Synthesis, in vitro and in silico anti-bacterial analysis of piperine and piperic ester analogues

Article abstract of DOI:10.1111/cbdd.13842

A set of 12 analogues of piperine was designed, replacing the amide functional group of the molecule with different aliphatic and aromatic ester functional groups. Molecular docking studies of these molecules with FDA-approved target proteins for anti-bacterial drugs were done. The binding energy of the proteins and the ligands were low and the analogues were found to be drug-like based on the ADME results; hence, the molecules were synthesized. The synthesized compounds were tested for their anti-bacterial property against six bacterial species via Agar well-diffusion method. Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus epidermidis were the strains tested. The overall susceptibility is higher in gram-positive. The analogues showed better activity than piperine. The analogues, propyl piperic ester (P3) and 2-fluorophenyl piperic ester (P9) and 4-fluorophenyl piperic ester (P10) showed comparatively bigger inhibition zones for all the strains.

Full text of DOI:10.1111/cbdd.13842

Received: 13 November 2020
DOI: 10.1111/cbdd.13842
Revised: 2 January 2021
Accepted: 23 March 2021
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R E S E A R C H A R T I C L E
Synthesis, in vitro and in silico anti-bacterial analysis of piperine
and piperic ester analogues
Arthi Sivashanmugam
|
Sivan Velmathi
Organic and Polymer Synthesis Laboratory,
Department of Chemistry, National Institute
of Technology, Tiruchirappalli, India
Abstract
A set of 12 analogues of piperine was designed, replacing the amide functional
group of the molecule with different aliphatic and aromatic ester functional groups.
Molecular docking studies of these molecules with FDA-approved target proteins for
anti-bacterial drugs were done. The binding energy of the proteins and the ligands
were low and the analogues were found to be drug-like based on the ADME results;
hence, the molecules were synthesized. The synthesized compounds were tested
for their anti-bacterial property against six bacterial species via Agar well-diffusion
method. Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus,
Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus epidermidis
were the strains tested. The overall susceptibility is higher in gram-positive. The ana-
logues showed better activity than piperine. The analogues, propyl piperic ester (P3)
and 2-fluorophenyl piperic ester (P9) and 4-fluorophenyl piperic ester (P10) showed
comparatively bigger inhibition zones for all the strains.
Correspondence
Sivan Velmathi, Organic and Polymer
Synthesis Laboratory, Department
of Chemistry, National Institute of
Technology, Tiruchirappalli 620015, India.
Emails: velmathis@nitt.edu; svelmathi@
hotmail.com
Funding information
MHRD
K E Y W O R D S
ADME, anti-microbial, bacteria, docking, piperine, piperine analogues
1
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INTRODUCTION
modulation property, anti-cancerous property, TRPV1 inhi-
bition etc. Focussing on the anti-bacterial activity, there have
A constant update in the library of anti-microbial is must
as bacteria tend to mutate. A mutated bacterium, otherwise
called a Super bug, is considered highly dangerous, and it can
withstand a broad range of anti-bacterial drugs at once. For
instance, mutated Staphylococcus aureus, methicillin resis-
tant S. aureus (MRSA), can be effectively encountered with
new antibiotics that the bacterium or its antecedent had not
fought with before. Introducing ‘Molecule with new pharma-
cophore’ as antibiotics is one of the efficient ways to address
this problem. Natural products that are known for their bio-
activity provides such perfect new molecular bases. Herein,
we report the bactericidal effect of a natural product piperine
and its structural analogues.
been quite a number of articles that discuss the synergistic
anti-microbial effect, the most studied bacteria for the syner-
gistic effect of piperine and its analogues are Staphylococcus
aureus with NorA efflux pump and MdeA efflux pump
(Khan et al., 2006; A. Kumar et al., 2008; Mgbeahuruike
et al., 2019; Mirza et al., 2011; Raja et al., 2015; Sangwan
et al., 2008) of piperine analogues and just very few reports
on the bactericidal effect of the compounds.
In 2013, the anti-bacterial activity of piperine analogues
against four Gram-negative and three Gram-positive bacteria
was tested by Umadevi et.al. They have targeted the piperidine
ring moiety and replaced it with 4-chloro aniline, 4-bromo an-
iline, histidine, phenylalanine and tryptophan amines. These
amide analogues are reported to have better activity than pip-
erine (Umadevi et al., 2013). When the piperidine moiety is
replaced with α-aminoacyl phenylalanine pinanediol boronic
Piperine is a bio-active component obtained from Indian
black pepper, Piper nigrum. Piperine is reported for its anti-
microbial property, bioavailability enhancement, GABA
Chem Biol Drug Des. 2021;00:1–11.
wileyonlinelibrary.com/journal/cbdd
© 2021 John Wiley & Sons A/S.
