Synthesis, biological activities and docking studies of piperazine incorporated 1, 3, 4-oxadiazole derivatives

Article abstract of DOI:10.1016/j.molstruc.2019.04.106

New series of 1, 3, 4-oxadiazoles incorporating piperazine scaffolds in a single molecular framework has been reported. The structures of the synthesized derivatives were assigned by IR, NMR and mass spectral techniques. The hybrid compounds were evaluated for their antimicrobial, antitubercular and antioxidant activities. The observed MIC values of anti-tubular activities for the molecule 3a, 3b, 3c, 3d and 3e were 6.25, 3.12, 3.12, 1.60 and 50.0 μg/ml respectively. As compared to ascorbic acid (IC₅₀ = 62.91 μg/ml), molecule 3a exhibited better antioxidant activities (IC₅₀ = 36.72 μg/ml). Also, all molecules have shown significant antimicrobial activities. In addition, docking simulations were performed to study ligand-protein interactions and to determine the probable binding conformations. In drug likeness model study compound 3b possessed maximum drug likeness model score (0.75) similar to the standard drug streptomycin. The compound 3a, 3b and 3c were emerged as potential derivatives in the series and could serve as lead compound for the development of potential therapeutic agents.

Full text of DOI:10.1016/j.molstruc.2019.04.106

Accepted Manuscript
Synthesis, biological activities and docking studies of piperazine incorporated 1, 3, 4-
oxadiazole derivatives
Shipra Bhati, Vijay Kumar, Simranjeet Singh, Joginder Singh
PII:
S0022-2860(19)30515-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.04.106
MOLSTR 26478
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 February 2019
Revised Date: 1 April 2019
Accepted Date: 24 April 2019
Please cite this article as: S. Bhati, V. Kumar, S. Singh, J. Singh, Synthesis, biological activities and
docking studies of piperazine incorporated 1, 3, 4-oxadiazole derivatives, Journal of Molecular Structure
(2019), doi: https://doi.org/10.1016/j.molstruc.2019.04.106.
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Graphical Abstract

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Synthesis, biological activities and docking studies of piperazine
incorporated 1, 3, 4-oxadiazole derivatives
Shipra Bhati,1# Vijay Kumar,2# Simranjeet Singh,3,4,5# Joginder Singh3*
1. Department of Chemistry, The Oxford College of Engineering, Bangalore (Karnataka) -
560068, India. (E-mail: shiprabhati@yahoo.com)
2. Department of Chemistry, Regional Ayurveda Research Institute for Drug
Development, Gwalior (M.P.) – 474009, India. (E-mail: vijaychem99@gmail.com)
3. Department of Biotechnology, Lovely Professional University, Jalandhar (Punjab) –
144111, India. (E-mail: simnav14@gmail.com)
4. Regional Advanced Water Testing laboratory, Mohali (Punjab) -160059, India.
5. Punjab Biotechnology Incubators, Mohali (Punjab) -160059, India.
# Equal contribution.
* Corresponding Author, Dr. Joginder Singh, (E-mail: simnav41@gmail.com)
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Synthesis, biological activities and docking studies of piperazine
incorporated 1, 3, 4-oxadiazole derivatives
Abstract
New series of 1, 3, 4-oxadiazoles incorporating piperazine scaffolds in a single molecular
framework has been reported. The structures of the synthesized derivatives were assigned by
IR, NMR and mass spectral techniques. The hybrid compounds were evaluated for their
antimicrobial, antitubercular and antioxidant activities. The observed MIC values of anti-
tubular activities for the molecule 3a, 3b, 3c, 3d and 3e were 6.25, 3.12, 3.12, 1.60 and 50.0
ꢀg/ml respectively. As compared to ascorbic acid (IC50 = 62.91 µg/ml), molecule 3a
exhibited better antioxidant activities (IC50 = 36.72 µg/ml). Also, all molecules have shown
significant antimicrobial activities. In addition, docking simulations were performed to study
ligand-protein interactions and to determine the probable binding conformations. In drug
likeness model study compound 3b possessed maximum drug likeness model score (0.75)
similar to the standard drug streptomycin. The compound 3a, 3b and 3c were emerged as
potential derivatives in the series and could serve as lead compound for the development of
potential therapeutic agents.
Keywords: Antimicrobial; anti-tubercular; antioxidant; bioavailability; docking simulations.
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1. Introduction
Antimicrobial resistant (AMR) and Tuberculosis (TB) are the global concern. The report
from WHO's Global Antimicrobial Resistance Surveillance System (GLASS), 2016-17 shows
that Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus
pneumoniae and Salmonella species are the most commonly reported resistant bacteria [1].
The report reveals widespread occurrence of antibiotic resistance. Emergence of
antimicrobial resistance in microorganisms is a natural phenomenon and to combat
antimicrobial resistance understanding of resistance mechanism, microorganism,
antimicrobial drug, host, and context; parallel to new drug discovery, broad ranging, and
multidisciplinary research is needed [2-5]. TB is a contagious disease caused by the
bacterium Mycobacterium tuberculosis. Multidrug-resistant TB (MDR-TB) remains a public
health crisis and a health security threat [6-7]. According to the WHO fact sheet on
tuberculosis, In 2017, 10 million people fell ill with TB, and 1.6 million including 2, 30, 000
children died from the disease [8]. There is a critical need of design and development of
novel chemotherapeutic agents to combat the emergence and increasing prevalence of
resistant pathogens.
