[11] while ribavirin (2) as antiviral agents,[12]
alprazolam (3) in anxiolytic treatment[13] and rizatriptan (4)
as antimigraine.[14] 1,2,4-Triazole derivatives have also
prevalent biological activities such as fungicidal,[15] insecti-
cidal,[16] antimicrobial,[17] antidepressant[18] and plant
growth regulators.[19] Similarly, the piperazine scaffold has
considerable importance in biology, which is an important
pharmacophore in numerous medicinal molecules such as
amoxapine (5) (antidepressant).[20] In addition, unsymmet-
rical urea and thiourea derivatives have diverse biological
activities including antibacterial, antifungal, antitubercular,
anti-inflammatory, antithyroid, antihelminitic, rodenticidal,
insecticidal and herbicidal properties.[21–24] Hence, we
focused, as in the part of our medicinal chemistry pro-
gramme, on the synthesis and biological evaluation of urea
and thiourea derivatives. Recently, we synthesized many
urea and thiourea compounds of valsartan[25] and lopinavir
intermediates,[26] diazaphospholes,[27] and piperazine doped
Results and discussion
Chemistry
The desired precursor intermediate 11 is not readily avail-
able, and hence attention has been focused initially to syn-
thesize it, using the reported procedure[32] with minor
modifications. The schematic of the whole procedure is
illustrated in Scheme 1.
The key starting material, 2-chloropyrazine (7), was
treated with hydrazine mono-hydrate in the presence of
base, sodium carbonate (Na2CO3) at harsh temperature
(120–130ꢀC) under optimized neat reaction conditions to
obtain desired 2-hydrazinopyrazine (8) with high yield
after isolation from mixture of water:isopropyl alcohol
(IPA) (2.0:4.5 v/v). The above reaction in the presence of
Na2CO3 resulted worthy reaction progress with improved
yield (77.8%) when compared with the reaction conducted
without base according to the previously reported proced-
ure (yield 53.6%).[32] The hydrazine intermediate 8 was
reacted with trifluoroacetyl chloride in the presence of
Na2CO3 to afford 2,2,2-trifluoro-N0-(pyrazin-2-yl)acetohy-
drazide (9) in optimum yield (86.5%). Compound 9 was
further cyclized in the presence of concentrated sulfuric
acid (H2SO4) to give 3-(trifluoromethyl)-1,2,4-triazolo[4,3-
a] pyrazine (10) as a pale brown solid. This cyclization
reaction in the presence of concentrated H2SO4 was
offered good yield of product 10 (67.3%) when compared
with the reaction in the presence of polyphosphoric acid
(PPA) (51.4%). During the isolation of product 10, the sul-
furic acid was quenched with aqueous sodium hydroxide
(NaOH) solution and the product was extracted with
dichloromethane (DCM) and then it was purified by
recrystallization from the mixture of acetone:cyclohexane
(1.5:6.5 v/v). The purification of product 10 using simple
recrystallization method, instead of laborious chromatog-
raphy technique used in the previous protocol, was an
febuxostat[28]
bial activities.
that exhibited promising antimicro-
Fused scaffolds, i.e., where two or more different pharma-
cophore units are combined together in one molecule, occu-
pied a unique place in the realm of medicinal chemistry.[29]
It has been recognized that 1,2,4-triazole fused compounds
are important scaffolds in pharmacologically active com-
pounds with significant antimicrobial and antitubercular
activities.[30–31] The precursor compound, 3-(trifluoro-
methyl)-5,6,7,8-tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine
(11) used in the present study is an interesting fused mol-
ecule of 1,2,4-triazole and piperazine, and is an active
pharmacophore of Sitagliptin drug (6) (Figure 1). It is well- additional merit of the described protocol in the present

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
3
N
N
N
N
Na2CO3, CF3COCl,
NH2NH2.H2O, Na2CO3,
120-130 oC, 1.5 h
H
CF3
DCM, 5-10 oC, 3.0 h
N
NH2
N
H
N
9
N
H
N
7
Cl
O
(86.5% yield)
8
(77.8% yield)
N
N
Con. H2SO4, 80-85 oC,
12.0 h, DCM, Aq. NaOH,
N
N
Pd/C, H2, MeOH,
40-45 oC, 24 h, IPA. HCl
NH
N
HCl
N
N
F3C
F3C
10
11
(82.9% yield)
(67.3% yield)
N
N
12(a-j)
R NCX = O, S
R
HN
Toluene, Et3N, 75-80 oC, 3 - 5 h
N
N
F3C
X = O,S
13(a-j)
(75.2-88.2% yield)
Cl
N
N
N
N
N
N
F
HN
O
Br
HN
O
HN
O
N
N
N
N
N
N
F3C
F3C
F3C
13b
13c
(80.9% yield;
4 h reaction time)
(76.6% yield;
13a
(88.2% yield;
5 h reaction time)
4 h reaction time)
F
N
N
N
N
N
N
F
HN
O
CH3
F3C
F
HN
HN
N
N
N
N
N
N
F3C
F3C
O
S
13e
13f
(75.2% yield;
5 h reaction time)
(81.8% yield;
4 h reaction time)
13d
(84.0% yield;
4 h reaction time)
Cl
Cl
N
N
N
N
N
N
N
Br
HN
Cl
HN
S
HN
S
N
N
N
N
N
N
F3C
F3C
F3C
F3C
S
13g
(75.4% yield;
13i
(82.9% yield;
13h
(84.1% yield;
5 h reaction time)
4 h reaction time)
34 h reaction time)
CF3
N
HN
S
N
N
13j
(80.3% yield;
5 h reaction time)
Scheme 1. Synthetic scheme to the preparation of title urea and thiourea derivatives.
study. Compound 10 was reduced with palladium-carbon 50% ethyl acetate:hexane mixture as the mobile phase based
(Pd/C) under hydrogen pressure to obtain crude 3-(tri- on the elution of the desired compound.
fluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo-[4,3-a]pyra-
zine and then treated with isopropyl alcohol saturated
with hydrogen chloride (IPA.HCl) to obtain the desired
Spectroscopic data
Structures of the newly synthesized compounds were eluci-
dated using spectroscopic data such as IR and NMR (1H
and 13C), mass and HRMS analyses. The precursor inter-
compound 11 in optimum yield (82.9%) as a white crystal-
line solid.
