Synthesis, biological evaluation and in silico studies of tetrazole-heterocycle hybrids

Article abstract of DOI:10.1016/j.molstruc.2018.07.114

The series of three different chemical entities of tetrazole-heterocycle hybrids such as thiophene, pyridine and quinoline tetrazoles were synthesized and characterized for the purpose to develop new lead molecules. Biological evaluations such as in vitro antimicrobial and anti-inflammatory activities were studied. Further, the in silico studies such as Molecular docking (with COX-1, COX-2 and 3TTZ), DFT calculations, the Molecular electrostatic potential (MEP) and ADME were investigated.

Full text of DOI:10.1016/j.molstruc.2018.07.114

Accepted Manuscript
Synthesis, biological evaluation and in silico studies of tetrazole-heterocycle hybrids
Rajendran Sribalan, Govindharasu Banuppriya, Maruthan Kirubavathi, Vediappen
Padmini
PII:
S0022-2860(18)30941-4
10.1016/j.molstruc.2018.07.114
MOLSTR 25519
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 22 February 2018
Revised Date: 30 July 2018
Accepted Date: 31 July 2018
Please cite this article as: R. Sribalan, G. Banuppriya, M. Kirubavathi, V. Padmini, Synthesis, biological
evaluation and in silico studies of tetrazole-heterocycle hybrids, Journal of Molecular Structure (2018),
doi: 10.1016/j.molstruc.2018.07.114.
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Synthesis, Biological evaluation and in silico studies of Tetrazole-Heterocycle
hybrids
Rajendran Sribalan, Govindharasu Banuppriya, Maruthan Kirubavathi, Vediappen Padmini*
Department of Organic Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai-625 021, TamilNadu, India.
*Corresponding author e-mail: padimini_tamilenthi@yahoo.co.in
Abstract
The series of three different chemical entities of tetrazole-heterocycle hybrids such as
thiophene, pyridine and quinoline tetrazoles were synthesized and characterized for the purpose
to develop a new lead molecules. Biological evaluations such as in vitro antimicrobial and anti-
inflammatory activities were studied. Further, the in silico studies such as Molecular docking
(with COX-1, COX-2 and 3TTZ), DFT calculations, the Molecular electrostatic potential (MEP)
and ADME were investigated.
Keywords: Hybrid tetrazole, antimicrobial, anti-inflammatory, Molecular docking,
Molecular orbitals.
1. Introduction
The studies of the biological importance of a single heterocyclic compounds and their
derivatives are interesting area in the field of medicinal chemistry [1]. This due to the fact that
75% of the available drugs in the market are heterocycle based drugs. Also, the introduction of
heterocycle on a molecule can change the physiochemical properties such as absorption,
distribution, metabolism and excretion [2]. Several heterocyclic compounds have been proved as
bioactive compounds [3]. Among various bioactive heterocyclic compounds, the tetrazoles and
their derivatives have attracted much attention in drug design [4]. Moreover, these derivatives
have several biological activities such as antimicrobial [5], antiviral [6], anti-inflammatory [7],
anticancer [8], antidiabetic [9] and antitumor [10]. Losartan, Irbesartan and Valsartan are the

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some of the drugs containing 5-aryl tetrazole unit. 1,5-disubstituted tetrazoles based drugs have
been used as an antibiotic (Flomoxef, cefazolin sodium and cefonicid) and anticonvulsant drug.
Another advantage of tetrazole is a bio-isosteres of carboxylic acids [11]. Based on the literature
reports, we have chosen 1,5-disubstituted tetrazole as a bioactive core in our research [12,13].
We expected the combination of another heterocycle on 5th position of tetrazole will contribute
to biological applications. So we designed and synthesis of the tetrazole derivatives with various
basic heterocycles such as thiophene, pyridine, quinoline and studied for their preliminary
biological evaluations such as antimicrobial and anti-inflammatory activities.
Many heterocyclic compounds are biologically potent compound, but active only in in
vitro level. Most of the compounds are failed in further pharmacological studies (like in vivo and
other pharmacological studies). So there is a various unresolved problem in drug design. The
main drawback of drug designing is that the bioactive compounds (in vitro level) are failed in
pharmacological studies such as absorption, distribution, metabolism, excretion and toxicity
(ADMET). And also some of them are not obeying Lipinski rule of five. On considering the
above facts, we carried out molecular docking studies against bacterial DNA Gyrase (3TTZ) and
inflammation responsible proteins namely 1PGG (cyclooxygenase-1) and 4COX
(cyclooxygenase-2)]. The possible binding orientations are identified with the DFT calculation
and Molecular electrostatic potential (MEP), which could explain the binding site of the ligand
and also it will be helpful to design new drugs for further improvement. Hence the in silico
ADMET studies were performed and presented here.
2. Experimental Section
2.1 General consideration

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All the chemicals and solvents (laboratory grade) were purchased from sigma Aldrich
or Merck. The solvents were used without any purification. Melting points were recorded
on sigma melting apparatus SL111140. IR spectrums were recorded in JASCO FT-IR410
using KBr pellet making method. 1H NMR & 13C NMR spectra were recorded on Bruker
300 MHz and 75 MHz instrument in CDCl3 with TMS as an internal standard or DMSO-
d6 for proton and carbon spectra. Chemical shift values are mentioned in δ (ppm) and
coupling constants are given in Hz. Mass spectra were recorded on AB SCIEX 3000 LC-
MS and LCQ Fleet mass spectrometer, Thermo Fisher Instruments Limited, US.
Electrospray ionization mass spectrometric method (ESI-MS). The progress of all
reactions was monitored by TLC on 2 × 5 cm pre-coated silica gel 60 F254 plates of a
thickness of 0.25 mm (Merck). The single crystal X-ray analysis was recorded in Bruker
Kappa APEXII single crystal X-ray diffractometer (Enraf Nonlus CAD4-MV31), SAIF,
IIT Madras. The elemental analyses were recorded in vario EL III CHNS element
analyzer.
2.2 Chemistry
2.2.1. General procedure for the synthesis of amides (1a-m)
To a DMF solution (3 mL) of carboxylic acid (2.00 mmol), was added at 0 °C carbonyl
diimidazole (2.2 mmol). The mixture was stirred for 30 minutes at room temperature. To that
solution corresponding amine (2.00 mmol) was added. The reaction mixture was stirred for 24
hours at room temperature. The completion of the reaction was monitored by TLC. The reaction
mixture was quenched with crushed ice (50 g) and extracted with ethyl acetate (100mL). The
organic layer was washed with water (100 mL) and brine solution (100 mL). The organic layer