Heterocyclic compounds are key components for development of chemotherapeutic
agents. Pyridine is the parent ring system of a large number of naturally occurring
products and important industrial, pharmaceutical and agricultural chemicals [10]. Pyridine-
based compounds have been playing a crucial role as agrochemicals or pesticides including
fungicides, insecticides/ acaricides and herbicides etc. [11]. Presently there are many drugs
available in the market like Pioglitazone HCl, Rosigitazone HCl, Isoniazid, Ethionamide,
Prothionamide , Prothionamide, Nikethamide, etc., that have pyridine ring in their structure
which is responsible for biological activities. Grombein and co-workers had presented 1-
Phenylsulfinyl-3-(pyridin-3-yl) naphthalen-2-ols derivatives as potent aldosterone synthase
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inhibitors [12]. Mohamed F.Abdel-Megeed and co-workers synthesized and evaluated
antimicrobial activity of
a
series of diphenyl 1-(aryl amino)-1-(pyridin-3-yl)
ethylphosphonates derivatives and found that the compounds showed high antimicrobial
activity against selected bacterial and fungal strains at low concentrations (10–100 ꢀg/mL).
Also these compounds showed significant cytotoxicity anticancer activities against liver
carcinoma cell line (HepG2) and human breast adenocarcinoma cell line (MCF7) [13].
Benzimidazole derivatives have been extensively studied in the field of medicinal
chemistry due to their versatile and eminent biological profile. A large number of activities
such as antimicrobial [14-16], anti-mycobacterial [17], analgesic [18], anticonvulsant [19] ,
antitumor [20] , antioxidant and anti-inflammatory [21], anti HIV activity [22], antiviral [23],
antimalarial [24] and anti-hypertension activity [25] etc. had been displayed by compounds
adjoining this heterocyclic system. The introduction of benzimidazole derivatives in
triphenylamine-based organic dyes can improve their photovoltaic performance because of
the extension of the π-conjugation structures in dye-sensitized solar cells [26].
Polybenzimidazole based nanocomposite membranes have also been reported as promising
electrolyte used for high temperature proton exchange membrane fuel cells [27]. Lijun Tang
and co-workers designed and synthesized benzimidazole-based fluorescent chemo sensor for
Cu2+ and sulfide anion detection in water [28].
Oxadiazole rings are commonly employed as bioisosteric replacement for carbonyl-
containing groups such as esters, amides, carbamates and hydroxamic esters and are
comparatively more stable in biological media and hence they are frequently used motif in
structure of drug candidates [29]. 1, 3, 4-oxadiazoles are nitrogen oxygen containing
heterocyclic compounds that are known for numerous therapeutic effects and are recognized
as important pharmacophore in drug discovery. 1, 3, 4-oxadiazole derivatives have occupied
prominent place in medicinal chemistry and are known for their diverse range of biological
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activities including antimicrobial [2-5,30,31], anti-mycobacterial [32] anti-inflammatory and
analgesic [33], anticancer [34,35], anti-allergic [36],antioxidant [37] insecticidal [38],
anticonvulsant [39] etc.
In the view of the facts mentioned above and in continuation to our research work, in
present report, the synthesis and characterization of some novel heterocyclic compounds
containing piperazine moiety has been carried out. The synthesized molecules were evaluated
for their antimicrobial, anti-tubercular and antioxidant potential. Further, molecular docking
studies were also carried out to understand binding interactions of ligands and selected
protein targets.
2. Experimental
2.1. Chemicals and Reagents
All chemicals and reagents used in current study were of analytical grade and were used
without further purification. Melting points were determined in open capillary using melting
point apparatus and are uncorrected. The IR spectra of all compounds were recorded on a
Perkin–Elmer FTIR spectrophotometer in KBr. 1H NMR and 13C NMR spectra were recorded
on a Bruker 300 MHz spectrometer using DMSO as a solvent and TMS as an internal
standard. Chemical shifts were reported in parts per million (ppm). Mass spectra were
recorded on JEOL SX 102/DA-6000 mass spectrometer data system using argon/Xenon (6
KV, 10 MA) as FAB gas. All the reactions were monitored by thin-layer chromatography
(TLC) using silica gel glass plates.
2.1.1. Synthesis of Ethyl (4-methyl piperazin-1-yl) acetate (1)
1-methyl piperazine (0.01 mol), ethyl chloroacetate (0.02 mol), anhydrous potassium
carbonate (2 g) were refluxed in 50 ml of dry acetone for 18 h. The inorganic solid was
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filtered off and the filtrate was concentrated under reduced pressure. Completion of the
reaction was monitored by TLC using mobile phase ethyl acetate/ hexane (6:4). Yield 80%,
m.p. 88–92 ˚C, IR (KBr) cm-1: 2978 (C-H str), 1720 (C=O), 1275 (C–O); 1H-NMR (DMSO)
ppm: 1.15 (t, 3H, Ester- CH3), 4.12 (q, 2H, Ester-CH2), 1.071 ( t, 4H, piperazine proton),
1.232 (s, 3H, N-CH3 piperazine), 2.502 ( dd, 4H, piperazine proton), 3.486 (s, 2H, N-CH2);
EI-MS: 186.14 (M+); Anal calcd for C9H18N2O2: C, 58.04; H, 9.74; N, 15.04; O, 17.18;
Found: C, 58.06; H, 9.72; N, 15.03; O, 17.17.