Finally, the synthesis of the title N-(substituted phenyl/
cyclohexyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1, 2, 4]tri- mediate 11 was synthesized according to reported procedure
azolo[4,3-a] pyrazine-7-carboxamides/carbothioamides 13a-j with modifications and its spectroscopic data matched to
were accomplished by the reaction of compound 11 with that reported data.[32] The characteristic absorption bands in
substituted isocyanate/thioisocyanate 12a-j in the presence the region of 3200–3350 cmꢁ1 in all the title products,
of excess triethylamine (TEA) under mild reaction condi- 1630–1690 cmꢁ1 in urea compounds and 1180–1300 cmꢁ1 in
tions. The crude products obtained after the workup were thiourea derivatives of IR spectra are mainly attributed due
purified by subjecting column chromatography using 10- to the presence of N-H, C ¼ O and C ¼ S linkages,

4
M. R. MANNAM ET AL.
Figure 2. Docking conformers of the active title products and compound 11 in the active site of adenosine-5-diphosphoribose enzyme (3V2B).
respectively. Likewise, the absorption bands around attached to carbonyl group in urea derivatives and thiocar-
1045–1130 cmꢁ1 confirms the presence of CF3 group as well
bonyl group in thiourea compounds, respectively. The doub-
as absorption bands in the range of 1520–1620 cmꢁ1 due to
let peaks at 115–119 ppm with
a coupling constant
C ¼ N linkage in triazole ring. The two protons resonated at
the chemical shift 10.58 ppm corresponding to NHHCl of
piperazine of precursor intermediate 11 was not appeared in
the newly synthesized urea and thiourea products, suggest-
ing that it involves in the formation of urea and thiourea
derivatives by interacting with cyanate/isocyanate groups. In
1H NMR spectra of title products, the chemical shift values
in the region of 9.00–9.98 ppm as singlets indicated the pres-
ence of NH proton attached to urea and thiourea functional-
ities. The peaks at 3.90–4.70 ppm as triplets and
4.80–5.00 ppm as singlet confirmed the protons –N-CH2-
CH2-N- and –N-CH2-C- present in the piperazine ring,
respectively. Moreover, the aromatic protons, based on the
structural orientation, resonated in the range of
6.80–7.95 ppm. The signals between 152–155 ppm and
260–270 Hz confirmed the presence of the –CF3 group.
Chemical shifts in the region of 40–46 ppm are correlated
with the carbons in the piperazine ring and other aromatic
carbons are observed in the range of 105–155 ppm. The
mass values found in ESI-MS in positive mode are well
coordinated with mass of the newly synthesized urea and
thiourea derivatives 13a-j and HRMS data provides add-
itional evidence in the structural characterization of
the products.
Biological activity
Because of the potential antimicrobial tendency of 1,2,4-
triazole fused molecules,[30] urea and thiourea deriva-
181–183 ppm in 13C NMR spectra are attributed the carbons tives,[24] and our continuing efforts in the development of

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
5
new class of antimicrobials,[25–28] we planned to investi- compounds against C. albicans (ATCC 90028). The urea
gate the antimicrobial potency of the newly synthesized
compounds 13a-j. American Type Culture Collection
(ATCC) bacterial strains such as Escherichia coli (ATCC
compounds 13a bearing with 4-fluorophenyl ring and 13d
bound with 2,4-difluorophenyl ring, and thiourea derivatives
such as 13g connected with 4-bromophenyl ring and 13j
associated with 3-trifluoromethylphenyl ring displayed
promising activity against both fungal strains, C neoformans
(ATCC 208821) and C. albicans (ATCC 90028) in the range
MIC values of 6.25-25.0 mg/mL. Thiourea derivative 13h
(MIC 12.50 mg/mL) and urea derivative 13c (MIC 25.0 mg/
mL) connected with 3-chlorophenyl ring showed good and
moderate activities against C. neoformans (ATCC 208821),
respectively.
Based on the overall biological activity observations, we
found that only a few compounds possess potential antibac-
terial and antifungal activities as well with MIC range
6.25–12.50 mg/mL, while most of the title products activity
against the pathogens such as E. coli (ATCC 25922), S. aur-
eus (ATCC 43300) and C. albicans (ATCC 90028) was poor,
and against P. aeruginosa (ATCC 27853), K. pneumonia
(ATCC 700603), A. baumannii (ATCC 19606) and C. neo-
formans (ATCC 208821) was moderate to very good. The
potential activity can be attributed to the presence of active
pharmacophore groups such as fluoro- and chloro- attached
to the phenyl ring. On the other hand, thiourea derivatives,
based on the activity results comparison of the same substi-
tuted set of compounds 13a/f, 13b/g and 13c/h, disclosed
better activity than that of urea compounds, suggesting the
thiourea functionality could also be a reason for the poten-
tial activity. Therefore, further investigation is required to
better understand the structural-activity relationship (SAR)
including altering the substituents on the phenyl ring, bond-
ing with other active pharmacophore unit like acyclic, cyclic
and heterocyclic, and structural modifications in basic
pharmacophore unit.
25922),
Klebsiella
pneumonia
(ATCC
700603),
Acinetobacter baumannii (ATCC 19606), Pseudomonas
aeruginosa (ATCC 27853) and Staphylococcus aureus
(ATCC 43300), and two fungi like Candida albicans
(ATCC 90028) and Cryptococcus neoformans (ATCC
208821) were used as test organisms. The preliminary
screening of the antimicrobial activity has been tested fol-
lowing the agar well diffusion method[33] and using the
antibiotics; Ciprofloxacin and fluconazole, as reference
drugs for bacterial and fungal activities, respectively. The
results were recorded for each tested compound as the
average diameter of zone of inhibition (ZOI) of microbial
growth around the disks in mm. The experimental results
obtained against bacteria and fungi are provided in Table
S1 and Table S2 (Supplemental Materials), respectively. A
few of the synthesized products exhibited potential growth
of zone of inhibitions. Hence, the minimum inhibitory
concentration (MICs) of the compounds, that is, the low-
est concentration showing no growth of inhibition was
evaluated. A Twofold serial dilution technique[34] was
used to screen MICs and experimental results are shown
in Table S3.