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was separated, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The
obtained product was taken to the next step.
2.2.2. General procedure for the synthesis of tetrazole (2a–m)
To an amide (1.01 mmol), phosphorus oxychloride (10.15 mmol) and sodium azide (4.06
o
mmol) was added. The reaction mixture was stirred for 9 hours at 80 C under a nitrogen
atmosphere and then it was cooled carefully quenched with ice water and neutralized with
saturated sodium bicarbonate solution. The product was extracted with ethyl acetate (75 mL),
washed with water (2 x 75 mL) and brine solution (75 mL). The organic layer was separated and
dried over anhydrous Na2SO4 and concentrated under reduced pressure.
2.2.2.1. 1-butyl-5-(thiophen-2-yl)-1H-tetrazole (2a)
Pale Yellowish oil, 1H NMR (300 MHz, CDCl3): δ ppm: 7.67 – 7.63 (m, 2H), 7.29-7.23 (m, 1H),
4.60 – 4.52 (m, 2H), 2.04 – 1.91 (m, 2H), 1.50 – 1.40 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H). 13C NMR
(75 MHz, CDCl3): δ ppm: 149.39, 130.25, 129.91, 128.40, 124.24, 48.12, 31.41, 19.66, 13.42.
ESI-LC/MS calculated m/z 208.2 found 209.1 (M++1). IR (KBr) ʋmax: 3103, 2960, 1572, 1475,
715. Anal.Calcd for: C9H12N4S: C, 51.90; H, 5.81; N, 26.90 %. Found: C, 51.93; H, 5.79; N,
26.89 %.
2.2.2.2. 1-(4-fluorophenyl)-5-(thiophen-2-yl)-1H-tetrazole (2b)
1
Colourless solid, Mp: 143-145 ºC, H NMR (300 MHz, CDCl3): δ ppm: 7.57 – 7.46 (m, 3H),
7.37 – 7.28 (m, 3H), 7.20-7.06 (m, 1H). 13C NMR (75 MHz, CDCl3): δ ppm: 165.62, 162.26,
150.02, 130.89, 130.73, 130.03, 128.71, 128.59, 128.21, 124.00, 117.50, 117.19. ESI-LC/MS
calculated m/z 246.0, found 247.1 (M++1). IR (KBr) ʋmax; 3076, 1568, 1269, 1155, 1097.
Anal.Calcd for: C11H7FN4S: C, 53.65; H, 2.87; N, 22.75 %. Found: C, 53.68; H, 2.88; N, 22.72
%.

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2.2.2.3. 1-phenyl-5-(thiophen-2-yl)-1H-tetrazole (2c)
Reddish Brown solid, Mp: 87-88 ºC, 1H NMR (300 MHz, CDCl3): δ ppm: 7.64 – 7.59 (m, 3H),
13
7.52 – 7.47 (m, 3H), 7.27 – 7.24 (m, 1H), 7.06 - 7.03 (m, 1H). C NMR (75 MHz, CDCl3): δ
ppm: 149.82, 134.01, 131.27, 130.71, 130.55, 130.10, 128.10, 126.35, 124.15. ESI-LC/MS
calculated m/z 228.0, found 229.2 (M++1). IR (KBr) ʋmax; 3078, 1551, 1267, 1123, 1095, 1020.
Anal.Calcd for: C11H8N4S: C, 57.88; H, 3.53; N, 24.54 %. Found: C, 57.89; H, 3.56; N, 24.51 %.
2.2.2.4. 1-(methylsulfonyl)-4-(5-(thiophen-2-yl)-1H-tetrazol-1-yl)piperidine (2d)
Colourless solid, Mp: 163-165 ºC, 1H NMR (300 MHz, CDCl3): δ ppm: 7.69 (dd, J = 5.1, 1.1 Hz,
1H), 7.64 (dd, J = 3.7, 1.1 Hz, 1H), 7.32 – 7.27 (m, 1H), 4.82 – 4,73 (m, 1H), 3.99 – 3.87 (m,
13
2H), 3.19 – 3.07 (m, 2H), 2.89 (s, 3H), 2.50 – 2.38 (m, 2H), 2.33 – 2.24 (m, 2H). C NMR (75
MHz, CDCl3): δ ppm: 148.69, 130.25, 129.82, 128.18, 123.18, 54.78, 43.95, 35.24, 30.97. ESI-
LC/MS calculated m/z 313.1 found 314.2 (M++1). IR (KBr) ʋmax; 2937, 1479, 1249, 1168, 1085,
1058, 949. Anal.Calcd for: C11H15N5O2S2: C, 42.16; H, 4.82; N, 22.35 %. Found: C, 42.15; H,
4.81; N, 22.35 %.
2.2.2.5. 3-(1-butyl-1H-tetrazol-5-yl)pyridine (2e)
1
Pale yellowish oil, H NMR (300 MHz, CDCl3): δ ppm: 8.94 – 8.85 (m, 2H), 8.10 - 8.07 (m,
1H), 7.56 (dd, J = 7.8, 4.9 Hz, 1H), 4.46 (t, J = 7.4 Hz, 2H), 2.05 – 1.90 (m, 2H), 1.38 - 1.33 (m,
2H), 0.93 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ ppm: 152.27, 152.00, 148.90,
136.51, 124.51, 48.19, 31.81, 19.67, 13.39. ESI-LC/MS calculated m/z 203.1, found 204.2
(M++1). IR (KBr) ʋmax: 2962, 2933, 1570, 1464, 707. Anal.Calcd for: C10H13N5: C, 59.10; H,
6.45; N, 34.46 %. Found: C, 59.13; H, 6.46; N, 34.47 %
2.2.2.6. 2-(1-cyclohexyl-1H-tetrazol-5-yl)pyridine (2f)