2.1.2. Synthesis of 2-(4-methyl piperazin-1-yl) acetohydrazide (2)
Ethyl (4-methyl piperazin-1-yl) acetate (0.01mol) and hydrazine hydrate (98%) (0.06 mol)
were refluxed in 20 ml ethanol for 10 h. Completion of the reaction was monitored by TLC
using mobile phase ethyl acetate/ hexane (8:4). After completion of the reaction, the mixture
was cooled and kept in refrigerator for overnight. The solid so obtained was filtered, washed
with cold water and recrystallized from methanol. Yield 70%; m.p.178–180 ˚C, IR (KBr) cm-
1: 3068.42 (-CONH), 2847.16 (C-H str.), 1687.35 (C=O) ; 1H-NMR (DMSO) ppm: 1.061 (
t, 4H, piperazine proton), 1.238 (s, 3H, N-CH3 piperazine), 2.507 ( dd, 4H, piperazine
proton), 3.47 (broad singlet, 2H, -NH2), 8.167 (s,1H, CONH) ; EI-MS: 172.13 (M+); Anal
calcd for C7H16N4O:C, 48.82; H, 9.36; N, 32.53; O, 9.29; Found: C, 48.80; H,9.30; N, 32.50;
O, 9.31.
2.1.3. General procedure for the synthesis of compounds (3a-3e)
Equimolar amount of 2-(4-methyl piperazin-1-yl) acetohydrazide (0.005 mol) and suitable
aromatic acid (0.005) were refluxed on a sand bath in the presence of cyclodehydration agent
POCl3 (10 ml) for 8-10 h. After completion of reaction, the mixture was cooled at room
temperature and poured onto crushed ice. On basification with sodium bicarbonate (10%), a
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solid mass was separated out which was filtered, dried and recrystallized from ethanol to give
the target compounds 3a-3e.
2.1.3.1. Synthesis of 1-methyl-4-[(5-phenyl-1, 3, 4-oxadiazol-2-yl)methyl] piperazine (3a).
Yield 70%; m.p. 190 ˚C , IR (KBr) cm-1: 3570.06 (CH arom), 2925.62 (CH aliph), 1660.13
(C=N ), 1260.77 (C-O-C asymm), 1170.12 (C-N piperazine), 1105.17 (C–O–Csymm), 1022.94
1
(N–N), 925.92, 837.12 (CH); H-NMR (DMSO) ppm: 1.067 ( t, 4H, piperazine proton),
1.236 (s, 3H, N-CH3 piperazine), 2.502 ( dd, 4H, piperazine proton), 3.427 ( dd, 4H,
piperazine proton), 4.27 (s, 2H, N-CH2), 6.98-7.88 (m, 5H, Ar-H); EI-MS: 258.14 (M+); Anal
calcd for C14H18N4O: C, 65.09; H, 7.02; N, 21.69; O, 6.19; Found: C, 65.07; H, 7.03;N,
21.66; O, 6.18.
2.1.3.2. Synthesis of 1-methyl-4-{[(5-(4-chloro phenyl)-1, 3, 4-oxadiazol-2-yl)methyl]
piperazine (3b). Yield 50%; m.p. 220 ˚C , IR (KBr) cm-1: 3580.07 (CH arom), 2921.25 (CH
aliph) , 1174.33 (C-N piperazine), 1680.08 (C=N ), 1282.74 (C-O-C asymm),1090.3 (C–O–
1
Csymm), 1013.28 (N–N), 926.23, 849.54 (CH ),759.15 (C-Cl); H-NMR (DMSO) ppm:
1.060 ( t, 4H, piperazine proton), 1.235 (s, 3H, N-CH3, piperazine), 2.507 ( dd, 4H,
piperazine proton), 3.437 ( dd, 4H, piperazine proton), 4.28 (s, 2H, N-CH2), 7.560-8.179 (m,
4H, aromatic); EI-MS: 292.11 (M+); Anal calcd for C14H17N4OCl: C, 57.44; H, 5.85; N,
19.14; O, 5.46; Found: C, 57.45; H, 5.87;N, 19.12; O, 5.47.
2.1.3.3. Synthesis of 1-methyl-4-{[(5-(4-hydroxy phenyl)-1, 3, 4-oxadiazol-2-yl)methyl]
piperazine (3c). Yield 40%; m.p. 195 ˚C , IR (KBr) cm-1: 3654.25 (OH), 3573.07 (CH arom),
2923.42 (CH aliph), 1162.20 (C-N piperazine), 1672.75 (C=N ), 1272.77 (C-O-C
asymm),1082.17 (C–O–Csymm), 1012.94 (N–N), 925.92, 837.12 (CH);1H-NMR (DMSO)
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ppm: 1.056 ( t, 4H, piperazine proton), 1.192 (s, 3H, N-CH3, piperazine), 2.502 ( dd, 4H,
piperazine proton), 3.42 ( dd, 4H, piperazine proton), 4.31 (s, 2H, N-CH2), 6.96-7.99 (m, 4H,
aromatic), 9.64 (s, 1H, Ar-OH); EI-MS: 274.14 (M+); Anal calcd for C14H18N4O2: C, 61.30;
H, 6.61; N, 20.42; O11.66; Found: C, 61.28; H, 6.60;N, 20.44; O, 11.67.