As depicted in Table S1 and Table S3, all the title prod-
ucts showed better antimicrobial activity than precursor
compound 11, demonstrating that the substituents and func-
tional groups such as urea and thiourea are enriching the
biological activity. In fact, urea compound 13d substituted
with 2,4-difluorophenyl ring, and the thiourea derivatives,
13i bearing 3,4-dichlorophenyl ring and 13j bound with 3-
trifluoromethylphenyl ring exhibited potential bacterial
activity against all the tested bacteria in the range MIC val-
ues of 6.25–25.0 mg/mL, although, the rest of the com-
pounds showed lower activity when compared with the
reference drug, Ciprofloxacin (MIC 1.562–3.125 mg/mL).
The compounds such as 13a linked with 4-fluorophenyl ring
against P. aeruginosa (ATCC 27853) (MIC 6.25 mg/mL), K.
pneumonia (ATCC 700603) (MIC 12.50 mg/mL) and A. bau-
mannii (ATCC 19606) (MIC 12.50 mg/mL), 13b bearing
with 4-bromophenyl ring (MIC 12.50 mg/mL) and 13f
bound with 4-fluorophenyl ring (MIC 12.50 mg/mL) against
K. pneumonia (ATCC 700603), 4-bromophenyl ring substi-
tuted thiourea 13g against A. baumannii (ATCC 19606)
(MIC 12.50 mg/mL) and E. coli (ATCC 25922) (MIC 25.0
mg/mL), and 3-chlorophenyl ring bound thiourea 13h
against A. baumannii (ATCC 19606) (MIC 12.50 mg/mL)
showed good antibacterial activity independently. These
results suggest that thiourea derivatives possess good anti-
bacterial activity than urea compounds.
Molecular docking study
ADP-ribosylation is a protein modification process that acts
in pathogenic mechanisms, intracellular signaling systems,
DNA repair, and cell division and that has been studied in
animals, plants, and bacteria.[35–36] Numerous bacterial tox-
ins are ADP-ribosyltransferases, which can add poly-ADP-
ribose (PARylation) and mono-ADP-ribose (MARylation) to
proteins as well as catalyze ADP-ribosylation by transferring
the ADP-ribose moiety of NAD to target protein.
Poly(ADP-ribose) polymerase (PARP) is a glutamate of a
target protein (initiation) like the other bacterial ADP-ribo-
syltransferases distinctly from mono-ADP-ribosylation.[37]
PARP is located in the nucleus of most eukaryotes and helps
to maintain genomic integrity in base excision repair, in
DNA recombination, amino acids modifications and in cel-
lular differentiation.[36,38] Therefore, the topics of PARP
proteins have attracted a new wave of interest. Recently,
urea and thiourea derivatives of 2-nitro-5-[thiophene-
2yl]benzenamine are reported as good antimicrobial agents
and showed high binding energies with poly (adp-ribose)
The antifungal activity (Table S2 and Table S3) revealed
that most of title products possess good growth of inhibition
against C. neoformans (ATCC 208821) than C. albicans
(ATCC 90028). All the compounds except compound 13f
showed good activity against C. neoformans (ATCC 208821)
at both the concentrations, 50 and 100 mg/mL, and only few polymerase 15 (3V2B) enzyme.[39]

6
M. R. MANNAM ET AL.
In order to interpret the binding mode of the synthesized acceptors ꢂ10, number of hydrogen bond donors ꢂ5, and
urea and thiourea derivatives 13a-j, the molecular docking number of rotatable bonds ꢂ10 and obeyed Lipinski Rule 5
without any violation, suggesting the synthesized title prod-
ucts are orally active chemotherapeutic agents with
good adsorption.
study was performed against three-dimensional structure of
poly(ADP-ribose) polymerase 15 (ARTD7, BAL3), macro
domain 2 in complex with adenosine-5-diphosphoribose
enzyme (3V2B), which retrieved from Protein Data Bank,
using the AutoDockVina Tools.[40–41] The protein was
refined with MD simulation which was carried out with the
Visual Molecular Dynamics (VMD) tool.[42] The CHARMM
27 field was used for parameterization, and the program
NAMD was used for energy minimization and molecular
dynamics (MD) simulations.[43] The precursor intermediate
11 and the title ligands were redocked at the crystal enzyme
structure of the protein and the best energy conformations
of poly(ADP-ribose) polymerase 15 (ARTD7, BAL3)-ligand
were studied. The analysis of the binding conformation has
furnished using a scoring function based on the free energy
of bindings.[44] The individual energies of the poly (ADP-
ribose) polymerase 15 (ARTD7, BAL3) with the ligands are
tabulated in Table S 4, and the active conformers are shown
in Figure 2 and Figure S1.
The pharmacokinetic properties and toxicities were pre-
dicted for all the synthesized compounds using OSIRIS
property explorer. LogS indicating the parameter of aqueous
solubility, and other properties like mutagenicity, tumorigen-
icity, irritation effect, and risk of reproductive effect are pre-
dicted for toxicity study. The drug score combines drug
likeness, cLogP, LogS, and toxicity risks in one value can
show overall potential to qualify drug. All the synthesized
compounds showed more aqueous solubility (-3.81 to
-5.50 mol./L), drug likeness (-10.4 to -19.8) and drug score
(0.18 to 0.31), and did not explored toxicity properties such
as mutagenicity, tumorigenicity, irritation effect, and risk of
reproductive effect which demonstrating the title products
having high bio-availability and possess good drug-like
properties. However, urea compounds such as 13b and 13c
and thiourea derivatives like 13h, 13i and 13j showed good
binding energies than other title compounds which provide
a good agreement to in vitro antimicrobial activity.