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1
White solid, Mp: 125-128 °C, H NMR (300 MHz, CDCl3): δ ppm: 8.94 – 8.78 (m, 2H), 8.07 –
7.99 (m, 1H), 7.58 – 7.53 (m, 1H), 4.37 – 4.26 (m, 1H), 2.26 – 2.02 (m, 5H), 2.02 – 1.93 (m,
2H), 1.47 – 1.30 (m, 3H). 13C NMR (75 MHz, CDCl3): δ ppm: 152.15, 151.28, 149.00, 136.64,
124.08, 121.20, 58.67, 33.34, 25.21, 24.72. ESI-LC/MS calculated m/z 229.2 found 230.2
(M++1). IR (KBr) ʋmax: 3078, 2933, 1568, 1462, 705. Anal.Calcd for: C12H15N5: C, 62.86; H,
6.59; N, 30.54 %. Found: C, 62.88; H, 6.58; N, 30.56 %.
2.2.2.7. 3-(1-(4-fluorophenyl)-1H-tetrazol-5-yl)pyridine (2g)
Colourless sloid, Mp: 120-121 ºC, 1H NMR (300 MHz, CDCl3): δ ppm: 8.76 - 8.74 (m, 2H), 8.00
– 7.93 (m, 1H), 7.48 – 7.38 (m, 3H), 7.27 (m, 2H).13C NMR (75 MHz, CDCl3): δ ppm: 165.32,
161.96, 152.32, 151.65, 149.26, 136.28, 130.19, 127.61, 127.49, 123.85, 120.17, 117.69, 117.39.
ESI-LC/MS calculated m/z 241.2 found 242.2 (M++1). IR (KBr) ʋmax; 3076, 1504, 1226, 1134,
1097, 1024, 993. Anal.Calcd for: C12H8FN5: C, 59.75; H, 3.34; N, 29.03 %. Found: C, 59.74; H,
3.36; N, 29.04 %.
2.2.2.8. 3-(1-benzyl-1H-tetrazol-5-yl)pyridine (2h)
Colourless solid, Mp: 98-100 ºC, 1H NMR (300 MHz, CDCl3): δ ppm: 8.82 – 8.72 (m, 1H), 7.99
– 7.90 (m, 1H), 7.52 – 7.44 (m, 1H), 7.36-7.25 (m, 3H), 7.17 – 7.13 (m, 2H), 5.67 (s, 2H). 13C
NMR (75 MHz, CDCl3): δ ppm: 152.35, 152.28, 149.04, 136.40, 133.43, 129.36, 129.03, 127.11,
123.86, 120.50, 51.70. ESI-LC/MS calculated m/z 237.2 found 238.1 (M++1). IR (KBr) ʋmax
:
3051, 2926, 1568, 1454, 704. Anal.Calcd for: C13H11N5: C, 65.81; H, 4.67; N, 29.52 %. Found:
C, 65.80; H, 4.69; N, 29.54 %.
2.2.2.9. 2-(1-butyl-1H-tetrazol-5-yl)quinoline (2i)
Pale yellowish oil, 1H NMR (300 MHz, CDCl3): δ ppm: 8.47 – 8.32 (m, 2H), 8.13 (d, J = 8.5 Hz,
1H), 7.92 (d, J = 8.1 Hz, 1H), 7.86 – 7.79 (m, 1H), 7.70 – 7.64 (m, 1H), 5.21 – 5.10 (m, 2H),

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2.12 – 1.99 (m, 2H), 1.50 - 1.45 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ
ppm: 151.58, 147.28, 144.69, 137.43, 130.39, 129.54, 128.19, 128.05, 127.79, 120.88, 118.68,
49.66, 31.83, 19.63, 13.52. ESI-LC/MS calculated m/z 253.3, found 254.4(M++1). IR (KBr) ʋmax
:
3061, 2960, 1599, 1498, 767. Anal.Calcd for: C14H15N5: C, 66.38; H, 5.97; N, 27.65 %. Found:
C, 66.39; H, 5.96; N, 27.64 %.
2.2.2.10. 2-(1-cyclohexyl-1H-tetrazol-5-yl)quinoline (2j)
White solid, Mp: 82-85 °C, 1H NMR (300 MHz, CDCl3): δ ppm: 8.44 – 8.34 (m, 2H), 8.13 (d, J
= 8.5 Hz, 1H), 7.92 (d, J = 7.4 Hz, 1H), 7.85 – 7.78 (m, 1H), 7.71 – 7.63 (m, 1H), 5.82 – 5.75 (m,
1H), 2.33 – 2.93 (m, 2H), 2.17 – 2.08 (m, 4H), 1.99 – 1.81 (m, 2H), 1.58 – 1.40 (m, 4H). 13C
NMR (75 MHz, CDCl3): δ ppm: 151.40, 147.51, 145.30, 137.57, 130.49, 129.92, 128.33, 127.97,
121.49, 60.12, 32.96, 25.71, 25.27. ESI-LC/MS calculated m/z 279.3 found 280.2 (M++1). IR
(KBr) ʋmax: 2929, 1502, 1462, 763. Anal.Calcd for: C16H17N5: C, 68.79; H, 6.13; N, 25.07 %.
Found: C, 68.76; H, 6.11; N, 25.08 %.
2.2.2.11. 2-(1-(1-(methylsulfonyl)piperidin-4-yl)-1H-tetrazol-5-yl)quinoline (2k)
1
Colourless solid, Mp: 208-210 ºC, H NMR (300 MHz, CDCl3): δ ppm: 8.47 – 8.38 (m, 2H),
8.09 (d, J = 8.4 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.88 – 7.81 (m, 1H), 7.72 – 7.67 (m, 1H), 6.00
– 5.96 (m, 1H), 4.08 – 3.99 (m, 2H), 3.23 – 3.11 (m, 2H), 2.93 (s, 3H), 2.52 – 2.44 (m, 4H). 13C
NMR (75 MHz, CDCl3): δ ppm: 151.67, 147.52, 145.03, 137.95, 130.78, 129.73, 128.64, 128.49,
128.14, 121.52, 56.86, 44.84, 36.45, 31.54. ESI-LC/MS calculated m/z 357.1 found 358.2
(M++1). IR (KBr) ʋmax: 3009, 1496, 1101, 1249, 1157, 1018, 966. Anal.Calcd for: C16H18N6O3S:
C, 51.33; H, 4.85; N, 22.45 %. Found: C, 51.35; H, 4.86; N, 22.47%.
2.2.2.12. 2-(1-(p-tolyl)-1H-tetrazol-5-yl)quinoline (2l)

