Synthesis and in vitro and in silico antimicrobial studies of novel piperine–pyridine analogs

Article abstract of DOI:10.1007/s11164-018-3324-1

Pepper is used as a food additive and preservatives because of its antimicrobial activity. Piperine, an alkaloid amide derived from pepper, is the cause of its biological activity. Aminopyridine is a well-known antimicrobial agent. In?silico studies proved that conjugation of piperine with substituted aminopyridine results in a new hybrid molecule with improved antimicrobial activity compared with the parent molecules. The present work describes design and synthesis of novel piperine analogs with substituted aminopyridine analogs (PY1–8). The synthesized compounds were characterized by ¹H and ¹³C nuclear magnetic resonance (NMR), infrared (IR), and mass spectroscopy and subjected to antimicrobial testing using bacterial strains Bacillus subtilis, Streptobacillus, Staphylococcus aureus, Escherichia coli, and Salmonella typhi and fungal strains Aspergillus niger, A.?flavus, and A.?fumigatus.

Full text of DOI:10.1007/s11164-018-3324-1

Res Chem Intermed
https://doi.org/10.1007/s11164-018-3324-1
Synthesis and in vitro and in silico antimicrobial studies
of novel piperine–pyridine analogs
1
1
•
Karteek Rao Amperayani
1
Uma Devi Parimi
•
Kottakki Naveen Kumar
Received: 7 November 2017 / Accepted: 5 February 2018
Ó Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract Pepper is used as a food additive and preservatives because of its
antimicrobial activity. Piperine, an alkaloid amide derived from pepper, is the cause
of its biological activity. Aminopyridine is a well-known antimicrobial agent.
In silico studies proved that conjugation of piperine with substituted aminopyridine
results in a new hybrid molecule with improved antimicrobial activity compared
with the parent molecules. The present work describes design and synthesis of novel
piperine analogs with substituted aminopyridine analogs (PY1–8). The synthesized
1
13
compounds were characterized by H and C nuclear magnetic resonance (NMR),
infrared (IR), and mass spectroscopy and subjected to antimicrobial testing using
bacterial strains Bacillus subtilis, Streptobacillus, Staphylococcus aureus, Escher-
ichia coli, and Salmonella typhi and fungal strains Aspergillus niger, A. flavus, and
A. fumigatus.
Keywords Piperine Á Aminopyridine Á Antimicrobial activity Á In silico
studies
Introduction
Piper nigrum, commonly known as black pepper, is considered as the king of spices
and is a traditional drug used for many diseases. Black pepper is used as a traditional
medicine and food preservative [1] due to its antibacterial and antifungal activities.
Black pepper is reported to possess antibacterial activity [2, 3]. Zou et al. [4] studied
the mechanism of antibacterial activity of black pepper chloroform extract (BPCE).
&
Uma Devi Parimi
umadevichemistry@gmail.com
1
Department of Chemistry, GITAM Institute of Sciences, GITAM University, Visakhapatnam,
Andhra Pradesh 530045, India
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K. R. Amperayani et al.
The biological activities and pungency of pepper are due to the presence of an
alkaloid, viz. piperine [5, 6], an amide of piperidine. On hydrolysis, it converts into
piperic acid and piperidine. Piperine alkaloid is a potent antibacterial agent against
Staphylococcus aureus, Salmonella typhi, Escherichia coli, and Proteus sp. [7, 8].
Antioxidant and antimicrobial activities of various solvent extracts of pepper,
piperine, and piperic acid have been explored [9]. Piperine analogs also show
excellent antibacterial activity compared with the parent molecule [10]. Zainab
et al. [11] evaluated the antimicrobial activity of piperine purified from Piper
nigrum. Piperine analogs were found to be more potent inhibitors of the NorA efflux
pump of Staphylococcus aureus compared with the parent molecule [12].
Quantitative structure–activity relationship (QSAR) analysis of piperine analogs
as inhibitors of efflux pump NorA from Staphylococcus aureus has been performed
to obtain a highly accurate model enabling prediction of inhibition of S. aureus [13].
