Design, synthesis, biological evaluation and computational study of novel triazolo [4,3-a]pyrazin analogues

Article abstract of DOI:10.1016/j.molstruc.2019.01.091

The triazolo [4,3-a]pyrazin analogues are of interest due to their potential activity against various infectious and non-infectious disease. In search of suitable potent drug candidate, we report here the design, synthesis, characterization, biological activities and computation study of novel triazolo [4,3-a]pyrazin analogues. The synthesized molecules were characterized by various spectroscopic studies such as IR, Mass, ¹H NMR, ¹³C NMR and elemental analysis. The newly synthesized compounds were evaluated for their in vitro biological activities such as anti-malarial, anti-tuberculosis, anti-bacterial and anti-fungal activities against plasmodium falciparum, H₃₇Rv, various bacterial and fungal strains, respectively. The molecular docking study was carried out with enzyme aspartic proteinase zymogen proplasmepsin II from plasmodium falciparum to analyze their binding orientation in the active site of the aspartic proteinase enzyme. The best docking complex was subjected to molecular dynamics simulation to illustrate the stability of these complexes and the most prominent interactions during the simulated trajectory. We have also calculated ADMET properties of all the synthesized compounds to predict the pharmacokinetic properties for the selection of the active and bioavailability of compounds.

Full text of DOI:10.1016/j.molstruc.2019.01.091

Accepted Manuscript
Design, synthesis, biological evaluation and computational study of novel triazolo
[4,3-a]pyrazin analogues
Divya J. Jethava, Prachi T. Acharya, Mahesh S. Vasava, Manoj N. Bhoi, Zeel A.
Bhavsar, Sanjay K. Rathwa, Dhanji P. Rajani, Hitesh D. Patel
PII:
S0022-2860(19)30109-7
DOI:
https://doi.org/10.1016/j.molstruc.2019.01.091
MOLSTR 26142
Reference:
To appear in: Journal of Molecular Structure
Received Date: 21 November 2018
Revised Date: 5 January 2019
Accepted Date: 28 January 2019
Please cite this article as: D.J. Jethava, P.T. Acharya, M.S. Vasava, M.N. Bhoi, Z.A. Bhavsar,
S.K. Rathwa, D.P. Rajani, H.D. Patel, Design, synthesis, biological evaluation and computational
study of novel triazolo [4,3-a]pyrazin analogues, Journal of Molecular Structure (2019), doi: https://
doi.org/10.1016/j.molstruc.2019.01.091.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, biological evaluation and computational study of novel
triazolo[4,3-a]pyrazin analogues
a
a
a
b
a
Divya J. Jethava , Prachi T. Acharya , Mahesh S. Vasava , Manoj N. Bhoi , Zeel A. Bhavsar ,
c
a*
Sanjay K. Rathwa, Dhanji P.Rajani , and Hitesh D. Patel
a
Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad, India.
b
Plot no 18, Piramal Enterprises Limited, Pharmez, Matoda, Ahmedabad, India.
c
Microcare Laboratory and TRC, Surat, India.
E-mail: drhiteshpatel1@gmail.com.
Tel.: +91-079-26300969; fax: +91-079-26308545.
Abstract
The triazolo[4,3-a]pyrazin analogues are of interest due to their potential activity against
various infectious and non-infectious disease. In search of suitable potent drug candidate, we
report here the design, synthesis, characterization, biological activities and computation study
of novel triazolo[4,3-a]pyrazin analogues. The synthesized molecules were characterized by
1
13
various spectroscopic studies such as IR, Mass, H-NMR, C-NMR and elemental analysis.
The newly synthesized compounds were evaluated for their in vitro biological activities such
as anti-malarial, anti-tuberculosis, anti-bacterial and anti-fungal activities against plasmodium
falciparum, H Rv, various bacterial and fungal strains, respectively. The molecular docking
37
study was carried out with enzyme aspartic proteinase zymogen proplasmepsin II from
plasmodium falciparum to analyze their binding orientation in the active site of the aspartic
proteinase enzyme. The best docking complex was subjected to molecular dynamics
simulation to illustrate the stability of these complexes and the most prominent interactions
during the simulated trajectory. We have also calculated ADMET properties of all the
synthesized compounds to predict the pharmacokinetic properties for the selection of the
active and bioavailability of compounds.
Keywords: ADMET, Anti-bacterial, Anti-malarial, Anti-tuberculosis, Biological evaluation,
Molecular docking, molecular dynamics simulation, Plasmodium spp., Structure activity
relationship (SAR), and triazolo[4,3-a]pyrazine

ACCEPTED MANUSCRIPT

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1
. Introduction
A microorganism such as viruses, fungi, bacteria, or parasites are responsible for
infectious disease (include HIV/AIDS, Tuberculosis, Schistosomiasis, Malaria, Chickenpox,
Lymphatic) that worsens individual's health.[1] Generally, they live in and on our bodies.
They are usually inoffensive or even may be helpful. However, some organisms may cause
disease under certain circumstances.[1] Among them, Malaria is one of the most infected
disease caused by parasites of the genus Plasmodium spp. It spread to people through the
bites of an infected female Anopheles mosquito. Plasmodium falciparum is the most
infectious species in human malarial parasite, which responsible for about 1 million deaths
every year.[2,3] As per the World Health Organization (WHO) 2015 report, around 3.2
billion people are at risk of malaria, principally in poor as well as emerging countries,
registering 250 million clinical cases of malaria and more than 400,000 deaths by the year,
mainly children under the age of five years as well as pregnant women.[4,5] There are mainly
five malaria parasite species (P. falciparum, P. vivax, P. ovale, P. malariae and P.knowlesi)
available across the world. Among them, the two most deadly forms of malaria are produced
by infection with not only P. falciparum but also P. vivax.[6] P. falciparum is the most lethal
form of malaria and gave around 50% contribution of the total cases.[7] The anti-malarial
drugs (Figure 1) are responsible for a grave problem to control disease when the resistance
development in P. falciparum.[8-16] Recently the artemisinins have recognised to show
resistance against P. falciparum in five countries such as Democratic Republic, Viet Nam,
Cambodia, Thailand, Lao People’s and Myanmar.[17] It is one of the greatest significant
constituent of the artemisinin-based incorporated treatments. If artemisinin’s resistance will
stretch out to Africa or India, then the world-wide humanity rate may increase as no other
drug is being presented with same level effectiveness and acceptability.[7, 18, 19] Thus,
therefore there is a crucial need of novel anti-malarial pharmacophores for the development
of inoffensive and quick acting anti-malarial candidates.
The chemistry of heterocycles is one of the significant field for research in therapeutic
chemistry as they have been found to display improved biological activity. It play significant
role for the development of numerous therapeutic agents in medicinal chemistry.
Heterocyclic compounds principally those with oxygen, nitrogen and Sulphur atoms work as
a unique and versatile scaffold for novel drug design and discovery.[20] However, a number
of alkaloids comprising both nitrogen and sulphur atom in natural products. Among them,
benzothiazole is one of the interesting terrestrial and rarely available marine natural products.