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1
Colourless solid, Mp: 126-128 ºC, H NMR (300 MHz, CDCl3): δ ppm:8.34 – 8.24 (m, 2H),
7.87 (d, J = 8.1 Hz, 1H), 7.75 - 7.71 (m, 2H), 7.65 – 7.58 (m, 1H), 7.44 (d, J = 8.3 Hz, 2H), 7.32
13
(d, J = 8.0 Hz, 2H), 2.49 (s, 3H). C NMR (75 MHz, CDCl3): δ ppm: 152.81, 147.57, 144.28,
140.35, 137.40, 133.36, 130.39, 130.05, 129.56, 128.40, 127.80, 126.16, 121.37, 21.44. ESI-
LC/MS calculated m/z 287.1, found 288.2 (M++1). IR (KBr) ʋmax; 3069, 1555, 1264, 1146, 1094,
1009. Anal.Calcd for: C17H13N5: C, 71.06; H, 4.56; N, 24.37 %. Found: C, 71.05; H, 4.60; N,
24.39 %.
2.2.2.13. 2-(1-(naphthalen-1-yl)-1H-tetrazol-5-yl)quinoline (2m)
1
Colourless solid, Mp: 110-113 ºC, H NMR (300 MHz, CDCl3): δ ppm: 8.39 – 8.24 (m, 2H),
8.12 - 7.99 (m, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.76 (dd, J = 6.0, 3.6 Hz, 1H), 7.66 – 7.59 (m, 2H),
7.57 – 7.51 (m, 1H), 7.51 – 7.45 (m, 2H), 7.44 – 7.37 (m, 1H), 7.21 (d, J = 8.5 Hz, 1H), 7.05 (dd,
J = 6.1, 3.6 Hz, 1H). 13C NMR (75 MHz, CDCl3): δ ppm: 154.11, 147.21, 143.41, 137.27,
133.95, 132.75, 130.87, 130.13, 129.91, 129.82, 128.31, 128.25, 128.13, 127.79, 127.55, 126.93,
125.39, 124.88, 122.17, 120.70. ESI-LC/MS calculated m/z 323.1, found 324.2 (M++1). IR
(KBr) ʋmax; 3059, 1595, 1253, 1145, 1109, 1039. Anal.Calcd for: C20H13N5: C, 74.29; H, 4.05; N,
21.66 %. Found: C, 74.30; H, 4.07; N, 21.66 %.
2.3. Biology and in silico studies
2.3.1 Antibacterial activity
The bacterial strains used for the examinations were K. pneumoniae (ATCC 13883), P.
aeruginosa (ATCC 10145), S. aureus (ATCC 11632), S. pyogenes (ATCC 12358) and C. albicans
(ATCC 66027) obtained from either American type culture collection or purchased from
Himedia, Mumbai. The experiments were repeated twice. The experimental technique used is as
followed by Sribalan et al [14].

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2.3.2. Anti-inflammatory activity
The synthesized compounds were tested for anti-inflammatory activity (bovine serum
albumin denaturation technique). The experimental technique used is as followed by Sribalan et
al [15].
2.3.3. Molecular docking study
Molecular docking of compounds into the bacterial enzyme, COX-1 and COX-2 enzymes
was carried out using the Auto-Dock software (version 4.2)[16,17]. Accelrys discovery studio
client 4.1 was used for the visualizing protein-ligand complex. Three dimensional structures of
synthesized derivatives were optimized using the Gaussian 09W software (for the ligand). The
crystal structure of the bacterial enzyme, COX-1 and COX-2 (3TTZ.pdb, 1PGG.pdb and
4COX.pdb) were downloaded from Protein Data bank. All bound water and ligand were
eliminated from the protein and polar hydrogen was added. Moreover all docking, a grid box size
of 60x60x60 points in X, Y and Z direction. A grid spacing of 0.375 Å and ten runs were
generated by using Lamarckian genetic algorithm searches.
2.3.4. Computational calculations
All the computational calculations including representation of Highest occupied
molecular orbital (HOMO) and Lowest unoccupied molecular orbital (LUMO) in the checkpoint
files was performed with the Gaussian 09W program using density functional theory [18]. The
chemical structure of the compound was optimized with B3LYP/6.311 ++ G (d,p) basis set. The
Gauss view software package was used to visualize the computed structures including HOMO,
LUMO and Molecular electrostatic potential (MEP) representations.
2.3.5. ADME calculations

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The pharmacological properties like molar volume (MV), topological polar surface area
(TPSA) and Lipinski rule of five were calculated using Molinspiration online tool [19]. The
Absorption (% ABS) was calculated by the formula %ABS = 109 - (0.345 X TPSA) [20] and the
aqueous solubility was calculated using online admetsar tools [21].
3. Results and Discussion
3.1. Chemistry
The designed heterocyclic tetrazoles were synthesized from their corresponding amide
precursors which are represented in Scheme 1. The amides can be prepared from their
corresponding acids and amines with the use of coupling agent like carbonyl diimidazole [22].
The reaction of amides with phosphorus oxychloride and sodium azide yielded tetrazoles [23].
1
The synthesized compounds were characterized by H NMR, 13C NMR, Mass and IR
spectroscopies. The disappearance of amide peak in proton NMR clearly indicates the
conversion of the amide. Similarly, the disappearance of amide carbonyl peak in the region 160
ppm and the appearance of tetrazolyl carbon in the region of 155 ppm clearly indicate the
tetrazoles formation. The ESI-Mass spectrum also attributed the tetrazoles formation. The mass
spectrum clearly showed the molecular ion peaks for the tetrazoles either in positive mode or in
negative mode. Similarly, the IR spectrum also gave the additional evidence for the
disappearance of the carbonyl stretching frequency (1600-1650 cm-1) and appearance new
absorbance which is due to the formation of tetrazoles.
For example, in the characterization of 2a, the disappearance of amide peak at 6.08 ppm
1
13
in H NMR and disappearance of amide carbonyl peak at 162.1 ppm in C NMR indicates the
complete conversion of the amide. The appearance of a new peak at 149.3 ppm in 13C NMR