Piperine was tested for use in combination with mupirocin for antimicrobial activity
against Staphylococcus aureus strains, including methicillin-resistant S. aureus
(
MRSA), which reduced the minimum inhibitory concentration (MIC) of mupirocin
and also lowered the mutation frequency [14]. Piperine was evaluated for its
immunomodulatory activity to enhance the efficacy of rifampicin in a murine model
of Mycobacterium tuberculosis infection [15]. In one study on the combination of
piperine with gentamicin prepared by dehydration–rehydration, novel strategies
were developed to prevent bacterial growth, revealing that the liposomal combi-
nation is a powerful nano-antibacterial agent to eradicate MRSA infection [16].
Inhibitory effects of curcumin, capsaicin, and piperine against Helicobacter pylori
were investigated, and the results suggested that capsaicin and piperine have
antiinflammatory effects on H. pylori-induced gastritis in gerbils, independent of
direct antibacterial effects [17]. The pro- and antioxidant and NOX-inhibiting
qualities of four phytochemicals, viz. celastrol, resveratrol, apigenin, and piperine,
were also studied [18]. Piper nigrum fruit isolations were prepared and their activity
in vivo studied by oral administration to guinea pigs, revealing antitussive effect
[
19]. Extract of Piper nigrum was given to rats and an ultraperformance liquid
chromatography–tandem mass spectrometry (UPLC-MS–MS) method developed to
determine their content in plasma, showing good linearity [20]. Piperine induced
increase of mammalian target of rapamycin complex 1 (mTORC1) activity in
resident peritoneal macrophages.
Pyridine is a versatile basic six-member aromatic heterocyclic molecule with
chemical formula C H N, containing one nitrogen and five carbon atoms. The
5
5
structure of pyridine is almost like that of benzene. Aminopyridines have been
found to possess a broad spectrum of biological activities. Aminopyridines are
potent pharmacophores with versatile applications in pharmaceutical industry.
Drugs including aminopyridine moiety such as tenoxicam, piroxicam [nonsteroidal
antiinflammatory drugs (NSAIDs)], sulfasalazine (used to treat rheumatoid arthritis,
ulcerative colitis, and Crohn’s disease), delavirdine [anti-human immunodeficiency
virus (HIV) drug], sulfapyridine (antibacterial drug), and tripelennamine (antihis-
tamine drug) are available on the market. 4-Aminopyridine is present in tacrine, a
centrally acting anticholinesterase. Reports suggest that aminopyridines exhibit
1
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Synthesis and in vitro and in silico antimicrobial studies of…
various biological applications [21]. Derivatives of aminopyridines possess
antibacterial and antimicrobial activity [22–30].
A recent approach for discovery of new pharmaceutical drugs involves linking
two molecules and development of hybrid or conjugate molecules with individual
essential activity into a single hybrid molecule with elevated biological activity.
Such hybrid or conjugate molecules can show enhanced biological activity
compared with both parent molecules and be very effective drugs. They have
different alkylation sites, which are essential to and play a vital role in tumor
treatment [31]. Literature review reveals that [32] designed and synthesized such
novel hybrid molecules for malaria treatment, with decreased side effects and
increased activity of the drug moiety. This approach of combining different active
molecules for enhanced bioavailability is encouraging. We employed this strategy
to synthesize piperine analogs [33], with positive results. Taking this as a precedent
and considering the antimicrobial nature of both piperine and aminopyridine, in this
work we designed and synthesized hybrid piperine–pyridine analogs as promising
antimicrobial agents. All target molecules were designed using ChemDraw software
and used for in silico molecular docking studies. To date, there are no reports on
synthesis of novel piperine–pyridine analogs or study of their antimicrobial activity.
Materials and methods
All reagents used were of analytical reagent (AR) grade and used directly without
further purification. Piperine was purchased from Sigma Aldrich chemicals with
9
7 % purity and used directly. Melting points were determined in open capillary
1
13
tube. H and C NMR spectra were recorded on Bruker Avance 400 and 100
spectrometer, respectively, with tetramethylsilane (TMS) as internal standard;
chemical shifts are given in parts per million (ppm). High-resolution mass data were
obtained using a Bruker micro TOF-Q II ESI instrument operating at ambient
temperature. An FTIR spectrum was recorded on a PerkinElmer FTIR spectropho-
tometer using KBr pellets. Reaction progress was monitored by thin-layer
chromatography on Merck TLC silica gel plates using hexane:ethylacetate (6:4)
as solvent. Spots were visualized under ultraviolet (UV) chamber.