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1

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SIVASHANMUGAM ANd VELMATHI
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ester in the place of piperidine ring, the anti-bacterial activity
reduces. Here, piperine shows better activity than the boronic
ester analogues (Venugopal, 2014). Then, using the theory
of Hybrids or Conjugate molecule, piperidine ring has been
replaced with substituted pyridine moieties and 1,2,4 triazole
rings retaining the amide functionality. These analogues ex-
ceeded the activity of piperine successfully against S. aureus
(ATR) spectroscopy. Reaction progress was monitored by
thin-layer chromatography on Merck TLC silica gel plates
with either hexane: ethyl acetate (6:4) or methanol: chloro-
form (1:9) as mobile phase. Spots were visualized under ul-
traviolet (UV) chamber. The anti-bacterial experiments were
carried out in Thermo Fischer Bio Safety Cabinet of B2 se-
ries, model number 1300. HiMedia Sterile disposable petri
plates were used to grow the culture.
(
Amperayani et al., 2018; Kumar et al., 2019).
In this study, the analogues were synthesized in an or-
derly fashion with alkyl and aryl esters. The focus is on the
bactericidal efficacy of the molecules and to find the plausi-
ble reason for the activity, and the synergism is not checked.
The objective is to change the functional group of the mol-
ecule and record the change in activity. Firstly, the tertiary
amide functional group in the molecule is converted to pip-
eric acid which is then converted to different aliphatic and
aromatic esters. Esters are labile functional groups that are
used as prodrugs and drugs (Lavis, 2008; Roll et al., 2007;
Wollina, 2011). Since there is no research explicitly on the
anti-bacterial properties of alkyl/aryl piperic carboxylic es-
ters, we took the opportunity to study them.
Six bacterial species, Acinetobacter baumannii,
Pseudomonas aeruginosa, Escherichia faecalis, Escherichia
coli, Staphylococcus epidermidis and Staphylococcus aureus,
were taken for the screening. The bactericidal activity of the
analogues is considerable against the third generation bacte-
ria, which are substantially stronger than the first sub-culture.
This proves the analogues to be potent. Also, all the ana-
logues are more potent than the mother molecule piperine.
2.1
|
Chemistry
The amide functional group of piperine was converted to a
better reactive carboxylic acid functional group via base hy-
drolysis, to piperic acid. Piperic acid is the first analogue of
piperine, and this molecule was used as the starting mate-
rial for the synthesis of all the other ester analogues in this
study. Piperic acid was treated with thionyl chloride to form
piperic acyl chloride which is then treated with alcoholic or
phenolic -OH to form respective esters. The structures of the
1
13
compounds are validated by H, C NMR and IR spectro-
scopic techniques.
2.2
|
Synthesis of piperic acid
Piperine (10 g) was dissolved in 200 ml of 15% ethanolic
NaOH in a single neck RB flask and refluxed. The reaction
was monitored through TLC. The reaction got over after
2
4 hr. The ethanol in the medium was completely evaporated,
and the resulting sodium piperate is dissolved in ice cold dis-
tilled water. To this, 0.1N HCl was added slowly, till the pre-
cipitation of piperic acid was complete. The precipitate was
then filtered and washed with distilled water. The product
was recrystallized in ethanol.
2
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MATERIALS AND METHODS
All reagents used were of analytical grade (AR) and used di-
rectly without further purification. Piperine was a kind gift
from the commercial producers Polyhedron Laboratories
Pvt. Ltd, Tamilnadu, India. AV-500-Bruker 500 MHz high-
resolution multinuclear FT-NMR spectrometer was used for
2.3
|
Synthesis of piperic esters
1
13
H & C Spectroscopic measurements, and chemical shifts
are given in parts per million (ppm). FTIR spectrum was re-
corded on a PerkinElmer FT-IR attenuated total reflectance
Scheme1representsthegeneralsyntheticprocedurefollowed.