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indicates the formation of tetrazolyl carbon. The multiplet at 7.66-7.64 ppm for 2 protons and
multiplet at 7.29-7.23 ppm for one proton indicates the presence of thiophene ring. The multiplet
around 4.62-4.50 ppm for 2 protons indicates the presence of CH2 unit which was attached to
tetrazole ring nitrogen. The remaining multiplets around 1.99–1.90 for 2 protons, 1.45–1.42 for 2
protons and 0.98 ppm for 3 protons indicates the presence of n-butyl unit. In the 13C NMR, the
peaks at 130.25, 129.91, 128.40 and 124.24 ppm indicate the presence of thiophene ring. The
peaks at 48.12, 31.41, 19.66 and 13.42 ppm indicate the presence of n-butyl unit. The ESI-Mass
clearly showed the peak at 209.1 in a positive mode which also confirms the product formation.
In the FT-IR spectrum, the band around 3103 cm-1 indicates the presence of aromatic C-H units
(thiophene C-H units). The band around 1572 cm-1 indicates the presence of C=N unit. Similarly,
the band around 1475 cm-1 indicates the presence of N=N unit. In addition, the compound 2g was
confirmed by single crystal X-ray analysis (Fig. 1) (CCDC No: 996387). This list of synthesized
compounds is represented in Fig. 2.
3.2. Biology
3.2.1. Antimicrobial activity
A series of heterocyclic tetrazoles were screened for antimicrobial activity against the
bacterias such as Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC
27853), Staphylococcus aureus (ATCC 25923), Streptococcus pyogenes (ATCC 12358) and
fungal pathogen Candida albicans (ATCC 66027). Except the compound 2f, all the compounds
showed the activity against Klebsiella pneumoniae. The compound 2e has the pyridyl ring and n-
butyl unit showed better inhibition than the standard drug (amikacin) and the compound 2k
bearing quinoline and piperidyl sulfonamide unit showed comparable inhibition to the standard.
The compounds 2b, 2c, 2d, 2g, 2h, 2j, 2l and 2m showed the moderate activity against the

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Klebsiella pneumoniae. The compound 2k showed the low activity against Pseudomonas
aeruginosa and the compound 2d, 2h and 2j exhibited the moderate activity against
Pseudomonas aeruginosa. The compound 2b bearing thiophene and p-fluoro phenyl unit showed
potent activity against Staphylococcus aureus. The compounds 2c, 2e, 2i, 2l and 2m showed low
to moderate activity against the Staphylococcus aureus. Similarly, the compound 2k, bearing the
quinoline and piperidyl sulphonamide unit showed good inhibition against Streptococcus
pyogenes. The compounds 2d, 2h, 2i, 2j and 2l showed moderate activity against Streptococcus
pyogenes. Some of the compounds showed the antifungal activity. Among those compounds, 2b
having the thiophene and phenyl ring showed good activity against Candida albicans. The
compounds 2a, 2c and 2g showed moderate activity against a fungal pathogen. Overall, the
synthesized compounds showed better to good zone of inhibition than parent thiophene,
quinoline, pyridine and tetrazole. The zone of inhibitions of the compounds, standard and parent
were listed in Table 1.
3.2.2. Anti-inflammatory activity
All the synthesized compounds were studied for their in vitro anti-inflammatory activity by
using inhibition of albumin denaturation technique with minor modification. The activities of the
compounds were checked with four different concentrations of 50 µg/mL, 100 µg/mL, 200
µg/mL and 400 µg/mL respectively. Diclofenac sodium was used as the standard drug. All the
compounds showed moderate to good anti-inflammatory activity. The compound 2b and the
compound 2i showed a good percentage of inhibition, which is almost equal to the standard
diclofenac sodium. Other than these, all the compounds showed moderate anti-inflammatory
activity. Overall, the synthesized compounds showed good to moderate anti-inflammatory

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activity than parent thiophene, quinoline, pyridine and tetrazole. The various concentrations of
compounds, standard, and parent’s percentage inhibitions were listed in Table 2.
3.2.3. ADME prediction
All the synthesized heterocyclic hybrids have been screened for in silico ADME
properties by using online tool Molinspiration and ADMETsar. The molecular weights of
compounds are in the range of 208-358 g/mol. The log P value (partition coefficient in octanol-
water) is 1.01-4.18. The numbers of hydrogen bond acceptor in the ligands are 4-8. A number of
hydrogen bond donors are zero. The Molar volumes of the synthesized compounds are in the
range of 186-283. Hence all the compounds are obeying the Lipinski rule of five. Also, the other
parameters have been checked such as TPSA (topological polar surface area), Q log p and
number of rotatable bonds. All the values calculated for the synthesized compounds are
comparable with the 95% ideal drugs in the market [24]. The percentage absorption is calculated
from topological polar surface area [%ABS = 109 – (0.345 x TPSA)]. Usually, the compounds
having more than 80% absorption can be considered as a good drug. Among these compound
except 2d (72%) and 2k (67%), all the other compounds have good absorption (>80%). Some of
the compounds showed around 93 % absorption. From all the above prediction, we theoretically
suggest that all the synthesized compounds have very good ADME properties. The ADME
properties for the synthesized compounds are represented in Table 3.
3.2.4. Molecular Docking studies
3.2.4.1. DNA gyrase inhibition (3TTZ)
DNA gyrase inhibition is one of the mechanisms for preventing the bacterial replication
process which induces bacterial death [25]. Some nitrogen based heterocyclic drugs are used as a
DNA gyrase inhibitor (Nalidixic acid, ciprofloxacin). On the basis of this idea, the enzyme 3TTZ