As seen in Table S4, the synthesized compounds are bind
well into the active site of enzyme with the best binding
energies (scores) in order of 13j (ꢁ9.8 kcal/mol)>13h
(ꢁ9.4 kcal/mol)>13i
(ꢁ9.3 kcal/mol)>13f
(ꢁ9.2 kcal/
Experimental
mol)>13g
(ꢁ8.9 kcal/mol)=13b
(ꢁ8.9 kcal/mol)>13c
Materials and methods
(ꢁ8.8 kcal/mol)>13e (ꢁ8.6 kcal/mol)>13a (ꢁ8.3 kcal/mol),
13d (ꢁ8.1 kcal/mol) and compound 11 (ꢁ6.1 kcal/mol).
Interestingly, all the synthesized urea and thiourea molecules
13a-j showed the better binding energies with enzyme than
a precursor intermediate 11. Thiourea derivatives, among
the same substituted set of compounds 13a/f, 13b/g and
13c/h, were bound well (> ꢁ9.0 kcal/mol) with enzyme than
that of urea derivatives (< ꢁ9.0 kcal/mol), and it is provided
a good agreement to in vitro antimicrobial activity. It dem-
onstrated that thiourea functionality perform as an active
pharmacophore unit to explore potential activity than urea
group. The active site residues such as Ser302, Ala385,
Val424, Gln427, Tnr388, Gly389 and Ala391 are play vital
role in the ligands interactions and could be involved in
antimicrobial activity.
Chemicals, solvents and reagents used in this study were
purchased from Sigma-Aldrich and sd Fine-Chem. Limited
and used directly without further purification. The progress
of reactions was monitored by thin layer chromatography
(TLC) (Merck silica plates), using visualization by UV light
or in iodine vapor. The column chromatography packed
with Merck 120 mesh silica gel as a stationary phase and a
mixture of different ratios of ethyl acetate (EtOAc) and hex-
ane, based on the elution of compounds, as a mobile phase
were used to purify the title crude products. Melting range
of the title products was recorded using open capillary tube
on Guna melting point apparatus with increasing tempera-
ture rate 3 ꢀC/min and is uncorrected. IR spectra were
recorded on Shimadzu IR Affinity-I FT-IR spectrophotom-
eter, using KBr dispersion method, over the range of
4000–400 cmꢁ1. One dimensional nuclear magnetic reson-
ance spectroscopy (1D NMR) (1H and 13C) was performed
on AscendTM Bruker 400 instrument which operated at
400 MHz frequency for 1H NMR and 100 MHz frequency
for 13C NMR in DMSO-d6 solvent, and tetramethylsilane
(TMS) was used as the internal standard. NMR results are
reported as chemical shifts (d) in ppm, multiplicity, coupling
constant values (J) in Hertz (Hz), number of protons and
proton’s position. Multiplicities are shown as the abbrevia-
tions: s (singlet), brs (broad singlet), d (doublet), t (triplet)
and m (multiplet). High-resolution mass spectra were
obtained by using a QTOF mass spectrometer. International
principles and regulations are followed during the screening
of biological activity. The Supplemental Materials contains
ADME calculations
A computational study has been carried out on the synthe-
sized products for the prediction of ADME properties. The
obtained results are summarized in Table S5 and Table S6
(Supplemental Materials). ‘‘Lipinski Rule of 5’’ stated that a
compound can more likely to be membrane permeable and
easily absorbed by the body if its molecular weight is less
than 500, lipophilicity would be expressed as a quantity
known as logP is less than 5, the number of groups in the
molecule that can donate hydrogen atoms to hydrogen
bonds is less than 5, the number of groups that can accept
hydrogen atoms to form hydrogen bonds is less than 10 and
the number of rotatable bonds is less than 10.[45] The data
displayed that all the synthesized compounds having logP ꢂ sample 1H and 13C NMR and mass spectra for products
5, molecular weight ꢂ500, number of hydrogen bond 13a-j (Figures S2 – S48).

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
7
Preparation of 2-hydrazinopyrazine (8)
Preparation of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-
1,2,4-triazolo-[4,3-a]pyrazine hydrochloride (11)
The mixture of 2-chloropyrazine (7) (20.0 g, 0.175 mol),
hydrazine hydrate (60 mL) and sodium carbonate (27.84 g, A mixture of compound 10 (12.0 g, 0.064 mol) and the cata-
ꢀ
0.264 mol) was stirred for 2 h at 120-130 C. After comple- lyst, Pd/C (10% w/w, 120.0 mg) in methanol (MeOH)
tion of the reaction that was checked by TLC, the reaction (120 mL) was charged into an autoclave (500 mL) at ambient
mass was allowed to cool at 50-60 ꢀC and then water temperature. The reaction mass was agitated for 24 h by
2
ꢀ
(60.0 mL) followed by isopropyl alcohol (IPA) (40.0 mL) maintaining 3.5-4.5 Kg/cm hydrogen pressure at 40-45 C.
were added. The reaction mass was maintained for 16 h at The completion of reaction was judged by TLC. The reac-
ꢀ
0-3 C and then the precipitated white solid was separated tion mass was concentrated under reduced pressure after
by filtration. The wet solid was dried under vacuum at 45- remove the Pd/C catalyst by passing through celite under
ꢀ
50 C to obtain desired 2-hydrazinopyrazine (14.8 g, 77.8%). nitrogen atmosphere. The residue was dissolved in IPA
1H NMR (400 MHz; CD3OD), d, ppm (J, Hz): 7.94-8.14 (24.0 mL) and then IPA.HCl (24.0 mL) was added slowly at
(2H, m, Ar-H); 7.69 (1H, s, Ar-H). ESI-MS m/z (rel, %): 0-5 ꢀC. The precipitated solid was separated by filtration,
111.07 (M þ Hþ) (100). HRMS (FAB) Calc.: C4H6N4: after one hour agitation of the reaction mass, to obtain 3-tri-
110.059246; Found: 111.0592 [M þ Hþ].
fluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo-[4,3-a]pyrazine
hydrochloride (11) as a white crystalline solid (12.1 g, 82.9%).
m.p.: 237-249 ꢀC (Lit. 236-246 ꢀC reported in chemical book).
