Synthesis and pharmacological evaluation of pyridinyl-1,3,4-oxadiazolyl-ethanone derivatives as antimicrobial, antifungal and antitubercular agents

Article abstract of DOI:10.1007/s00044-017-2098-0

Abstract: Nicotinic acid was converted into different substituted acetylated nicotinic acid derivatives by sequential transformation involving formation of hydrazide, Schiff’s base and finally acylated oxadiazole derivatives. The synthesized compounds were characterized by spectroscopic techniques and evaluated in vitro for the antimicrobial, antifungal, as well as antitubercular activity. Among all the synthesized derivatives, compounds 6b, 6d, 6e, 6g, and 6j demonstrated excellent antimicrobial activity on Bacillus subtilis. The compounds 6d, 6j and compounds 6b, 6f, 6h, and 6i exhibited maximum zone of inhibition against fungi Candida albicans as well as Aspergillus niger, respectively at the concentration of 500 μg/mL. The antitubercular activity exhibited by 6f, 6g, and 6d with minimum inhibitory concentration (MIC) values of 1.2, 3.1, and 7.8 μg/mL, respectively. The synthesized compounds were studied by molecular docking through Autodock Vina to evaluate their interaction at respective proteins. Further the effect of synthesized derivatives on surface morphology of human erythrocytes as well as hemolysis was also evaluated. The results demonstrated lesser extent of hemolytic toxicity.

Full text of DOI:10.1007/s00044-017-2098-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2098-0
ORIGINAL RESEARCH
Synthesis and pharmacological evaluation of pyridinyl-1,3,4-
oxadiazolyl-ethanone derivatives as antimicrobial, antifungal and
antitubercular agents
Mohammad Hashim Mansoori1 Gopal L. Khatik2 Vijay Mishra3
●
●
Received: 12 June 2017 / Accepted: 12 October 2017
© Springer Science+Business Media, LLC 2017
Abstract Nicotinic acid was converted into different sub-
stituted acetylated nicotinic acid derivatives by sequential
transformation involving formation of hydrazide, Schiff’s
base and finally acylated oxadiazole derivatives. The syn-
thesized compounds were characterized by spectroscopic
techniques and evaluated in vitro for the antimicrobial,
antifungal, as well as antitubercular activity. Among all the
synthesized derivatives, compounds 6b, 6d, 6e, 6g, and 6j
demonstrated excellent antimicrobial activity on Bacillus
subtilis. The compounds 6d, 6j and compounds 6b, 6f, 6h,
and 6i exhibited maximum zone of inhibition against fungi
Candida albicans as well as Aspergillus niger, respectively
at the concentration of 500 µg/mL. The antitubercular
activity exhibited by 6f, 6g, and 6d with minimum inhibi-
tory concentration (MIC) values of 1.2, 3.1, and 7.8 µg/mL,
respectively. The synthesized compounds were studied by
molecular docking through Autodock Vina to evaluate their
interaction at respective proteins. Further the effect of
synthesized derivatives on surface morphology of human
erythrocytes as well as hemolysis was also evaluated. The
results demonstrated lesser extent of hemolytic toxicity.
Graphical abstract
●
●
●
Keywords Tuberculosis Nicotinic acid Schiff base
●
●
Antibacterial activity Antifungal activity Hemolytic
toxicity
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2098-0) contains supplementary
material, which is available to authorized users.
* Vijay Mishra
Introduction
vijaymishra2@gmail.com
vijay.20352@lpu.co.in
Currently microbial diseases are major concern due to
developing resistance against the existing antimicrobial
agents. Among the microbial diseases bacterial and fungal
infections are more likely to cause the high rate of morbidity
and mortality worldwide. Tuberculosis (TB), a respiratory
transmitted disease representing a formidable challenge to
global public health, is caused by the infection with various
strains of the air-born pathogen of genus Mycobacterium
1
Department of Medicinal Chemistry, Adina Institute of
Pharmaceutical Sciences, Sagar, MP 470002, India
2
Department of Pharmaceutical Chemistry, Lovely Institute of
Technology (Pharmacy), Lovely Professional University,
Phagwara, Punjab 144411, India
3
Department of Pharmaceutics, Lovely Institute of Technology
(Pharmacy), Lovely Professional University, Phagwara, Punjab
144411, India

Med Chem Res
consisting chloroform and methanol in 1:1 ratio. All che-
micals used were of analytical grade purchased from
respective manufacturers. Open capillary method was
employed for the determination of melting points and were
uncorrected.
Fourier transformed infrared spectroscopic data was
recorded on spectrophotometer (Perkin Elmer 783, Pyrogen
1000 Spectrophotometer, USA) employing KBr disc
method. Mass spectra of all the synthesized compounds
were recorded on a JEOL GC Mass spectrometer (Japan).