ACCEPTED MANUSCRIPT
(DNA gyrase enzyme) was chosen for docking. All the synthesized compounds were docked
with the DNA gyrase enzyme 3TTZ to identify the possible protein-ligand interaction.
Generally, the ligands are binding with protein via various interactions such as pi-alkyl
interaction, pi-donor hydrogen bond interaction and conventional hydrogen bond interaction.
Nitrogen in the synthesized compounds can form hydrogen bond with the amino acid of
the enzyme. Similarly, the thiophene based derivatives also showed the conventional hydrogen
bond interactions with the amino acid. But the nitrogen present in either pyridine derivative or
quinoline derivative did not show any conventional hydrogen bond interactions. Most of the
compounds showed hydrogen bond with the amino acid GLY85 of the enzyme 3TTZ. But the
compound 2d showed the hydrogen bond with the amino acid GLU58 and the compound 2f
showed the hydrogen bond with the amino acid SER129. In the compound 2f, the tetrazole
nitrogen form the hydrogen bond with hydroxyl hydrogen (N---HO) of SER129. In the
compounds 2c, 2e, 2h and 2i, the tetrazole nitrogen interacted by the hydrogen bond with peptide
nitrogen of GLY85 (N---HN). The entire group of synthesized compounds exhibited very good
bind energies and inhibition constants. The binding energies are in the range of -5.97 to -9.06
kcal/mol and inhibition constant values in the range of 51.96 to 0.22 µM. Among the various
compounds, the compound 2m quinoline tetrazoles containing naphthalene ring showed highest
binding energy and inhibition constant. Overall, the quinoline tetrazoles derivative showed very
good binding interactions with DNA gyrase enzyme than the thiophene and pyridyl tetrazoles
derivatives. The model protein-ligand complex (2l with 3TTZ) is represented in Fig. 3. The
Docking energy, inhibition constant and hydrogen bonding of ligand-protein complexes were
represented in Table 4.
3.2.4.1. Cyclooxygenase inhibition

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The synthesized compounds exhibited in vitro anti-inflammatory activity.
Cyclooxygenase is one of the enzymes responsible for producing prostaglandin. The
cyclooxygenase enzyme inhibition will provide pain relief and cure inflammation [26]. On the
basis of this, the protein 1PGG and 4COX were chosen for docking. All the synthesized
compounds were docked with COX-1(1PGG) and COX-2(4COX) enzyme. Similar to the DNA
gyrase, the docking against cyclooxygenase showed several interactions with COX-1 (1PGG)
enzyme. Among the 13 synthesized hybrids, the compound 2b and 2c containing n-butyl and p-
fluoro phenyl substituents showed hydrogen bonding with SER530 of 1PGG. Similarly, the
compounds 2e, 2f and 2g containing n-butyl, cyclohexyl and p-fluoro phenyl substituents
showed hydrogen bond with SER530 of 1PGG. The compound 2m quinoline tetrazoles
containing naphthalene ring showed hydrogen bond with TYR385. The hydrogen bonds are Pi-
donor hydrogen bonds which were formed between aromatic ring of the synthesized compounds
and the hydrogen of the amino acid (SER530 or TYR385). All the compounds showed very good
binding energies (-7.11 to -11.35 kcal/mol) and inhibition constants (6.11 to 0.004 µM). Within
this series, the quinoline based tetrazoles 2j, 2k, 2l and 2m showed highest binding energy which
is -11.35, -10.25, -10.76 and -10.87 kcal/mol and their inhibition constants are 0.004, 0.032,
0.012 and 0.010 µM respectively. In the overall docking of the COX-1 enzyme with the
synthesized compounds, quinoline based tetrazoles derivative showed better docking results than
thiophene and pyridyl tetrazoles. Similarly, the synthesized compounds were docked with a
COX-2 enzyme (4COX). Most of the compounds showed hydrogen bond interaction with amino
acid of the COX-2 enzyme as similar to COX-1. The thiophene and pyridine based tetrazoles
(2a-h) showed the hydrogen bond with SER530. The compound 2a thiophene tetrazole
containing n-butyl substituents exhibited the conventional hydrogen bond interaction between

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tetrazole nitrogen and hydroxyl group of SER530 (N---HO). The compound 2h containing
benzyl substituent showed both conventional and pi-donor hydrogen bond interactions with
SER530 (N---HO, Tet---HO). In the compounds 2b, 2c, 2f and 2i, the tetrazole rings have shown
the pi-donor hydrogen bond interaction with hydroxyl of SER530 (Tet---HO). In the case of
compound 2a and 2f, two hydrogen bond interactions were formed with two amino acids of
TYR385 and SER530. The compound 2k containing piperidine sulphonamide unit showed
hydrogen bond with ARG120. The entire list of compounds showed very good binding energies
(-6.85 to -10.41kcal/mol) and inhibition constants (9.47 to 0.023 µM). Among these derivatives,
the compounds 2j, 2k, 2l and 2m quinoline tetrazoles showed the highest binding energies (-
9.37, -10.41, -9.48 and -10.16 kcal/mol) and their inhibition constants were 0.135, 0.023, 0.112
and 0.356 µM respectively. Overall, quinoline based tetrazoles derivative showed better docking
results than thiophene and pyridyl tetrazoles. The model protein-ligand complexes (2l with
1PGG and 2l with 4COX) are represented in Fig. 3 & 4. The binding energy, inhibition constant
and hydrogen bonding of COX-1 and COX-2 with amino acids were represented in Table 5 & 6.
3.5. Computational Studies
3.5.1. Frontier molecular orbitals
The highest occupied molecular orbital and lowest unoccupied molecular orbital can be
used to predict the most reactive position of the molecule in pi-electron systems. In an organic
molecule, the electrons are not assigned to individual covalent bond, which is moving under the
whole molecule by the influence of nuclei of the molecule. So the electron density of the
molecule has not same in all the region of the molecule. The electron cloud plays a major role in
the intermolecular interaction. The protein-ligand docking method depends on various
intermolecular interactions such as pi-donor hydrogen bond interaction, pi-alkyl interaction, pi-