In a vacuum dried RBF, piperic acid (500 mg, 2.29 mmol)
SCHEME 1 General Scheme of synthesis of ester analogues of piperine

SIVASHANMUGAM ANd VELMATHI
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dissolved in 15 ml of DCM was added. An ice bath was used
anti-bacterial targets. The proteins were prepped by remov-
ing the water molecules and the ligands from their crystal
structures. Flexible blind docking was employed. The target
sites in the protein structures were confirmed via Metapocket
2.0 (Zhang et al., 2011). Here, the receptor is protein mol-
ecule, and the ligand is piperine analogue. Ligands were
drawn using ChemDraw suite.
Autodock Vina with Python GUI was used for the Docking
studies (Trott & Olson, 2010). Conjugate gradient algo-
rithm and MMFF94 were the forcefield used for the energy
minimization of the ligands, throughout the docking stud-
ies (Developed by the Resource for Biocomputing; Morris
et al., 2009). The exhaustiveness was set to 8, total number
of steps involved in the energy minimization to 200 steps and
the process to end when the energy difference between the
structures was found to be <0.1 for the whole process. For
each protein used, the dimension of the Grid box differed.
The binding score of the docked model with the least Root
Mean Square Deviation (RMSD) was considered. Discovery
Studio was used to view the Autodock results.
The ADME properties of the molecules were calculated
theoretically. The theoretical calculations of physicochem-
ical properties like partition co-efficient (Log P) (Cheng
et al., 2007; Wildman & Crippen, 1999), Topological Polar
Surface Area (TPSA), number of hydrogen Bonds, number of
hydrogen acceptors, molar refractivity, gastro intestinal ab-
sorption, cytochrome P450 interaction along with the com-
pliance or violation of the drug-likeness rules of Lipinski,
Ghose, Veber, Egan and Muegge were considered before syn-
thesis (Daina et al., 2017).
to cool this set up, and SOCl (1.5 mmol) was added slowly.
2
After the addition of SOCl , the temperature was gradually
2
raised to room temperature. The formation of acyl chloride
was confirmed through TLC. The reaction was completed
after 7 hr. In a separate vacuum-dried RBF, alcohol/phenol of
interest (1.1 mmol) and tri ethyl amine (1 mmol) were taken
and dissolved in 10 ml of DCM. These contents were trans-
ferred slowly into the RBF containing acyl chloride at 0°C.
The reaction was let to run and monitored via TLC. After the
completion, the reaction was quenched with water and the
organic layer was collected. The collected DCM layer was
washed with water, brine solution, dried with magnesium
sulphate and evaporated to get respective esters. The crude
product was then column chromatographed using hexane and
ethyl acetate (9:1) to get pure compounds. Figure 1 enlists the
structure of the analogues.
2
.4
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Computational analysis
The analogues were studied theoretically for their anti-
bacterial property via chemometric studies. The compounds
were docked with the crystal structures of 7 proteins ob-
tained from RCSB Protein Data bank (Berman et al., 2000).
Uridylate kinase, Penicillin-binding protein 1B, Membrane-
bound lytic murein transglycosylase F, Acyl carrier protein,
tRNA dimethyl allyl transferase and Teichoic acid D-alanine
hydrolase proteins (PDB ID: 4a7x, 3vma, 5a5x, 6dfl, 3crm,
5
zh8, 1amp) were taken as targets, which are popular
P2
P3
P1
P4
P7
P5
P8
P6
P9
P11
P12
P10
FIGURE 1 Structure of Piperine analogues

4
SIVASHANMUGAM ANd VELMATHI
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2
.5
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Anti-bacterial assay
Commercially obtained three gram-positive and three gram-
negative bacterial strains were taken for the anti-bacterial test.
Escherichia coli, Acinetobacter baumannii, Staphylococcus
aureus, Staphylococcus epidermidis, Pseudomonas aerugi-
nosa and Enterococcus faecalis were the bacteria. The ob-
tained bacteria were sub-cultured to their third generations
and then used. Agar well-diffusion method was employed.