1H NMR (400 MHz; DMSO-d6), d, ppm (J, Hz): 3.65 (2H, t,
J ¼ 5.6, Piperazine); 4.46 (2H, t, J ¼ 5.6, Piperazine); 4.61
(2H, s, Piperazine); 10.58 (2H, br, NH2þ). 13C NMR
(100 MHz; DMSO-d6) d, ppm (J, Hz): 39.09 (-CH2-CH2-),
40.14 (-CH2-CH2-), 40.90 (-CH2-), 117.17 (d, J ¼ 268.4, CF3),
142.29 (C triazole), 148.73 (C triazole). LC-MS m/z (rel, %):
193.08 (M þ H)þ (100).
Preparation of 2,2,2-trifluoro-N0-(pyrazin-2-yl)
acetohydrazide (9)
To a stirred reaction mixture of 2-hydrazinopyrazine (8)
(14.0 g, 0.127 mol) and sodium carbonate (16.2 g, 0.153 mol)
in dichloromethane (DCM) (112 mL), the solution of tri-
fluoroacetyl chlorideꢀ (18.5 g, 0.14 mol) in DCM (28 mL) was
added slowly at 0-5 C. The reaction mixture was agitated at
ꢀ
5-10 C until completion of the reaction that was judged by
TLC. Water (140 mL) was added to the reaction mass to
remove water soluble components and then it was allowed
to stand to attain the partition between DCM and water.
General procedure for the synthesis of title urea and
thiourea derivatives (13a-j)
The organic layer was concentrated under atmospheric con- To the stirred reaction mixture of 3-(trifluoromethyl)-
ꢀ
ditions at below 45 C. The crude residue was recrystallized 5,6,7,8-tetrahydro-1,2,4-triazolo-[4,3-a]pyrazine hydrochlor-
from mixture of EtOAc:hexane to obtain 2,2,2-trifluoro-N0- ide (11) (300 mg, 1.32 mmol), TEA (0.55 mL, 3.95 mmol)
(pyrazin-2-yl)acetohydrazide (9) as off-white solid (22.5 g, and toluene (10 mL), substituted cyanates 12(a-e)/isocya-
1
86.5%). H NMR (400 MHz; CD3OD), d, ppm (J, Hz): 7.94- nates 12(f-j) (1.32 mmol) were added at ambient tempera-
ꢀ
8.22 (2H, m, Ar-H); 7.66 (1H, s, Ar-H). ESI-MS m/z (rel, ture. The reaction mass was agitated at 75-80 C until the
%): 207.12 (M þ Hþ) (100).
completion of the reaction that was monitored by TLC. The
reaction mass was allowed to cool at ambient temperature
and it was washed sequentially with 3% aqueous HCl
(5.0 mL) and then water (5.0 mL). The organic fraction was
concentrated under vacuum at 50-55 ꢀC to obtain crude
product. It was purified by column chromatography using
10-50% of EtOAc:hexane mixture as a mobile phase.
Preparation of 3-(trifluoromethyl)-1,2,4-triazolo[4,3-a]
pyrazine (10)
To the solution of concentrated H2SO4 (20.0 mL), com-
pound 9 (20.0 g, 0.097 mol) was added in a portion-wise at
ꢀ
0-5 C and then the reaction mass was stirred for 12 h at
ꢀ
80-85 C. After completion of the reaction that was checked
N-(4-Fluorophenyl)-3-(trifluoromethyl)-5,6,7,8-
tetrahydro-[1, 2, 4]triazolo [4,3-a]pyrazine-7-
carboxamide (13a)
by TLC, DCM (120 mL) was added to the reaction mass at
ambient temperature and then the reaction mass was
quenched with 30% aqueous NaOH solution by maintaining
temperature at -5 to 5 C (Highly exothermic) up to attain White crystalline solid; m.r.: 206-208 ꢀC. IR (KBr, ꢀ~/cmꢁ1):
ꢀ
the mass pH to 8.0-10.0. The inorganic salts were separated 3360 (N-H, str); 1674 (C=O, str); 1544 (C=N, str); 1163
1
by filtration, and the solvent was removed from the organic (-CF3, str). H NMR (400 MHz; DMSO-d6), d, ppm (J, Hz):
layer under atmospheric conditions at below 45 ꢀC. The 3.95-3.98 (2H, t, J ¼ 5.2, Piperazine), 4.22-4.24 (2H, t,
crude residue product was recrystallized from acetone:cyclo- J ¼ 5.2, Piperazine), 4.95 (2H, s, Piperazine), 7.08-7.13 (2H,
hexane (1.5:6.5 v/v) to obtain, 3-(trifluoromethyl)-1,2,4-tria- m, Ar-H), 7.43-7.79 (2H, m, Ar-H), 8.9 (1H, s, NH). 13C
zolo[4,3-a]pyrazine (10) as a pale brown solid (12.4 g, NMR (100 MHz; DMSO-d6), d, ppm (J, Hz): 39.98 (-CH2-
1
67.3%). H NMR (400 MHz; CDCl3) d, ppm (J, Hz): 8.12- CH2-); 40.87 (-CH2-CH2-); 43.30 (CH2-C-), 114.79 (d,
8.22 (2H, m, Ar-H); 9.43 (1H, s, Ar-H). ESI-MS m/z (rel, J ¼ 22.0, CF3), 117.12 (d, J ¼ 268.0, C-F Ar), 121.68 (d,
%): 189.03 (M þ H)þ (100). HRMS (FAB) Calc.: C6H3F3N4: J ¼ 8.0, C Ar), 136.14, 151.09 (-C=O), 154.59 (C Ar), 156.50
188.0309816199; Found: 189.0309 [M þ Hþ].