Synthesis of piperine analogs
Hydrolysis of piperine to piperic acid
Piperine (10 g) and 300 ml 20 % ethanolic KOH were taken in a round-bottomed
flask and refluxed for 10 h. The resulting potassium piperate was filtered and
washed with anhydrous ethanol. The precipitate was dissolved in distilled water,
and 0.1 M HCl was added to the solution, followed by filtering and washing with
distilled water. Yellow crystals of piperic acid were recrystallized from ethanol [34]
(
yield 86.6 %, m.p. 214–216 °C).
123

K. R. Amperayani et al.
Coupling of piperine with aminopyrimidine
Exactly weighed 2.18 g piperic acid in 20 ml dimethylformamide (DMF) was taken
in a round-bottomed flask and stirred at 0–5 °C, followed by addition of 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) with
continuation of stirring at 0–5 °C for 30 min. To this mixture, 2.60 ml N,N-
diisopropylethylamine (DIPEA) and 2 g substituted aminopyridine were added
dropwise, followed by stirring at room temperature for 5 h; reaction completion was
monitored by TLC using 6:4 hexane:ethylacetate. After reaction completion, the
crude mixture was washed with HCl then NaOH solution and concentrated to give
crude product. The crude product was column chromatographed using hexane:ethy-
lacetate to obtain pure compounds.
PY1
amide M.p. 248 °C, yield: 65 %, brown crystalline solid. H NMR (500 MHz,
chloroform-d) d 9.44 (s, 1H), 8.50–8.45 (m, 2H), 7.71–7.66 (m, 2H), 7.09–7.00 (m,
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(pyridin-4-yl)penta-2,4-dien-
1
2
H), 6.90 (dd, J = 8.4, 2.0 Hz, 1H), 6.80–6.72 (m, 2H), 6.69 (ddd, J = 14.1, 7.0,
.8 Hz, 1H), 6.04 (d, J = 2.4 Hz, 1H), 5.99 (d, J = 2.4 Hz, 1H), 5.56 (dd,
0
1
3
J = 14.6, 1.0 Hz, 1H) C NMR (125 MHz, common NMR solvents) d 165.29,
1
50.44, 147.52, 147.39, 143.61, 140.82, 138.78, 130.71, 125.85, 123.16, 123.08,
13.65, 108.52, 106.90, 101.00. Mass spectrum: m/z 295.1 (100 %), 296.11 (20 %).
1
IR spectrum: 1710 (amide-CONH), 3100–3500 (amide NH stretching).
PY2
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(pyridin-2-yl)penta-2,4-dien-
1
amide M.p. 238 °C, yield: 70 %, pale-yellow crystalline solid. H NMR
(500 MHz, chloroform-d) d 8.70 (dd, J = 3.5, 1.5 Hz, 1H), 8.55 (s, 1H), 8.23
(dd, J = 7.9, 1.4 Hz, 1H), 7.95 (td, J = 7.7, 1.6 Hz, 1H), 7.38–7.28 (m, 2H), 7.04
(d, J = 2.0 Hz, 1H), 6.90 (dd, J = 8.4, 2.0 Hz, 1H), 6.80–6.72 (m, 2H), 6.69 (ddd,
J = 14.1, 7.1, 0.9 Hz, 1H), 6.04 (d, J = 2.4 Hz, 1H), 5.99 (d, J = 2.4 Hz, 1H),
1
3
5
1
1
.63–5.56 (m, 1H). C NMR (125 MHz, common NMR solvents) d 166.30, 152.75,
47.92, 147.52, 147.39, 140.82, 138.78, 138.19, 130.71, 125.85, 123.16, 122.92,
18.85, 115.24, 108.52, 106.90, 101.00. Mass spectrum: m/z 295.1 (100 %), 296.11
(
20 %). IR spectrum: 1675 (amide-CONH), 3100–3300 (amide NH stretching).