1
The H nuclear magnetic resonance (NMR) spectra of the
synthesized acetylated schiffs base of nicotinic acid were
recorded in terms of chemical shifts (δ, ppm) on JEOL GSX
400 spectrometer (Japan) using trimethyl silaxane as inter-
nal standard and dimethyl sulphoxide (DMSO) as solvent
(Supplementary data).
Fig. 1 Nicotinic acid, isonicotinic acid and its antimicrobial derivative
(isoniazid)
mainly Mycobacterium tuberculosis (M. tbc) (Nookala and
Subash 2013). In 2015, as per World Health Organization
(WHO) statistics, an estimated 10.4 million new TB cases
and 1.4 million TB deaths were reported worldwide. During
the period of year 2000 and 2015, the deaths due to TB cut
down by 22%, however TB stayed as one of the top 10
causes of death worldwide in 2015 (WHO 2015).
General procedure of Schiff’s base
Mixture of nicotinic acid (0.03 mol, 1.0 equiv) and PCl5,
(0.05 mL, 1.5 equiv) in 20 mL anhydrous CCl4 was refluxed
for 2 h. Solvent was distilled off and nicotinoyl chloride was
obtained (Navarrete-Vázquez et al. 2007). Further the
hydrazine hydrate (0.1 mol, 3.3 equiv) was added in nico-
tinoyl chloride (0.03 mol, 1.0 equiv) drop wise at 0 °C, the
resultant mixture was separated out and washed with aqu-
eous sodium carbonate (10% w/v) and dried under vacuum.
The crystalline nicotinoyl hydrazide was obtained by
recrystallization with methanol (Kalia et al. 2007). Further
nicotinoyl hydrazide (0.01 mol, 1.0 equiv) and aromatic
aldehyde (0.01 mol, 1.0 equiv) were mixed in tetra-
hydrofuran (25 mL) and heated gently for 2 h at 60 °C. The
reaction mixture was then poured into ice cold water and
filtered. The pure compound, Schiff base was obtained by
recrystallization with methanol (Boovanahalli et al. 2007).
Although a number of drugs are available for the treatment
of TB but most of them fail to treat TB completely, because
the interruption of antibiotics course and insufficient level of
drug in the body are unable to kill 100% of mycobacteria.
Among all the drugs, high-dose of isoniazid, pyrazinamide,
and ethambutol (the first-line drugs) are the first choice for the
treatment of TB, while fluoroquinolones (levofloxacin,
ciprofloxacin, ofloxacin) are the second choice (Slayden et al.
2000; Caminero et al. 2010), but isoniazid and flor-
oquinolones have limited use due to its hepatotoxicity and
nephrotoxicity. These drawbacks suggest that the design of
newer and more potent antitubercular (anti-TB) drugs with
minimal toxic effects for improved treatment of drug-resistant
and drug-sensitive TB is imperative (Punkvang et al. 2010).
From the past few decades the chemical research revealed
that the nicotinic acid (pyridine-3-carboxylic acid) and iso-
nicotinic acid (pyridine-4-carboxylic acid) derivatives like
isoniazid (Fig. 1), a well known anti-TB drug showed potent
antimicrobial activity (Jo et al. 2004; Lourenço et al. 2007;
Navarrete-Vázquez et al. 2007; Desai et al. 2008).
Synthesis of acetylated nicotinic acid derivatives (6a–j)
Acetic anhydride (10 mL) and Schiff base (0.01 mol) were
dissolved in 25 mL of ethanol and refluxed for 2 h. Then
reaction mixture was concentrated and allowed to cool
resulting in acetylated product, which was filtered, washed
with water and recrystallized using methanol (Pandey et al.
2005). This final step resulted in the synthesis of different
acetylated nicotinic acid derivatives (compounds 6a–j)
(Table 1), which were characterized using various spectro-
scopic techniques.
The present investigation was aimed to find out newer
acetylated derivatives of nicotinic acid with potent anti-
bacterial, antifungal, and antitubercular activities.