FIGURE 2 Structure of P2 with H numbering
2
0 ml of Muller–Hinton Agar (MHA) was spread on Petri
is another characteristic of CO stretching. These peaks are
observed in all the analogue's IR spectrum, which is one of
the clear indications for the formation of the compounds. In
plates and led to solidify in the room temperature. The cul-
5
tured bacteria were diluted with sterile solution to 10 CFU/
1
ml prior to the even application on the MHA Petri plates. The
stock solution of piperine and the analogues was prepared
in the concentration of 50 mg/ml in DMSO solvent. Serial
dilution was avoided to reduce the volume of DMSO used in
the well. The concentration variation of the compounds was
achieved by using different volumes (30 µl = 1.5 mg/well,
H NMR, the unsaturated chain protons and the benzene pro-
tons come in the aromatic region of δ 7.43–δ 6.67; breaking
that down, quartet at δ 7.43 ppm is due to H with J = 15 Hz
8
indicating trans coupling with the protons H and H ; singlet
6
7
at δ 6.99 ppm is due to H ; a doublet with coupling constant
3
10 Hz indicates cis coupling of H with H ; peaks by H and
4
5
5
4
0 µl = 2 mg/well, 50 µl = 2.5 mg/well) of the same stock
H are merged to give peaks around δ 6.79 ppm, with cou-
6
solution, which is one of our major variations from the litera-
ture. Wells of 6 mm diameter were made in the MHA petri
plates. The target compounds were added to the wells and in-
cubated at 37°C for 24 hr. Gentamycin and Ampicillin (30 µg/
ml) were used as reference drugs. A blank well (DMSO) was
kept for reference. The activity of the target compounds was
measured based on the measure of the zone of inhibition after
pling constant values 15, 20 and 5 Hz respectively; H gives
7
a quartet at δ 6.72–δ 6.67 with coupling constant 15 Hz indi-
cating trans coupling; the high trans coupling constant values
indicate the flexible unsaturated chain; further, singlet at δ
5.98 ppm is due to H and H ; H gives rise to a doublet at
1
2
9
δ 5.939 away from the other alkyl chain protons due to its
position near the carbonyl group which de-shields the H ;
9
2
4 hr of incubation. The diameter of the zone of inhibition
a quartet at δ 4.24–δ 4.20 ppm is due to the ethyl protons
was measured in millimetres.
H and H with J = 10 Hz; and triplet at δ 1.31 ppm is due to
1
1
10
H , H and H of the ethyl protons, the coupling constant
1
1
12
13
value 10Hz indicated vicinal coupling. The same pattern can
3
3
|
RESULTS AND DISCUSSION
Spectroscopic analysis
be observed in all the other analogues too.
1
3
For C NMR (Figure 2), the carbonyl carbon in P2 gives
its peak at δ 167 ppm; C and C give peaks at δ 148.55 and
.1
|
2
3
1
48.29 ppm respectively; C gives peak at δ 140 ppm; C ,
7 8 9
10 6
C , C and C give peaks at δ 130.59, 124.56, 122.94 and
The spectral data of the synthesized compounds clearly show
the formation of the analogues. The shift of peak in IR from
120.46 ppm; C and C at δ 108.55 and 105.88 ppm; C at
4
5
1
-
1
1
674 for piperic acid to around 1,700 cm for the ester ana-
δ 101.40 ppm and the ethyl carbons C and C came at δ
13 14
logues is clear indication of formation of carboxylic ester
functional group, while the peak for dioxy methylene group
60.31 and δ 14.35 ppm, respectively. The trend is observed in
all the analogues which proves formation of the compounds.
-1
1
appears around 1,492 cm . In the H NMR, the dioxy meth-
ylene group (-O-CH -O-) gives a singlet around δ 6 ppm, as
2
the alkyl chain is in conjugation with the aromatic ring, the
peaks for the protons in the alkyl chain falls under the aro-
matic region too, except for one proton next to the carbonyl
3.2
|
Computational analysis
The analogues were given ester functional group, as esters
are well-known prodrugs. The pharmacophore of the mol-
ecule maintained for this work is given in Figure 3. The phar-
macophore contains three hydrogen donors (HYD) separated
1
3
group which gives a doublet around δ 6 ppm. In C NMR,
the peak around δ 167 ppm indicates the carboxylic ester
functional group and peak around δ 101 ppm indicates the
0
dioxy methylene (-O-CH -O-) carbon.
by the distance of 1.2 A : two aromatic ring moieties (AR) at
2
0
Structure of P2 in Figure 2 is taken for single compound
distance 2.1 A from each other and one aromatic ring from
−1
0
discussion, 1,703 cm indicates carbonyl ester stretch-
the HYD is at the distance 2 A , and all these are surrounded
−1
ing, peak around 1,489 cm indicates -CH - bending from
by 4 hydrogen acceptors (ACC). Similar arrangement could
be found in molecules like gingerol, curcumin and quercetin
which are known for their anti-microbial properties.