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A benzothiazole containing alkaloids are comparatively very rare in nature such as D-
luciferin,[21-23]rifamycins P and Q[24] and thiazinotrienomycin F and G [25] bisabolane
type sesquiterpenoid, diterpenes erythrazoles A and B[26] (Figure 2). Benzothiazole
derivatives have gained significant attention in synthetic organic chemistry with valuable
applications in the pharmaceutical industry. Current literature based on benzothiazole
derivatives reveals about numerous biological activities such as anti-tuberculosis,[27],
trypanocidal agent,[28] COX inhibitor,[29] hypoglycemic,[30] antitumor,[31,32]
antidiabetic,[33] anti-urease,[34] inhibitor of α-glucosidase,[35] antimicrobial.[36-38] anti-
tubercular,[39] anti-HIV,[40] anticancer,[40] anti-schizophrenic,[41] anti-inflammatory,
analgesic and anti-convulsing activity.[42] It is also a part of some renowned drugs such as
pramipexole, riluzole, ethoxzolamide.[38]
The chemistry of fused ring containing heterocycles is recognized to be an important
field of investigation in medicinal chemistry.[43-46] Consequently, they have been
establishing to show enhanced biological activity. Among them, The scaffold, triazolo[4,3-
a]pyrazine which have gained significant attention in synthetic organic chemistry with
valuable importance in drug discovery program especially in diabetes. From the present
literature survey, triazolo[4,3-a]pyrazine analog have shown numerous biological activities
such as histamine H4 receptor (H4R),[47] Anti-diabetic,[48] blood-brain barrier
permeability,[49] good Jak inhibitor,[50] Brain Penetrant Phosphodiesterase 2 (PDE2)
inhibitor,[51] non-xanthine adenosine receptor,[52] anticonvulsant activity,[53] human renin
activity,[54-56] Adenosine Human Receptor,[57] DPP-IV inhibitor,[58] neurokinin-3
receptor (NK-3)[59] in medicinal chemistry and in other wide range of industrial production
application.
Inspire by the significant biological activity of the structurally diverse heterocycles
with fused heterocyclic systems along with active heterocyclic analogous. As a result of the
aforementioned description, triazolo[4,3-a]pyrazine analogues were designed and synthesized
to investigate of the potential ability of novel designed hybrid molecules using the basic
reaction condition with a good practical yield by conventional method. We have evaluated
their anti-malarial activity against P. falciparum, anti-tuberculosis against H Rv stain, anti-
3
7
bacterial activity against Gram-positive bacteria Staphylococcus aureus (MTCC-96),
Staphylococcus pyogenus (MTCC-442), Gram-negative bacteria Escherichia coli. (MTCC-
4
43), Pseudomonas aeruginosa (MTCC-1688) and anti-fungal efficacy against fungal strains.
We have carried out molecular docking studies helped in revealing the mode of action of
these compounds through their interactions with the active site of enzyme aspartic proteinase

ACCEPTED MANUSCRIPT
zymogen proplasmepsin II from plasmodium falciparum. Molecular dynamics (MD)
simulations may assistance to explain the binding site and time dynamics of interacting
amino acid residues and to extend the information obtained from docking studies. We have
also carried out in silico ADMET prediction and Lipinski rules of 5 of synthesized
compounds.
2
. Material and Methods
All the required chemicals and solvents were purchased from commercial sources
Spectrochem and Avra chemical). The reactions were monitored by thin layer
(
chromatography (TLC). TLC was visualized by UV radiation, exposure to iodine vapor and
spray reagent. Pre-coated aluminum sheets (Silica gel G 60 F , Merck Germany) were
2
54
employed for thin-layer chromatography (TLC). The melting points recorded on optimelt
automated melting point system which were uncorrected. IR spectra was recorded on a
-1
Perkin-Elmer 377 spectrophotometer in KBr with absorption in cm . Elemental analysis was
1
performed on vario MICRO cube, elementar CHNS analyser serial no.: 15084053. H- NMR
1
3
and C-NMR spectra were recorded on Bruker AV 400 and 100 MHz using DMSO-d as
6
solvent and TMS as an internal standard. Splitting patterns are designated as follows; s,
singlet; d, doublet; m, multiplet dd, double doublet. Chemical shift values are given in ppm.
Mass spectra was recorded on Advion Expression CMS, USA, using Methanol: Water:
Formic acid (80: 20: 0.1) as mobile phase.
2
2
.1. Chemistry
.1.1. Synthesis of N’-(chloroacetyl)-2,2,2-trifluoroacetohydrazide[60] (5):
To a stirred solution of 60% w/v Hydrazine hydrate 2 (3.0 g (2.92 mL), 59.92 mmol,
.0 eq.) in acetonitrile (35 mL) was added slowly ethyl trifluoroacetate 1 (8.51 g, (7.12 mL),
9.92 mmol, 1eq.) over a period of 10 min at 10 °C. The internal temperature was increased
1
5
to 25 ̊C from 10 °C. The resulting reaction mixture was stirred at 22 - 25 °C for 1h. Reaction
completion was checked by TLC (30% ethyl acetate: hexane). The resulting solution was
cooled to 7 °C. 2.4 g of 50 wt% NaOH (59.92 mmol) and chloro acetyl chloride 4 (6.77 g,
(4.77 mL), 59.92 mmol) were added simultaneously over 2.5h at <15 °C. When the reaction
was monitored by TLC (30% ethyl acetate: hexane), the reaction mixture was directly
distilled out to remove unwanted liquid (water and ethanol) at 27-30 °C under high vacuum.
During the distillation, acetonitrile (20 mL) were added slowly to maintain constant volume
(10-15 mL). After the distillation, the slurry was filtered to remove NaCl and the cake was
rinsed with about 2 x 5 mL of acetonitrile. The filtrate was concentrated at reduced pressure

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to give bishydrazide 5 product (3.0 g, 14.67 mmol, 69.5% yield, m.p. 139-140 °C) which was
used as such in the next step.
2
.1.2. Synthesis of 2-(chloromethyl)-5-(trifluoromethyl)-1,3,4-oxadiazole[60] (6):
Phosphorus oxychloride (2.25 g (1.4 mL), 13.27 mmol. 1 eq.) was added to the stirred
solution of bishydrazide 5 (3.0 g, 14.67 mmol. 1 eq.) in acetonitrile (20 mL). The reaction
mixture was heated at 80 °C for 24h. The progress of the reaction was monitored by TLC
(
50% ethyl acetate: hexane). In a separate vessel, (1:1) MTBE and H O were mixed and
2
cooled to 0 °C. The reaction mixture was cooled to room temperature and added to the
quench solution keeping the internal temperature below 10 °C. After the addition was
complete, the mixture was agitated vigorously for 30 min., the temperature was warmed to 25
°
C and the aqueous layer was cut. The organic layer was then washed with 2 x 20 mL of 5%
wt NaHCO and with (2 x 15 mL) of 20% wt brine solution. Solvents were removed by
3
distillation at 45 °C to obtain 6 as an orange oil in 76.8% yield (2.1 g, 11.26 mmol, b.p. 55-60
°
C. This oil was used directly in the preparation of 8.
2
.1.3. Synthesis of 2,2,2-trifluoro-N’-[(2Z)-piperazin-2-ylidene]acetohydrazide[60] (8):
To a stirred solution of ethylenediamine 7 (1.96 g, 2.2 mL, 32.65 mmol) in methanol
(20 mL) cooled at -20 °C was added the crude oxadiazole 6 (2.1 g, 11.25 mmol) keeping the
internal temperature below -20 °C. After the addition was completed, the resulting slurry was
aged at -20 °C for 1h and the progress of the reaction was monitored by TLC. Ethanol (15
mL) was then charged and the slurry slowly warmed to -5 °C. After 60 min at -5 °C, the
slurry was filtered and washed with 2 x 5 mL of ethanol at -5 °C and dried under a vacuum
flow to afford (Z)-2,2,2-trifluoro-N'-(piperazin-2-ylidene)acetohydrazide 8 as a white solid in
o
6
3.4% yield (1.5 g, 7.14 mmol., m.p. 141-143 C,)
2
.1.4. Synthesis of 3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazine
Hydrochloride salt[60] (9):
To a stirred solution of (Z)-2,2,2-trifluoro-N'-(piperazin-2-ylidene)acetohydrazide 8
1.5 g, 7.14 mmol) in methanol (20 mL) was warmed to 55 °C. To this, HCl (0.2 mL, 7.81
(
mmol) was charged over 1 hr at this temperature. All solids dissolved during the course of the
addition. The reaction was aged for 30 min and monitored by TLC. The solution was cooled
to 20 °C and aged at this temperature until a seed bed formed (10 min to 1 hour). MTBE (15
mL) was charged at 20 °C over 10 minutes. After charging all the MTBE, the resulting slurry
was cooled to 2 °C, aged for 30 min and filtered. The white solids obtained were washed with
ethanol : MTBE (1:3 v/v) and dried under vacuum at 45 °C to give 3-(trifluoromethyl)-
5
,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine hydrochloride salt 9 (1.36 g, 7.08 mmol).