Materials and methods
1-(2-Phenyl-5-(Pyridin-3-yl)-1,3,4-oxadiazole-3 (2H)-yl)
Chemistry
ethanone (6a)
The synthesized compounds were purified by thin layer
Yield: 62%; mp 145–147 °C; IR (KBr) νmax 3009, 2901,
1
chromatography technique using
a
solvent system
1686, 1602, 1584, 1329, 1323 cm−1: H NMR (DMSO-d6,

Med Chem Res
Table 1 Synthesis of nicotinic acid derivatives (6a–j)
Synthesized compounds
R1
R2
R3
R4
R5
Yield (%)
6a
6b
6c
6d
6e
6f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
62
65
63
67
61
68
66
64
65
63
H
N(CH3)2
H
H
H
H
H
Cl
NO2
H
H
H
H
H
OCH3
NO2
H
H
Cl
H
H
H
6g
6h
6i
NO2
OCH3
H
H
H
OH
H
H
CH3
OCH3
H
6j
OCH3
H
H
400 MHz): 7.3–9.2 (m, 9H), 8.1 (s, 1H), 2.4 (s, 3H); 13C
NMR (DMSO-d6, 100 MHz) δ: 23.4 (CH3), 74.5 (CH),
123.9 (CH), 126.3 (C), 126.8 (CH), 127.0 (CH), 128.6
(CH), 137.2 (CH), 140.4 (C), 151.5 (CH), 152.0 (CH),
155.0 (C), 168.6 (C); EIMS m/z 267.17 (M+); Anal
C15H13N3O2 calcd. C, 67.4, H, 4.9, N, 15.72. Found C,
67.1, H, 4.6, N, 15.64.
(CH), 123.9 (CH), 126.3 (C), 126.7 (CH), 128.2 (CH),
128.4 (CH), 128.7 (CH), 132.3 (C), 137.2 (CH), 142.8 (C),
151.5 (CH), 152.0 (CH), 155.0 (C), 168.6 (C); EIMS m/z
301.51 (M+); Anal C15H12ClN3O2 calcd. C, 59.71, H, 4.01,
N, 13.93. Found C, 59.68, H, 4.0, N, 35.84.
1-(2-(2-Nitrophenyl)-5-(pyridine-3-yl)-1,3,4-oxadiazole-3
(2H)-yl) ethanone (6d)
1-(2-(4-dimethylamino)phenyl)-5-(pyridine-3-yl)-1,3,4-
oxadiazole-3(2H)-yl) ethanone (6b)
Yield: 67%; mp 189–191 °C; IR (KBr) νmax:: 3029, 2849,
1669, 1651, 1593, 1420, 1343 cm−1; 1H NMR (DMSO-d6,
400 MHz): 7.6–9.2 (m, 8H), 8.1 (s, 1H), 2.4 (s, 3H); 13C
NMR (DMSO-d6, 100 MHz) δ: 23.4 (CH3), 65.9 (CH),
120.9 (CH), 123.9 (CH), 126.3 (C), 127.7 (CH), 127.9
(CH), 134.7 (CH), 137.2 (CH), 137.5 (C), 147.0 (C), 151.5
(CH), 152.0 (CH), 155.0 (C), 168.6 (C); EIMS m/z 312.71
(M+); anal C15H12N4O4 calcd. C, 57.69, H, 3.87, N, 17.94.
Found C, 57.61, H, 3.82, N, 17.83.
Yield: 65%; mp 170–172 °C; IR (KBr) νmax: 3080, 2924,
1
1701, 1632, 1597, 1421, 1361 cm−1; H NMR (DMSO-d6,
400 MHz): 6.6–9.2 (m, 8H), 8.1 (s, 1H), 2.4 (s, 3H), 2.9 (s,
6H); 13C NMR (DMSO-d6, 100 MHz) δ: 23.4 (CH3), 40.3
(CH3), 74.5 (CH), 114.1 (CH), 123.9 (CH), 126.3 (C),
127.9 (CH), 129.9 (CH), 137.2 (CH), 147.6 (C), 151.5
(CH), 152.0 (CH), 155.0 (C), 168.6 (C); EIMS m/z 310.25
(M+); Anal C17H18N4O2 calcd. C, 65.79, H, 5.85, N, 18.05.
Found C, 65.68, H, 5.76, N, 18.02.
1-(2-(4-Methoxyphenyl)-5-(pyridine-3-yl)-1,3,4-oxadiazole-
3(2H)-yl) ethanone (6e)
1-(2-(2-chlorophenyl)-5-(pyridine-3-yl)-1,3,4-oxadiazole-3
(2H)-yl) ethanone (6c)
Yield: 61%; mp 173–175 °C; IR (KBr) νmax cm−1: 3072,
1
2924, 1701, 1632, 1597, 1421, 1361, 1293 cm−1; HNMR
(DMSO-d6, 400 MHz): 6.8–9.2 (m, 8H), 8.1 (s, 1H), 2.4 (s,
3H), 3.7 (s, 3H); 13C NMR ((DMSO-d6, 100 MHz) δ: 23.4
(CH3), 55.9 (CH3), 74.5 (CH), 114.1 (CH), 123.9 (CH),
126.3 (C), 132.7 (C), 137.2 (CH), 151.5 (CH), 152.0 (CH),
155.0 (C), 158.7 (C), 168.6 (C); EIMS m/z 297.33 (M+);
Yield: 63%; mp 180–182 °C; IR (KBr) νmax:: 3058, 2885,
1673, 1638, 1594, 1419, 1360, 762 cm−1
;
1H NMR
(DMSO-d6, 400 MHz): 7.2–9.2 (m, 8H), 8.1 (s, 1H), 2.4 (s,
3H); 13C NMR (DMSO-d6, 100 MHz): δ: 23.4 (CH3), 65.4

Med Chem Res
anal C16H15N3O3 calcd. C, 64.64, H, 5.09, N, 14.13. Found
C, 64.56, H, 5.06, N, 14.08.
anal C16H15N3O2 calcd. C, 68.31, H, 5.37, N, 14.94. Found
C, 68.26, H, 5.15, N, 14.67.