2
−1
1
,3-benzodioxole ring, peak around 1,253 cm is the indi-
−
1
cation of asymmetric -CO stretching and peak at 930 cm

SIVASHANMUGAM ANd VELMATHI
5
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FIGURE 3 Pharmacophore of the
analogues generated by overlapping the
1
5 analogues and the Piperine molecule.
Here, Acc is Acceptor, AR is Aromatic
Ring, HYD is H Donor, Bond length is
in A0
TABLE 1 Binding energies in kcal/mol
of the analogues with different proteins
Compound
4a7x
3vma
5a5x
6dfl
3crm
5zh8
1amp
Pip
PA
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
−7.3
−6.1
−7.2
−6.1
−6.2
−6.2
−6.4
−6.4
−7.2
−7.5
−7
−8.7
−6.9
−7
−5.9
−7.7
−7.5
−7.7
−7.4
8.5
−9.1
−8.9
−8.9
−8.8
−8.3
−7.3
−7.3
−7.5
−7.1
−7.3
−6.9
−6.8
−7.2
−7.6
−8.6
−8.2
−9.3
−8.5
−8
−7.8
−9.2
−9.2
−6.4
−9.5
−9.8
−6.5
−7.9
−11.3
−8
−8.2
−7.3
−7.2
−6.9
−7.2
−7.2
−7
−7.6
−6.9
−7.3
−6.6
−6.8
−6.9
−6.8
−7.9
−7.9
−7.6
−7.9
−8.1
−8.9
−9.2
−6.7
−5.4
−5.1
−5.1
−5.8
−6.1
−5.2
−5.8
−6.6
−6.7
−6.3
−6.4
−6.8
−6.6
−6.7
−8
−8.5
−7.9
−8.1
−8.4
−8.2
−9.2
−10.5
−11.8
−10.7
−7.1
−7.1
−7.2
The compounds were docked with 7 proteins with PDB ID:
a7x, 3vma, 5a5x, 6dfl, 3crm, 5zh8 and 1amp. UridylateAQ6
analogues are predicted to be CYP inhibitors which again
supports the drug bioavailability. It is noted that the mole-
cules follow the Rule of five by Lipinski, drug-like rules by
Ghose, Veber, Egan and Muegge. As the ADME properties
were satisfactory and the Binding Energy of the molecules
was found to be good, it was proceeded to the synthesis of
the molecules. The binding energy of the compounds is in
Table 1, and the ADME results are furnished in the support-
ing information.
4
kinase is involved in UDP synthesis. Penicillin-binding pro-
tein 1B is involved in the inhibition of cell wall synthesis.
Membrane-bound lytic murein transglycosylase F was in-
volved in cell wall synthesis. Acyl carrier and lipopolysac-
charide core heptose (I) kinase protein, tRNA dimethylallyl
transferase and Teichoic acid D-alanine hydrolase proteins
were taken as targets.
From Swiss ADME, the ADME properties of the com-
pounds were calculated and it was made sure the analogues
were drug-like theoretically. We have taken WLogP and
XLogP3 (atomistic LogP values), which are proven to be
most relevant to the Experimental LogP (Pyka et al., 2006).
The XLogP3 of the analogues ranges from 3.23 to 6.44, while
WLogP ranges from 1.96 to 4.78. The bioavailability score
predicted by the software is 0.55–0.56 which is equivalent
to the predicted bioavailability of the commercial antibiotics
The binding of the ligands that showed highest inhibition
zones and nil inhibition zones against Protein (PDB ID: 6dfl)
whose binding energy best correlated with the experimen-
tal results is exhibited in Figures 4–7. The aliphatic and the
aromatic esters are divided and analysed for better under-
standing. Overall, in the aliphatic ester analogues P3 and P6
were the highest and nil inhibition zone producers respec-
tively in almost all the strains. Similarly, P9 and P10 are best
among aromatic esters, while P8 being the least inhibition
zone producer in the aromatic esters. The analogues are not
similar in action for all the bacterial strains. For the strains
E. faecalis and P. aeruginosa, A. baumannii analogue P3 has
(
for example, Ciprofloxacin: WLogP –1.18, Bioavailability
Score- 0.55, with GI absorption, Ampicillin: WLogP -
0.39, Bioavailability Score- 0.55, with GI absorption). The
−

6
SIVASHANMUGAM ANd VELMATHI
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FIGURE 4 Binding mode of P6 with protein (PDB ID: 6dfl). (a) 3d binding mode (b) 2d binding mode
FIGURE 5 Binding mode of P8 with protein (PDB ID: 6dfl). (a) 3d binding mode (b) 2d binding mode
produced comparatively higher inhibition zone. Similarly for
the strains S. epidermidis and S. aureus, the F substituted an-
alogues P9 and P10 have shown better inhibition zones. It is
to be noted that A. baumanni and P. aeruginosa fall under the
order Pseudomonadales and strains S. epidermidis and S. au-
reus fall under the order Bacillales.
