Novel N-thioamide analogues of pyrazolylpyrimidine based piperazine: Design, synthesis, characterization, in-silico molecular docking study and biological evaluation

Article abstract of DOI:10.1016/j.molstruc.2018.08.018

Utilizing molecular hybridization approach, a progression of novel pyrazolylpyrimidine based N-thioamide derivatives of piperazine were identified in an effort to develop newer antibacterial and antitubercular agents against the cumulative bacterial resistance. Spectral analysis using Mass, ¹H NMR and ¹³C NMR spectral techniques have been studied in order to affirm the structure of synthesized end molecules. Biological evaluation of all synthesized molecules were studied in-vitro for their antibacterial, antituberculosis and antimalarial efficacy against various bacterial and fungal strains, H37Rv and Plasmodium falciparum respectively. Molecular docking and ADME properties prediction study were also carried out for better insights of responsible proteins with the synthesized molecules. Interestingly, some of the pyrazolylpyrimidine based piperazine N-thioamide derivatives exhibited potential antibacterial, antifungal and antimalarial potency.

Full text of DOI:10.1016/j.molstruc.2018.08.018

Accepted Manuscript
Novel N-thioamide analogues of pyrazolylpyrimidine based piperazine: Design,
synthesis, characterization, in-silico molecular docking study and biological evaluation
Mayur K. Vekariya, Dhaval B. Patel, Pranav A. Pandya, Rajesh H. Vekariya, Prapti U.
Shah, Dhanji P. Rajani, Nisha K. Shah
PII:
S0022-2860(18)30961-X
10.1016/j.molstruc.2018.08.018
MOLSTR 25539
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 13 July 2018
Revised Date: 6 August 2018
Accepted Date: 6 August 2018
Please cite this article as: M.K. Vekariya, D.B. Patel, P.A. Pandya, R.H. Vekariya, P.U. Shah, D.P.
Rajani, N.K. Shah, Novel N-thioamide analogues of pyrazolylpyrimidine based piperazine: Design,
synthesis, characterization, in-silico molecular docking study and biological evaluation, Journal of
Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.08.018.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel N-thioamide analogues of Pyrazolylpyrimidine based Piperazine:
Design, Synthesis, Characterization, In-silico molecular docking study and
Biological evaluation
Mayur K. Vekariyaa, Dhaval B. Patela, Pranav A. Pandyaa, Rajesh H. Vekariyab,
Prapti U. Shahc, Dhanji P. Rajanid, Nisha K. Shaha*
aDepartment of Chemistry,Gujarat University, Ahmedabad, Gujarat 380009,
India.
bBlueORB Solution LLC,1100 Corporate Square Drive Suite 123, Creve Coeur,
Saint Louis, MO 63132, USA.
cDepartment of Environmental Science, Gujarat University, Ahmedabad,
Gujarat 380009, India.
dMicrocare Laboratory and TRC, Surat, Gujarat 395003, India
* Author for correspondence
Dr. Nisha K. Shah
Professor & Head
Department of Chemistry,
Gujarat University,
Ahmedabad, Gujarat 380009, India.
Tel : (M) +91 9825312095, (O) +91 79 26300969
Email: nish_chem2004@yahoo.com

ACCEPTED MANUSCRIPT
Abstract:
Utilizing molecular hybridization approach, a progression of novel pyrazolylpyrimidine based
N-thioamide derivatives of piperazine were identified in an effort to develop newer
antibacterial and antitubercular agents against the cumulative bacterial resistance. Spectral
analysis using Mass, 1H-NMR and 13C-NMR spectral techniques have been studied in order to
affirm the structure of synthesized end molecules. Biological evaluation of all synthesized
molecules were studied in-vitro for their antibacterial, antituberculosis and antimalarial
efficacy against various bacterial and fungal strains, H37Rv and Plasmodium falciparum
respectively. Molecular docking and ADME properties prediction study were also carried out
for better insights of responsible proteins with the synthesised molecules. Interestingly,
some of the pyrazolylpyrimidine based piperazine N-thioamide derivatives exhibited
potential antibacterial, antifungal and antimalarial potency.
Keywords: Antimicrobial agents, docking, hybrids, piperazines, pyrazole, pyrimidine
Introduction
As indicated by WHO report 2016, more cases of tuberculosis occurs in the world than is
beforehand expected[1]. As per this report sum of 10.4 million new cases of tuberculosis
(TB) were stated in 2016. Thus, TB is on ninth rank in the list of death causing diseases
worldwide. Recent approach to cure from tuberculosis involves DOTS (directly observed
treatment, short-course) treatment. DOTS treatment consist of six month usage of four first-
line TB-drugs i.e. Isoniazid, Rifampicin, Ethambutol and Pyrazinamide[2]. However, the
growth of XDR and MDR TB forms turn out to be a dangerous issue leads to an increment of
complications to cure from TB and therefore there is a need to find novel anti-TB
pharmacophores for the development of harmless and fast acting antitubercular
candidates.[3] Apart from TB, Malaria is also stated as most perilous life affecting disease as
21.60 Crores malaria cases were added in WHO report 2016[4]. Thus, both of TB and malaria
remained as dangerous diseases till date and current research lead towards the
identification of novel effective and safe antimalarial candidates to fight against the issue.
Moreover, many classes of antibiotics were failing to show their efficacy due to the
continuous increment of bacterial resistance occurred mainly because of random
prescription of antibiotic drugs and its non-compliance treatment. To overcome from an
existing situation obtained because of bacterial resistance, there is a need for the
advancement of novel antibacterial and antifungal candidates.
Molecular hybridization is well known concept for the design and development of drug
molecules which includes the combination of different pharmacophores of bioactive natural
or synthetic substances into a single new hybrid compound with improved affinity and
potency as compared to the parent drug molecules.[5] Additionally, this approach can result
in compounds having different and/or dual modes of action, altered selectivity profile and
decreased undesired side effects.[6] In the medicinal research field, major significance was

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attained by Pyrazole derivatives among all heterocycles by presenting wide range of
biological potency [7–10] like antitumor[9,11,12], antiviral[13,14], and anti-inflammatory
agents[15]. Along with pyrazole, pyrimidine moiety came across in fame from various
literature as it was stated as a core unit in thymine, cytosine and uracil i.e. building blocks of
nucleic acid. Further, it’s derivatives were stated as broad spectrum of biological active
agents such as anti-HIV[16], antimicrobial[16], antidiabetic[17] and antiinflammatory[18].
Apart from these, several antiviral (idoxuridine and trifluridine), antibacterial (trimethoprim,
sulphamethiazine and sulfadiazine), antimalarial (sulphadoxin), anti HIV (zidovudine,
Retrovir and stavudine), antibiotic (Bacimethrin), antitubercular (Viomycin) and anticancer
(5-fluorouracil) drugs comprises of pyrimidine motif in their structural formula.
Furthermore, N-heteroaryl compounds have been the focus of interest as pharmaceutical
agents and as agrochemicals in recent decades due to their ability to demonstrate a
significant role in certain biological process. In particular, Pyrimidine moieties attached with
nitrogen atom of pyrazole are the best example as N-Pyrazolyl pyrimidines have been
reported as hA2A receptor antagonists with excellent aqueous solubility[19], as antidiabetic
agents (agonistic activity against human GPR142)[20], as anti-inflammatory agents[21], as
kinase inhibitors[22,23], as antiulcer agents[24,25]. Thus, N-Pyrazolyl pyrimidine fragment
was used for Molecular hybridization in the present research.
Furthermore, Piperazine is the most common heterocyclic secondary amine candidate as
many piperazine comprised compounds have demonstrated extensive range of biological
activities [26] including anticancer[27], antianginal[28,29], antidepressant[30–32],
antihistamine[33], antipsychotic[34,35], analgesic and anti-inflammatory[36]. From
literature survey, it was also observed that among piperazine containing compounds, N-
thioamide derivatives of piperazine exhibited a wide range of biological activities as these
derivatives were reported as potent DNA as gyrase inhibitors[37–39], as potent β-
glucuronidase inhibitor[40], as potent arora kinase inhibitor[41], as potent antimicrobial
agent[42], as potent phosphoglycerate dehydrogenase inhibitor[43], as potent FLT-3
inhibitor[44], as selective PDGFR inhibitor[45] and as Potent bacterial Sfp-PPTase
inhibitor[46]. In all of these potent piperazine N-thiomide derivatives, one hydrophobic core
unit attached with the other nitrogen of piperazine was found which may lead it towards
more efficacy. So, piperazine N-thioamides were used as second fragment for Molecular
hybridization. In addition, many of above bioactive piperazines, pyrimidine was found to be
attached with piperazine. So in light of the aformentioned facts, pyrazolyl pyrimidine was
selected as hydrophobic moiety along with N-thioamides of pipearzine to obtain new potent
antimicrobial chemical entities through Molecular hybridization.
<<<<< Insert Figure 1>>>>>
Finally, N-pyrazolylpyrimidine based piperazine N-thioamide motif was selected in order to
design novel bioactive piperazine derivatives. As a result of the aforementioned delineation
(Figure 1), N-pyrazolylpyrimidine based piperazine N- thioamide analogues were designed
and synthesized to investigate of the potential ability of novel designed hybrid molecules.