1-(2-(3-Chloro-4-nitrophenyl)-5-(pyridine-3-yl)-1,3,4-
1-(2-(3,4-Dimethoxyphenyl)-5-(pyridine-3-yl)-1,3,4-
oxadiazole-3(2H)-yl) ethanone (6f)
oxadiazole-3(2H)-yl) ethanone (6j)
Yield: 68%; mp 182–184 °C; IR (KBr) νmax: 3000, 2900,
Yield: 63%; mp 140–142 °C; IR (KBr) νmax:: 3210, 2915,
1
1700, 1640, 1596, 1468, 1423, 1375, 738 cm−1; H NMR
1698, 1652, 1596, 1417, 1373, 1301 cm−1 1H NMR
;
(DMSO-d6, 400 MHz): 7.6–9.2 (m, 7H), 8.1 (s, 1H), 2.4 (s,
3H); 13C NMR (DMSO-d6, 100 MHz) δ: 23.4 (CH3), 74.0
(CH), 122.3 (CH), 123.9 (CH), 126.0 (CH), 126.3 (C),
127.7 (CH), 128.7 (C), 137.2 (CH), 151.5 (CH), 152.0
(CH), 155.0 (C), 146.8 (C), 147.9 (C), 168.6 (C); EIMS m/z
346.22 (M+); anal C15H11ClN4O4 calcd. C, 51.96, H, 3.2,
N, 16.16. Found C, 51.85, H, 3.1, N, 16.04.
(DMSO-d6, 400 MHz): 7.5–9.2 (m, 7H), 8.1 (s, 1H), 2.4 (s,
3H), 3.6 (s, 6H); 13C NMR (DMSO-d6, 100 MHz) δ: 23.4
(CH3), 56.2 (CH3), 74.8 (CH), 112.2 (CH), 115.1 (CH),
120.3 (CH), 123.9 (CH), 126.3 (C), 133.7 (C), 137.2 (CH),
147.8 (C), 149.6 (C), 151.5 (CH), 152.0 (CH), 155.0 (C),
168.6 (C); EIMS m/z 327.92 (M+); Anal C17H17N3O4
calcd. C, 62.38, H, 5.23, N, 12.84. Found C, 62.29, H, 5.11,
N, 12.74.
1-(2-(3-Nitro-phenyl)-5-pyridine-3-yl-(1,3,4) oxadiazol-3-
yl) ethanone (6g)
Antimicrobial activity
Yield: 66%; mp 191–193 °C; IR (KBr) νmax: 3195, 2923,
1682, 1651, 1596, 1475, 1387 cm−1; 1H NMR (DMSO-d6,
400 MHz): 7.2–9.2 (m, 8H), 8.1 (s, 1H), 2.4 (s, 3H); 13C
NMR (DMSO-d6, 100 MHz) δ: 23.4 (CH3), 73.5 (CH),
119.1 (CH), 122.2 (CH), 123.9 (CH), 126.3 (C), 129.5
(CH), 133.1 (CH), 137.2 (CH), 141.3 (C), 148.2 (C), 151.5
(CH), 152.0 (CH), 155.0 (C), 168.6 (C); EIMS m/z 312.88
(M+); anal C15H12N4O4 calcd. C, 57.69, H, 3.87, N, 17.94.
Found C, 57.61, H, 3.79, N, 17.85.
The cup-plate method was adopted in the present investi-
gation. Briefly, the nutrient agar medium, containing stan-
dard bacterial inoculums was filled in cups or disks of
standard diameter. After introducing the test compounds
into the disks, the diameter of zone of inhibition was
measured. All synthesized compound were dissolved in
dimethyl formamide (DMF) to form the concentration of
125, 250, and 500 μg/mL. Ciprofloxacin and ketoconazole
were used as the standard for antibacterial and antifungal
studies, respectively.