2.1.3.4. Synthesis of 1-{[(5-(4-methoxy phenyl)-1, 3, 4-oxadiazol-2-yl]methyl}-4-methyl
piperazine (3d). Yield 60%; m.p. 204 ˚C , IR (KBr) cm-1: 3583.06 (CH arom), 2920.82 (CH
aliph), 1172.55 (C-N piperazine), 1682.75 (C=N ), 1260.77 (C-O-C asymm),1105.17 (C–O–
Csymm), 1022.94 (N–N), 925.92, 837.12 (CH);1H-NMR (DMSO) ppm: 1.058 ( t, 4H,
piperazine proton), 1.196 (s, 3H, N-CH3 piperazine), 2.506 ( dd, 4H, piperazine proton),
3.437 ( dd, 4H, piperazine proton), 3.818 (s,3H,Ar-OCH3), 4.29 (s, 2H, N-CH2), 6.99-8.04
(m, 4H, aromatic); 13C-NMR (DMSO) ppm: 39.336, 39.545, 39.754, 39.962, 40.171,
55.968, 114.25, 115.285, 128.856, 162.44, 163.29, 163.94; EI-MS: 288.15 (M+); Anal calcd
for C15H20N4O2: C, 62.48; H, 6.99; N, 19.43; O, 11.10; Found: C, 62.49; H, 6.96; N, 19.42;
O, 11.11.
2.1.3.5. Synthesis of 1-methyl-4-{[(5-(3-nitro phenyl)-1, 3, 4-oxadiazol-2-yl)methyl]
piperazine (3e). Yield 30%; m.p. 210 ˚C , IR (KBr) cm-1: 3588.06 (CH arom), 2922.84 (CH
aliph), 1175.11 (C-N piperazine), 1692.05 (C=N ), 1260.77 (C-O-C asymm),1125.17 (C–O–
Csymm), 1517.98 (N=O asymm), 1317.38 (N=O symm),1064.12 (C–O–Csymm), 1025.12 (N–
1
N), 940.67, 742.12 (CH); H-NMR (DMSO) ppm: 1.053 ( t, 4H, piperazine proton), 1.192
(s, 3H, N-CH3 piperazine), 2.502 ( dd, 4H, piperazine proton), 3.427 ( dd, 4H, piperazine
proton), 4.37 (s, 2H, N-CH2), 7.56-8.82 (m, 4H, aromatic); EI-MS: 303.13 (M+); Anal calcd
for C14H17N5O3: C, 55.44; H, 5.65; N, 23.09; O, 15.82; Found: C, 55.46; H, 5.64;N, 23.08; O,
15.80.
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2.2. Prediction of Molecular Properties Descriptors
Prediction of molecular properties descriptors is significant in the discovery and development
of potential therapeutics. In the present study Molinspiration program [40] was used for
calculating molecular properties such as milogP (partition coefficient), TPSA (topological
polar surface area), MW (molecular weight), nON ( number of hydrogen bond acceptors),
nOHNH ( number of hydrogen bond donors), Nrotb (number of rotatable bonds), Nvio
(Number of violations), Natoms (range of atoms) and Volume. The bioactivity scores of
GPCR ligand, ion channel modulator, nuclear receptor ligand, kinase inhibitor, protease
inhibitor and enzyme inhibitor were also calculated using molinspiration server.
2.3. Antimicrobial Activity
The synthesized compounds 3a-3e were screened in vitro for their antibacterial activity
against four bacterial strains i.e. Pseudomonas aeruginosa ( MTCC 4673), Staphylococcus
aureus (MTCC 7443), Salmonella enterica (MTCC 164), Streptococcus mutans (MTCC 497)
and one fungal strain A parasiticus (MTCC 646) by following standard literature reported
procedure of disk-diffusion method [41-45]. The cultures used in this experiment were
ordered from MTCC Chandigarh (India). The media used for this purpose are nutrient agar
media and potato dextrose media. 28 grams of nutrient agar and 39.0 grams of Potato
dextrose agar was added to the 1 liter of double distilled water separately, it was mixed
thoroughly and pH was adjusted at 7.5 ± 0.2. The solution was heated to dissolve the
ingredients completely after which the media was autoclaved at 121˚C for 45 minutes at
15lbs pressure. After autoclaving, 15-20 ml of that media was poured into petridish for
studying antimicrobial activities. Whattman no. 1 filter paper disks were sterilized by
autoclaving at 160˚C for 1 h. Then the sterile disks were impregnated with the test
compounds of different concentrations (50 ppm, 250 ppm and 500 ppm). Cultures having 105
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CFU/mL were used against each concentration levels. The impregnated disks were placed on
the medium suitably spaced apart and the plates were incubated at 37˚C for 24 h for bacterial
strains and 48 h for fungal species. Streptomycin and ketoconazole were used as standard
drug for antibacterial and anti fungal activity respectively. After incubation, the degree of
inhibition was observed by formation of zones. The diameters of the zones were measured in
mm.
2.4. Antitubercular Activity
The antitubercular activity of compounds 3a-3e was assessed against Mycobacteria
tuberculosis (Vaccine strain, H37 RV strain, ATCC-27294) using Microplate Alamar Blue
Assay (MABA) [46,47]. This methodology is non-toxic, uses a thermally stable reagent and
shows good correlation with propotional and BACTEC radiometric method. 200 ꢀl of sterile
deionzed water was added to all outer perimeter wells of sterile 96 wells plate to minimized
evaporation of medium in the test wells during incubation. The 96 wells plate received 100 ꢀl
of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on plate.
The final drug concentrations tested were 100 to 0.2 ꢀg/ml. Plates were covered and sealed
with parafilm and incubated at 37 ºC for five days. After this time, 25ꢀl of freshly prepared
1:1 mixture of Almar Blue reagent and 10% tween 80 was added to the plate and incubated
for 24 hrs. Pyrazinamide, Ciprofloxacin and Streptomycin were used as standard drugs. A
blue color in the well was interpreted as no bacterial growth, and pink color was scored as
growth. The MIC was defined as lowest drug concentration which prevented the color change
from blue to pink.