ACCEPTED MANUSCRIPT
White solid, m.p. 263-265 ºC; Anal. Calcd for C H ClF N : C, 31.52; H, 3.53; Cl, 15.51; F,
6
8
3
4
1
2
4.93; N, 24.51 %; found C, 31.65; H, 3.66; N, 24.62 %; H NMR (400 MHz, DMSO) δ
0.56 (s, 1H), 4.61 (s, 2H), 4.47 – 4.44 (t, J = 11.2 Hz, 2H), 3.66 -3.63 (t, J = 11.6 Hz, 2H);
1
ESI-MS (m/z): 192.74 [M+1].
.1.5. General process of Synthesis of N-(benzo[d]thiazol-2-yl)-2-chloroacetamide
derivative[61] (11a-11e):
A solution of various derivative of 2-amino benzothiazole derivatives (1 eq.) in
2
CHCl (5 mL) were cooled at 0 °C. Triethylamine (3.0 eq., d = 0.726 g/mL, 1.4 mL) and
3
chloro acetyl chloride 4 (1.1 eq.) were sequentially added to the reaction mixture at 0 °C. The
reaction mixture was stirred at 0 °C for 30 minutes and allowed to reach to room temperature.
The reaction mixture was further refluxed for 18h. The progress of the reaction was
monitored by TLC (5% MeOH : DCM). The reaction mixture was diluted with CHCl (10
3
mL) and washed with saturated bicarbonate solution (2 x 15 mL). The organic phase was
washed with brine, dried over Na SO and concentrated under reduced pressure to afford N-
2
4
(benzo[d]thiazol-2-yl)-2-chloroacetamide derivatives (11a-11e) with good yield which is
used in the next step without further purification.
2
.1.6. General process of Synthesis of N-(benzo[d]thiazol-2-yl)-2-(3-(trifluoromethyl)-
,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)acetamide derivatives (12a-12e):
A solution of 3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazine
5
hydrochloride salt (1.0 eq.) in dimethylformamide (DMF, 2-3 mL) was added potassium
carbonate (2.5 eq.) at and stirred for 15 minutes at room temperature. To this, N-
(
benzo[d]thiazol-2-yl)-2-chloroacetamide derivatives (1.0 eq.) in DMF was added to the
reaction mixture at room temperature. The resulting reaction mixture was heated at 70 °C for
8h. The completion of the reaction was monitored on TLC (using 10% MeOH: DCM and
1
ammonia as a modifier as mobile phase) which confirmed that the reaction got completed
after 18h. The reaction mixture was poured into ice-cold water and extracted with ethyl
acetate (2 x 30 mL). Organic layers were combined and the combined organic layer was dried
over sodium sulfate and concentrated under reduced pressure to afford crude desired product
and crystallized it from methanol to get N-(benzo[d]thiazol-2-yl)-2-(3-(trifluoromethyl)-5,6-
dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7 (8H)-yl)acetamide derivatives (12a-12e) in good
practical yield.
N-(benzo[d]thiazol-2-yl)-2-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-
7
(8H)-yl)acetamide (12a):

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Solid, m.p. 198-200 ºC; Anal. Calcd for C H F N OS: C, 47.12; H, 3.43; F, 14.91; N,
1
5
13
3
6
-
2
1.98; O, 4.18; S, 8.39 %; found C, 47.35; H, 3.65; N, 22.15; S, 8.52 %; IR (KBr) (υmax, cm
1
)
: 3089 (=C-H ), 2945 (C-H ), 1662 (C=O ), 1587 (C=C ), 1326 (C-N ), 1203 (C-F );
str str str str str str
1
H NMR (400 MHz, DMSO) δ 10.05 (s, 1H, -NH), 8.29 – 7.88 (m, 2H, C & C ), 7.56 –
2
3
26
7
.39 (m, 2H, C24&25), 4.04 (dt, J = 8.6, 5.5 Hz, 2H, C ), 3.82 (s, 1H, C ), 3.56 (d, J = 14.7
10 6
13
Hz, 2H, C2&6), 3.39 (s, 1H, C ), 3.10 (t, J = 5.4 Hz, 1H, C ), 2.67 (t, J = 5.4 Hz, 1H, C ); C
2
9
9
NMR (100 MHz, DMSO) δ 169.53 (C ), 153.43 (C , 151.3 (C ), 144.39 (C ), 134.41 (C ),
1
19)
21
5
22
1
5
32.73 (C ), 124.30 (C ), 122.86 (C ), 121.34 (C ), 118.71 (C ), 58.69, (C ), 52.34 (C ),
13 14 24 23 26 2 6
1.18 (C ), 43.09 (C ) ; ESI-MS (m/z): 383.3 [M+1].
9
10
N-(6-chlorobenzo[d]thiazol-2-yl)-2-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]tri- azolo[4, 3-
a]pyrazin-7(8H)-yl)acetamide (12b):
Solid, m.p. 220 ºC; Anal. Calcd for C H ClF N OS: C, 43.22; H, 2.90; Cl, 8.51; F, 13.67;
1
5
12
3
6
N, 20.16; O, 3.84; S, 7.69 %; found C, 43.61; H, 3.10; N, 20.42; S, 7.79 %; IR (KBr) (υmax
,
-1
cm ): 3051 (=C-H ), 2890 (C-H ), 1645 (C=O ), 1589 (C=C ), 1352 (C-N ), 1152 (C-
str
str
str
str
str
1
F ); H NMR (400 MHz, DMSO) δ 10.25 (s, 1H, -NH), 8.24 (d, J = 1.4 Hz, 1H, C ), 8.00
str
23
(
d, J = 7.5 Hz, 1H, C ), 7.51 (dd, J = 7.5, 1.6 Hz, 1H, C ), 4.04 (dt, J = 24.9, 5.5 Hz, 2H,
26 25
C ), 3.74 (d, J = 4.6 Hz, 2H, C ), 3.60 (s, 1H, C ), 3.53 (s, 1H, C ), 3.04 (t, J = 5.5 Hz, 1H,
10
6
2
2
C ), 2.85 (t, J = 5.5 Hz, 1H, C ); ESI-MS (m/z): 417.8 [M+1].
9
9
N-(6-methoxybenzo[d]thiazol-2-yl)-2-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]tri- azolo[4,3-
a]pyrazin-7(8H)-yl)acetamide (12c):
Solid, m.p. 235-237 ºC; Anal. Calcd for C H F N O S: C, 46.60; H, 3.67; F, 13.82; N,
1
6
15
3
6
2
-1
2
0.38; O, 7.76; S, 7.77 %; found C, 46.89; H, 3.79; N, 20.56; S, 7.86; IR (KBr) (υmax, cm ):
3
042 (=C-H ), 2984 (C-H ), 1612 (C=O ), 1572 (C=C ), 1250 (C-N ), 1145 (C-F ); 1H
str
str
str
str
str
str
NMR (400 MHz, DMSO) δ 10.10 (s, 1H, -NH), 8.04 (d, J = 7.5 Hz, 1H, C ), 7.77 (d, J = 1.4
26
Hz, 1H, C ), 7.13 (dd, J = 7.5, 1.4 Hz, 1H, C ), 4.05 (t, J = 5.4 Hz, 2H, C ), 3.81 (s, 3H, -
23
25
10
OMe), 3.75 (s, 1H, C ), 3.66 (s, 1H, C ), 3.55 (s, 1H, C ), 3.38 (s, 1H, C ), 3.06 (t, J = 5.4 Hz,
6
2
6
2
1
H, C ), 2.79 (t, J = 5.4 Hz, 1H, C ); ESI-MS (m/z): 413.4 [M+1].
9 9
N-(6-fluorobenzo[d]thiazol-2-yl)-2-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazolo[4 ,3-
a]pyrazin-7(8H)-yl)acetamide (12d):
Solid, m.p. 210-213 ºC; Anal. Calcd for C H F N OS: C, 45.00; H, 3.02; F, 18.98; N,
1
5
12
4
6
-
1
2
3
0.99; O, 4.00; S, 8.01 %; found C, 45.25; H, 3.20; N, 21.23; S, 8.35; IR (KBr) (υmax, cm ):
1
075 (=C-H ), 2964 (C-H ), 1645 (C=O ), 1575 (C=C ), 1263 (C-N ), 1356 (C-F ); H
str
str
str
str
str
str
NMR (400 MHz, DMSO) δ 9.62 (s, 1H, -NH), 8.06 (dd, J = 7.5, 5.0 Hz, 1H, C ), 7.97 (dd, J
2
6
=
7.9, 1.4 Hz, 1H, C ), 7.26 (td, J = 7.9, 1.4 Hz, 1H, C ), 4.09 – 3.98 (m, 2H, C ), 3.71 (d, J
23 25 10