1-(2-(4-Hydroxy-3-methoxyphenyl)-5-(pyridine-3-yl)-1,3,4-
oxadiazole-3(2H)-yl)ethanone (6h)
Antibacterial activity
Yield: 64%; mp 150–152 °C; IR (KBr) νmax: 3383, 3063,
Different microorganisms like S. aureus, B. aureus, E. coli,
and P. aeruginosa used for determining the anti-bacterial
activity of synthesized compounds were confirmed by
biochemical test and maintained on nutrient agar (Muller
Hinton Agar, HiMedia, Mumbai) slopes at pH 7.4. Briefly
the required amount of test solution (40 μL of each tested
concentration of synthesized compounds) and standard
solution (40 μL) were added into each cup and kept
undisturbed for at least 2 h at room temperature to allow
proper diffusion of the test and standard solution into the
nutrient agar medium. The petri plates were incubated at 37
1 °C for 24 h and the diameter of zone of inhibition was
measured. Simultaneously controls were maintained using
DMF for the observation of solvent effects.
1
2957, 1698, 1651, 1596, 1475, 1374, 1304, 1202 cm−1; H
NMR (DMSO-d6, 400 MHz): 7.6–9.2 (m, 7H), 8.1 (s, 1H),
2.4 (s, 3H), 2.1 (s, 1H), 3.8 (s, 3H);13C NMR (DMSO-d6,
100 MHz) δ: 23.4 (CH3), 56.2 (CH3), 74.8 (CH), 112.4
(CH), 116.7 (CH), 120.7 (CH), 123.9 (CH), 126.3 (C),
134.0 (C), 137.2 (CH), 143.7 (C), 151.2 (C), 151.5 (CH),
152.0 (CH), 155.0 (C), 168.6 (C); EIMS m/z 313.09 (M+);
anal C16H15N3O4 calcd. C, 61.34, H, 4.83, N, 13.41. Found
C, 61.24, H, 4.76, N, 13.34.
1-(5-Pyridine-3-yl)-2-p-tolyl-1,3,4-oxadiazole-3(2H)-yl)
ethanone (6i)
Yield: 65%; mp 157–159 °C; IR (KBr) νmax: 3212, 3010,
1699, 1636, 1574, 1418, 1361, 1293 cm−1
;
1H NMR
Antifungal activity
(DMSO-d6, 400 MHz): 7.3–9.2 (m, 8H), 8.1 (s, 1H), 2.3 (s,
3H), 2.4 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ: 23.4
(CH3), 24.3 (CH3), 74.5 (CH), 123.9 (CH), 126.9 (CH),
126.3 (C), 128.9 (CH), 136.4 (C), 137.2 (CH), 151.5 (CH),
152.0 (CH), 155.0 (C), 168.6 (C); EIMS m/z 281.42 (M+);
Fungi C. albicans and A. niger were employed for the
estimation of antifungal activity of synthesized compounds.
Sabouraud dextrose agar (SDA; HiMedia, Mumbai) was
used for the cultivation of fungi, particularly pathogenic

Med Chem Res
fungi associated with skin infections. The 40 μL of solution
of each test compounds at the concentration of 125, 250,
and 500 μg/mL prepared in DMF was placed in the cups
under laminar air flow. The DMF and Ketoconazole were
used as control and standard, respectively. Zones of inhi-
bition (mm) were measured after 24 h of incubation.
Kamanna and Kashyap 2007)-
Percentinhibition ¼ 1 À ðtest well FU=mean FU of
triplicateB wellsÞ Â 100
ð1Þ
Evaluation of effect on surface morphology of human
erythrocytes
In vitro antitubercular activity
This study was performed to evaluate whether the synthe-
sized compounds exhibited any detrimental effect on the
surface morphology of human erythrocytes. This study was
performed according to the previously reported method, with
slight modification (Mishra et al. 2010). Briefly, blood of a
healthy donor obtained from a local blood bank was kept in
a blood collection tube (Piove Di Sacco, Italy) containing K3
EDTA anticoagulant. Erythrocytes separated by centrifuga-
tion were washed with phosphate buffer saline (PBS) pH 7.4
and resuspended in PBS pH 7.4. Erythrocytes were used
immediately after isolation. Suspension of erythrocytes in
PBS was treated with different synthesized compounds at a
fixed concentration (1 mg/mL). Then cell samples were
viewed under a Leica optical microscope (Leica, DMLB,
Switzerland) using a magnification of 400× for the mor-
phological condition of erythrocytes (Mishra and Jain 2014).