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2.5. Antioxidant Activity
The synthesized compounds 3a-3e were evaluated for their antioxidant activity by using
DPPH (2, 2-diphenyl-1-picrylhydrazyl), free radical scavenging method according to the
reported method with slight modifications [48,49]. Briefly, 1 mg/ml solutions of compounds
and ascorbic acid were prepared by dissolving them into DMSO. 25, 50, 75 and 100 µL of
each was added separately to 10.0 mL amber color volumetric flasks containing 2.0 ml of
0.01mM DPPH (prepared in ethanol). The final volume was made up to 3.0 ml and after 30
minutes absorbance was checked at 517 nm by using UV-visible spectrophotometer. Pure
DPPH solution (0.01mM) was used as a control and ethanol was as a blank. The decrease of
in absorbance equates the DPPH radical scavenging capacity. The above process was
repeated three times for ascorbic acid (positive control) and compounds. The radical
scavenging ability was calculated according to the formula: Radical scavenging activity =
(A0- AT/ A0) × 100; where, A0 is the absorbance of pure DPPH solution (0.01mM), and AT is
the absorbance of (DPPH) and compounds. The IC50 was defined as half maximal inhibitory
concentration of the compounds that inhibit DPPH radical.
2.6. Docking Simulations
The molecular docking simulations were performed to analyze drug-target interactions of the
synthesized compounds using the Autodock 4.2 software [50]. The chemical structures of the
compounds were drawn and their 3D structures were optimized in the ACD Labs
ChemSketch 12.0 software. Docking study of antimicrobial activity was carried out against
three different protein targets i.e. Isocitrate lyase (P. aeruginosa, PDB ID: 6g1o),
Dihydrofolate reductase (S. aureus, PDB ID: 5isp) and Noranthrone synthase (A. parasiticus,
PDB ID: 5kbz). Methionine aminopeptidase (M. Tubeculosis, PDB ID: 5yxf) and tyrosine-
protein kinase (Homosapiens, PDB ID: 6il3) were selected for in-silico docking simulations
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of antitubercular and antioxidant activity respectively of the synthesized compounds. The X-
ray crystallographic structure of the target proteins were retrieved from the protein data bank
[51] . Prior to the simulations all bound ligands, cofactors, and water molecules were
removed from the proteins. Gasteiger charges were computed, and the Auto Dock atom types
were defined using Auto Dock version 4.2. The three dimensional grid boxes were created,
the spacing between grid points was 0.375 angstroms, and the grid maps representing the
intact ligand in the actual docking target site were calculated with Auto Grid algorithm.
Finally, Auto Dock was used to calculate the binding free energy of a given inhibitor
conformation in the macromolecular structure. Lamarckian Genetic Algorithm (LGA)
parameters were set to default settings, which includes 150 runs, 150 conformational
possibilities, 50 populations and 2,50,0000 energy evaluations. During the docking process, a
maximum of 10 conformers was considered for each compound. The results were evaluated
based on the binding compatibility i.e. binding energy in kcal/mol and inhibition constant.
The resultant protein-ligand complex structures were explored using Pymol program [52].
3. Results and Discussion
3.1. Chemistry
The synthesis protocol of the title compounds (3a-3e) was outlined in Scheme – 1 and
Supplementary Data S1-S8. First, Ethyl (4-methyl piperazin-1-yl) acetate (1) was synthesized
via the reaction of 1-methyl piperazine with ethyl chloro-acetate in the presence of potassium
carbonate. In the next step compound (1) was refluxed with hydrazine hydrate in ethanol to
obtain 2-(4-methyl piperazin-1-yl) aceto-hydrazide (2). Finally the cyclization reaction of
compound (2) with various aromatic carboxylic acids in the presence of POCl3 gave the titled
compounds (3a-3e). The structure of the synthesized compounds was enlightened by FTIR,
1H NMR, C13 NMR and mass spectral analysis.
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In the IR spectra of compound (1), characteristic stretching vibration at 1720 cm-1 was
observed, which confirmed the presence of carbonyl group as a part of an ester linkage.
Subsequent reaction with hydrazine hydrate resulted in the formation of compound (2) which
gave a slightly lower frequency signal at 1687.35 cm-1 and a new frequency mode at 3068.42
cm-1 supported aceto-hydrazide formation. The aceto-hydrazide when further treated with
aromatic acids in POCl3 underwent cyclisation yielding compounds 3a-3e. The characteristic
C=N band (1660–1692ꢁcm-1), C-O-C bands (1260-1282 ꢁcm-1 and 1105-1090 cm-1) and N-N
bands (1012-1517 cm-1) of medium-strong intensity were identified in each IR spectra;
indicating the formation of an oxadiazole ring. The 1H NMR spectra of compound (1) gave a
triplet at δ 1.15 and a quartet at δ 4.12 integrating for three and two proton respectively. Such
a pattern is indicative of the fact that an ethyl group is present. A downfield signal at δ 3.486
1
was observed integrating for two protons confirmed the presence of a methylene group. H
NMR spectra of compound (2), an up field signal at δ 8.167 and a broad singlet at 3.47
accounting for one and two protons respectively , were characterized as –CONH and –NH2
group. In the 1H NMR spectra of the compounds 3d a singlet at δ 4.29 and δ 3.80 integrating
for two and three protons respectively were identified as methylene group and methoxy
group. The other aliphatic and aromatic protons were observed at the expected regions.