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Biological evaluation of all synthesized molecules were studied in-vitro for their antibacterial
efficacy against two gram positive bacterial strains (Staphylococcus aureus MTCC 96,
Streptococcus pyogenes MTCC 442), two gram negative bacterial strains (Escherichia coli
MTCC 443,Pseudomonas aeruginosa MTCC 1688) as well as three fungal strains (Aspergillus
clavatus MTCC 1323, Candida albicans MTCC 227 and Aspergillus niger MTCC 282),
antituberculosis efficacy against H37Rv and antimalarial efficacy against Plasmodium
falciparum respectively. On the basis of biological assay results, structure-activity
relationship (SAR) and molecular interaction studies of N-pyrazolylpyrimidine based
piperazine N- thioamide analogues were also described.
Experimental protocol:
Material and Methods
All the required reagents and crude materials were procured from known marketed sources
and were utilized without any refinement. Reaction progress was checked on TLC of Merck
(pre-covered silica gel 60F254 on aluminium sheets) and envisioned by UV light and iodine.
Melting point determination using Optimelt MPA100, an automated apparatus and
spectroscopic analysis i.e. Mass and NMR spectra were utilized to characterize all
synthesized compounds. Mass spectra were analyzed by Water's SQD detector, Waters USA
using 10mM ammonium acetate in water:Methanol(60:40) as mobile phase with electron
1
spray ionization (ESI). H-NMR spectra were recorded on Bruker AV 400MHz spectrometer
(Bruker Avance III, Germany) using DMSO-d6 and TMS as a solvent and internal reference
solvent respectively. Similarly 13C-NMR spectra were recorded on a Bruker AV 100 MHz
spectrometer DMSO-d6 and TMS as a solvent and internal reference solvent respectively.
ADME prediction was done on QikProp and Molecular docking study was performed using
Glide on Schrodinger Maestro 11.
Synthetic protocol for Targeted molecules
Ethyl 2-((dimethylamino)methylene)-3-oxobutanoate (Enaminone Intermediate)
Ethylacetoacetate (26 g, 200 mmol) and DMF-DMA (26.2 g, 220 mmol) were charged in 100
ml three-necked flask at room temperature and stirred for 5 h. The reaction mixture was
then treated with n-pentane to evacuate unreacted material in n-pentane. Product
formation was affirmed with boiling point determination and on TLC plate visualization in
iodine. Obtained crude dark brown liquid was used further without any purification as
Enaminone Intermediate. Yield 95%; b.p. 180°C.
4-chloro-6-hydrazinylpyrimidine (A)
A solution of 4,6-Dichloropyrimidine (15 g, 100 mmol) in ethanol was cooled to 0-5°C in 250
ml three-necked flask. To the above flask Hydrazine hydrate (4.7 ml, 120 mmol) was added
dropwise under cooling atmosphere. After that, the reaction mixture was stirred for 90
minutes at room temperature. Reaction progress was checked on TLC and poured into

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water to get crude product. Pure 4-chloro-6-hydrazinylpyrimidine (A) as Pale yellow
solids[47] obtained by recrystallization in ethanol. Yield 95%; Yellowish white solid; Rf = 0.5
(hexane:ethyl acetate, 4:1); m.p. 164 °C, 1H NMR (d6-DMSO, 400 MHz) δ = 8.83 (s, 1H), 8.17
(s, 1H),6.76 (s, 1H), 4.50 (s, 2H); EI-MS,(m/z): 145.02 (M+1).
Ethyl 1-(6-chloropyrimidin-4-yl)-5-methyl-1H-pyrazole-4-carboxylate (B)
Intermediate-A (13.0 mL, 69.2 mmol) and 1N HCl (10mL) were added dropwise in the
0
solution of C (10.0 g, 69.2 mmol) in ethanol (100 mL) at 0-5 C and was stirred for 1h.
Completion of reaction was confirmed on TLC after 1h. Then reaction mixture was poured
on ice water to afford crude product. Filtration and recrystallization from ethanol of crude
product yielded ethyl 1-(6-chloropyrimidin-4-yl)-5-methyl-1H-pyrazole-4-carboxylate (B) as
1
white crystals.[47] Yield 90%; Rf = 0.8 (hexane:ethyl acetate, 4:1); m.p. 180°C; H NMR 8.94
(s, 1H), 8.42 (s, 1H), 7.238 (s, 1H), 4.216 (q, J=7.2 Hz, 2H), 2.782 (s, 3H), 1.312 (t, J=7.2 Hz,
3H). EI-MS (m/z): 268.07 (M+2).
Ethyl 5-methyl-1-(6-(piperazin-1-yl)pyrimidin-4-yl)-1H-pyrazole-4-carboxylate (C)
To a solution of B (20.0g, 74.9 mmol) in DMF (50 mL) was added piperazine (7.6 mL, 75
0
mmol) dropwise at 0-50C and the mixture was stirred at 80 C for 1 h. After complete
consumption of the reactant B observed on TLC, reaction mixture was poured on ice-cold
water. Resultant white precipitates were filtered off, dried and recrystallized with ethanol
followed by tituration with hexane to afford Ethyl 5-methyl-1-(6-(piperazin-1-yl)pyrimidin-4-
yl)-1H-pyrazole-4-carboxylate (C) as white solid. Yield 80%; Rf = 0.35 (hexane:ethyl acetate,
1
1:1); m.p. 188-192°C; H NMR 8.52 (s, 1H, pyridine CH), 8.07 (s, pyrazol, 1H CH), 7.10 (s,
pyrimidine, 1H CH), 4.81 (s, 1H, piperazine NH), 4.25 (q, Jꢀ=ꢀ6.8ꢀHz, 2H, ethoxy CH2), 3.82-
3.43 (m, 8H, piperazine CH2), 2.88 (s, 3H, CH3-Pyrazole), 1.29 (t, Jꢀ=ꢀ7.2ꢀHz, 3H, ethoxy CH3);
EI-MS (m/z): 338.47 (M+1).
Novel N-thiomide analogues of Ethyl 5-methyl-1-(6-(piperazin-1-yl)pyrimidin-4-yl)-1H-
pyrazole-4-carboxylate (D1-D24)
To the solution of C (1.0 mmol) in 5 mL DMF and corresponding isothiocyanates (1.0 mmol)
were added dropwise in 25-mL RBF in cooling atmosphere. After addition reaction mixture
was stirred at room temperature for 2-3 h. Reaction progress was monitored by TLC plate
by using hexane: ethylacetate solvent system as mobile phase. After completion of reaction
mixture was poured into cold water (30 mL) and the resulting solid was filtered off, washed
with cold water. Crude products were recrystallized from ethanol (95%) to afford pure
compounds. The product formation were further confirmed by spectral data (1H–NMR, 13C–
NMR and ESI-MS).
Ethyl 5-methyl-1-(6-(4-(phenylcarbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-1H-pyrazole-4-
carboxylate (D1)