In the 3D view, we can observe that the better working an-
alogues P3, P9 and P10 pass through the hydrophobic tunnels
of the α-helix system of the protein. The binding mode of
P3, P9 and P10 is similar in the 1,3-benzodioxole ring region
of the analogue. In the figures, an inactive analogue from
aliphatic ester (P6) and an inactive analogue from aromatic
ester (P8) are shown to highlight the difference in the binding
modes. In all active analogues, P3, P9 and P10, Pi–Pi staking
is observed between the 1,3-benzodioxole ring and the pocket
atoms PHE A:227 and TYR A:211. Similarly, these analogues
show a Pi–alkyl interaction between 1,3-benzodioxole ring
and Leu A:210 of the protein molecule. The fluoro phenyl
ring of the P9 and P10 is observed to form a Pi–alkyl interac-
tion with LYS A:224 and ARG A:221, along with a Pi–sigma
interaction with LEU A:223. The residues LEU A:219, VAL
A:144, LEU A:240, GLU A:251, LEU A:228, MSE A:250
and ALA A:214 are involved in the Van der waal's interaction
with the molecule. In P3, other than the common interaction
observed as with P9, alkyl interactions between the propyl
chain and the residues LEU A:123, LEU A:143 and TRP

SIVASHANMUGAM ANd VELMATHI
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FIGURE 6 Binding mode of P9 with protein (PDB ID: 6dfl). (a) 3d binding mode (b) 2d binding mode
FIGURE 7 Binding mode of P3 with protein (PDB ID: 6dfl). (a) 3d binding mode (b) 2d binding mode
A:130 can be observed. Also VAL A:147, VAL A:144, LEU
A:207, MSE A:250, LEU A:228, LYS A:224, ALA A:214,
ILE A:217, ILE A:166, SER A:127 and PHE A:126 are in-
volved in the Van der waal's interaction with the molecule P3.
an extra protection to the cell making it less susceptible. Here,
we used third generation bacteria which are substantially
stronger that increases the ability of the bacteria to mutate or
resist the drug much better, than the weaker bacteria. The re-
sults are given in the Tables 2 and 3. The Anti-bacterial assay
plates after 24 hr of incubation with compounds P1, P2, P3
and P4 numbered as 1, 2, 3 and 4 respectively with Positive
3
.3
|
Anti-bacterial activity
(
+) and Negative (−) controls against the six bacterial strains
Having the piperine binding sites in mind, we started the work
by classifying the bacteria based on the thickness of their cell
walls. Three gram-positive and three gram-negative bacteria
were taken, and the action of the analogues over the bacteria
was observed. Gram-positive bacteria have thick peptidogly-
can layer while the gram-negative bacteria have thinner layer,
but gram-negative bacteria have lipid bilayer which provides
are shown in Figure 8.
Based on our anti-bacterial result, we observed that,
1. The analogues show better activity than piperine at any
concentration.
2. Piperine showed no inhibition zones for any strains but for
S. aureus and P. aeruginosa.

8
SIVASHANMUGAM ANd VELMATHI
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SIVASHANMUGAM ANd VELMATHI
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1
0
SIVASHANMUGAM ANd VELMATHI
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FIGURE 8 Anti-bacterial assay
plates after 24 hr of incubation. Results of
compounds P1, P2, P3 and P4 numbered
as 1, 2, 3 and 4 respectively with Positive
+) and Negative (−) controls against the
six bacterial strains
(
A.baummanni
E. faecalis
S. aureus
S. epidermidis
P. aeruginosa
E. coli
3
4
. PA, P3, P9 and P10 showed the best activity among all the
analogues for all the bacteria.
model of the ligands to the protein PDB ID: 6dfl shows simi-
lar binding pattern among the best active analogues P3, P9
and P10 at 1,3-benzodioxole ring of the molecules along with
the increased number of non-covalent interaction overall. This
could be due to the optimum flexibility, bond energy and the
rotatable bonds in the analogue. Additionally, the analogue P3
produces better inhibition zones for the bacterial strains under
the order Pseudomonadales, while the fluoro phenyl esters P9
and P10 work best for the strains under the order Bacillales.