PY3 (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(pyridin-3-yl)penta-2,4-dien-
1
amide M.p. 262 °C, yield: 65 %, grey crystalline solid. H NMR (500 MHz,
chloroform-d) d 8.75 (t, J = 1.8 Hz, 2H), 7.90 (dt, J = 7.9, 1.9 Hz, 2H), 7.77 (dt,
J = 3.5, 1.7 Hz, 2H), 7.51 (dd, J = 7.8, 3.6 Hz, 2H), 7.35 (dd, J = 14.7, 7.1 Hz,
2
6
H), 7.04 (d, J = 2.0 Hz, 2H), 6.90 (dd, J = 8.4, 2.0 Hz, 2H), 6.80–6.72 (m, 4H),
.69 (ddd, J = 14.1, 7.1, 0.9 Hz, 2H), 6.04 (d, J = 2.4 Hz, 2H), 5.99 (d,
1
3
J = 2.4 Hz, 2H), 5.56 (dd, J = 14.6, 1.0 Hz, 2H). C NMR (125 MHz, common
NMR solvents) d 164.94, 147.52, 147.39, 144.56, 143.89, 140.82, 138.78, 133.36,
1
30.71, 127.01, 125.85, 124.38, 123.16, 123.08, 108.52, 106.90, 101.00. Mass
spectrum: m/z 295.1 (100 %), 296.11 (20 %). IR spectrum: 1650 (amide-CONH),
100–3300 (amide NH stretching).
3
1
23

Synthesis and in vitro and in silico antimicrobial studies of…
PY4 (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(6-methylpyridin-2-yl)penta-2,4-di-
1
enamide M.p. 256 °C, yield: 70 %, brownish crystalline solid. H NMR
(
500 MHz, chloroform-d) d 8.40 (s, 1H), 7.54–7.43 (m, 2H), 7.33 (dd, J = 14.6,
7
.1 Hz, 1H), 7.06–6.97 (m, 2H), 6.90 (dd, J = 8.4, 2.0 Hz, 1H), 6.80–6.72 (m, 2H),
.69 (ddd, J = 14.1, 7.1, 0.9 Hz, 1H), 6.04 (d, J = 2.4 Hz, 1H), 5.99 (d,
6
J = 2.4 Hz, 1H), 5.63–5.56 (m, 1H), 2.42 (d, J = 0.7 Hz, 3H). C NMR
1
3
(
125 MHz, common NMR solvents) d 166.21, 156.90, 151.07, 147.52, 147.39,
1
1
40.82, 138.78, 138.46, 130.71, 125.85, 123.16, 122.92, 118.68, 111.87, 108.52,
06.90, 101.00, 24.46. Mass spectrum: m/z 309.1 (100 %), 310.12 (20 %). IR
spectrum: 1700 (amide-CONH), 3100–3500 (amide NH stretching).
PY5 (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(3-hydroxypyridin-2-yl)penta-2,4-
1
dienamide M.p. 222 °C, yield: 63 %, colorless crystalline solid. H NMR
(
500 MHz, chloroform-d) d 8.98 (s, 1H), 8.27 (dd, J = 3.5, 1.8 Hz, 1H), 7.88 (s,
1H), 7.39 (dd, J = 14.6, 7.2 Hz, 1H), 7.33 (dd, J = 7.9, 1.8 Hz, 1H), 7.25 (dd,
J = 7.9, 3.5 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 6.90 (dd, J = 8.4, 2.0 Hz, 1H),
6
5
.80–6.72 (m, 2H), 6.69 (ddd, J = 14.1, 7.1, 0.9 Hz, 1H), 6.04 (d, J = 2.4 Hz, 1H),
1
.99 (d, J = 2.4 Hz, 1H), 5.60 (dd, J = 14.7, 0.9 Hz, 1H). C NMR (125 MHz,
3
common NMR solvents) d 166.36, 147.52, 147.39, 144.95, 141.60, 140.82, 139.06,
38.78, 130.71, 125.85, 123.16, 122.83, 121.96, 120.57, 108.52, 106.90, 101.00.
Mass spectrum: m/z 311.10 (100 %), 312.10 (20 %). IR spectrum: 1675 (amide-
1
CONH), 3100–3300 (amide NH stretching).