(C Ar), 158.87 (C Ar). LC-MS, m/z, (rel, %): 330.09

8
M. R. MANNAM ET AL.
[M þ H]þ (100). HRMS (FAB) Calc.: C13H11F4N5O: 348.13 [M þ H]þ (100). HRMS (FAB) Calc.: C13H10F5N5O:
329.08997; Found: 330.0967 [M þ Hþ].
347.08055; Found: 348.0952 [M þ Hþ].
N-(4-Bromophenyl)-3-(trifluoromethyl)-5,6,7,8-
tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine-7-
carboxamide (13b)
N-(4-Methylcyclohexyl)-3-(trifluoromethyl)-5,6,7,8-
tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine-7-
carboxamide (13e)
Off-white solid; m.r.: 218-221 ꢀC. IR (KBr, ꢀ~/cmꢁ1): 3407 Pale brown powder; m.r.: 216-219 ꢀC. IR (KBr, ꢀ~/cmꢁ1):
(N-H, str); 1668 (-C=O, str); 1533 (C=N, str); 1147 (CF3, 3302 (N-H, str); 1637 (C=O, str); 1550 (C=N, str); 1113
1
str); 835 (C-Br, str). 1H NMR (400 MHz; DMSO-d6), d, (CF3, str). H NMR (400 MHz; DMSO-d6), d, ppm, (J, Hz):
ppm, (J, Hz): 3.96-3.98 (2H, t, J ¼ 5.2, Piperazine); 4.22-4.24 0.85-0.86 (3H, m, Cyclohexyl); 0.88- 0.99 (2H, m,
(2H, t, J ¼ 5.2, Piperazine); 4.96 (2H, s, Piperazine); 7.42- Cyclohexyl); 1.16-1.32 (3H, m, Cyclohexyl); 1.63-1.66 (2H,
7.48 (4H, m, Ar-H); 8.99 (1H, s, NH). 13C NMR (100 MHz; m, Cyclohexyl); 1.75- 1.77 (2H, m, Cyclohexyl); 3.34-3.42
DMSO-d6), d, ppm, (J, Hz): 40.03 (-CH2-CH2-); 40.92 (1H, m, Cyclohexyl); 3.80-3.83 (2H, t, J ¼ 5.2, Piperazine);
(-CH2-CH2-); 43.32 (-CH2-); 113.88 (C Ar); 117.14 (d, 4.12-4.14 (2H, t, J ¼ 5.2, Piperazine); 4.77 (2H, s,
J ¼ 268.0, CF3); 121.70 (C Ar); 131.19 (C Ar); 139.41 (C Ar); Piperazine); 6.60 (1H, s, NH). 13C NMR (100 MHz; DMSO-
142.33 (C Ar); 151.07 (C=O); 154.34 (C Ar). LC-MS m/z d6), d, ppm, (J, Hz): 22.17 (C-Cyclohexane); 31.59 (C-
(rel, %): 390.00 [M þ H]þ (100); 392.00 [M þ H þ 2]þ (97). Cyclohexane);
32.71
(C-Cyclohexane);
33.81
(C-
HRMS (FAB) Calc.: C13H11BrF3N5O: 389.00990; Found: Cyclohexane); 40.12 (-CH2-CH2-); 40.60 (-CH2-CH2-); 43.25
390.0154 [M þ Hþ].
(-CH2-); 49.48 (C-Cyclohexane); 117.10 (d, J ¼ 269.0, CF3);
142.25 (C, Ar); 151.33 (C=O); 156.07 (C, Ar). LC-MS m/z
(rel, %): 332.25 [M þ H]þ (100). HRMS (FAB) Calc.:
C14H20F3N5O: 331.16199; Found: 332.1682 [M þ Hþ].
N-(3-Chlorophenyl)-3-(trifluoromethyl)-5,6,7,8-
tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine-7-
carboxamide (13c)
N-(4-Fluorophenyl)-3-(trifluoromethyl)-5,6,7,8-
tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine-7-
carbothioamide (13f)
Off-white powder; m.r.: 166-168 ꢀC. IR (KBr, ꢀ~/cmꢁ1): 3315
(N-H, str); 1664 (C=O, str); 1527 (C=N, str); 1143 (CF3,
str), 777 (C-Cl, str). 1H NMR (400 MHz; DMSO-d6), d,
ppm, (J, Hz): 3.96-3.99 (2H, t, J ¼ 5.2, Piperazine); 4.22-4.25 White solid; m.r.: 204-206 ꢀC. IR (KBr, ꢀ~/cmꢁ1): 3232 (N-H,
(2H, t, J ¼ 5.2, Piperazine); 4.96 (2H, s, Piperazine); 7.01- str); 1552 (C ¼ N, str); 1274 (C ¼ S, str); 1122 (CF3, str);
1
7.04 (1H, m, Ar-H); 7.27-7.31 (1H, t, J ¼ 8.0, Ar-H); 7.40-7- 1020 (C-F, str). H NMR (400 MHz; DMSO-d6), d, ppm, (J,
42 (1H, d, J ¼ 8.4, Ar-H); 7.66 (1H, s, Ar-H); 9.04 (1H, s, Hz): 4.31-4.33 (2H, t, J ¼ 5.2, Piperazine); 4.41-4.44 (2H, t,
NH). 13C NMR (100 MHz; DMSO-d6), d, ppm, (J, Hz): J ¼ 5.2, Piperazine); 5.38 (2H, s, Piperazine); 7.15-7.19 (2H,
40.03 (-CH2-CH2-); 40.9 (-CH2-CH2-); 43.25 (-CH2-); m, Ar-H); 7.31-7.34 (2H, m, Ar-H); 9.73 (1H, s, NH). 13C
117.11 (C Ar); 117.92 (d, J ¼ 268.0, -CF3); 119.09 (C Ar); NMR (100 MHz; DMSO-d6), d, ppm, (J, Hz): 42.94 (-CH2-
121.84 (C Ar); 130.04 (C Ar); 132.75 (C Ar); 141.53 (C Ar); CH2-); 44.04 (-CH2-CH2-); 44.82 (-CH2-); 114.70 (d,
142.31 (C Ar); 151.01 (C=O); 154.26 (C Ar). LC-MS m/z J ¼ 22.0, C Ar); 117.08 (d, J ¼ 268, CF3); 128.10 (d, J ¼ 9.0, C
(rel, %): 346.11 [M þ H]þ (100); 348.06 [M þ Hþþ2] (34). Ar); 136.74 (d, J ¼ 3.0, C Ar); 142.28 (d, J ¼ 38.0, C Ar);
HRMS (FAB) Calc.: C13H11ClF3N5O: 345.06042; Found: 150.95 (C Ar); 158.37 (d, J ¼ 240.0, C Ar); 182.42 (C=S).