ACCEPTED MANUSCRIPT
=
18.1 Hz, 2H, C2&6), 3.60 (d, J = 5.7 Hz, 2H, C2&6), 3.08 (t, J = 6.0 Hz, 2H, C ); ESI-MS
9
(m/z): 401.3 [M+1].
N-(6-hydroxybenzo[d]thiazol-2-yl)-2-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazolo[4,3-
a]pyrazin-7(8H)-yl)acetamide (12e):
Solid, m.p. 197-198 ºC; Anal. Calcd for C H F N O S: C, 45.23; H, 3.29; F, 14.31; N,
1
5
13
3
6
2
-
2
1.10; O, 8.03; S, 8.05 %; found C, 45.45; H, 3.42; N, 21.26; S, 8.25 %; IR (KBr) (υmax, cm
1
)
: 3089 (=C-H ), 2945 (C-H ), 1656 (C=O ), 1523 (C=C ), 1316 (C-N ), 1245 (C-F );
str str str str str str
1
H NMR (400 MHz, DMSO) δ 10.25 (s, 1H, -NH), 7.90 (dd, J = 4.4, 3.0 Hz, 2H, C & C ),
2
3
26
7
.09 (dd, J = 7.5, 1.4 Hz, 1H, C ), 4.04 (t, J = 5.2 Hz, 2H, C ), 3.82 (s, 1H, C ), 3.63 (s, 1H,
25 10 6
-
C ), 3.54 (s, 1H, C ), 3.41 (d, J = 4.6 Hz, 1H, C ), 2.91 (t, J = 5.2 Hz, 1H, C ), 2.76 (t, J =
2 6 2 9
5
.2 Hz, 1H, C ), 1.75 (s, 1H, -OH); ESI-MS (m/z): 399.3 [M+1].
9
2
.1.7. General process of Synthesis of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]
triazolo[4,3-a]pyrazine derivatives (12f-12j):
A solution of 3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazine
hydrochloride salt 9 (1.0 eq.) in dimethylformamide (DMF, 2-3 mL) was added potassium
carbonate (2.5 eq.) at 5 °C and stirred for 15 minutes at room temperature. To this, various
chloride derivatives (1.0 eq.) in DMF was added to reaction mixture at same temperature.
The resultant reaction mixture was allowed to stir at room temperature for 24h. The
completion of the reaction was monitored on TLC (using 10% MeOH: DCM and ammonia as
a modifier as mobile phase). The reaction mixture was poured into ice-cold water and
extracted with ethyl acetate (2 x 15 mL). Organic layers were combined and the combined
organic layer was dried over sodium sulfate and concentrated under reduced pressure to
afford crude material and crystallized it from methanol to get 3-(trifluoromethyl)-5,6,7,8-
tetrahydro[1,2,4]triazolo[4,3-a]pyrazine derivatives (12f-12j) in good practical yield.
(4-fluorophenyl)(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-
yl)methanone (12f):
Solid, m.p. 179-185 ºC; Anal. Calcd for C H F N O: C, 49.69; H, 3.21; F, 24.18; N, 17.83;
1
3
10
4
4
-1
O, 5.09 %; found C, 49.82; H, 3.28; N, 17.92 %; IR (KBr) (υmax, cm ): 3023 (=C-H ), 2964
str
1
(
C-H ), 1642 (C=O ), 1574 (C=C ), 1321 (C-N ), 1245 (C-F ); H NMR (400 MHz,
str
str
str
str
str
DMSO) δ 7.89 – 7.78 (m, 2H, -Ar), 7.17 (t, J = 7.8 Hz, 2H, -Ar), 4.53 (s, 1H, C ), 4.35 (s,
6
1
H, C ), 4.30 – 4.20 (m, 2H, C ), 3.81 (t, J = 5.4 Hz, 1H, C ), 3.71 (t, J = 5.4 Hz, 1H, C );
6 10 9 9
ESI-MS (m/z): 315.2 [M+1].
furan-2-yl(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-
yl)methanone (12g):

ACCEPTED MANUSCRIPT
Solid, m.p. 180-183 ºC; Anal. Calcd for C H F N O : C, 46.16; H, 3.17; F, 19.91; N, 19.58;
1
1
9
3
4
2
-1
O, 11.18 %; found C, 46.32; H, 3.26; N, 19.63 %; IR (KBr) (υmax, cm ): 3041 (=C-H ), 2981
str
1
(
C-H ), 1665 (C=O ), 1557 (C=C ), 1312 (C-N ), 1251 (C-O ), 1190 (C-F ); H NMR
str
str
str
str
str
str
(400 MHz, DMSO) δ 7.49 (dd, J = 7.5, 1.4 Hz, 1H, -Ar), 6.99 (dd, J = 7.5, 1.6 Hz, 1H, -Ar),
6
.46 (t, J = 7.5 Hz, 1H, -Ar), 4.43 (s, 1H, C ), 4.22 (t, J = 5.6 Hz, 2H, C ), 4.13 (s, 1H, C ),
6 10 6
3
.82 (t, J = 5.6 Hz, 1H, C ), 3.60 (t, J = 5.6 Hz, 1H, C ); ESI-MS (m/z): 287.2 [M+1].
9
9
p-tolyl(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone
12h):
Solid, m.p. 181-183 ºC; Anal. Calcd for C H F N O: C, 54.19; H, 4.22; F, 18.37; N, 18.06;
(
1
4
13
3
4
-1
O, 5.16 %; found C, 54.32; H, 4.36; N, 18.26 %; IR (KBr) (υmax, cm ): 3039 (=C-H ), 2974
str
1
(
C-H ), 1652 (C=O ), 1587 (C=C ), 1356 (C-N ), 1178 (C-F ); H NMR (400 MHz,
str
str
str
str
str
DMSO) δ 7.79 (d, J = 7.5 Hz, 2H, -Ar), 7.30 (d, J = 7.5 Hz, 2H, -Ar), 4.53 (s, 1H, C ), 4.34
6
(
s, 1H, C ), 4.20 (t, J = 5.3 Hz, 2H, C ), 3.80 (t, J = 5.3 Hz, 1H, C ), 3.71 (t, J = 5.3 Hz, 1H,
6 10 9
C ), 2.33 (s, 3H, -CH ); ESI-MS (m/z) 311.2 [M+1].
9
3
7
-((4-methyl-3-nitrophenyl)sulfonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]
triazolo[4,3-a]pyrazine (12i):
Solid, m.p. 182-186 ºC; Anal. Calcd for C H F N O S: C, 39.90; H, 3.09; F, 14.56; N,
1
3
12
3
5
4
-
1
7.90; O, 16.35; S, 8.19 %; found C, 40.10; H, 3.26; N, 18.21; S, 8.26 %; IR (KBr) (υmax, cm
1
)
: 3056 (=C-H ), 2989 (C-H ), 1563 (C=C ), 1535 & 1345 (N-O ), 1345 (C-N ), 1245
str str str str str
1
(
C-F ); H NMR (400 MHz, DMSO) δ 8.76 (d, J = 1.4 Hz, 1H, -Ar), 8.26 (dd, J = 7.5, 1.4
str
Hz, 1H, -Ar), 7.62 (d, J = 7.5 Hz, 1H, -Ar), 4.57 (s, 1H, C ), 4.51 (s, 1H, C ), 4.07 (t, J = 5.2
6
6
Hz, 2H, C ), 3.83 (t, J = 5.2 Hz, 1H, C ), 3.75 (t, J = 5.2 Hz, 1H, C ), 2.38 (s, 3H, -CH );
10
9
9
3
ESI-MS (m/z): 392.3 [M+1].
,4-dimethyl-5-((3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-
yl)methyl)thiazole (12j):
Solid, m.p. 185-187 ºC; Anal. Calcd for : C H F N S: C, 45.42; H, 4.45; F, 17.96; N, 22.07;
2
1
2
14
3
5
-1
S, 10.10 %; found C, 45.62; H, 4.54; N, 22.27; S, 10.21 %; IR (KBr) (υmax, cm ): 3045 (=C-
1
H ), 2975 (C-H ), 1545 (C=C ), 1345 (C-N ), 1235 (C-F ); H NMR (400 MHz, DMSO)
str
str
str
str
str
δ 4.05 (dd, J = 6.1, 4.7 Hz, 3H, C &-CH ), 3.76 (s, 1H, C ), 3.53 (s, 2H, C &-CH ), 3.11 (t,
1
0
2
6
6
2
J = 5.4 Hz, 1H, C ), 2.78 (t, J = 5.4 Hz, 1H, C ), 2.63 (s, 3H, -CH ), 2.33 (s, 3H, -CH ); ESI-
9
9
3
3
MS (m/z) :318.3 [M+1].
2
.2. In-vitro Biological screening
We have carried out biological screening at Microcare Laboratory and TRC, Surat, India as