Microplate Alamar Blue Assay (MABA) was used to
determine the antimycobacterial activity of the synthesized
compounds. Briefly, 100 mL of Middle brook 7H9 broth
(Difco, Detroit, USA) supplemented with 0.2% glycerol,
OADC (oleic acid, albumin, dextrose, catalase), and 0.05%
Tween 80 (Complete medium was referred as 7H9GC-T80)
was used to inoculate bacterial strains M. tuberculosis
H37Rv (ATCC 27294). Rifampicin was used as positive
control. Minimum inhibitory concentration (MIC) was
determined post 7 days incubation at 37 1 °C. For mini-
mizing the background fluorescence Anti-TB susceptibility
testing was executed in black, clear-bottomed 96-well
microplates. Initial dilutions of synthesized compound were
prepared in DMSO and 0.1 mL of H12 media in microplates
was used for subsequent two fold dilutions. The H37 Rv
was diluted in 7H9 media to reach approximately 2 × 105
cfu/mL and 0.1 mL was added to wells. At 7th day of
incubation, 20 µL of Alamar Blue solution and 12.5 mL of
20% Tween 80 were added to all the wells and plates were
re-incubated for 24 h. For measuring fluorescence Victor II
multilabel fluorometer (Perkin Elmer Life Sciences Inc.,
Boston, MA) was operated at 530 and 590 nm. The MIC
was taken as the lowest drug concentration affecting an
inhibition of ≥ 90% and percent inhibition was determined
by following equation (Table 2) (Lourenço et al. 2007;
In vitro hemolytic toxicity
For studying the effects of synthesized compounds on the
lysis of erythrocytes, the suspension of erythrocytes (0.1
mL; 10% hematocrit value) and synthesized compounds
(0.9 mL; 1 mg/mL in PBS, pH 7.4) were incubated for 30
min at 37 1 °C and then the mixtures were centrifuged at
3000 rpm for 10 min to remove nonlysed erythrocytes. The
supernatant was analyzed spectrophotometrically (1601
UV–Vis spectrophotometer, Shimadzu, Japan) at 540 nm (n
= 6). To obtain 0 and 100% hemolysis, erythrocytes sus-
pension (0.1 mL) was mixed with NaCl solution (0.9 mL;
0.9% w/v) and distilled water (0.9 mL), respectively (Mishra
et al. 2010; Mishra and Jain 2014). The degree of hemolysis
was determined by using the following equation:
Table 2 In vitro antitubercular activity of synthesized compounds
(6a–j)
Entry
Compounds
% Inhibition (100 µg/mL)
MIC (µg/mL)
1
6a
101
101
100
101
48
10.4
8.6
2
6b
3
6c
11.7
7.8
Hemolysisð%Þ¼ðAbsS À Abs0Þ=ðAbs100 À Abs0Þ Â 100
ð2Þ
4
6d
5
6e
>100
1.2
6
6f
101
100
98
where AbsS, Abs0, and Abs100 are the absorbance of
sample, a solution showing 0% hemolysis and a solution
showing 100% hemolysis, respectively.
7
6g
3.1
8
6h
53.9
>100
46.3
0.0047
9
6i
78
10
11
6j
101
100
Molecular modeling
Rifampicin
For the identification of potentially active ligands, the
Test concentration: 100 µg/mL; strain: M. tuberculosis H37Rv;
method: MABA; stock concentration: 10 mg/mL
designed molecules were analyzed by molecular docking

Med Chem Res
using Autodock Vina 1.5.6 software, a molecular docking
software (Trott and Olson 2010; Chaurasiya et al. 2016;
Kaur and Khatik 2016; Khatik et al. 2011, 2012). For the
extraction and preparation of ligands, the desired proteins
were downloaded from protein data bank (www.rcsb.org/
pdb/home/home.do). The selected protein was validated by
the extraction of ligand and docking it in a same manner as
actual ligand. For preparation of protein it was reloaded and
various problems were fixed such as missing bonds or
atoms, and removed extraneous structures like water
molecules. Polar hydrogens were added along with the
Kollaman charges. After saving the macromolecule (as
pdbqt file) the ligand.pdbqt was loaded and set it as map
type by choosing ligand and grid box was generated. The
compounds were drawn by ChemDraw Ultra and converted
to 3D structures. Geometry of all compounds was optimized
by semiemperical MM2 method. Molecular docking was
performed on optimized structure of protein.