PY6 (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(5-chloropyridin-2-yl)penta-2,4-di-
1
enamide M.p. 268 °C, yield: 74 %, pale-brown crystalline solid. H NMR
(
500 MHz, chloroform-d) d 8.98 (s, 1H), 8.27 (dd, J = 3.5, 1.8 Hz, 1H), 7.88 (s,
1H), 7.39 (dd, J = 14.6, 7.2 Hz, 1H), 7.33 (dd, J = 7.9, 1.8 Hz, 1H), 7.25 (dd,
J = 7.9, 3.5 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 6.90 (dd, J = 8.4, 2.0 Hz, 1H),
6
5
.80–6.72 (m, 2H), 6.69 (ddd, J = 14.1, 7.1, 0.9 Hz, 1H), 6.04 (d, J = 2.4 Hz, 1H),
1
.99 (d, J = 2.4 Hz, 1H), 5.60 (dd, J = 14.7, 0.9 Hz, 1H). C NMR (125 MHz,
3
common NMR solvents) d 166.36, 147.52, 147.39, 144.95, 141.60, 140.82, 139.06,
38.78, 130.71, 125.85, 123.16, 122.83, 121.96, 120.57, 108.52, 106.90, 101.00.
Mass spectrum: m/z 329.06 (100 %), 331.06 (35 %), 330.07 (20 %). IR spectrum:
680 (amide-CONH), 3200–3500 (amide NH stretching).
1
1
PY7 (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(3-methylpyridin-2-yl)penta-2,4-di-
1
enamide M.p. 232 °C, yield: 75 %, colorless crystalline solid. H NMR
(
500 MHz, chloroform-d) d 8.52–8.43 (m, 2H), 7.66 (ddd, J = 7.9, 1.8, 0.8 Hz,
1
H), 7.37 (dd, J = 14.7, 7.1 Hz, 1H), 7.27 (dd, J = 7.9, 3.5 Hz, 1H), 7.04 (d,
J = 2.0 Hz, 1H), 6.90 (dd, J = 8.4, 2.0 Hz, 1H), 6.80–6.72 (m, 2H), 6.69 (ddd,
J = 14.1, 7.1, 0.9 Hz, 1H), 6.04 (d, J = 2.4 Hz, 1H), 5.99 (d, J = 2.4 Hz, 1H), 5.60
1
dd, J = 14.6, 0.9 Hz, 1H), 2.34 (d, J = 0.7 Hz, 3H). C NMR (125 MHz,
3
(
common NMR solvents) d 166.31, 150.46, 147.52, 147.39, 145.47, 140.82, 138.78,
1
1
38.37, 130.71, 125.85, 123.16, 123.07, 122.83, 118.49, 108.52, 106.90, 101.00,
6.34. Mass spectrum: m/z 309.1 (100 %), 310.12 (20 %). IR spectrum: 1670
(
amide-CONH), 3100–3500 (amide NH stretching).
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K. R. Amperayani et al.
PY8 (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(5-nitropyridin-2-yl)penta-2,4-di-
1
enamide M.p. 252 °C, yield: 72 %, grey crystalline solid. H NMR (500 MHz,
chloroform-d) d 9.42 (d, J = 1.8 Hz, 2H), 9.13 (s, 2H), 8.52 (dd, J = 8.4, 2.0 Hz,
2
H), 7.82 (d, J = 8.4 Hz, 2H), 7.33 (dd, J = 14.7, 7.1 Hz, 2H), 7.04 (d,
J = 2.0 Hz, 2H), 6.90 (dd, J = 8.4, 2.0 Hz, 2H), 6.80–6.72 (m, 4H), 6.69 (ddd,
J = 14.1, 7.1, 0.9 Hz, 2H), 6.04 (d, J = 2.4 Hz, 2H), 5.99 (d, J = 2.4 Hz, 2H),
1
3
5
1
1
3
.63–5.56 (m, 2H). C NMR (125 MHz, common NMR solvents) d 166.30, 155.57,
47.52, 147.39, 146.13, 140.82, 138.78, 138.29, 134.49, 130.71, 125.85, 123.16,
22.92, 113.78, 108.52, 106.90, 101.00. Mass spectrum: m/z 340.09 (100 %),
41.09 (20 %). IR spectrum: 1690 (amide-CONH), 3100–3400 (amide NH
stretching).