ACCEPTED MANUSCRIPT
per earlier reported methods.[62-70] 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), anti-tuberculer activity against H Rv and anti-malarial efficacy against
37
Plasmodium falciparum respectively. In antibacterial evaluation, ampicillin, was used
whereas in antifungal evaluation, greseofulvin and nystatin were used as standard control
drugs. Likewise, Isoniazid for anti-tubercular and quinine for anti-malarial evaluation were
used as standard control drug.[62-70]
2
2
.3. Computational study
.3.1. In-silico ADMET prediction and drug likeness study
The pharmacokinetic parameters such as absorption, distribution, metabolism and excretion
ADME) were evaluated by using Qikprop tool of Schrodinger software for forecasting the
(
drug-likeness properties of all synthesised molecules.[71] This software package used for the
exact possessions of molecules as compared with 95% of known drugs. It also assesses the
fittingness of the compounds based on Lipinski’s rule of five for checking the drug like
properties[72] Some other significant properties such as lipinski’s rule of 5 (molecular
weight, hydrogen bond donor (Donor-HB), hydrogen bond acceptor (Accept-HB),
octanol/water partition coefficient (QlogPo/w), aqueous solubility (QPlogS)) and ADMET
parameter such as brain and blood partition co-efficient (QlogBB), percent human oral
absorption were reflected for our present study to evaluate the potentiality of the drug.
2
.3.2. Molecular docking study
From the RCSB Protein Data Bank, protein structure coordinates (PDB Code 1PFZ[73],
CJN [74] & 2H7I [75] were procured. The molecular docking study was performed as per
4
previously reported method.[69-70]
The Glide score is calculated by using the following equation:[76]
GScore = 0.065*vdW + 0.130*Coul + Lipo + Hbond + Metal + BuryP + RotB +Site
Where, vdW denotes the van der Waals energy;
Coul is the coulomb energy; Lipo denotes the lipophilic contact term;
Hbond is the hydrogen-bonding term;
Metal is the metal binding term;
BuryP represents the penalty for buried polar groups;

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RotB is the penalty for freezing rotatable bonds and Site is the polar interactions at the
active site.
2
.3.3. Molecular dynamics (MD) simulations
The coordinates of the best docking configurations of selected 1PFZ-12C complexes
were subjected to molecular dynamics simulation using Desmond simulation package of
Schrödinger Materials Science Suite 2015-4.[77-78] to study the conformational changes and
binding stability of these complex. We have carried out molecular dynamics (MD)
simulations using the NPT ensemble using 300 K temperature and 1.0325 bar a pressure for
this run. The simulation length was 50 ns with a relaxation time 1 ps for the ligand 12c. We
have used OPLS_2005 force field parameters in this simulations.[79] A cut-off radius of 9.0
Å was applied for short-range van der Waals and Coulomb interactions. The Nosé-Hoover
[
80] and Martyna-Tuckerman-Klein[80] chain coupling scheme were used for the thermostat
and barostat methods (with a coupling constant 2.0 ps). The trajectories were saved at 4.8 ps
intervals for analysis. The performance and binding between the ligands and protein were
analyzed using the Simulation Interaction Diagram tool implemented in Desmond MD
package. The stability of molecular dynamics simulations was observed by looking on the
RMSD of the ligand and protein atom positions in time as normally employed for studies of
protein-ligand interactions.[81-83] The long-range electrostatic interactions were
calculated using the particle mesh Ewald method.[84] The water molecules were explicitly
described using the simple point charge model.[85] The input as well as output files were
operated by Maestro graphical user interface application for Schrödinger Materials Science
Suite 2015-4.
3
. Result and discussion
3
.1. Design of novel molecules
Synthesis of new and potent compounds has an endless demand. Keeping in this view,
a great deal of research work is being devoted to recognize newer, potent and hit molecular
entities which are active against the different bacterial strains. Generally, The four important
molecular design strategies are (1) computer aided drug design-based on ligand-protein
interaction,[86] (2) molecular hybridization of two active pharmacophoric units into a single
hybrid molecular frame,[87] (3) structural modification of the known drug molecule,[88] and
(4) blind or random screening of different structural units and proceeding in an observed

ACCEPTED MANUSCRIPT
window of particular biological activity.[89] All these above approaches are being
considered as promising methods for development of effective drugs.[90]
Among them, Molecular hybridization which is a one of the valuable structural
modification approach that comprises the amalgamation of two or more pharmacophores into
a single entity. Hybrid drug design has emerged as a prime tool for the discovery of
innovative different therapies that can potentially overcome most of the pharmacokinetic
drawbacks encountered when using conventional various drugs in the last couple of
years.[91] The structural modification of the known drug molecule is one of the way to
identify newer molecular entities which are active against the bacterial strains.[92] We
therefore visualized that integrating triazolo[4,3-a]pyrazine and benzothiazole moieties in one
molecular platform could potentially produce novel hybrid compound with enhanced affinity
and potency as compared to the parent drug molecules (Figure 3).[93]
3
.2. Chemistry
The synthesis of triazolo[4,3-a]pyrazine-benzothiazole hybrid derivatives were
accomplished by the synthetic route illustrated in schemes 1-3. In Scheme 1, the starting
compound, Hydrazine hydrate 2 first reacted with ethyl trifluoroacetate 1 using acetonitrile as
solvent at lower temperature to form intermediate 3 in good yield. Then, intermediate 3 was
further in situ reacted with chloroacetyl chloride 4 using basic medium (10% NaOH) at
ambient temperature to get N’-(chloroacetyl)-2,2,2-trifluoroacetohydrazide 5. The cyclisation
reaction of intermediate-5 was performed using phosphorus oxychloride (POCl ) to get 1, 3,
3
4
-oxadiazole derivative 6 in step-3. The 1, 3, 4-oxadiazole derivative 6 was further cyclised
using ethylene-diamine 7 using methanol as solvent at lower temperature yielded the
corresponding intermediate-8 which was used in next step for preparation of hydrochloride
salt of 3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazine[60] 9 in good
1
practical yield. The synthesised scaffold analysed with H-NMR and mass analysis for
structure conformation.
In scheme 2, various derivative of 2-amino benzothiazole reacted with chloro acetyl
chloride in the presence of Triethylamine using chloroform as solvent to furnish various
derivative of N-(benzo[d]thiazol-2-yl)-2-chloroacetamide derivative[61] (11a-11e) which
was used in next step as such. In the next step, the reaction between Int-11a-11e and 3-
(
trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazine 9 using K CO as base
2 3
using DMF solvent via conventional heating at 70 ºC to give desired product (12a-12e) in
good practical yield (69-81%).