346.0668 [M þ Hþ], 348.0628 [M þ Hþþ2].
LC-MS m/z (rel, %): 346.05 [M þ H]þ (100). HRMS (FAB)
Calc.: C13H11F4N5S: 345.06712; Found: 346.0735 [M þ Hþ].
N-(2,4-Difluorophenyl)-3-(trifluoromethyl)-5,6,7,8-
tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine-7-
carboxamide (13d)
N-(4-bromophenyl)-3-(trifluoromethyl)-5,6,7,8-
tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine-7-
carbothioamide (13g)
Pale brown solid; m.r.: 156-158 ꢀC. IR (KBr, ꢀ~/cmꢁ1): 3242
(N-H, str); 1645 (C=O, str); 1516 (C=N, str); 1141 (CF3, Pale brown solid; m.r.: 153-155 ꢀC. IR (KBr, ꢀ~/cmꢁ1): 3240
str); 1097 (C-F, str). 1H NMR (400 MHz; DMSO-d6), d, (N-H, str); 1528 (C ¼ N, str); 1274 (C ¼ S, str); 1159 (CF3,
ppm, (J, Hz): 3.95-3.98 (2H, t, J ¼ 5.2, Piperazine); 4.2-2- str); 783 (C-Br, str). 1H NMR (400 MHz; DMSO-d6), d,
4.25 (2H, t, J ¼ 5.2, Piperazine); 4.93 (2H, s, Piperazine); ppm, (J, Hz): 4.33-4.34 (2H, t, J ¼ 4.8, Piperazine); 4.40-4.43
7.02-7.06 (1H, m, Ar-H); 7.25-7.30 (1H, m, Ar-H); 7.38-7.44 (2H, t, J ¼ 4.8, Piperazine); 5.38 (2H, s, Piperazine); 7-28-
(1H, m, Ar-H); 8.75 (1H, s, NH). 13C NMR (100 MHz; 7.60 (4H, m, Ar-H); 9.82 (1H, s, NH). 13C NMR (100 MHz;
DMSO-d6), d, ppm, (J, Hz): 40.12 (-CH2-CH2-); 40.89 DMSO-d6), d, ppm, (J, Hz): 42.93 (-CH2-CH2-); 44.18
(-CH2-CH2-); 43.27 (-CH2-); 103.83 (C Ar); 110.77 (dd, (-CH2-CH2-); 44.99 (-CH2-); 117.10 (d, J ¼ 269.0, CF3);
J ¼ 18.0 Hz, 4.0, C, Ar); 117.11 (d, J ¼ 269.0, CF3); 123.24 (C 120.52 (C Ar); 124.49 (C Ar); 127.58 (C Ar); 128.21 (C Ar);
Ar); 128.03 (C Ar); 142.32 (C Ar); 151.02 (C=O); 154.52 (C 142.16 (C Ar); 150.91 (C Ar); 182.14 (C=S). LC-MS m/z
Ar); 156.99 (C Ar); 160.14 (C Ar). LC-MS m/z (rel, %): (rel, %): 405.97 [M-H]þ (100); 407.95 [M-H þ 2]þ (97).

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
9
HRMS (FAB) Calc.: C13H11BrF3N5S: 404.98706; Found: 125.43 (C Ar); 128.30 (C Ar); 128.62 (d, J ¼ 32.0, C Ar);
405.9857 [M þ Hþ].
128.94 (C Ar); 129.23 (C Ar); 141.34 (C Ar); 141.90 (C Ar);
150.89 (C Ar); 182.16 (C=S). LC-MS m/z (rel, %): 396.07
[M þ H]þ (100). HRMS (FAB) Calc.: C14H11F6N5S:
395.06393; Found: 396.0711 [M þ Hþ].
N-(3-Chlorophenyl)-3-(trifluoromethyl)-5,6,7,8-
tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine-7-
carbothioamide (13h)
Conclusion
Off-white solid; m.r.: 137-139 ꢀC. IR (KBr, ꢀ~/cmꢁ1): 3219
(N-H, str); 1546 (C ¼ N, str); 1273 (C ¼ S, str), 1161 (CF3,
str); 715 (C-Cl, str). 1H NMR (400 MHz; DMSO-d6), d,
ppm, (J, Hz): 4.33-4.34 (2H, t, J ¼ 5.2, Piperazine); 4.41-4.43
(2H, t, J ¼ 5.2, Piperazine); 5.38 (2H, s, Piperazine); 7.21-
7.22 (1H, d, J ¼ 7.6, Ar-H); 7.30-7.38 (2H, m, Ar-H); 7.47
(1H, s, Ar-H); 9.82 (1H, s, NH). 13C NMR (100 MHz;
DMSO-d6), d, ppm, (J, Hz): 42.92 (-CH2-CH2-); 44.19
(-CH2-CH2-); 44.98 (-CH2-); 114.41 (C Ar); 117.10 (d,
J ¼ 268.0, CF3); 122.46 (C Ar); 124.05 (C Ar); 125.37 (C Ar);
129.73 (C Ar); 132.19 (C Ar); 141.90 (C Ar); 150.90 (C Ar);
182.16 (C=S). LC-MS m/z (rel, %): 361.98 [M þ H]þ (100);
363.95 [M þ Hþþ2]þ (35). HRMS (FAB) Calc.:
C13H11ClF3N5S: 361.03758; Found: 362.0314 [M þ H]þ,
364.0297 [M þ Hþþ2].