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0
Yield; 95%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 231-233 C; 1H NMR (400
Hz, DMSO-d6) δ: 9.391 (s, -NHCS, 1H), 8.561 (s, pyrimidine, 1H), 8.084 (s, pyrazol, 1H),
7.342-7.287 (m, 4H), 7.138-7.108 (m, 2H), 4.265 (q, J= 7.2 Hz, 2H), 4.072 (broad s, 4H), 3.852
(broad s, 4H), 2.892 (s, 3H), 1.304 (t, J = 7.2 Hz, 3H); 13C NMR (100 Hz, DMSO-d6) δ:
181.04(CS), 162.51, 158.46, 157.27, 145.38, 142.35, 133.31, 132.69, 129.88, 124.51, 113.91,
93.29, 59.84(CH2 ester), 47.01(piperazine), 42.77(piperazine), 14.21(Me-pyrazole),
13.08(Me-ester); EI-MS (m/z): 359.5 (M+1).
Ethyl 5-methyl-1-(6-(4-(methylcarbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-1H-pyrazole-4-
carboxylate (D2)
0
Yield; 95%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 215-217 C; 1H NMR (400
Hz, DMSO-d6) δ: 8.541 (s, -NHCS, 1H), 8.078 (s, pyrimidine, 1H), 7.780 (s, pyrazol, 1H), 7.077
(s, pyrimidine, 1H), 4.261 (q, J= 7.6 Hz, 2H), 3.928 (broad s, 4H), 3.773 (broad s, 4H), 2.940 (s,
3H), 2.880 (s, 3H), 1.304 (t, J = 7.6 Hz, 3H); 13C NMR (100 Hz, DMSO-d6) δ: 181.91, 162.51,
158.43, 157.24, 145.35, 142.21, 113.87, 93.29, 59.82, 45.94, 44.49, 42.77, 32.51, 14.19,
13.05; EI-MS (m/z): 359.5 (M+1).
Ethyl
1-(6-(4-(ethylcarbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-pyrazole-4-
carboxylate (D3)
Yield; 95%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 182-184 C; 1H NMR (400
Hz, DMSO-d6) δ: 8.544 (s, -NHCS, 1H), 8.080 (s, pyrimidine, 1H), 7.730 (s, pyrazol, 1H), 7.077
(s, pyrimidine, 1H), 4.262 (q, J= 6.8 Hz, 2H), 3.930 (broad s, 4H), 3.772 (broad s, 4H), 3.540 (s,
2H), 2.880 (s, 3H), 1.302 (t, J = 7.6 Hz, 3H), 1.117 (t, J = 6.4 Hz, 3H); 13C NMR (100 Hz, DMSO-
d6) δ: 180.99, 162.51, 158.43, 157.25, 145.35, 142.22, 113.88, 93.35, 59.83, 45.94, 42.78,
14.34, 13.03; EI-MS (m/z): 359.5 (M+1).
0
Ethyl 5-methyl-1-(6-(4-(propylcarbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-1H-pyrazole-4-
carboxylate (D4)
0
Yield; 95%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 166-168 C; 1H NMR (400
Hz, DMSO-d6) δ: 8.547 (s, -NHCS, 1H), 8.084 (s, pyrimidine, 1H), 7.740 (s, pyrazol, 1H), 7.080
(s, pyrimidine, 1H), 4.262 (q, J= 7.2 Hz, 2H), 3.933 (broad s, 4H), 3.772 (broad s, 4H), 3.458
(q, J= 6.4 Hz, 2H), 2.880 (s, 3H), 1.560 (q, J= 7.2 Hz, 2H), 1.301 (t, J = 7.2 Hz, 3H), 0.856 (t, J =
7.2 Hz, 3H); 13C NMR (100 Hz, DMSO-d6) δ: 181.21, 162.55, 158.43, 157.27, 145.35, 142.33,
113.88, 93.40, 59.83, 47.07, 46.00, 42.77, 21.88, 14.20, 13.02, 11.32; EI-MS (m/z): 359.5
(M+1).
Ethyl
1-(6-(4-(butylcarbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-pyrazole-4-
carboxylate (D5)
Yield; 85%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 160-162 C; 1H NMR (400
Hz, DMSO-d6) δ: 8.542 (s, -NHCS, 1H), 8.079 (s, pyrimidine, 1H), 7.713 (s, pyrazol, 1H), 7.074
(s, pyrimidine, 1H), 4.262 (q, J= 6.8 Hz, 2H), 3.933 (broad s, 4H), 3.768 (broad s, 4H), 3.505
(q, J= 6.4 Hz, 2H), 2.880 (s, 3H), 1.543 (q, J= 7.2 Hz, 2H), 1.320 – 1.248 (m, 5H), 0.894 (t, J =
7.6 Hz, 3H); 13C NMR (100 Hz, DMSO-d6) δ: 181.19, 162.58, 158.43, 157.24, 145.34, 142.21,
0

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113.88, 93.33, 59.82, 46.00, 45.04, 42.77, 30.81, 19.57, 14.19, 13.78, 13.03; EI-MS (m/z):
359.5 (M+1).
Ethyl 1-(6-(4-(isopropylcarbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-pyrazole-
4-carboxylate (D6)
0
Yield; 85%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 182-184 C; 1H NMR (400
Hz, DMSO-d6) δ: 8.548 (s, -NHCS, 1H), 8.085 (s, pyrimidine, 1H), 7.370 (s, pyrazol, 1H),
7.078(s, pyrimidine, 1H), 4.571-4.522 (m, 1H),4.263 (q, J=7.2 Hz, 2H), 3.927 (broad s, 4H),
3.767 (broad s, 3H), 2.880 (s, 3H), 1.301 (t, J=7.2 Hz, 3H), 1.160 (d, J=6.4 Hz, 6H); 13C NMR
(100 Hz, DMSO-d6) δ: 181.21, 162.55, 158.43, 157.27, 145.35, 142.33, 113.88, 93.40, 59.83,
47.07, 46.00, 43.87, 21.88, 14.20, 13.02; EI-MS (m/z): 359.5 (M+1).
Ethyl 1-(6-(4-(tert-butylcarbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-pyrazole-
4-carboxylate (D7)
0
Yield; 85%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 160-162 C; 1H NMR (400
Hz, DMSO-d6) δ: 8.548 (s, -NHCS, 1H), 8.088 (s, pyrimidine, 1H), 7.059 (s, pyrazol, 1H),
6.073(s, pyrimidine, 1H), 4.264 (q, J=7.6 Hz, 2H), 3.894 (broad s, 4H), 3.765 (broad s, 3H),
2.882 (s, 3H), 1.465 (s, 9H), 1.300 (t, J=6.8 Hz, 3H), 1.160 (d, J=6.4 Hz, 6H); 13C NMR (100 Hz,
DMSO-d6) δ: 181.19, 162.58, 158.43, 157.24, 145.34, 142.21, 113.88, 93.33, 59.82, 46.00,
45.04, 42.77, 30.81, 13.78, 13.03; EI-MS (m/z): 359.5 (M+1).
Ethyl
1-(6-(4-(allylcarbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-pyrazole-4-
carboxylate (D8)
Yield; 95%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 191-193 C; 1H NMR (400
Hz, DMSO-d6) δ: 8.543 (s, -NHCS, 1H), 8.075 (s, pyrimidine, 1H), 7.910 (s, pyrazol, 1H), 7.079
(s, pyrimidine, 1H), 5.900 (dt, J= 16.8, 5.6 Hz, 1H), 5. 146 (d, J= 17.2 Hz, 1H), 5.072 (d, J= 10.0
Hz, 1H), 4.288-4.200 (m, 4H), 3.961 (broad s, 4H), 3.781 (broad s, 4H), 2.880 (s, 3H), 1.303 (t,
J= 6.8 Hz, 3H); 13C NMR (100 Hz, DMSO-d6) δ: 181.37, 162.58, 162.48, 158.44, 157.25,
145.33, 142.22, 135.40, 115.38, 113.88, 93.35, 59.83, 47.67, 46.17, 42.77, 14.20, 13.03; EI-
MS (m/z): 359.5 (M+1).
0
Ethyl 1-(6-(4-(cyclohexylcarbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-pyrazole-
4-carboxylate (D9)
0
Yield; 92%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 196-199 C; 1H NMR (400
Hz, DMSO-d6) δ: 8.539 (s, -NHCS, 1H), 8.075 (s, pyrimidine, 1H), 7.337 (s, pyrazol, 1H), 7.069
(s, pyrimidine, 1H), 4.270-4.236 (m, 3H), 3.929 (broad s, 4H), 3.763 (broad s, 4H), 2.878 (s,
3H), 1.877-1.589 (m, 5H), 1.318-1.079 (m, 8H); 13C NMR (100 Hz, DMSO-d6) δ: 180.24,
162.58, 162.47, 158.42, 157.24, 145.34, 142.21, 113.88, 93.32, 59.82, 54.51, 42.77, 40.12,
32.05, 25.19, 14.19, 13.03; EI-MS (m/z): 359.5 (M+1).
Ethyl 1-(6-(4-(benzylcarbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-pyrazole-4-
carboxylate (D10)
0
Yield; 93%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 182-184 C; 1H NMR (400
Hz, DMSO-d6) δ: 8.544 (s, -NHCS, 1H), 8.332 (s, pyrimidine, 1H), 8.079 (s, pyrazol, 1H),