Antimicrobial activity
Antimicrobial activity was investigated against Gram-positive bacteria (Staphylo-
coccus aureus, Bacillus subtilis, and Bacillus cereus), Gram-negative bacteria
(
Escherichia coli, Enterococcus faecalis, Klebsiella pneumonia, and Pseudomonas
aeruginosa), and fungal strains (Aspergillus niger, A. flavus, A. fumigatus, and
Candida albicans). The test was conducted by agar well diffusion method. In this
method, 20 ml M u¨ ller–Hinton medium was spread on Petri plates, and these plates
were spread with 24-h-cultured 108 colony-forming units (CFU)/ml of microbial
strains. Wells of 6 mm diameter were made in the culture medium. Target
compounds (50 ll) were added to the wells and incubated at 37 °C for 24 h.
Streptomycin and tetracycline (30 lg/ml each) were taken as reference drugs for
antibacterial activity, and carbendazim for antifungal activity. A positive control
was kept for reference. The activity of the target compounds was measured based on
the inhibition zone formed around the well. The assay was carried out in triplicate,
and mean values were taken as readings. The diameter of the zone of inhibition was
measured in millimeters.
In silico docking studies
Possible docking modes of the piperine–pyridine analogs with 5XGT (Staphylo-
coccus aureus) [35], 3ZIH (Bacillus subtilis) [36], 5NWT (Escherichia coli) [37],
and 2W7Q (Pseudomonas aeruginosa) [38] were studied using AutoDock 2.0
software [39]. The target molecules were designed using ChemDraw software and
used to analyze the binding affinity with the bacterial proteins. Crystal structures of
all the bacterial proteins were downloaded from the Protein Data Bank website
(
http://www.rcsb.org) in PDB format and converted to PDBQT format. We chose
the Lamarckian genetic algorithm (LGA) for ligand conformations. An AutoDock
tool was used for creating PDBQT files [40]. The protocol was given in AutoDock
Tools software. AutoDock was run several times to obtain various docked con-
formations and used to analyze the predicted docking energy. AutoDock Tools
provide various parameters to analyze the results of docking simulations, such as
binding energy, ligand efficiency, inhibition constant, and intramolecular energy.
1
23

Synthesis and in vitro and in silico antimicrobial studies of…
Scheme 1 Preparation of piperic acid from piperine
Scheme 2 Coupling of pyridine with piperic acid
Scheme 3 Mechanism of amide bond formation
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K. R. Amperayani et al.
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23

Synthesis and in vitro and in silico antimicrobial studies of…
For each ligand, ten best conformations were generated and scored using Auto-
Dock 4.2 scoring functions [41].
Results and discussion
Chemistry
Considering our interest in piperine and its analogs and our previous work on
piperine analogs, piperine–pyridine analogs were designed using ChemDraw
software and synthesized. Piperine was first hydrolyzed to piperic acid (Scheme 1),
then coupled to different substituted aminopyridines using coupling agent HBTU
and DIPEA as shown in Scheme 2. The mechanism of amide bond formation is
shown in Scheme 3. The structures of all the synthesized compounds are presented
1
13
in Table 3. All novel compounds were characterized using H and C NMR and
1
mass spectra. The H NMR spectrum of compound PY1 showed a signal at
chemical shift of d 9.44, corresponding to amide N–H protons as singlet. The
Table 2 Antifungal activity of piperine–pyridine analogs (zone of inhibition in mm)
S. no. Compound
Aspergillus niger A. flavus A. fumigatus Candida albicans
1
2
3
4
5
6
7
8
9
PY1
PY2
PY3
PY4
PY5
PY6
PY7
PY8
15
25
22
13
20
20
24
13
18
22
22
19
30
27
12
6
16
18
11
20
25
12
14
15
28
24
16
13
15
23
16
21
20
30
Carbendazim (30 lg/ml) 30
35
Bold values indicate the highest activity than the standard drug
Fig. 1 Zone of inhibition for Staphylococcus aureus
123