ACCEPTED MANUSCRIPT
Similarly in reaction scheme 3, Various chloride derivative was directly reacted with
3
-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo [4,3-a]pyrazine 9 using K CO as base
2
3
using DMF solvent by conventional method at room temperature to give desired product
12f-12j) in good 71-80% yield. Various chemical structure and % yield of synthesized
(
compound was abridged in Table 1.
A synthesized compounds were characterized by different spectroscopic techniques
1
13
like elemental analysis, IR, H-NMR, C-NMR and mass spectrometry and were in full
agreement with proposed structures before evaluating for In vitro biological activities. IR
-1
spectra of compound 12a showed various absorption bands at 3089, 2945, and 1662 cm due
to alkene (=C-H ), Alkane (C-H ), Amide (C=O ) group. Absorption band at 1587, 1326,
str
str
str
-1
1
203 cm over the range was due to stretching vibrations of aromatic (C=C ), (C-N ), and
str str
1
(
C-F ) respectively. The characteristic signals in H-NMR of compound 12a, Aromatic ring
str
protons resonated at 8.29-7.88, and 7.56-7.39 δ ppm value. The scaffold triazolo[4,3-
a]pyrazin and other protons shoed peaks at 4.04 (dt, J = 8.6, 5.5 Hz, 2H), 3.82 (s, 1H), 3.56
(d, J = 14.7 Hz, 2H), 3.39 (s, 1H), 3.10 (t, J = 5.4 Hz, 1H), 2.67 (t, J = 5.4 Hz, 1H) δ ppm
1
3
value. The C NMR spectrum is in good agreement with the structure assigned.
1
3
Characteristic peaks at δ value 169.53 ppm which was most deshielded signal in C NMR
confirmed the presence of >C=O in amide type ketone in side chain between two scaffold.
The mass spectrum of (12a) showed a molecular ion peak at m/z = 383.3 [M+1]
corresponding to a molecular formula C H F N OS.
1
5
13
3
6
Taking into consideration the entire outcome, a plausible mechanistic pathway for the
synthesis of 12a is illustrated in Scheme 4 and 5.
3
3
.3. In-vitro Biological screening
.3.1. Anti-Bacterial & Anti-Fungal Activity
The micro dilution vulnerability test in broth dilution method was used to evaluate the
anti-bacterial activity of synthesised compounds. All compounds were screened against
common bacterial strains, viz. Gram-positive bacteria Staphylococcus aureus (MTCC-96),
Staphylococcus pyogenus (MTCC-442), Gram-negative bacteria (Escherichia coli. (MTCC-
4
43), Pseudomonas aeruginosa (MTCC-1688)) and fungi Candida albicans (MTCC-227),
Aspergillus niger (MTCC-282) and Aspergillus clavatus (MTCC-1323) strains for
determination of their antibacterial as well as antifungal efficacy.
All compounds were evaluated against the individual minimal inhibitory
concentration (MIC, µg/mL) values of test compounds against the test microbes are listed in

ACCEPTED MANUSCRIPT
Table 2 (Figure 4) along with MIC values of reference compounds ampicillin (for bacteria)
and griseofulvin (for fungi).
It is worth mentioning that all molecules exhibited excellent inhibition activity against
bacterial strain S. aureus and significant activity against S. pyogenus and good to moderate
activity against E. coli and P. aeruginosa strains. As per the biological result from the table 2,
the most of the synthesized compounds showed variable degrees of inhibition against the test
microorganisms. Six of synthesized compounds i.e. 12a (100 µg/mL), 12b (100 µg/mL), 12c
(100 µg/mL), 12i (125 µg/mL), 12j (100 µg/mL), and 12i (62.5 µg/mL) found to be more
potent anti-bacterial agent against the gram-positive bacteria S. aureus organisms as compare
to Ampicillin. While Compound 12i, and 12j showed admirable MIC value (100 µg/mL,
6
2.5µg/mL) against the gram-positive bacteria S. pyogenus. Compound 12e showed excellent
activity (50 µg/mL) against Gram-negative bacteria E. coli, while 12e showed excellent
activity (62.5 µg/mL) against P. aeruginosa comparable to that of the reference drug (Table
2
, Figure 4).
MIC values of antifungal activity, showed that most compounds exhibited good
inhibition against the C. albicans fungal strains and moderate activity against A. niger and A.
clavatus strains. Among them, 12b (250 µg/mL), 12c (250 µg/mL), 12d (250 µg/mL), 12e
(250 µg/mL), 12f (500 µg/mL), 12g (500 µg/mL), 12h (250 µg/mL), 12i (500 µg/mL) and
1
2i (500 µg/mL) exhibited strong inhibition against C. albicans as compare to standard drug
greseofulvin (Table 2, Figure 5). All newly synthesised compounds showed lesser response
against other two fungi against A. niger and A. clavatus strains.
3
.3.2. Anti-tubercular activity
All new compounds were screened against M. tuberculosis H Rv strain using the
3
7
well-known Lowenstein–Jensen (L-J) method. Isoniazid was used as a positive drug standard.
The results of the in vitro Anti-tubercular activity screening are given in Table 2, (Figure 6).
The results of Anti-tubercular activity indicated that most of the synthesized
compounds exhibited low to good activity against M. tuberculosis strains in vitro. As seen in
Table 2, (Figure 6), the Synthesized compounds showed divers Anti-tubercular activity.
Compounds 12a, 12d, 12e and 12j were known to show same MIC value (500 µg/mL) of the
targeted pathogenic strain of Mycobacterium H Rv. Compound 12g which have furan ring
3
7
directly attached with scaffold (triazolo[4,3-a]pyrazine) proved to be best Anti-tubercular
activity agent shows MIC value 62.5 µg/mL and whereas compound 12i which possessed -
CH and –NO group on aromatic ring to be the least effective in possessing anti-tuberculosis
3
2
activity (MIC value 1000 µg/mL). In the case of Compound 12f which have fluoro (-F) group

ACCEPTED MANUSCRIPT
at para position displayed second highest MIC value 100 µg/mL as compare to compound
1
2h which possess methyl (-CH ) at para position showed MIC value 125 µg/mL) due to
3
high electronegativity of fluorine atom with respect to standard drug isoniazid.
3
.3.3. Anti-malarial activity
All the ten synthesized compounds were evaluated for their in vitro anti-malarial
activity against Plasmodium spp. strains of P. falciparum using quinine as reference drug.
The biological screening results of anti-malarial activity are given in Table 2 (Figure 7).
th
Among the series, compounds 12a, 12c, 12d, and 12h having -H, -OMe, -F group on 6
position of benzothiazole ring and Benzoyl showed lesser anti-malarial activity (0.79 µg/ml),
(0.45 µg/ml), (0.98 µg/ml), (0.79 µg/ml) respectively as compare to standard drug Quinine
which were found to be the most active against P. falciparum strain. Compound 12f, 12g, 12i,
and 12e were found to be five-fold lesser active (1.42 µg/ml), (1.05 µg/ml), (1.04 µg/ml) and
(1.18 µg/ml) individually than reference drug. Compounds 12b, and 12i exhibit significant
ten-fold less activity as per reference standard drug.
3
1
.3.4. Structure-activity relationship (SAR)
As per our above biological discussion, for the synthesis of newly design compounds
2a-12j, we have incorporated triazolo[4,3-a]pyrazine rings with benzothiazole nucleus,
phenyl ring, furan and thiazole ring. We have introduced different electron withdrawing
th
groups and electron donating groups on different ring system such as benzothiazole ring (6
position group such as -H, -Cl,-OMe, -F, -OH), phenyl ring (-NO , -CH , -F), furan ring and
2
3
thiazole ring (2,4-dimethyl group) during synthesis of bioactive triazolo[4,3-a]pyrazine
derivatives. The electronic configuration of the various functional group led to promising
biological activity. We have illustrated the outcome of SAR study in graphical form in Figure
8
-9.
th
Compounds possessing electron donating group excluding -OMe group on 6 position
on benzothiazole ring (12c) led to excellent anti-malarial activity. However, it was also
observed that -H and –F group on sixth substitution of benzothiazole ring (12a & 12d) led to
same bioactivity. Sixth substitution with -Cl and -OH group which were decreasing anti-
malarial activity. While compounds containing -CH (12f) and -F (12h) group to phenyl ring
3
and furan ring (12g) as substituents led to good anti-tubercular activity however, its showed
less anti-malarial activity. Furthermore, Compounds possessing m-NO , p-CH on phenyl ring
2
3
and 2,4-dimethyl group on thiazole ring as substituents resulted in decreasing anti-malarial
activity.