Fig. 2 Pictorial representation of synthesized nicotinic acid derivatives (6a–j)
Scheme 1 Synthesis of
acetylated nicotinic acid
derivatives

Med Chem Res
Results and discussion
Chemistry
synthesized nicotinyl derivatives was achieved in good yield
by reacting aromatic aldehydes i.e., benzaldehyde (4a),
dimethylaminobenzaldehyde (4b), 2-chlorobenzaldehyde
(4c), 2-nitrobenzaldehyde (4d), 4-methoxybenzaldehyde
(4e), 3-chloro-4-nitrobenzaldehyde (4f), 3-nitrobenzaldehyde
(4g), 4-hydroxy-3-methoxybenzaldehyde (4h), 4-methylben-
zaldehyde (4i), and 3,4-dimethoxybenzaldehyde (4j) with
Different acetylated nicotinic acid derivatives (6a–j)
(Table 1; Fig. 2) were synthesized by the acetylation of
Schiff base with different substituents. The desired
Fig. 3 Antibacterial activity of nicotinic acid derivatives (6a–j)
Fig. 4 Antifungal activity of nicotinic acid derivatives (6a–j)

Med Chem Res
Fig. 5 Erythrocytes
photographs (400×) showing
surface morphology under
influence of i normal saline, ii
ciprofloxacin, iii ketoconazole,
and iv compound 6g (1 mg/mL)
Table 3 Molecular docking study of synthesized compounds (6a–j)
Entry Compounds
Binding affinity (Kcal/mol)
Antimicrobial Antifungal Antitubercular
2xct
3gw9
3hnt
1
6a
6b
6c
6d
6e
6f
−8.3
−8.7
−8.3
−8.3
−8.5
−9.5
−9.9
−8.4
−7.8
−9.0
−7.1
−8.0
−7.1
−7.7
−7.6
−8.6
−8.0
−7.8
−8.4
−8.4
–
−6.4
−6.4
−6.5
−6.1
−6.4
−6.6
−6.6
−6.4
−6.5
−6.5
–
2
3
4
5
6
7
6g
6h
6i
Fig. 6 Hemolytic toxicity profile of different compounds. Results are
represented as mean SD (n = 6)
8
9
10
11
12
13
6j
nicotinoyl hydrazide in ethanol and acetic anhydride
(Scheme 1). The nicotinic acid was converted to nicotinoyl
chloride by refluxing with phosphorus pentachloride (PCl5)
in carbon tetrachloride (CCl4) and thereafter it was reacted
with hydrazine at low temperature to afford nicotinoyl
hydrazide. The synthesized derivatives were characterized
by elemental analysis, mass, IR, and NMR spectroscopy.
Ciprofloxacin −10.4
Ketoconazole
Rifampicin
–
–
−8.5
–
–
−6.8
albicans and Aspergillus niger in terms of zone of inhibi-
tion (Fig. 4). At the selected maximum concentration (500
µg/mL), compounds 6a, 6b, 6e, 6f, 6g, and 6i exhibited
maximum zone of inhibition (antibacterial activity) against
B. subtilis. Apart from this compounds 6c and 6d displayed
maximum zone of inhibition against S. aureus and P.
aeuroginosa, respectively at concentration of 500 µg/mL
while compound 6j exhibited similar antibacterial activity
against other strain of bacteria i.e., E. coli and S. aureus at
Antibacterial and antifungal activity
The in vitro antimicrobial activities of all synthesized
derivatives (6a–j) were performed by cup-plate method
using various bacterial strains like Staphylococcus aureus,
Bacillus subtilis, Escherichia coli, and Pseudomonas aer-
uginosa, (Fig. 3) as well as fungal strains like Candida

Med Chem Res
the 500 µg/mL concentration. Interestingly, compounds 6h
and 6i showed maximum antibacterial activity against E.
coli and P. aeuroginosa, respectively at concentration of
250 µg/mL (Fig. 3).
Further, all synthesized compounds (6a–j) were also
evaluated for in vitro antifungal activity against selected
fungi i.e., C. albicans and A. niger. At the concentration of
500 µg/mL, compounds 6d and 6j exhibited maximum
antifungal activity against C. albicans while compounds 6b,
6f, 6h, and 6i exhibited maximum zone of inhibition against
A. niger. Interestingly, compounds 6e and 6g showed
similar antifungal activity against both, C. albicans and A.
niger. The compounds 6a and 6c were devoid of any
antifungal activity at various tested concentrations ranging
from 125 to 500 µg/mL (Fig. 4).
For investigating the antitubercular activity, all the syn-
thesized compounds 6a–j were screened against M. tuber-
culosis H37Rv strain. The result of antitubercular activity is
represented in Table 2 in terms of % inhibition and MIC
values. All the compounds revealed significant inhibitory
activity against M. tuberculosis H37Rv at 100 µg/mL with
100% inhibition except compounds 6e and 6i, which
showed less than 90% inhibition. The compounds sub-
stituted with nitro groups (6f, 6g, and 6d) showed higher
antitubercular activity as compared to other compounds.
Out of all the synthesized derivatives, compounds 6f, 6g,
and 6d exhibited excellent antitubercular activity with MIC
values of 1.2, 3.1, and 7.8 µg/mL, respectively. The position
of nitro substituent greatly affected the antitubercular
activity (Lourenço et al. 2007; Kamanna and Kashyap
2007). The nitro group at para position (compound 6f)
showed more potency than others.