Further the mass spectrum of 3d showed a (M+) peak at m/z 288.15; (100%), which is in
agreement with its molecular formula C15H20N4O2. 13C NMR data also supported the
structures of the compound 3d.
3.2 Molecular properties descriptors
The drug likeness of the synthesized compounds (3a-3e) was estimated by calculating
molecular descriptors using Molinspiration tool and results were presented in Table - 1.
Lipinski’s rule of five [53] predicts the molecular properties related to the pharmacokinetics
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of molecules. According to this rule, the compound that have miLogP<5, molecular weight
<500, the number of hydrogen bond acceptors<10 and number of hydrogen bond donors<5,
possess good oral bioavailability. The compounds were in accordance with Lipinski’s rule of
five showing good drug likeness for drug targets with no violations. The bioactivity scores of
the compound 3a-3e were also predicted by calculating the activity score of GPCR ligand,
ion channel modulator, nuclear receptor legend, kinase inhibitor, protease inhibitor and
enzyme inhibitor by Molinspiration and were presented in Table - 2. A molecule having
bioactivity score more than 0.00, it is presumed to be active, while values -0.50 to 0.00 are
expected to be moderately active and if score is less than -0.50, it is considered to be inactive
[40,41]. The compounds were moderately active against GPCR ligand, ion channel
modulator, Kinase inhibitor, nuclear receptor ligand, protease inhibitor and Enzyme inhibitor.
Compound 3c was found to be active against kinase inhibitor with the bioactivity scores of
0.03. None of the compound was inactive against the selected drug targets. Drug likeness
model scores were computed for all the compounds by molsoft software. The observed scores
for the molecule 3a, 3b, 3c, 3d, 3e, Streptomycin, Ketoconazole and Ascorbic acid were
0.37, 0.75, 0.68, 0.54, 0.21 0.75, 1.32 and 0.84 respectively. Compound 3b possessed
maximum drug likeness model score (0.75) similar to the standard drug streptomycin,
followed by compound 3c (0.68) in the series. Figure - 1 depicts graphical representation of
the drug likeness model scores of compound 3b and 3c. The green color indicates non drug-
like behavior and those fall under blue color are considered as drug-like.
Prediction of ADMET properties was done using admetSAR tool and the calculated
ADMET parameters were illustrated in Table - 3. The results indicated that all the
synthesized compounds 3a-3e could be absorbed through intestinal tract when administrated
orally. The compounds showed penetration across Blood-Brain Barrier (BBB) and also
possessed Caco-2 Permeability (except compound 3e). In terms of metabolism, compound
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3a, 3b and 3c showed low CYP Inhibitory Promiscuity whereas compound 3d and 3e were
found to have high CYP Inhibitory Promiscuity. The AMES toxicity test results revealed that
except compound 3e, the derivatives were found to be nontoxic which means that these
compounds were non-mutagenic. The synthesized compounds 3a-3e were non carcinogenic
similar to the standard drugs. The computed LD50 doses of the synthesized compounds in rat
model were found between the range 2.5899 to 2.8526 mol/Kg. Compound 3c was found to
be most lethal in the series showing lowest LD50 dose value i.e. 2.5899 mol/Kg.
3.3. Antimicrobial Activity
The synthesized compounds (3a-3e) were screened for their in-vitro antibacterial activity
against two gram negative bacterial strains i.e. Pseudomonas aeruginosa, Salmonella enterica
and two gram positive bacterial strain i.e. Staphylococcus aureus, Streptococcus mutans by
taking Streptomycin as standard drug while in vitro antifungal activity was carried out against
a fungal strain Aspergillus parasiticus by taking Ketoconazole as standard drug at
concentration 50, 250 and 500 ppm by disk diffusion method. The results of zone of
inhibition measured for antibacterial and fungal activity were presented in Table - 4 and
Supplementary Data S9&S10. The majority of the compounds were found to be active
against selected bacterial and fungal strains. Maximum degree of inhibition was observed at
500 ppm concentration. All the molecules have shown better anti-bacterial activities.
Compound 3b has shown prominent activities against all bacterial strains. The results of
antifungal activity revealed that compounds 3a and 3b (3a<3b) were found to be active
against Aspergillus parasiticus while compound 3c, 3d and 3e were inactive.
The minimum inhibitory concentration (MIC) values of all compounds were
evaluated and reported under Table – 3. The MIC values were ranged over 12.5 to 200 mg/l.
As compared to the standards, molecules 3a and 3b have shown prominent antimicrobial
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activities over ranged from 12.5 to 25 mg/l (Table – 3). The results of antimicrobial screening
revealed that the compounds showed significant activity in comparison of standard drugs.
Compounds 3a and 3b emerged as potent antimicrobial derivative in the series against
selected bacterial and fungal strains.
3.4. Antitubercular Activity
The synthesized compounds 3a-3e was screened for their in vitro antitubercular activity
against Mycobacteria tuberculosis at concentration ranging from 0.8-100 ꢀg/ml (Table – 5
and Supplementary Data S11) by using Micro-plate Alamar Blue assay (MABA).