From this, it is clear that the molecule has not lost its activity
with the change in the functional group, that is, from amide
to ester, but enhancement in the activity is observed. Yet, to
increase the activity of the molecules to be equivalent to the
commercial drugs, further modifications have to be done.
. The inhibition zones for the Gram-positive bacteria are
slightly higher than the Gram-negative bacteria. The inhi-
bition of gram-negative is usually higher due to its thick
outer lipid layer.
5
. P3 works best for the strains under the order
Pseudomonadales, and P9 and P10 for the strains under
the order Bacillales
The theoretical interaction of the analogues with the
Hydrolases protein (PDP ID: 6dfl) in Figures 4–7 shows the
possible interaction mode of the ligands. From which it is
clear that P3, P9 and P10 have comparatively more number
of non-covalent interactions with the α-helix structure of the
protein than other analogues.
ACKNOWLEDGEMENT
One of the authors A.S gratefully acknowledges MHRD for
fellowship.
4
|
CONCLUSION
CONFLICTS OF INTEREST
The synthesized structural analogues of piperine were tested
against six bacterial strains, A. baumannii, E. coli, S. aureus,
S. epidermidis, P. aeruginosa and E. Faecalis. The cultures
used were of third generation (3rd sub-culture), which con-
siderably increases the strength of the bacteria than the first
sub-culture. To maintain low carrier solvent level, the con-
centration variation was achieved by using different volume
of dissolved analogues in the agar well from the same stock
solution. Most of the analogues show better activity than pip-
erine. The activity of the analogues increases with increase in
the concentration. Proteins involved in the Hydrolases show
better correlation with the activity of the molecules. Propyl
substituted piperic ester analogue among the aliphatic esters
category and fluoro phenyl piperic esters among the aromatic
esters category show better activity. The theoretical binding
The authors declare no conflicts of interest.
ORCID
Arthi Sivashanmugam
https://orcid.org/0000-0002-7487-
7142
Sivan Velmathi
https://orcid.org/0000-0001-7303-0698
REFERENCES
Amperayani, K. R., Kumar, K. N., & Parimi, U. D. (2018). Synthesis
and in vitro and in silico antimicrobial studies of novel piperine–
pyridine analogs. Research on Chemical Intermediates, 44(5),
3
549–3564. https://doi.org/10.1007/s11164-018-3324-1
Berman, H. M., Feng, J. W. Z., Gilliland, G., Bhat, T. N., Weissig, H.,
Shindyalov, I. N., & Bourne, P. E. (2000). The protein data bank
nucleic acids research. Retrieved from rcsb.org. rcsb.org

SIVASHANMUGAM ANd VELMATHI
11
|
Cheng, T., Zhao, Y., Li, X., Lin, F. U., Xu, Y., Zhang, X., Li, Y., Wang,
R., & Lai, L. (2007). Computation of octanol-water partition coef-
ficients by guiding an additive model with knowledge. Journal of
Chemical Information and Modeling, 47(6), 2140–2148. https://doi.
org/10.1021/ci700257y
Roll, A., Reich, K., & Boer, A. (2007). Use of fumaric acid esters in pso-
riasis. Indian Journal of Dermatology, Venereology and Leprology,
73(2), 133–137. https://doi.org/10.4103/0378-6323.31908
Sangwan, P. L., Koul, J. L., Koul, S., Reddy, M. V., Thota, N., Khan,
I. A., Kumar, A., Kalia, N. P., & Qazi, G. N. (2008). Piperine ana-
logs as potent Staphylococcus aureus NorA efflux pump inhibitors.
Bioorganic & Medicinal Chemistry, 16(22), 9847–9857. https://doi.
org/10.1016/j.bmc.2008.09.042
Trott, O., & Olson, A. J. (2010). AutoDock Vina: Improving the speed
and accuracy of docking with a new scoring function, efficient opti-
mization and multithreading. Journal of Computational Chemistry,
31, 455–461.
Umadevi, P., Deepti, K., & Venugopal, D. V. R. (2013). Synthesis, an-
ticancer and antibacterial activities of piperine analogs. Medicinal
Chemistry Research, 22(11), 5466–5471. https://doi.org/10.1007/
s00044-013-0541-4
Venugopal, D. V. R. (2014). Synthesis, of novel piperine analogs of
dipeptidyl boronic acid as antimicrobial and anticancer agents.