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7.322-7.078 (m, 5H), 7.078 (s, pyrimidine, 1H), 4.833 (s, 2H), 4.254 (broad s, 2H), 4.008
(broad s, 4H), 3.801 (broad s, 4H), 2.884 (s, 3H), 1.302 (broad s, 3H); 13C NMR (100 Hz,
DMSO-d6) δ: 181.69, 162.58, 162.46, 158.43, 157.22, 145.37, 142.22, 139.59, 128.04,
127.21, 126.54, 113.88, 93.24, 59.82, 48.34, 46.28, 42.79, 14.19, 13.07; EI-MS (m/z): 359.5
(M+1).
Ethyl 1-(6-(4-((2-methoxyphenyl)carbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-
pyrazole-4-carboxylate (D11)
0
Yield; 96%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 191-193 C; 1H NMR (400
Hz, DMSO-d6) δ: 8.863 (s, -NHCS, 1H), 8.553 (s, pyrimidine, 1H), 8.075 (s, pyrazole, 1H),
7.279-6.918 (m,5H), 4.261 (broad s, 2H), 4.074 (broad s, 4H), 3.841-3.774 (m, 7H), 2.891 (s,
3H), 1.305 (broad s, 3H); 13C NMR (100 Hz, DMSO-d6) δ: 182.02, 162.59, 162.48, 158.46,
157.25, 154.11, 145.37, 142.22, 129.32, 129.25, 126.94, 119.79, 113.90, 111.67, 93.31,
59.83, 55.47, 46.74, 42.81, 14.20, 13.06; EI-MS (m/z): 359.5 (M+1).
Ethyl 1-(6-(4-((3-methoxyphenyl)carbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-
pyrazole-4-carboxylate (D12)
0
Yield; 93%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 175-177 C; 1H NMR (400
Hz, DMSO-d6) δ: 9.381 (s, -NHCS, 1H), 8.562 (s, pyrimidine, 1H), 8.089 (s, pyrazol, 1H), 7.208
(t, J=8.0 Hz, 1H), 7.108 (s, 1H), 6.964 (s,1H, pyrazole), 6.927 (d, J=8.0Hz, 1H), 6.699 (d, J=7.2
Hz, 1H), 4.264 (q, J=7.2 Hz, 2H), 4.056 (broad s, 4H), 3.847 (broad s, 4H), 3.734 (s, 3H), 2.891
(s, 3H), 1.303 (q, J=7.2 Hz, 3H); 13C NMR (100 Hz, DMSO-d6) δ: 181.82, 162.65, 162.58,
158.86, 157.45, 154.31, 145.35, 142.42, 129.29, 129.05, 126.83, 119.69, 113.50, 111.46,
93.21, 59.53, 55.87, 46.84, 42.40, 14.30, 13.08; EI-MS (m/z): 359.5 (M+1).
Ethyl 1-(6-(4-((4-methoxyphenyl)carbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-
pyrazole-4-carboxylate (D13)
0
Yield; 91%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 221-223 C; 1H NMR (400
Hz, DMSO-d6) δ: 9.275 (s, -NHCS, 1H), 8.562 (s, pyrimidine, 1H), 8.090 (s, pyrazol, 1H), 7.192
(d, J=8.4 Hz, 2H), 7.107 (s, 1H), 8.879 (d, J=8.4 Hz, 2H), 4.264 (q, J=6.8 Hz, 2H), 4.061 (broad
s, 4H), 3.842 (broad s, 4H), 3.748 (s, 3H), 2.891 (s, 3H), 1.305 (t, J=6.8 Hz, 3H); 13C NMR (100
Hz, DMSO-d6) δ: 182.02, 162.59, 162.48, 158.46, 157.25, 154.11, 145.37, 142.22, 129.32,
129.25, 126.94, 119.79, 113.90, 111.67, 93.31, 59.83, 55.47, 46.74, 42.81, 14.20, 13.06; EI-
MS (m/z): 359.5 (M+1).
Ethyl
1-(6-(4-((3-chlorophenyl)carbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-
pyrazole-4-carboxylate (D14)
Yield; 90%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 206-208 C; 1H NMR (400
Hz, DMSO-d6) δ: 9.497 (s, -NHCS, 1H), 8.566 (s, pyrimidine, 1H), 8.092 (s, pyrazol, 1H), 7.473
(s, 1H), 7.322-7.115 (m, 4H), 4.266 (q, J=6.8 Hz, 2H), 4.072 (broad s, 4H), 3.859 (broad s, 4H),
2.893 (s, 3H), 1.304 (t, J=6.8 Hz, 3H); 13C NMR (100 Hz, DMSO-d6) δ: 181.95, 162.18, 162.66,
158.37, 157.17, 145.28, 142.14, 139.17, 128.22, 127.64, 126.70, 113.61, 93.21, 59.73, 48.46,
46.88, 42.72, 14.19, 13.06; EI-MS (m/z): 359.5 (M+1).
0