Pyrazinamide, Ciprofloxacin and Streptomycin were used as reference drugs. The compounds
showed significant activity compared to the standard drugs. The minimum inhibitory
concentration (MIC) was determined as lowest drug concentration which prevented the color
change from blue to pink. The results indicated that compound 3d having methoxy group at
phenyl ring of oxadiazole ring was emerged as most effective analog with good
antitubercular activity (MIC value 1.6 ꢀg/ ml) in the series. Besides, Compound 3b and 3c
showed MIC value 3.12 ꢀg/ ml while compound 3a demonstrated MIC value 6.25 ꢀg/ ml. A
high value of MIC i.e. 50 ꢀg/ ml was observed for compound 3e.
3.5. Antioxidant Activity
The in vitro antioxidant activity of the compound 3a-3e was determined
spectrophotometrically by DPPH free radical scavenging method at concentration ranging
from 25 µL to 100 µL and the results were summarized in Table - 6. Ascorbic acid was used
as positive control. The reduction capacity of DPPH radicals was determined by the decrease
in its absorbance at 517ꢁnm, which is induced by antioxidants. The compounds showed
moderate scavenging activity on DPPH compared to the standard. It was observed that
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antioxidant potential of the compounds increased with increase in the concentration. The
compounds showed significant scavenging activity on DPPH compared to the standard. It
was observed that antioxidant potential of the compounds increased with increase in the
concentration. Compound 3a appeared to be the most potent derivative in the series with IC50
value of 36.76 µg/ml followed by compound 3b with IC50 value of 52.42 µg/ml. Compound
3a and 3b showed better activity compared to the standard drug ascorbic acid which showed
IC50 value of 62.91 µg/ml
3.6. Docking Simulations
Docking is a computational simulation approach which is used to determine the affinity of
binding of a small molecule to a receptor molecule to form a stable complex. To support
results obtained from in vitro antimicrobial, antitubercular and antioxidant activities, it was
thought worth-while to conduct in sillico docking study. AutoDock 4.2, the docking program
was employed to estimate binding affinity of ligand-receptor complex in the receptor binding
site. The binding energy values were calculated based on the total intermolecular energies
(Kcal/mol) including hydrogen bond energy, Van Der Waals energy, desolvation energy and
electrostatic energy. The receptor was kept rigid, while ligands were set flexible to rotate and
explore most probable binding poses. For docking study of antibacterial activity all the
synthesized derivatives 3a-3e were docked with ATP-binding casette(ABC) transporter
ComA protein (PDB ID: 3vx4). Compound 3e showed minimum binding energy (-7.81
Kcal/mol, Ki; 1.87 µM), followed by compound 3a (-7.51 Kcal/mol, Ki; 3.14 µM). Protein
target Aflatoxin biosynthesis polyketide synthase (PDB ID: 3ils) was selected for docking
study of antifungal activity. It was observed that compound 3e showed strong affinity
towards selected protein target with binding energy, - 8.31 Kcal/mol in the series. The results
of docking study of antimicrobial activity were presented in Table -7. Docking studies
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revealed that the synthesized compounds 3a-3e showed good binding energy towards
selected protein targets compared to the standard drugs. The derivatives 3a-3e were also
subjected to three dimensional molecular docking on crystal structure of enoyl-acyl carrier
protein (EACP) reductase enzyme (PDB ID: 2nsd) and Protein-tyrosine kinase 2-β (PDB ID:
5tob) for Docking simulation of antitubercular activity and anti-oxidant activity. The results
of docking of antitubercular and antioxidant activities were summarized in Table - 8. The
results suggested that the derivatives 3a-3e showed better binding energy compared to the
standard drugs against the selected protein targets. Compound 3e (-9.22 Kcal/ mol, Ki-173.83
nM), 3b (-8.44 Kcal/ mol, Ki-652.56 nM) and 3a (-8.24 Kcal/ mol, Ki-905.77 nM) were best
ligands in the series in terms of binding energy and inhibition constant against protein target
2nsd. Docking results of antioxidant activity revealed that compound 3e (-7.49 Kcal/mol, Ki-
3.23 µM), 3b (-7.35 Kcal/mol, Ki-4.08 µM) and 3c (-7.24 Kcal/mol, Ki-4.97 µM) showed
strong affinity with protein target 5tob in the series. From the in silico docking simulations it
is quite evident that all the synthesized compounds 3a-3e had great potential as antimicrobial,
antitubercular and antioxidant agents. Figure – 2 & 3 depicts binding mode of best conformer
of compound 3a, 3b and 3c against selected protein targets for docking studies.
4. Conclusion
Herein we report the synthesis of 1-methyl-4-[(5-phenyl/substituted phenyl-1, 3, 4-oxadiazol-
2-yl) methyl] piperazine derivatives (3a-3e). The structures of the synthesized compounds
1
were confirmed by IR, HNMR, 13C NMR and mass spectral techniques. The synthetic
compounds were evaluated for their in vitro antimicrobial, antitubercular and antioxidant
potential. The derivatives showed remarkable activity compared to the standard drugs. The
derivatives were in accordance with Lipinski’s rule of five and had good bioavailability.
Docking simulations were performed to gain an insight into the binding modes of the
18

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synthesized derivatives and good binding affinity was recorded against selected protein
targets. A good correlation was observed between the experimental results and theoretical
predictions. Among the synthesized compounds, 1-methyl-4-[(5-phenyl-1,3,4-oxadiazol-2-
yl)methyl]
piperazine
(3a),
1-methyl-4-{[(5-(4-chloro
phenyl)-1,3,4-oxadiazol-2-
yl)methyl]piperazine (3b) and 1-methyl-4-{[(5-(4-hydroxy phenyl)-1,3,4-oxadiazol-2-
yl)methyl]piperazine (3c) emerged as promising lead compound in the series and can be
consider for further in vivo validation. Structural modification of these compounds could
possibly bring more potent compounds.