We have synthesized a new series of urea 13a-e and thiourea
13f-j derivatives of a precursor compound, 3-(trifluoro-
methyl)-5,6,7,8-tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine (11).
An efficient and robust procedure has been developed for
the preparation of a precursor compound 11 with high
yield (overall yield 37.3%) from 2-chloropyrazine through
the chemical transformations such as hydrazine substitu-
tion, trifluoroacetyl group induction, cyclization and pyra-
zine ring reduction. The improved yield, simple work-up
procedure and purification of intermediates from simple
recrystallization processes are the advantageous of the pre-
sent protocol. The antimicrobial screening data displayed
that the derivatives 13d, 13i and 13j against bacteria and
13a, 13d, 13g and 13j against fungi possess significant
potential activity with MIC range 6.25-25.0 mg/mL. The
electronegative functional groups such as fluoro, trifluoro-
methyl and chloro substituents on phenyl ring might be a
reason to provide promising antimicrobial activity. The
activity assessment of the same substituted set of com-
pounds 13a/f, 13b/g and 13c/h demonstrated that thiourea
derivatives possess better activity than urea compounds,
suggesting the thiourea functionality could be a reason for
high potential activity. Molecular docking studies showed
good binding energies (-8.1 to -9.8 kcal/mol) of the synthe-
sized compounds with in the active site of poly (ADP-
ribose) polymerase 15 (ARTD7, BAL3), macro domain 2
in complex with adenosine-5-diphosphoribose enzyme
(3V2B). Also, the compounds possess good solubility, drug
likeness, drug score and followed the Lipinski’s rule of five
without any violation which demonstrate the synthesized
products are orally active chemotherapeutic agents with
good adsorption and bio-availability. The preliminary bio-
assay data and modeling studies described in the present
study, confirms these class of molecules might be used as
antimicrobial lead compounds on further developments
with structural modifications.
N-(3,4-Dichlorophenyl)-3-(trifluoromethyl)-5,6,7,8-
tetrahydro-[1, 2, 4]triazolo[4,3-a]pyrazine-7-
carbothioamide (13i)
Off-white solid; m.r.: 187-188 ꢀC. IR (KBr, ꢀ~/cmꢁ1): 3215
(N-H, str); 1537 (C ¼ N, str); 1273 (C ¼ S, str); 1159 (CF3,
str); 717 (C-Cl, str). 1H NMR (400 MHz; DMSO-d6), d,
ppm, (J, Hz): 4.33-4.34 (2H, d, J ¼ 4.8, Piperazine); 4.41-4.42
(2H, d, J ¼ 5.2, Piperazine); 5.38 (2H, s, Piperazine); 7.35-
7.37 (1H, m, Ar-H); 7.58-7.60 (1H, d, J ¼ 8.8, Ar-H); 7.67
(1H, m, Ar-H); 9.86 (1H, s, NH). 13C NMR (100 MHz;
DMSO-d6), d, ppm, (J, Hz): 42.87 (-CH2-CH2-); 44.21
(-CH2-CH2-); 45.0 (-CH2-); 117.07 (d, J ¼ 268.0, CF3);
125.58 (C Ar); 126.78 (C Ar); 127.11 (C Ar); 129.91 (C Ar);
130.14 (C Ar); 140.70 (C Ar); 142.27 (d, J ¼ 39.0, C Ar);
150.84 (C Ar); 182.08 (C=S). LC-MS m/z (rel, %): 396.00
[M þ H]þ (100); 398.01 [M þ Hþþ2] (65); 400.04
[M þ Hþþ4] (15). HRMS (FAB) Calc.: C13H10Cl2F3N5S:
394.99860;
Found:
396.0059
[M þ H]þ,
398.0017
[M þ Hþþ2], 399.9978 [M þ Hþþ4].
3 -(Trifluoromethyl)-N-[3-(trifluoromethyl)phenyl]-
5,6,7,8-tetrahydro[1, 2, 4]triazolo[4,3-a]pyrazine-7-
carbothioamide (13j)
Acknowledgements
We appreciate the great support of Srimurugan S and Pramod Kumar,
Micro Labs Ltd., API Division, Bangalore, India for giving unlimited
accesses to use analytical techniques, and Madhu Sudhana Saddala,
Department of Bioinformatics, Sri Venkateswara University, Tirupati,
India providing the support in the evaluation of docking studies.
Pale brown solid; m.r.: 171-173 ꢀC. IR (KBr, ꢀ~/cmꢁ1): 3246
(N-H, str); 1562 (C ¼ N, str), 1217 (C ¼ S, str); 1124 (CF3,
str); 1072 (CF3, str). 1H NMR (400 MHz; DMSO-d6), d,
ppm, (J, Hz): 4.35-4.36 (2H, d, J ¼ 4.8, Piperazine); 4.43-4.46
(2H, d, J ¼ 5.2, Piperazine); 5.51 (2H, s, Piperazine); 7.49-
7.51 (1H, d, J ¼ 7.6, Ar-H); 7.55-7.59 (1H, t, J ¼ 8.0, Ar-H);
7.67-7.69 (1H, d, J ¼ 8.0, Ar-H); 7.74 (1H, s, Ar-H), 9.92
(1H, s, NH). 13C NMR (100 MHz; DMSO-d6), d, ppm, (J,
Hz): 42.92 (-CH2-CH2-); 44.16 (-CH2-CH2-); 44.97 (-CH2-);
117.09 (d, J ¼ 268.0, CF3); 121.24 (C Ar); 122.72 (C Ar);
Funding
This study was in part supported by the 985 Project of National Key
Universities, Tianjin University, China and Foreign Youth Talent
Introduction Programme (WQ20180020), China.

10
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ORCID
Subba Rao Devineni
http://orcid.org/0000-0002-3046-9833
http://orcid.org/0000-0002-2955-474X
http://orcid.org/0000-0001-6566-2118
http://orcid.org/0000-0002-8087-954X
Chandra Mouli Pavuluri
Naga Raju Chamarthi
Raja Sekhara P. Kottapalli
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