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sigma interaction and pi-anion interaction. In all compounds, the electron density is more in
tetrazole and heterocyclic rings. In some cases (2c, 2g, 2h, 2l and 2m) the additional aromatic
side chains also have more electron density in either HOMO or LUMO. So these regions should
be responsible for interactions with the protein. The same results were obtained in molecular
docking studies, where the interactions were found between the aromatic rings (tetrazole,
heterocycle and phenyl ring) and amino acids. Especially the tetrazolyl nitrogen makes the
conventional hydrogen bond and pi-donor hydrogen bond with particular amino acids (GLY 85,
GLU 58, SER129, SER530, TYR385, TRP387 and ARG120).
Similarly, the thiophene sulphur makes the conventional hydrogen bonding with an
amino acid (GLY85, GLU 58, SER 530, TYR 385 and TRP 387). The aliphatic side chains do
not have more electron cloud. So there is less interaction with the protein. Comparatively,
quinoline tetrazoles have more electron density than the thiophene and pyridine tetrazoles.
Probably the quinoline tetrazole 2i, 2j, 2k, 2l and 2m have the more interaction with proteins
than the others. Among the quinoline tetrazoles, the compounds having the aromatic side chains
2l and 2m has better interaction than the others heterocyclic hybrids.
HOMO-LUMO band gap ∆E plays an important role in the chemical stability of the
molecule. The band gap values of the synthesized compounds (2a-m) are in the range of 4.07 to
5.69 eV. The band gap values clearly indicate the heterocyclic tetrazoles are chemically stable.
Similarly, the chemical potential and the global hardness are higher. The chemical potential and
hardness of the compounds are in the range of –4.31 to -4.90 eV and 2.03 to 2.84 eV
respectively. Also, the global softness of the synthesized compound is 0.17 to 0.24 eV-1. From
the results, we suggest the heterocyclic tetrazoles are hard material and chemically stable
compound. Comparatively, within this series the stability order is pyridine tetrazoles>thiophene

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tetrazole>quinoline tetrazole. The electrophilicity index is measured from chemical hardness and
chemical potential of the molecule.
The Electrophilicity is the possible descriptor of biological activity [27] which is the
capacity to accept the electrons from the environment. So, higher the electrophilicity index
higher will be the binding interaction with the environmental molecule. The electrophilicity
index of the synthesized compounds (2a-m) is in the range of 3.75 to 5.28. Comparatively the
quinoline tetrazole has the more electrophilicity index value than thiophene and pyridine
tetrazole derivative. The molecular docking studies also exhibited the similar results, the
quinoline tetrazoles having more interaction with the proteins. The global molecular reactive
descriptors such as HOMO, LUMO, band gap, chemical potential, global hardness, global
softness and electrophilicity index of the heterocycle tetrazole hybrids are represented in Table 7
and coherent with the commercially available drug (paracetamol)[28]. From the DFT results, we
suggest that the tetrazole heterocycle hybrids could be better molecules. The Frontier molecular
orbital of compound 2l is represented in Fig. 6.
The above DFT parameters were calculated from the given equations.
Band gap
Chemical potential µ = (EHOMO + ELUMO)/2
∆E = ELUMO – EHOMO
Global hardness
Global softness
ƞ = (ELUMO-EHOMO)/2
ζ = 1/2ƞ
Electrophilicity index ω = µ2/2ƞ
3.5.2. Molecular Electrostatic potential

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Molecular electrostatic potential (MEP) displays the charge distribution of molecule in
surfaces which can be helpful to find the electrophilic and nucleophilic site of the molecule. And
also this is the well suited method for analyzing the process of binding interaction and hydrogen
bonding interaction of biomolecules with a ligand which will be helpful to the biological activity
of the molecule [29]. The electrophilic site is visually represented in red colour which indicates
the negative region of the molecule. The nucleophilic site is blue in colour which indicates the
positive region of the molecule. The negative region of the molecule is more important because
which is ready to make the hydrogen bonding with protein. Here, all the 13 compounds were
studied for molecular electrostatic potential. The MEP clearly showed the negative region is
localized over tetrazolyl nitrogen which is probably responsible for hydrogen bonding. The
molecular docking studies also proved, the tetrazole ring showed more hydrogen bond with
enzymes. The surface nitrogen N2, N3 and N4 of tetrazoles are making the conventional
hydrogen bonding with amino acids. In the pyridine tetrazoles (2e, 2f, 2g and 2h), the pyridyl
nitrogen showed little negative charge which is not involving in hydrogen bonding with the
protein (results from molecular docking). The quinoline nitrogen (2i, 2j, 2k, 2l and 2m) has not
shown any negative charge and there is no hydrogen bonding with the enzymes (results from
molecular docking). The aromatic ring surfaces are the nucleophilic site. Especially the quinoline
ring surface clearly showed blue colour which is positive charge region. This may be responsible
for the other interactions with the enzyme. Overall the quinoline tetrazoles have both negative
and positive charged surface which make this series of molecules to have more interactions with
the amino acids of the enzyme. The Molecular electrostatic potential mapping of compound 2l is
represented in Fig. 7.
Conclusions

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The tetrazole-heterocycle hybrids exhibited excellent binding with enzymes in both
bacterial and inflammatory studies. In the molecules, the tetrazoles are the responsible unit
which makes more interaction with the enzyme. The in silico pharmacological results for the
hybrids were very good which is comparable to the regular drugs. The DFT parameters and MEP
clearly explains the tetrazole unit and aromatic regions are more responsible units in biological
interaction. Within the series, the quinoline tetrazole exhibited very good results than pyridyl and
thiophene tetrazole hybrid. So the tetrazole-heterocycle hybrids may be the lead molecule in
further studies. Further the current research is focused to study the experimental binding of the
enzyme with heterocyclic tetrazoles.
Acknowledgments
The authors are grateful to UGC and DST, New Delhi for financial support and DST-
IRHPA program for providing high resolution NMR facility.
Figures and captions:
Scheme 1. Synthesis of tetrazoles-heterocycle hybrid.
Fig 1. ORTEP diagram of compound 2g.
Fig .2. List of synthesized compounds (2a-m).
Fig. 3. Interaction of compound 2l with 3TTZ.
Fig. 4. Interaction of compound 2l with 1PGG.
Fig. 5. Interaction of 2l with 4COX.
Fig. 6. The HOMO and LUMO of Compound 2l.
Fig. 7. Molecular electrostatic potential mapping of compound 2l.
Tables and captions:

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Table 1. Antimicrobial activity of heterocyclic tetrazoles 2a-m (Zone of inhibition in mm).
Table 2. Anti-inflammatory activity of heterocyclic tetrazoles 2a-m (% inhibition).
Table 3. In silico physicochemical pharmacokinetic parameters important for good oral
bioavailability of synthesized compounds (2a-m).
Table 4: Molecular docking interaction of synthesized compounds (2a-m) against DNA gyrase.
Table 5: Molecular docking interaction of synthesized compounds (2a-m) against 1PGG.
Table 6. Molecular docking interaction of synthesized compounds (2a-m) against 4COX.
Table 7: DFT calculations of synthesized compound (2a-m).
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Table: 1
Entry
K.Pneum P.aerugin
S.
S.pyogen
C.
oniae
osa
aureus
es
albicans
2a
13.2±0.4
10.2±0.4
10.2±0.2
-
-
-
-
-
11.2±0.3
2b
15.0±0.1
-
16.1±0.3
2c
4.1±0.2
-
8.9±0.2
2d
14.2±0.2 10.2±0.3
-
8.0±0.3
-
-
2e
17.2±0.2
-
-
7.2±0.3
-
2f
-
-
-
-
2g
10.1±0.1
7.8±0.5
10.4±0.3
-
-
-
8.2±0.1
2h
10.2±0.1
-
-
12.0±0.1
8.0±0.2
12.2±0.3
15.9±0.2
8.2±0.3
-
-
2i
2j
7.1±0.2
-
12.2±0.2 12.0±0.3
-
-
2k
15.1±0.1
10.1±0.3
12.1±0.1
5.3±0.2
6.7±0.2
5.8±0.1
8.0±0.3
3.9±0.2
-
-
2l
-
4.6±0.3
4.2±0.4
-
-
2m
-
-
Thiophene
Quinoline
Pyridine
Tetrazole
S1
-
4.2±0.2
5.6±0.2
-
-
-
5.2±0.2
-
-
4.2±0.2
5.2±0.2
4.2±0.2
7.0±0.3
21.2
4.6±0.3
10.1±0.2
S2
17.2
17.0
18.2
18.1
Control – Dimethylsulfoxide: Standard – S1:Ketoconazole for fungi, S2:Amikacin for bacteria.

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Table :2
Entry
50µg/mL
100µg/mL
200µg/mL
400µg/mL
53.61±0.45
74.44±1.02
60.21±0.34
57.11±0.65
58.61±0.12
60.21±0.33
58.04±0.23
55.87±0.12
73.31±0.54
61.21±0.54
55.87±0.12
49.83±0.12
53.89±0.65
50.98±1.0
53.12±0.67
48.90±0.98
52.57±0.45
79.03
2a
11.15±0.80 21.13±0.67 45.77±0.21
17.60±0.41 41.93±0.43 63.68±0.23
12.41±0.52 28.71±0.31 45.77±0.21
11.49±0.34 20.62±0.51 48.32±0.44
11.30±0.81 20.21±0.31 47.62±0.65
12.60±0.21 25.96±0.44 49.67±1.05
12.01±0.32 22.23±0.21 49.83±0.34
12.66±0.11 28.71±0.23 47.26±0.78
16.30±1.51 37.93±0.87 59.87±0.65
13.05±0.45 27.69±0.34 50.64±0.13
12.63±0.41 25.96±0.23 49.83±0.76
11.06±0.56 21.02±1.20 40.39±0.21
12.35±0.67 25.96±0.45 48.14±0.11
2b
2c
2d
2e
2f
2g
2h
2i
2j
2l
2k
2m
Thiophene
Quinoline
Pyridine
Tetrazole
S3
7.01±0.32
11.01±0.44 22.48±0.33 41.27±0.54
6.98±0.54 14.49±0.45 31.92±0.65
10.02±0.32 18.62±0.87 39.25±0.65
14.00 20.42 63.61
15.98±0.23 33.55±0.45
Standard-S3: diclofenac sodium

ACCEPTED MANUSCRIPT
Table: 3
Compound
Abs
%
TPS
A
(A2)
<140
MV
n-
ROT
B
MW
<500
208.2
mi
log
P
n-ON
acceptors OHNH
donars
n-
QPlogs
Lipinski
violations
Ideal Range
in 95% of
drugs
-
-
<15
<5
<10
<5
-6.5-0.5
<1
2a
93.9
93.9
93.9
72.0
80.5
80.5
80.5
80.5
80.5
80.5
67.6
80.5
80.5
43.61 186.03
43.61 195.40
43.61 190.47
80.99 253.03
56.50 191.16
56.50 214.19
56.50 200.53
56.50 212.40
56.50 235.15
56.50 258.18
93.88 302.15
56.50 256.15
56.50 283.59
4
2
4
3
4
2
2
3
4
2
3
2
2
2.66
4
4
4
7
5
5
5
5
5
5
8
5
5
0
0
0
0
0
0
0
0
0
0
0
0
0
-1.53
-2.78
-1.64
-2.17
-1.53
-1.98
-3.40
-1.78
-2.78
-2.50
-3.18
-2.75
-2.88
0
0
0
0
0
0
0
0
0
0
0
0
0
2b
2c
2d
2e
2f
246.27 2.66
228.28 2.49
313.41 1.01
203.35 1.80
229.29 2.27
241.23 1.80
237.27 1.96
253.31 2.97
279.35 3.44
358.43 1.33
287.33 3.26
323.36 4.18
2g
2h
2i
2j
2k
2l
2m