ACCEPTED MANUSCRIPT
Ethyl
1-(6-(4-((4-chlorophenyl)carbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-
pyrazole-4-carboxylate (D15)
Yield; 95%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 214-216 C; 1H NMR (400
Hz, DMSO-d6) δ: 9.461 (s, -NHCS, 1H), 8.559 (s, pyrimidine, 1H), 8.084 (s, pyrazol, 1H), 7.365
(m, 4H), 7.105 (s, pyrimidine, 1H), 4.266 (q, J=6.8 Hz, 2H), 4.077 (broad s, 4H), 3.855 (broad
s, 4H), 2.893 (s, 3H), 1.306 (t, J=6.8 Hz, 3H); 13C NMR (100 Hz, DMSO-d6) δ: 181.25, 162.58,
162.46, 158.47, 157.27, 145.38, 142.24, 139.87, 128.32, 127.84, 126.90, 113.91, 93.31,
59.83, 48.56, 46.96, 42.80, 14.19, 13.06; EI-MS (m/z): 359.5 (M+1).
0
Ethyl
1-(6-(4-((4-fluorophenyl)carbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-
pyrazole-4-carboxylate (D16)
Yield; 90%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 218-220 C; 1H NMR (400
Hz, DMSO-d6) δ: 9.388 (s, -NHCS, 1H), 8.560 (s, pyrimidine, 1H), 8.084 (s, pyrazol, 1H),
7.329-7.107 (m, 5H), 4.266 (q, J=6.8 Hz, 2H), 4.078 (broad s, 4H), 3.855 (broad s, 4H), 2.892
(s, 3H), 1.305 (t, J=6.4 Hz, 3H); 13C NMR (100 Hz, DMSO-d6) δ: 181.50, 162.58, 160.41,
158.46, 157.27, 145.38, 142.24, 137.15, 127.72, 114.48, 93.31, 59.83, 46.83, 42.77, 14.19,
13.07; EI-MS (m/z): 359.5 (M+1).
0
Ethyl 1-(6-(4-((2,4-dichlorophenyl)carbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-
pyrazole-4-carboxylate (D17)
0
Yield; 94%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 215-217 C; 1H NMR (400
Hz, DMSO-d6) δ: 9.362 (s, -NHCS, 1H), 8.566 (s, pyrimidine, 1H), 8.088 (s, pyrazol, 1H), 7.656
(s, 1H), 7.420 (d, J= 7.6 Hz, 1H), 7.345 (d, J=8.4 Hz,1H), 7.121 (s, pyrimidine, 1H), 4.267 (q,
J=6.8 Hz, 2H), 4.089 (broad s, 4H), 3.863 (broad s, 4H), 2.893 (s, 3H), 1.305 (t, J=6.8 Hz, 3H);
13C NMR (100 Hz, DMSO-d6) δ: 181.64, 162.55, 158.48, 157.28, 145.38, 142.25, 137.49,
133.02, 132.27, 128.79, 127.36, 113.91, 93.43, 59.84, 46.96, 42.82, 14.21, 13.06; EI-MS
(m/z): 359.5 (M+1).
Ethyl
1-(6-(4-((3-iodophenyl)carbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-
pyrazole-4-carboxylate (D18)
Yield; 92%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 228-230 C; 1H NMR (400
Hz, DMSO-d6) δ: 9.454 (s, -NHCS, 1H), 8.560 (s, pyrimidine, 1H), 8.086 (s, pyrazol, 1H), 7.747
(s, 1H), 7.468 (d, J= 6.8 Hz, 1H), 7.398 (d, J= 7.2 Hz, 1H), 7.109 (s, 2H), 4.264 (q, J=6.4 Hz, 2H),
4.067 (broad s, 4H), 3.853 (broad s, 4H), 2.892 (s, 3H), 1.305 (t, J=6.4 Hz, 3H); 13C NMR (100
Hz, DMSO-d6) δ: 181.04, 162.52, 158.46, 157.27, 145.38, 142.30, 133.31, 132.69, 129.88,
124.51, 113.91, 93.29, 59.84, 47.01, 42.77, 14.21, 13.08; EI-MS (m/z): 359.5 (M+1).
0
Ethyl 5-methyl-1-(6-(4-(p-tolylcarbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-1H-pyrazole-4-
carboxylate (D19)
0
Yield; 93%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 233-235 C; 1H NMR (400
Hz, DMSO-d6) δ: 9.303 (s, -NHCS, 1H), 8.555 (s, pyrimidine, 1H), 8.079 (s, pyrazol, 1H),
7.203-7.101 (m, 5H), 4.273 (q, J=6.4 Hz, 2H), 4.059 (broad s, 4H), 3.841 (broad s, 4H), 2.890
(s, 3H), 2.281(s, 3H), 1.304 (t, J=6.8 Hz, 3H); 13C NMR (100 Hz, DMSO-d6) δ: 181.48, 162.54,

ACCEPTED MANUSCRIPT
158.47, 157.28, 145.37, 142.24, 138.25, 133.61, 128.46, 125.56, 113.90, 93.36, 59.83, 46.85,
42.82, 40.12, 20.51, 14.20, 13.05; EI-MS (m/z): 359.5 (M+1).
Ethyl 1-(6-(4-(benzoylcarbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-pyrazole-4-
carboxylate (D20)
0
Yield; 95%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 180-182 C; 1H NMR (400
Hz, DMSO-d6) δ: 10.954 (s, -NHCS, 1H), 8.564 (s, pyrimidine, 1H), 8.085 (s, pyrazol, 1H),
7.970 (t, J=7.2 Hz, 2H), 7.623 (d, J=7.6 Hz, 1H), 7.526 (t, J= 7.2 Hz, 2H), 7.092 (s, 1H), 4.260 (q,
J=7.2 Hz, 2H), 3.943 (broad s, 4H), 3.815 (broad s, 4H), 2.891 (s, 3H), 1.299 (t, J=7.2 Hz, 3H);
13C NMR (100 Hz, DMSO-d6) δ: 180.12, 164.00, 162.58, 162.41, 158.49, 157.49, 157.29,
145.41, 142.28, 132.52, 128.43, 127.03, 113.93, 93.34, 59.83, 49.44, 48.78, 42.88, 14.20,
13.08; EI-MS (m/z): 359.5 (M+1).
Ethyl
5-methyl-1-(6-(4-((4-nitrophenyl)carbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-1H-
pyrazole-4-carboxylate (D21)
Yield; 94%; Yellow solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 265-267 0C; 1H NMR (400
Hz, DMSO-d6) δ: 9.298 (s, -NHCS, 1H), 8.562 (s, pyrimidine, 1H), 8.175 (d, J=8.0 Hz, 2H),
8.081 (s, pyrazol, 1H), 7.658 (d, J=8.0 Hz, 2H), 7.114 (s, pyrimidine, 1H), 4.266 (q, J=6.4 Hz,
2H), 4.099 (broad s, 4H), 3.877 (broad s, 4H), 2.895 (s, 3H), 1.308 (t, J=6.4 Hz, 3H); 13C NMR
(100 Hz, DMSO-d6) δ: 180.74, 162.49, 158.48, 157.26, 147.81, 145.40, 142.25, 123.89,
122.69, 113.85, 93.27, 59.83, 47.51, 44.65, 42.78, 14.18, 13.08; EI-MS (m/z): 359.5 (M+1).
Ethyl 1-(6-(4-((2,3-dichlorophenyl)carbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-
pyrazole-4-carboxylate (D22)
0
Yield; 90%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 203-205 C; 1H NMR (400
Hz, DMSO-d6) δ: 9.488 (s, -NHCS, 1H), 8.571 (s, pyrimidine, 1H), 8.091 (s, pyrazol, 1H), 7.545
(d, J=7.6 Hz, 1H), 7.382-7.304 (m, 3H), 7.126 (s, pyrimidine, 1H), 4.267 (q, J=6.8 Hz, 2H),
4.094 (broad s, 4H), 3.871 (broad s, 4H), 2.895 (s, 3H), 1.305 (t, J=6.8 Hz, 3H); 13C NMR (100
Hz, DMSO-d6) δ: 163.61, 161.23, 157.79, 143.90, 140.60, 139.24, 120.14, 117.54, 95.30,
65.5, 56.4, 43.8, 40.1, 13.16; EI-MS (m/z): 359.5 (M+1).
Ethyl 1-(6-(4-((2,6-dichlorophenyl)carbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-
pyrazole-4-carboxylate (D23)
0
Yield; 90%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 253-255 C; 1H NMR (400
Hz, DMSO-d6) δ: 9.366 (s, -NHCS, 1H), 8.564 (s, pyrimidine, 1H), 8.087 (s, pyrazol, 1H), 7.548
(d, J=8.0 Hz, 2H), 7.382-7.304 (m, 2H), 7.112 (s, pyrimidine, 1H), 4.267 (q, J=6.8 Hz, 2H),
4.089 (broad s, 4H), 3.863 (broad s, 4H), 2.893 (s, 3H), 1.305 (t, J=6.8 Hz, 3H); 13C NMR (100
Hz, DMSO-d6) δ: 181.64, 162.55, 158.48, 157.28, 145.38, 142.25, 137.49, 133.02, 132.27,
128.79, 127.36, 113.91, 93.43, 59.84, 46.96, 42.82, 14.21, 13.06; EI-MS (m/z): 359.5 (M+1).
Ethyl
1-(6-(4-((2-chlorophenyl)carbamothioyl)piperazin-1-yl)pyrimidin-4-yl)-5-methyl-1H-
pyrazole-4-carboxylate (D24)