Acknowledgement
The authors are thankful to the Head, SAIF (Sophisticated Analytical Instrument Facility)/
1
CDRI (Central Drug research Institute), Lucknow for FTIR, H NMR and mass spectral
analysis. Thanks are also due to Dr. Kishore G. Bhat, MD, Microbiology, Maratha Mandal's
Central Research Laboratory, Belgaum for antitubercular activity and Head, Department of
Biotechnology, Lovely Professional University, Jalandhar for antioxidant activity.
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Captions of Tables, Schemes and Figures
Table - 1: Prediction of molecular properties descriptors of the titled compounds (3a-3e).
Table - 2: Prediction of bioactivity score of the titled compounds (3a-3e).
Table - 3: Prediction of ADMET profile of the title compounds (3a-3e).
Table - 4: Antimicrobial activities of the synthesized compounds (3a-3e) with Minimum
inhibitory concentration MIC mg/l (MBC/MFC) values.
Table - 5: Anti-tubercular activity of the synthesized compounds (3a-3e).
Table - 6: Antioxidant activity of the synthesized compounds (3a-3e).
Table - 7: Docking simulations for antimicrobial activity of the synthesized compounds (3a-
3e).
Table - 8: Docking simulations for antitubercular and antioxidant activity of the synthesized
compounds (3a-3e).
Scheme - 1: Synthetic route of compounds from 1 to 3 including 3a to 3e.
Figure - 1: Graph showing drug-likeness model score of compound 3b (a) and 3c (b)
respectively.
Figure - 2: Binding modes of compound 3a with protein targets 3vx4 (a), 3ils (b), 2nsd (c)
and 5tob (d) respectively visualized using Pymol software.
Figure - 3: Binding modes of compound 3b with protein targets 3vx4 (a), 3ils (b), 2nsd (c)
and 5tob (d) respectively visualized using Pymol software.
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Table - 1: Prediction of molecular properties descriptors of the titled compounds (3a-3e).
Comp.
miLogP
TPSA
MW
n-ON n-OH
or n-NH
Nrotb
Nvio
Volume
Natom
(mol/g)
258.32
292.77
274.32
288.35
303.32
581.58
531.44
176.12
1.06
1.73
0.58
1.11
0.99
-5.35
3.77
-1.40
45.40
45.40
65.63
54.63
91.22
336.45
69.08
107.22
5
5
6
0
0
1
3
3
3
4
4
9
7
2
0
0
0
0
0
3
1
0
244.00
257.54
252.02
269.55
267.34
497.25
452.47
139.71
19
20
20
21
22
40
36
12
3a
3b
3c
3d
3e
X
6
0
8
0
19
8
16
0
Y
Z
6
4
Here, X = Streptomycin, Y = Ketoconazole, Z = Ascorbic acid, logarithm of compound partition coefficient between n-octanol and water
(miLogP), topological polar surface area (TPSA), percentage of absorption (% ABS), molecular weight (MW), number of hydrogen bond
acceptors (n-ON), number of hydrogen bond donors (nOH/NH), number of rotatable bonds (Nrotb), Number of violations (Nvio).
Table - 2: Prediction of bioactivity score of the titled compounds (3a-3e).
Comp.
3a
3b
3c
3d
3e
X
Y
Z
GPCR
-0.20
-0.15
-0.09
-0.16
-0.25
0.09
ICM
KI
NRL
-0.73
PI
EI
-0.25
-0.25
-0.19
-0.32
-0.30
-0.16
-0.14
-0.24
-0.07
-0.05
0.03
-0.03
-0.10
-0.17
-0.17
-1.09
-0.29
-0.29
-0.22
-0.25
-0.31
0.65
-0.16
-0.18
-0.07
-0.19
-0.24
0.38
-0.67
-0.47
-0.59
-0.64
-0.18
-0.32
-1.01
0.26
-0.53
-0.21
-0.81
0.15
0.20
Here, X = Streptomycin, Y = Ketoconazole, Z = Ascorbic acid, Ligand (GPCR, GPCR), Ion channel modulator (ICM), Kinase inhibitor
(KI), Nuclear receptor ligand (NRL), Protease inhibitor (PI), Enzyme inhibitor (EI).
Table - 3: Prediction of ADMET profile of the title compounds (3a-3e)
Compd.
(BBB)
BBB+
BBB+
BBB+
BBB+
BBB+
BBB-
BBB+
BBB+
(HIA)
HIA+
HIA+
HIA+
HIA+
HIA+
HIA-
CaP
CIP
Low
Low
Low
High
High
Low
High
Low
AT
No
No
No
No
Toxic
Toxic
No
C
LD50 (mol/Kg)
2.8526
2.8073
2.5899
2.6038
2.6791
1.8409
3.4739
1.3059
Caco2+
Caco2+
Caco2+
Caco2+
Caco2-
Caco2-
Caco2+
Caco2-
No
No
No
No
No
No
No
No
3a
3b
3c
3d
3e
X
Y
Z
HIA+
HIA+
No
Here, X = Streptomycin, Y = Ketoconazole, Z = Ascorbic acid, Blood-Brain Barrier (BBB), Human Intestinal Absorption (HIA), Caco-2
Permeability (CaP), CYP Inhibitory Promiscuity(CIP), AMES toxicity (AT), Carcinogenicity (C), Rat Acute Toxicity LD50 mol/Kg (LD50).
2