Medicinal Chemistry, 4(9), 606–610. https://doi.org/10.4172/216
1-0444.1000201
Wildman, S. A., & Crippen, G. M. (1999). Prediction of physico-
chemical parameters by atomic contributions. Journal of Chemical
Information and Computer Sciences, 39(5), 868–873. https://doi.
org/10.1021/ci990307l
Wollina, U. (2011). Fumaric acid esters in dermatology. Indian
Dermatology Online Journal, 2(2), 111–119. https://doi.
org/10.4103/2229-5178.86007
Zhang, Z., Li, Y., Lin, B., Schroeder, M., & Huang, B. (2011).
Identification of cavities on protein surface using multiple compu-
tational approaches for drug binding site prediction. Bioinformatics,
27(15), 2083–2088. https://doi.org/10.1093/bioinformatics/btr331
Daina, A., Michielin, O., & Zoete, V. (2017). SwissADME: A free
web tool to evaluate pharmacokinetics, drug-likeness and medici-
nal chemistry friendliness of small molecules. Scientific Reports, 7,
4
2717. https://doi.org/10.1038/srep42717
Khan, I. A., Mirza, Z. M., Kumar, A., Verma, V., & Qazi, G. N. (2006).
Piperine, a phytochemical potentiator of ciprofloxacin against
Staphylococcus aureus. Antimicrobial Agents and Chemotherapy,
5
0(2), 810–812. https://doi.org/10.1128/AAC.50.2.810-812.2006
Kumar, A., Khan, I. A., Koul, S., Koul, J. L., Taneja, S. C., Ali, I., Ali,
F., Sharma, S., Mirza, Z. M., Kumar, M., Sangwan, P. L., Gupta,
P., Thota, N., & Qazi, G. N. (2008). Novel structural analogues of
piperine as inhibitors of the NorA efflux pump of Staphylococcus
aureus. Journal of Antimicrobial Chemotherapy, 61(6), 1270–1276.
https://doi.org/10.1093/jac/dkn088
Kumar, K. N., Amperayani, K. R., Sankar Ummdi, V. R., & Parimi,
U. D. (2019). Synthesis and antimicrobial activity of piper-
ine analogues containing 1,2,4-triazole ring. Asian Journal
of Chemistry, 31(5), 1077–1080. https://doi.org/10.14233/
ajchem.2019.21876
Lavis, L. D. (2008). Ester bonds in prodrugs. ACS Chemical Biology,
3
(4), 203–206. https://doi.org/10.1021/cb800065s
Mgbeahuruike, E. E., Stalnacke, M., Vuorela, H., & Holm, Y. (2019).
Antimicrobial and synergistic effects of commercial piperine and
piperlongumine in combination with conventional antimicrobials.
Antibiotics, 8(2), 55. https://doi.org/10.3390/antibiotics8020055
Mirza, Z. M., Kumar, A., Kalia, N. P., Zargar, A., & Khan, I. A. (2011).
Piperine as an inhibitor of the MdeA efflux pump of Staphylococcus
aureus. Journal of Medical Microbiology, 60(Pt 10), 1472–1478.
https://doi.org/10.1099/jmm.0.033167-0
SUPPORTING INFORMATION
Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew,
R. K., Goodsell, D. S., & Olson, A. J. (2009). Autodock4 and
AutoDockTools4: Automated docking with selective receptor flexi-
blity. Journal of Computational Chemistry, 16, 2785–2791.
Pyka, A., Babuska, M., & Zachariasz, M. (2006). A Comparison of the-
oritical methods of calculation of partition coefficients for selected
drugs. Analysis, 63, 159–167.Retrieved from https://www.ptfarm.pl/
pub/File/Acta_Poloniae/2006/3/159.pdf
Additional supporting information may be found online in
the Supporting Information section.
How to cite this article: Sivashanmugam A,
Velmathi S. Synthesis, in vitro and in silico anti-
bacterial analysis of piperine and piperic ester
analogues. Chem Biol Drug Des. 2021;00:1–11.
https://doi.org/10.1111/cbdd.13842
Raja, A., Abdul Kapur, M., Fijju, M., & Mohamed Salique, S. (2015). In
vitro studies on Efflux pump inhibition of Catharanthus roseus and
piperine against ofloxacin resistant M. tuberculosis. International
Journal of Pharmaceutical Science Invention, 4(9), 32–37.