ACCEPTED MANUSCRIPT
0
Yield; 92%; white solid; Rf = 0.4 (hexane:ethyl acetate, 1:1); m.p.: 228-230 C; 1H NMR (400
Hz, DMSO-d6) δ: 9.454 (s, -NHCS, 1H), 8.560 (s, pyrimidine, 1H), 8.086 (s, pyrazole, 1H),
7.279-6.918 (m,5H), 4.273 (q, J=6.4 Hz, 2H), 4.059 (broad s, 4H), 3.841 (broad s, 4H), 2.890
(s, 3H), 1.304 (t, J=6.8 Hz, 3H); 13C NMR (100 Hz, DMSO-d6) δ: 181.64, 162.55, 158.48,
157.28, 145.38, 142.25, 137.49, 133.02, 132.27, 128.79, 127.36, 113.91, 93.43, 59.84, 47.01,
42.77, 14.21, 13.08; EI-MS (m/z): 359.5 (M+1).
In-vitro Biological screening
Biological screening has been performed at Microcare Laboratory and TRC, Surat, India as
per earlier reported methods.[47–50] All synthesized compounds were evaluated in-vitro
for their antimicrobial efficacy against two gram positive bacterial strains (Staphylococcus
aureus MTCC 96, Streptococcus pyogenes MTCC 442), two gram negative bacterial strains
(Escherichia coli MTCC 443,Pseudomonas aeruginosa MTCC 1688) as well as three fungal
strains (Aspergillus clavatus MTCC 1323, Candida albicans MTCC 227 and Aspergillus niger
MTCC 282), antituberculosis efficacy against H37Rv and antimalarial efficacy against
Plasmodium falciparum respectively. In antibacterial evaluation, ampicillin, ciprofloxacin
and chloramphenicol were used whereas in antifungal evaluation, nystatin and greseofulvin
were used as standard control drugs. Similarly, isoniazide for antituberculosis and
Chloroquine as well as quinine for antimalarial evaluation were used as standard control
drugs.
Docking protocol
From the RCSB Protein Data Bank, protein structure coordinates (PDB Code 4DPD[51],
4CJN[52] & 1HNJ[53]) were procured. Docking study was performed as per previously
reported route.[47]
Results and Discussion
Synthesis and structural characterization
In present work, novel distinct N-thioamide derivatives of pyrazolylpyrimidine based
piperazine are prepared through a plausible conversion way. The synthesis route for the N-
thioamides of pyrazolylpyrimidine based piperazine is delineated below as Scheme 1.
As depicted in Scheme 1, 4,6-dichloropyrimidine selected as a primary reactant which when
reacted with hydrazine hydrate yielded Compound A. Enaminone intermediate were
obtained by the reaction of N,N-Dimethylformamide dimethyl acetal (DMF-DMA) [54] with
Ethylacetoacetate which was key step to formulate Compound B i.e. ethyl 1-(6-
chloropyrimidin-4-yl)-5-methyl-1H-pyrazole-4-carboxylate. Compound B was formed by the
reaction of Enaminone intermediate with Compound A catalysed by dilute HCl.[55]
Nucleophilic substitution of Compound B by piperazine resulted in formation of
monosubstitued
Ethyl
5-methyl-1-(6-(piperazin-1-yl)pyrimidin-4-yl)-1H-pyrazole-4-

ACCEPTED MANUSCRIPT
carboxylate (C) without using any base as catalyst. Pyrazole ring formation from enaminone
intermediate using dilute HCl and monosubstitution of piperazine without using any base
are the best protocol developed here to get products in high yield without further
refinement as previously reported methods associated with the formation of low yielded
product. Different isothiocyanates were used in final step to afford to the final targeted
compounds (D1-D24) with good yield and easy workup processes.
<<<<< Insert Scheme 1>>>>>
1
Structures of all synthesized compounds (D1-D24) were confirmed by their H-NMR, 13C-
NMR and Mass spectra analysis, and the obtained spectra for each compound are found as
per the proposed molecular structures. In mass spectra, molecular ion peak i.e. M+1 or M+2
peak confirms the formation of product as it was found appropriate to the molecular weight
1
of the compound. In Figure 2, δ values found from H-NMR and 13C-NMR spectra which
were denoted are in good accordance with those of specific protons and specific carbons of
common scaffold found in all compounds. In case of aromatic thioamide derivatives, all
protons were detected more deshielded as compared to aliphatic thioamides. In 13C-NMR,
carbon of thioamide group and carbonyl carbon of ester group (except compound D20)
were observed more deshielded than the other carbons present in respective compound at
around 181ppm and 162ppm respectively. Overview of NMR spectral date are represented
1
below as Figure 2 and spectral data i.e. H-NMR, 13C-NMR and Mass spectra of all
compounds were provided in supporting data.
<<<<< Insert Figure 2>>>>>
In-silico ADMET prediction and Drug likeness study
For drug discovery in earlier clinical step, Lipinski rule of five becomes the most influential concept
to check the Drug likeness properties of novel molecules.[56] Drug likeness properties of
synthesized compounds (D1-D24) were calculated by free Molinspiration-molecular
property calculation services online. Marvinsketch tool was used to obtain molar refractivity
of all novel designed target molecules using 3d structure followed by energy minimization.
Obtained results were depicted in supporting data as Table A. All compounds displayed
good drug likeness parameters without Lipinski’s rule of five violation and thereby shown
decent bioavailability. Alternative to this rule certain other pharmacokinetic parameters or
molecular descriptors like polar surface area were also becomes the powerful concept for
drug discovery in preclinical stage.
Thus, ADME properties were also predicted by Jorgensen’s Method[57] using Qikprop tool
(Shrodinger) for novel molecules and the obtained values are provided in supporting data as
Table B. All molecules found to have acceptable pharmacokinetic properties (ADME). All
compound have depicted decent human oral absorption percentage with in permissible
range (75.57–100%) and also good blood–brain barrier permeability (QP log BB) values
within acceptable range of -3 to 1.2. Similarly all compounds exhibited great intestinal