ACCEPTED MANUSCRIPT
As per the biological activity results, we have observed that when we have introduced
various electron donating and withdrawing group such as –H, -Cl, -OMe, -F, and –OH on
benzothiazole at sixth position in compound 12a-12e, it has furnished excellent activity
against S. aureus gram positive bacterial stain. When benzoyl ring with various function
group such as -F, m-NO , p-CH , furan ring and thaizole with 2,4-dimethyl group containing
2
3
compounds 12f, 12g, 12i and 12j, showed excellent activity against S. aureus stain. In
addition, compound possessing -CH group on phenyl ring decreased bacterial inhibition. An
3
excellent activity was observed against gram positive S. pyogenus stain when we introduced
electron donating substituents like -CH and withdrawing group -NO at para and meta
3
2
position respectively and 2,4-dimethyl on thiazole ring in compound 12i and 12j,
In a similar way, it is our observation that the incorporation of electron withdrawing
group like -Cl (sixth position on benzothiazole) and -F group (para position on benzoyl ring)
in compound 12b and 12f, it has yielded an excellent activity against gram negative E. coli.
bacterial stain. Compounds possessing various functional group different ring system such as
th
benzothiazole ring (6 position having group such as -H, -OMe, -F, -OH), phenyl ring (-NO ,
2
-
CH ), furan ring and thiazole ring (2,4-dimethyl group) comparatively lower inhibition
3
against E. coli. i.e. Gram negative bacterial strain. By using -F and -OH groups at sixth
positon on benzothiazole ring with compounds 12d, and 12e displayed excellent activity
against P. Aeruginosa are observed. When we introduced various group on benzothiazole,
phenyl, furan and thiazole ring, the anti-bacterial activity decreased against P. Aeruginosa
and S. Pyogenus (except compound 12i and 12j) due to function group.
In case of anti-fungal biological screening, we have observed that the introduction of
electron withdrawing group like -Cl and -F and electro donating group like -OH and -OMe at
sixth position of compound 12b, 12d, 12e and 12c, the outstanding activity against
C. albicans is observed. In the similar way, when we have used benzoyl ring with various
function group such as -F, m-NO , p-CH and furan ring in compound 12f, 12h and 12i and
2
3
1
2g with an excellent activity against C. albicans is furnished. Additionally, functional group
like, 2,4-dimethyl with thiazole ring in compound 12j, we have observed an excellent similar
activity against C. albicans as compare to standard references drug. Moreover, compound
1
2a possessing -H only at sixth position on benzothiazole ring resulted in decreasing anti-
fungal efficacy.
We know that substituted on final targeted compounds at diverse position on various
ring system such as benzothiazole ring, phenyl ring, furan ring and thiazole found to affect
biological potency in anti-tubercular activity. The electronic configuration of the various

ACCEPTED MANUSCRIPT
functional group led to auspicious effect on bioactivity. Compounds (12g) comprising furan
ring system with main scaffold led to best anti-tubercular activity. Furthermore, para
substitution of -CH (12f) and -F (12h) group on phenyl ring were led to be good bio-potency
3
against H Rv. Moreover, compounds possessing various functional groups -H, -Cl, -OMe, -
3
7
F, and -OH on benzothiazole ring system at sixth position like electron donating and
withdrawing groups resulted in decreasing anti-tubercular efficacy. Compounds with
different ring system such as phenyl (with functional group m-NO , p-CH ) and thiazole ring
2
3
(2,4-dimethyl group) resulted in decreasing anti-tubercular activity. SAR studies indicate that
electronic effects contribute to enzyme substrate interactions.
3
.3.5. In-silico ADMET prediction and drug likeness study
We have predicted the ADME properties of synthesized compounds and used
reference compound Quinine, ampicillin and isoniazid for the pharmaceutically relevant
properties to assess the drug likeness and pharmacokinetic properties. We used Qikprop
v3.5[71] for the assessment of some important absorption, distribution, metabolism and
excretion (ADME) parameters and its acceptable ranges are listed in Table 3 and 4. All the
newly synthesised compounds were prepared in neutralizing form for the calculation of
pharmacokinetic properties by Maestro Build module and LigPrep, saved in SD format. In
the present study, to find out the drug-likeness characteristics, molecules were assessed using
Lipinski’s rule of five showing zero violation of the rule (drug molecule should have
molecular weight ≤ 500, hydrogen bond acceptors, ≤ 10 and donor ≤ 5, log P ≤ 5, polar
surface area ≤ 140 Å.)[94] As rule of five compliance ensures the bioavailability, the
molecules in the designed series were assumed to have better duodenal penetrability (Table
3
). Alternate to this rule certain other molecular descriptors such as polar surface area was
also becomes the dominant concept for drug discovery in earlier stage.
The ADME properties were also foretold by Jorgensen’s Method[95] using Qikprop
tool (Schrodinger) for newly synthesised molecules along with standard references drugs and
the obtained values are provided in Table 4. All molecules found to have acceptable
pharmacokinetic properties. The value of both human serum albumin binding (QPlog Khsa)
range: -0.575 to -0.045) descriptor for the brain-blood partition coefficient (QPlog BB, range:
-1.36 to 0.451) and the blood-brain barrier mimic MDCK cell permeability (QPPMDCK,
range: 152.079 to 3755.179) show acceptable extrapolations for all the test compounds. The
descriptor QPPCaco representative Caco-2 cells penetrability, a model used for the gut blood
-1
barrier, showed good values (range: 49.726 to 1118.842 nm s ) for all the test compounds.
All compound have portrayed decent Percent human oral absorption with in allowable range

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(66.454 to 100%). The lipophilicity of all synthesized molecules were reported in the form of
log P and indicated that compounds should have possess enough passing capacity through
cell membrane. Likewise, other descriptor models like the number of rotatable bonds as well
as polar surface area (PSA, range: 50.101 to 116.548), used as an indicator of bioavailability
for the test compounds, fall in the probable ranges. Therefore, these ligands have a good
potential for ensuing development as an oral agents and can be potentially active drug
candidates.
3
.3.6. Molecular docking study
In order to find out the binding mode for the designed patent compounds, we have
selected in silico computational chemistry approaches such as molecular docking practice to
find out the bonding probable interaction of the most active designed bioactive molecules as
ligands with the parental protein i.e. enzyme. Molecular docking of the synthesized
molecules were studied against responsible enzymes i.e. enzyme aspartic proteinase
zymogen proplasmepsin II from plasmodium falciparum, S. aureus hydrolase and
Mycobacterium tuberculosis enoyl reductase (InhA) using PDB ID 1PFZ [73], 4CJN [74] and
2
H7I [75] correspondingly as respective inhibitors of such enzymes are found accountable for
the potency.
Before performing docking study, we have to validate our docking tool (Schrödinger
molecular modelling package) and PDB target that we used for present study. The validation
step is prerequisite for any successful experiment. There are a number of methods available
in literature [96] for validating docking programs and scoring functions. [97-98] Pose
selection is one of the most common method whereby docking programs (Schrödinger
molecular modelling package) is used to re-dock into the active site of the protein target with
native co-crystallized ligand.[96] As a result, the docked position was paralleled to the crystal
structure position by calculating RMSD value. As per the validation method, if the RMSD of
the best docked conformation should be ≤ 2.0 Å from the experimental, the used scoring
function can be considered as successful.[99]We have carried out re-docking of co-
crystallized native ligands into the active site of 1PFZ, 2H7I and 4CJN respectively using
schrodinger software for the validation of the docking process. The RMSD values of the
native co-crystallized ligand after docking were 0.035 (1PFZ), 0.755 (2H7I) and 1.184
(4CJN) Å, respectively which confirms the reliability of Schrodinger for docking compounds
under study (Figure S1 (supporting file).[99]