K. R. Amperayani et al.
multiplet signal in the range of 8.50–8.45 and 7.71–7.66 for four protons is related
to aromatic pyridine ring. The doublet signal at d 6.80–6.72 for two protons is
related to CH –O group, and the 5.56 doublets are assigned to CH–CO group. The
2
proton peaks for trans-double bond appear at 5.99, 6.90, and 6.69 ppm. The other
C–H protons of benzene ring appeared as doublet at d 6.90 ppm. Two protons on
benzene ring appeared as multiplet located at 7.09–7.00 ppm. The C NMR
1
3
spectrum of the compound exhibited signals at different d-chemical shifts including
1
1
3
65.29, 150.44, 147.52, 147.39, 143.61, 140.82, 138.78, 130.71, 125.85, 123.16,
23.08, 113.65, 108.52, 106.90, and 101.00 ppm. The IR spectrum showed bands at
100–3500 for amide N–H stretching, 3048 for aromatic C–H stretching, 1710 for
C=O amide stretching, 1594 for aromatic C=C, 1640 for C=C aliphatic double bond
-
stretching, and 1125 cm for C–O group.
1
Antimicrobial activity
Table 1 summarizes the antibacterial results of the tested compounds. The values
are zones of inhibition measured in mm. All tested compounds were active against
Fig. 2 Zone of inhibition for Escherichia coli
Fig. 3 Zone of inhibition for Aspergillus niger
1
23

Synthesis and in vitro and in silico antimicrobial studies of…
all tested organisms. Compounds PY1, PY4, PY8 were very active against Gram-
negative bacteria E. coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa,
whereas compound PY6 was only active against Gram-positive bacteria. Com-
pounds PY6 and PY7 showed good activity against Staphylococcus aureus (20 and
2
2 mm). For Bacillus subtilis, compounds PY2 and PY5 were most active.
Compounds PY3 and PY6 were active against Bacillus cereus. Among all target
compounds, PY5 showed very strong antibacterial activity, especially against
Enterococcus faecalis and Klebsiella pneumoniae, which might be due to presence
Fig. 4 Zone of inhibition for Streptobacillus
Fig. 5 Zone of inhibition for Bacillus subtilis
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K. R. Amperayani et al.
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Synthesis and in vitro and in silico antimicrobial studies of…
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K. R. Amperayani et al.
of ring-activating hydroxyl group. Most of the compounds were active against
Pseudomonas aeruginosa, with compound PY4 showing the highest activity
(
24 mm). The Gram-negative bacterium Enterococcus faecalis showed large
inhibition zone (26 mm) for the applied standard drug. The antifungal activity of
all target molecules is summarized in Table 2. All compounds were moderately
active towards all fungi. Compound PY5 with ring-activating hydroxyl group
showed the highest antifungal activity against Aspergillus flavus, A. fumigatus, and
Candida albicans, with compound PY6 being the next most active against
Aspergillus flavus. The zones of inhibition are shown in Figs. 1–5.
Fig. 6 Binding interactions for different amino acids with the target molecule
Fig. 7 In silico binding mode of piperine-pyridine analogs to protein molecule
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23

Synthesis and in vitro and in silico antimicrobial studies of…
Fig. 8 Ball and stick model for the interactions of amino acids to the analogs
In silico studies
In silicostudiesofdockingto5XGT(Staphylococcusaureus), 3ZIH(Bacillussubtilis),
5
NWT (Escherichia coli), and 2W7Q (Pseudomonas aeruginosa) were carried out
using AutoDock 2.0 software. The binding energies of the target compounds with all
microorganisms are expressed in kcal/mol in Table 3. Compound PY5 showed the
highest binding energy with all the organisms. Due to presence of hydroxyl group, this
compound might bond to protein via strong hydrogen bonding (Figs. 6–8).
Conclusions
A novel series of piperine–pyridine analogs (PY1–8) were designed and synthesized
by coupling piperic acid with substituted aminopyridines using HBTU as coupling
agent. All synthesized molecules were novel and tested for their antibacterial and
antifungal activities. In silico studies were also conducted to determine the binding
efficiency of each synthesized molecule to proteins of microorganisms. All tested
compounds showed moderate to high activity towards all tested organisms. Among
all the compounds, PY5 with ring-activating hydroxyl group exhibited the highest
activity against both bacteria and fungi. There are no reports on synthesis of
piperine–pyridine analogs, so this represents the first report on synthesis of novel
piperine–pyridine analogs and their antimicrobial activity.
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