ACCEPTED MANUSCRIPT
Absorption Prediction as predicted value of Caco-2 cell permeability (QPPCaco) for the all
compounds were found greater than 500nm/sec. All compound shown admirable range IC50
value for HERG K+ channel blockage (QP logHERG) below -5. PSA values for designed
molecules were also obtained in the range of 82.367-140.333 Å which guarantees good
influence on bioavailability of molecules.[58] Morever, the predicted results were found to
be differ in case of aqua solubility parameter (QPlogS) from the acceptable range i.e. -6.5 to
0.5. Number of methods available like Particle size reduction, pH adjustment, Hydrotrophy,
Solid dispersion etc to improve the aqua solubility of drug content.[59] The essential
pharmacokinetic parameters are delineated along with their permissible ranges in Table B
(Supporting Data). ADME prediction may found to facilitate assessment of the eligible
molecules.
In-vitro Biological evaluation:
All compounds were evaluated in-vitro for their biological potency against various
microorganisms and the resulted MIC or IC50 values were presented in Table 1 and Table 2.
As from the bioassay results, Compound D19 and D9 demonstrated good efficacy against
malarial organism with IC50 values of 0.07 and 0.09 μg/mL respectively as compare to
standard drugs i.e quinine and chloroquine. As compared to Quinine many of synthesized
thioamide derivatives exhibited good potency with IC50 values ranges from 0.15 - 0.26 μg/mL
against P.falciparum.
<<<<< Insert Table 1>>>>>
Amongst all compounds, two Compounds i.e. D15 and D19 against E.Coli, one compound i.e.
D20 against P. aeruginosa and S. pyogenes, two Compounds i.e. D20 and D10 against S.
aureus emerged out from in-vitro antimicrobial assay as more potent antibacterial agents as
compare to all three standard drugs i.e. Ampicillin, Ciprofloxacin and Chloramphenicol
(Table 2). Compound D20 exhibited excellent antibacterial activity against all bacterial strain
except E.Coli compared to standard drugs i.e. Ampicillin, Chloramphenicol and Ciprofloxacin.
Compound D15 demonstrated excellent activity with MIC value of 25 µg/mL equipotent to
standard drug ciprofloxacin. Among the three fungal strains, C. Albicans was only found to
be sensitive towards the targeted compounds (Table 2). Four of synthesized compounds i.e.
D5, D6, D19 and D20 found to be more potent antifungal agent against the C. Albicans
organisms as compare to Greseofulvin. D9 and D19 were emerged out as potent antimalarial
agents with IC50 value of 0.09 and 0.07 µg/mL respectively. Thus, these compounds found to
be more potent than standard antimalarial drug Quinine. In case of anti-tubercular assay, all
thioamides were found to have moderate MIC values ranging from 25 to 1000 μg/mL. Only
compound D6 exhibited good antitubercular activity (Table 1). From all biological assay, the
results revealed that synthesized N-thioamides of pyrazolylpyrimidine based piperazine
showed significant antibacterial and antimalarial activity.
<<<<< Insert Table 2>>>>>
In-silico Molecular docking studies

ACCEPTED MANUSCRIPT
To recognise the probable interaction of the bioactive molecules as ligands with the parent
protein i.e. enzyme, Molecular docking of the synthesized molecules were studied against
responsible enzymes i.e. P. falciparum dihydrofolate reductase (Pf-DHFR), β-Ketoacyl-acyl
carrier protein (ACP) synthase i.e. E.coli FabH and S.Aureus hydrolase using PDB ID 4DPD,
1HNJ & 4CJN respectively as respective inhibitors of such enzymes are found responsible for
the potency. Further, favourable interaction of many bioactive molecules against these
enzymes were found in literature[60–62] which encouraged us for the selection of the
aforementioned enzymes in present research. Enzyme i.e. protein 3D structures were taken
from protein data bank and Glide tool in Maestro 11 (Schrodinger, LLC, New York, NY, 2015)
was used to calculate docking scores of all synthesized molecules. Docking scores for all
synthesized compounds obtained were shown in supplementary data. Docking poses in 2D
and 3D view of most potent molecules in the active site of key enzymes are depicted in
Figures 3-5. 3D docking poses were generated only with those amino acid residue which are
in 4 Å range from ligand with the use of custom preset option. All potent ligands exhibited
strong interaction with respective enzymes with good docking scores. Common π-π
interaction with amino acid residue TRP 32 was observed by potent D15 and D19 in case of
FabH. Likewise D10 and D20 also interacted with amino acid residue TYR 105 via common π-
π interaction. Ligand protein interaction for some potent compounds with the respective
key enzyme were represented in Table 3 along with the docking score. Overall, the in-vitro
results found in accordance with the molecular docking results and the docked ligands
shown good docking score by showing good interactions with amino acid residues of the
corresponding enzymes. Hence, the potential behavior of these bioactive molecules (D9,
D19, D10, D15 and D20) may expected due to their inhibitory efficacy against responsible
enzymes. Various ligand protein interactions obtained were depicted along with docking
score and bond length in Table 3.
<<<<< Insert Figure 3>>>>>
<<<<< Insert Figure 4>>>>>
<<<<< Insert Figure 5>>>>>
<<<<< Insert Table 3>>>>>
Structure-activity relationship (SAR)
Substituted Arylamine at distinct position found to affect biological potency. The electronic
configuration of the various functional group led to promising effect on bioactivity.
Compounds comprising -OCH3 like electron-releasing groups on aromatic ring led to better
antitubercular activity. Additionally, ortho substitution of chloro group to phenyl ring was
led to be more biopotency. Moreover, compounds possessing -NO2 like electron
withdrawing groups attached on benzene ring resulted in decreasing antitubercular efficacy.
Compounds possessing electron-withdrawing and halogenated groups excluding iodo group
on aromatic ring led to increasing the antimalarial activity. It was also observed that ortho

ACCEPTED MANUSCRIPT
and para substitution of the methyl and methoxy groups on phenyl ring led to increasing
bioactivity. Meta substitution of iodo group phenyl ring exhibited excellent anti-malarial
activity.
Among aliphatic amine substitution, ethylamine and methylamine substitution led to
decrease in antitubercular and antimalarial efficacy. While compounds containing isopropyl
amine and butylamine substituents led to excellent antitubercular activity but less
antimalarial activity. Compounds possessing cyclohexyl amine and propyl amine
substituents led to excellent antimalarial activities. Furthermore, less antitubercular potency
was detected by isobutyl amine and propylamine substituted compounds.
Most compounds found active against S. aureus bacterial strain. Compounds comprising of
electron-releasing groups (like halogen) on aromatic ring found to be potent against Gram
positive bacterial strains. Another side, an electron withdrawing group like nitro on aromatic
ring directed increasing Gram positive bacterial inhibition. Para subtitution of the fluoro
group on aromatic ring with the lowest lipophilicity (Lowest Log P) and benzoyl substituent
led to an increase inhibition against both Gram negative as well as Gram positive bacterial
strains. In addition, compound possessing –CH3 and –OCH3 groups on phenyl ring decreased
bacterial inhibition.
Compounds possessing halogenated groups like -Cl and -F on aromatic ring contained
excellent inhibition against E. coli i.e. Gram negative bacterial strain. Compounds comprising
substituents like isobutyl amine, prop-2-en-1-amine and cyclohexyl amine led to more
potent Gram positive antibacterial activity while compounds comprising butyl amine,
isopropyl amine and propyl amine led to decrease inhibition against Gram positive bacterial
strain. Furthermore, compounds with methyl amine substitution expressed excellent Gram
negative E. coli bacterial inhibition activity. Figure 6 demonstrates the outcome of SAR study
in graphical form.
<<<<< Insert Figure 6>>>>>
Conclusion:
The present study demonstrated synthesis, spectroscopic evaluation, molecular docking
studies and in-vitro biological evaluation i.e. antimicrobial, antimalarial and anti-tubercular
assay of N-thioamide derivatives of pyrazolylpyrimidine based piperazine. A convenient and
most efficient synthetic pathway had been reported to synthesize novel hybrid molecules of
pyrazole, pyrimidine and piperazine. All compounds were obtained in good yield and high
purity. The formation of all compounds were unambiguously seen from the spectral data.
Notably, antibacterial studies demonstrate that four compounds D10, D15, D19 and D20
exhibit superior antibacterial activity against distinct bacterial strain. Compound D20 i.e.
benzoyl thioamide derivative was found most potent against all bacterial strains except E.
Coli. Furthermore, Compound D9 and D19 exhibit superior antimalarial activity among all