Normally, human erythrocytes are regular biconcave
disks and the deformability of normal human erythrocytes
geometry affects their ability to survive in the micro-
circulation. The toxicity of synthesized nicotinic acid deri-
vatives were evaluated by observing the changes in surface
morphology of erythrocytes, which reflected the toxicity.
All the compounds were studied at the concentration of
1 mg/mL. The erythrocytes cells displayed lesser
Fig. 7 Interaction of 6g (blue color) as an antimicrobial on protein
2xct (DNA gyrase) in line model (6g) ribbon structure (2xct) (color
figure online)
Fig. 8 The interaction of 6f
(blue color) as an antifungal on
protein 3gw9 in ball and stick
model (a, b) and ribbon
structure (c) (color figure online)

Med Chem Res
deformities in shape as compared with normal saline control
group as shown in Fig. 5. The compound 6g caused least
deformation to the erythrocytes as compared to the standard
ciprofloxacin and ketoconazole.
negligible toxicity, which advocated that the synthesized
acetylated nicotinic acid derivatives could be developed for
exploring its further biomedical applications.
Along with the changes in contour of RBCs, the hemo-
lysis of erythrocytes was also studied. Erythrocytes lysis is
an easy method to study any possible interaction between
synthesized compound and RBCs membrane. This can
provide qualitative as well as quantitative indication of
potential damage to RBCs by administered compound. It
gives a quantitative measure of hemoglobin (Hb) release
(Mishra et al. 2010; Mishra and Jain 2014). The compound
6g exhibited significantly very less hemolytic toxicity at
different concentrations i.e., 125, 250, and 500 µg/mL as
compared to the standards ciprofloxacin and ketoconazole
at concentration of 125 µg/mL (Fig. 6).
Molecular docking studies
For the identification of molecular interaction and insight
mechanism all the synthesized compounds 6a–j were
screened by Autodock Vina 1.5.6 software (version 1.5.6,
The SCRIPPS Research Institute, Germany) (Trott and
Olson 2010). The proteins PDB 2xct, 3gw9 and 3hnt were
downloaded from protein data bank (http://www.rcsb.org/
pdb/home/home.do). The structures of the compounds were
drawn by ChemBioDrwa Ultra 12.0 (CambridgeSoft) and
converted to 3D structures. Energy minimizations and
geometry optimization were performed by MM2 Interface
program on ChemBio3D Ultra 12.0. Further the interactions
of these compounds were evaluated on 2xct (DNA gyrase),
On the basis of effects on surface morphology of ery-
throcytes and hemolytic toxicity investigations, it could be
concluded that the synthesized compounds exhibited
Fig. 9 The interaction of 6f
(green color) as an antitubercular
with 3hnt protein (a, b) and
ribbon structure (c)

Med Chem Res
Acknowledgements Authors are obliged to Sophisticated Analytical
Instrument Facility, Indian Institute of Technology, Chennai, India for
IR, NMR and mass spectroscopy of compounds.
3gw9 and 3hnt protein for the antimicrobial, antifungal and
antitubercular activity, respectively.
The results of binding affinity obtained by molecular
docking are shown in Table 3. The binding affinity corre-
lates with their in vitro activity. In-depth analysis was done
for the compounds, which showed the best activity in
in vitro assay as well as good binding affinity in molecular
docking study. Among the all, compound 6g showed better
binding affinity (−9.9 Kcal/mol) as compared to that of the
ciprofloxacin (−10.4 Kcal/mol; entry 7, Table 3). Similarly
compound 6g showed good in vitro antimicrobial activity
against S. aureus and P. aeuroginosa at concentration of
500 µg/mL. The interaction of compound 6g with protein
2xct (DNA gyrase), the binding site was shown in Fig. 7.
Under in vitro antifungal activity the compounds 6b, 6f,
6h, and 6i exhibited maximum zone of inhibition and good
binding affinity to 3gw9 protein. The compound 6f com-
pound has good binding affinity (−8.6 Kcal/mol), which
was comparable to ketoconazole (−8.5 Kcal/mol) (entry 6,
Table 3). The interaction of compound 6f with 3hnt protein
was depicted in Fig. 8 showing hydrophobic interaction of
compound 6f with various amino acid residues i.e.,
PHE110, TYR109, THR214, VAL461 at binding site.
The compounds substituted with nitro groups (6f, 6g, and
6d) showed higher in vitro antitubercular activity and same
was reflected in the binding affinity obtained from mole-
cular docking. The compound 6f with para nitro group
showed more potency and high binding affinity (−6.6 Kcal/
mol; entry 6, Table 3). In-depth binding interaction for 6f
compound (Fig. 9), showed hydrophobic interaction with
various amino acid residues i.e., PHE105, TRP47 at binding
site.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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Conclusion
The acetylated nicotinic acid derivatives (6a–j) were syn-
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