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The biological experimental observations were followed up with molecular docking
studies, in which the most active compounds base on biological activities such as anti-
malarial (12a, 12c, & 12d), Anti-bacterial (12a and 12e) and anti-tuberculosis activity (12f,
1
2g) along with standard references drug (Quinine, Ampicillin & Isoniazid respectively) were
docked into the active site of aforementioned responsible enzymes (Figure 10-12). Further,
favorable interaction of many bioactive molecules against these enzymes were found in
literature[68-70, 100-103] which encouraged us for the selection of the aforementioned
enzymes in current research. The docking studies were performed with Glide (Grid-Based
Ligand Docking With Energetics) program[100] incorporated in the Schrödinger molecular
modeling package (Schrödinger, LLC, New York, NY, Maestro version 11.4.011, 2015).
Docking scores for all active bioactive compounds obtained were shown in Table 5. Docking
poses in 2D and 3D view of most potent molecules in the active site of key enzymes are
illustrated in Figures 10-12. We have taken 3D docking poses only with those amino acid
residue which are in 4 Å range from ligand with the use of custom preset option [69, 103] All
potent ligands exhibited strong interaction with respective enzymes with good docking scores
as compare to standard references drugs.
While comparatively reliable binding were observed for all the potent biologically
active molecules such as anti-malarial (12a, 12c, & 12d), Anti-bacterial (12a and 12e) and
Anti-Tuberculosis activity (12f, 12g) in regards to triazolo[4,3-a]pyrazine ring, it was the
interaction of the benzothiazole nucleus, and other aromatic ring in side chain and mid of side
chain (-CO-) with the nearby amino acid residues that produced the variation in the observed
binding affinity for these derivatives. The best docked poses were chosen on the basis of
scoring function and their binding characteristics comparing with standard references drug.
Ligand protein interaction for some potent compounds with the respective key enzyme were
represented in Table 5 along with the docking score.
Among them, compound 12c seems to be the most promising lead molecule which
shows a docking score of -8.550 kcal/mol with the P. falciparum. Docking results clearly
indicate that, the interactions between potent molecules with P. falciparum are impressive in
comparison to Quinine as standard references drug. The interaction diagrams of highest score
compounds 12c are shown in Figure 10. The highest energy docking pose of 12c revealed the
presence of four decisive hydrogen bonding interactions with Tyr111 (2.25 Å), Gly112 (2.70
Å), Ser215 (2.75 Å), and Ser317 (1.88 Å) through -NH group of benzothiazole moiety and -
CO group of mid side chain. It also shows one π – π interaction with Tyr111. Compound 12e
shows hydrophobic interactions with the amino acid residues present in the P. falciparum

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such as Gly104, Ile105, Gly106, Gly110, Tyr111, Gly112, Trp131, Val134, Ser215, Leu216,
Ala217, Asn218, Leu265, Thr266, Tyr267, Ala312, Gly313, Pro314, Leu315, Ser317,
Ala319, Ala320 and Ile369.(Table 5).
While we used Quinine as standard reference drug for this docking study which
shows docking score -4.953 kcal/mol as compare with potent newly molecules. It shows one
hydrogen bond with amino acid Tyr111 and one π – π interaction with Tyr111. It shows
hydrophobic interactions with the amino acid residues present in PDB receptor such as
Gly104, Ile105, Gly106, Asp107, Gly110, Tyr111, Gly112, Trp131, Hie214, Ser215,
Leu216, Ala217, Tyr267, Thr266, Leu265, Lys285, Leu315, Ser317, Arg318, Ala319, and
Ala320 (Table 5) (Figure 10).
Generally, the molecular docking results found in agreement with In-vitro results and
the docked ligands shown good docking score by displaying good binding interactions with
amino acid residues of the aforesaid enzymes. Hence, the possible behaviour of these
bioactive molecules (12a, 12c, 12d, 12e, 12f, and 12g) may anticipated because of their
inhibitory efficacy against accountable enzymes. Various ligand protein interactions obtained
were depicted along with docking score and bond length in Table 5.
The docking interaction exposed that the primary driving forces for mechanical
interlocking between these triazolo[4,3-a]pyrazine derivatives and the active site of the with
P. falciparum were the π – π interaction, hydrogen bonding and hydrophobic
complementarities. It could also deliver a quantitative insight into various non-bonded
interactions responsible for the variation observed in the binding affinity of these molecules
which can guide site specific modification in these molecules to arrive at potent anti-malarial
candidacy.
3
.3.7. Molecular dynamics simulation
The stability of the docked complex in the Plasmodium spp. (PDB: 1PFZ) was
validated by execution MD simulation of the best docked configurations of promising ligand
(12c) using Desmond.[104] To understand to reach of the real physiological situation, we
+
have done MD simulations in water system and neutralized with Na counter ions, keeping
the salt concentration at 0.15 M. The values of RMSD[105] with respect to the time
development are often exploited to study whether or not a simulated system attains stability.
A plot of protein-ligand RMSD with the time progression is illustrated in Figure 13.[106]
The protein showed RMSD values which portrayed on left Y-axis is based on all
backbone Cα atoms relative to the matching starting structures of all trajectories for this

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simulated 12c-1PFZ system.[106] The right handed Y-axis indicates the RMSD values of
ligand which showed how stable the ligand is with regard to the protein and its binding site or
pocket. The plot, ‘Lig fit Prot’ shows the RMSD of a ligand when the protein-ligand complex
is first allied on the protein mainstay of the reference and then the RMSD value of the ligand
heavy atoms is measured. In all the cases, the RMSD values augmented rapidly during the
first 2-5 ns and then reach plateaus, which imply that the simulated systems attain
stability.[106]
Furthermore, the orientation of side chain of each residue, Cα, heavy atoms,
backbone, and β-factor of the protein shown in the plot of root-mean-square fluctuations
(RMSF) in the MD trajectory were also calculated and portrayed in Figure 14-15. RMSF for
most of the residues of all the simulated complexes were within 5.4 Å. The lower atomic
fluctuation for residues, β-factor, Cα, backbone atoms and its heavy atoms is due to small
conformational vicissitudes during the simulation.
The results in the form of stacked bar plots which were standardized during the course
of the trajectory are represented in Figure 16. Protein interactions with the ligand can be
checked throughout the simulation. These interactions can be categorized by type and
summarized, as shown in Figure 16. Protein-ligand interactions are mainly categorized into
four types: hydrogen Bonds, hydrophobic, ionic and water bridges. Each interaction type
contains more specific subtypes, which can be explored through the 'Simulation Interactions
Diagram' panel. At the end of the simulation, the system was individually inspected based on
the average value of the interaction occupancies of the binding site residues.
In the case of sulindac-bound system, approximately 160% of the simulation time
Lys285 was involved in the ionic bonding and hydrogen bonding with the ligand. Tyr111 and
Ser215 were showed 90% and 70% of the simulation time representative an aggregate of
hydrophobic interaction as well as hydrogen bonding respectively.

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4
. Conclusion
The focal point of the present study was on the development of new bioactive based on
hybrid triazolo[4,3-a]pyrazin analogues with the hope of generating novel bioactive chemical
moieties that could be useful as potent anti-malarial, antibacterial and anti-tubercular agents.
We have synthesized hybrid triazolo[4,3-a]pyrazin based analogues and characterized by
different spectroscopic methods. Most of the synthetic analogues were found to be active
against gram +ve and gram -ve bacterial strains and also demonstrated good in vitro
antibacterial activity. All the compounds showed excellent anti-bacterial activity against
distinct bacterial strain (S. aureus) as compare to Ampicillin. Most of the compounds
demonstrated good anti-fungal activity against C. Albicans stain except compound 12a.
Amongst the screened compounds, 12a (MIC 0.79 µg/ml), 12c (MIC 0.45 µg/ml) and 12d
(MIC 0.79 µg/ml) were found to be superior anti-malarial agents against P. falciparum. We
have also carried out anti-tuberculosis activity of synthesized analogue. Compound 12g (MIC
value 62.5 µg/ml), 12f and 12h (MIC value 100 and 125 µg/ml) showed good anti-
tuberculosis activity against Mycobacterium tuberculosis H Rv strain. The structure activity
3
7
relationships discussed above may provide valuable information for further design and
synthesis of compounds with optimized anti-malarial activity. In silico molecular docking
studies outcomes show that all these hit compounds were also found to have promising
interaction with the accountable enzymes in terms of good potency of compounds with good
docking score. Results of molecular dynamics simulation of selected 1PFZ-12c system in
terms of RMSD, RMSF and per-residue interaction analyses validate the stability of the
studied complexes throughout the simulated trajectories. Further, simulation studies with

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longer time period could probably provide more insights of ligand interactions in protein
environment. The calculated ADMET parameters validated good pharmacokinetic properties
of synthesized compounds. This study could identify new anti-bacterial, anti-malarial, and
anti-tubercular agents which found to be used as lead molecules for further optimization and
development for infectious disease.
Acknowledgement
We are thankful to UGC-Info net and INFLIBNET Gujarat University for providing e-source
facilities and also thankful to department of the chemistry for providing necessary facility
during work.
Conflicts of interest: Authors have declared that there is not any conflict of interest
.
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