ACCEPTED MANUSCRIPT
compounds. Molecular docking studies results show that all these potent compounds were
also found to have favourable interaction with the responsible enzymes that means
compounds with good docking scores have shown good potency. Thus, the in-silico studies
and the biological assay can be specifically related with each other and optimization of
potent molecules identified in the present study can be conveyed out for further
advancement.
Acknowledgement
Chemistry Department, Gujarat University, Ahmedabad is acknowledged for providing the
required facilities. UGC, India and GUJCOST are acknowledged by Mayur K. Vekariya and
Nisha K. Shah for providing financial support under UGC-BSR Fellowship (Grant No: F./25-
1/2013- 14 (BSR) / 7-74/2007 (BSR) dated 30-05- 2014) and Minor Research Project.
Conflicts of interest
The authors declare that they have no competing interests.
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Figures and Scheme captions
Scheme 1: Reagents and conditions: (i) NH2NH2.H2O, EtOH, rt;
(ii) Enaminone intermediate, 1N HCl, EtOH, rt;
(iii) Piperazine, DMF, 800 C;
(iv) different isothiocynates, DMF;
Figure 1: hybridization approach to design targeted molecules
Figure 2: Overview of NMR spectral data for all compounds
Figure 3: Docking pose of D9 and D19 with pf-DHFR (4DPD)
Figure 4: Docking pose of D15 and D19 with E.Coli FabH (1HNJ)
Figure 5: Docking pose of D10 and D20 with S. Aureus Hydrolase (4CJN)

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Figure 6: Graphical presentation of Structure-activity relationship study
Table 1: Antitubercular and Antimalarial activity of D1-D24
Antitubercular
activity
Antimalarial
R
activity
Group
Comp.
H37Rv
MTCC200
MICs,(µg/mL)
P.falciparum
(IC50, µg/mL)
D1
D2
D3
100
125
100
0.26
1.57
1.12
D4
D5
250
50
0.16
1.12
D6
D7
D8
D9
25
0.97
1.62
0.84
0.09
100
500
125
D10
D11
100
250
0.53
0.15
D12
62.5
0.38

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D13
D14
50
0.43
1.08
250
D15
D16
100
250
0.26
0.68
D17
1000
0.23
D18
D19
500
125
0.20
0.07
D20
250
0.94
D21
D22
100
250
1.47
0.30
D23
D24
125
0.32
0.47
62.5
Drug
Drug
Drug
0.20
--
--
Isoniazide
Chloroquin
Quinine
0.02
0.26
--

ACCEPTED MANUSCRIPT
Table 2 : Antibacterial and antifungal activity (MICs, µg/ml)
Antibacterial activity
Antifungal activity
Gram negative
bacteria
Gram positive
bacteria
Compounds
E.C.
P.A.
S.A.
S.P.
C.A.
A.N.
A.C.
MTCC MTCC MTCC MTCC
MTCC MTCC MTCC
443
1688
96
442
227
282
1323
D1
62.5
62.5
250
100
100
125
125
100
250
100
100
125
100
100
25
500
250
500
500
250
100
100
250
500
500
500
250
250
500
500
250
500
500
200
12.5
100
100
500
250
100
50
250
250
500
500
500
500
100
62.5
100
50
500
500
250
500
500
500
250
100
125
500
500
500
500
250
250
125
200
200
250
25
500
500
500
250
250
500
1000
1000
500
1000
500
500
500
500
500
500
500
500
200
250
500
1000
1000
500
-
1000
1000
1000
1000
1000
1000
500
1000
1000
1000
1000
1000
1000
500
D2
D3
D4
D5
D6
D7
D8
1000
1000
1000
1000
1000
1000
1000
1000
1000
500
1000
1000
1000
1000
1000
1000
1000
1000
1000
500
D9
D10
D11
250
100
500
500
250
250
100
200
250
50
D12
D13
D14
D15
D16
62.5
500
250
50
D17
D18
500
500
D19
1000
100
500
D20
100
100
62.5
62.5
200
100
50
100
D21
200
100
125
250
250
50
50
-
-
100
500
500
250
100
50
50
-
-
1000
1000
1000
500
1000
1000
1000
500
D22
D23
D24
-
-
-
-
-
-
Ampicillin
Chloramphenicol
Ciprofloxacin
Nystatin
Greseofulvin
-
-
25
-
-
25
-
-
100
500
100
100
100
100

ACCEPTED MANUSCRIPT
Table 3 : Various Ligand protein Interactions
Protein
(PDB)
Comp
Code
Dock
Score
H-bond
Aminoacidb
π – π
interaction
Atoma
B.L. (Å)c
H atom (piperazine)
H atom (Cyclohexyl)
H atom (piperazine)
H atom (NHCS)
H atom (NHCS)
N atom (pyrazole)
H atom (Phenyl)
H atom (Phenyl)
H atom (piperazine)
Phenyl ring
O(OH) of SER 11
H(Ph) of PHE 58
O(OH) of SER 108
O(CO) of ILE 164
H(OH) of ILE 164
H(OH) of SER 108
O(OH) of THR 28
H(OH) of THR 28
O(CO) of GLY152
--
2.37
2.40
2.31
2.12
1.98
2.56
2.36
1.48
2.30
--
--
--
D9
-7.460
-7.320
-7.175
--
Pf-DHFR
(4DPD)
--
--
D19
D15
--
--
--
--
E.coli
FabH
(1HNJ)
TRP32
H atom (tolyl)
O(OH) of THR 28
H(OH) of THR 28
O(CO) of GLY152
--
2.34
1.45
2.40
--
--
H atom (tolyl)
--
D19
D10
D20
-6.551
-3.726
-3.384
H atom (piperazine)
Phenyl ring
--
TRP32
--
N atom (pyrimidine)
H atom (ethyl)
H(OH) of TYR 105
O(CO) of ASP 295
H(NH2) of LEU 147
H(NH3) of LYS 273
--
2.15
2.40
2.64
1.97
N atom (pyrazole)
H atom (ethyl)
--
--
S.Aureus
hydrolase
(4CJN)
pyrimidine ring
N atom (pyrimidine)
O atom (CO)
TYR105
--
H(OH) of TYR 105
H(NH3) of LYS 316
--
2.15
2.01
--
pyrimidine ring
Pyrazole ring
aAtom of ligand participate in interaction with residue
bInteractive amino acid residue
cBond distance
TYR105
TYR295
--

ACCEPTED MANUSCRIPT
Figure 1

ACCEPTED MANUSCRIPT
Scheme 1
Figure 2

ACCEPTED MANUSCRIPT
Figure 3
Figure 4

ACCEPTED MANUSCRIPT
Figure 5
R = Isobutyl, Prop-2-en and cyclohexyl groups = Increased
Gram positive bacterial activity
R = butyl, isopropyl and propyl groups = Decreased
bacterial activity
Only methyl groups= Increased E . Col i Gram
negative bacterial activity
Most of electron withdrawing groups on aromatic
ring = Decreased antitubercular activity
Only methoxy grop at 3rd and 4th position of aromatic
ring = Increased anti-tubercularactivity
Electron donating (halogenated) groups on aromatic
ring = Increased Gram positive bacterial activity
Only fluoro agrops at 4th position of aromatic ring and
benzoyl substitute = Increased bacterial activity
Aliphatic Amines
Electron withdrawing group and haloginated groups
expect iodo group = Decreased anti-malarial activity
Methyl group at 4th position of aromatic ring =
A
r
o
m
a
t
i
Electron withdrawing groups like nitro group =
Increased Gram positive bacterial activity
c
Incrased anti-malarial activity
A
m
in
e
s
s
mine
atic A
Arom
Iodo group at 3rd position and Methoxy group at 2nd
position of aromatic ring = Incrased anti-malarial
activity
Methyl and methoxy groups on aromatic ring =
Decreased bacterial activity
Halogenated groups like F and Cl on aromatic ring =
Increased E . C ol i Gram negative bacterial activity
Only chloro grop at 2nd position of aromatic ring =
Increased anti-tubercularactivity
s
e
n
i
m
A
c
i
t
a
h
p
i
l
R
A
ethyl, methyl, butyl, Ios-propyl groups = Decreased
anti-malarial activity
NH
S
Cyclohexyl and propyl groups = Increased anti-
malarial activity
N
Compound 20, having
benzoyl substitution
Butyl and iso-propyl groups = Increased anti-tubercular
activity
ethyl, methyl, Iso-butyl, propyl groups = Decreased
anti-tubercular activity
N
O
possess excellent
N
O
Antifungal Activity
N
N
N
Targeted compounds
(D1-D24)
Figure 6