Synthesis and antimicrobial evaluation of piperic acid amides and their lower homologues

Article abstract of DOI:10.1002/ddr.21630

Seven piperic acid amides along with their lower homologs (12) were synthesized using HATU-DIPEA coupling reagent. All the synthesized derivatives were evaluated for their antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, and vancomycin-resistant P. aeruginosa. They were found to be more active on P. aeruginosa than on S. aureus. However, they did not exhibit potent activity on Vancomycin resistant P. aeruginosa. Among the tested compounds, methylenedioxycinnamic acid amide of anthranilic acid (MDCA-AA, 2a) was found to be most active against S. aureus with MIC of 3.125 μg/ml. The PAS and INH amides of piperic acid were screened against Mycobacterium tuberculosis H37R_a strain. They were found to be most active among all the tested compounds but were found to be less active than the standard drug, isoniazid.

Full text of DOI:10.1002/ddr.21630

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Received: 24 June 2019
DOI: 10.1002/ddr.21630
Revised: 19 November 2019
Accepted: 21 November 2019
R E S E A R C H A R T I C L E
Synthesis and antimicrobial evaluation of piperic acid amides
and their lower homologues
Prabhakar S. Achanta1 | Sneha Raj2 | Soyar Horam2 | Jesu Arockiaraj3
Ravi Kumar Bobbala1 | Raghuram Rao Akkinepally1 | Mukesh Pasupuleti2
Appa Rao V. N. Achanta1
|
|
1University College of Pharmaceutical Sciences, Kakatiya University, Warangal Urban, Telangana, India
2Department of Microbiology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India
3Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Chennai, Tamil Nadu,
India
Correspondence
Abstract
Appa Rao V. N. Achanta, University College of
Pharmaceutical Sciences, Kakatiya University,
Seven piperic acid amides along with their lower homologs (12) were synthesized using
Warangal Urban 506009, Telangana, India.
HATU-DIPEA coupling reagent. All the synthesized derivatives were evaluated for their
antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, and
vancomycin-resistant P. aeruginosa. They were found to be more active on
P. aeruginosa than on S. aureus. However, they did not exhibit potent activity on
Vancomycin resistant P. aeruginosa. Among the tested compounds, methylenedio-
xycinnamic acid amide of anthranilic acid (MDCA-AA, 2a) was found to be most active
against S. aureus with MIC of 3.125 μg/ml. The PAS and INH amides of piperic acid were
screened against Mycobacterium tuberculosis H37Ra strain. They were found to be most
active among all the tested compounds but were found to be less active than the stan-
dard drug, isoniazid.
Email: avnapparao@yahoo.com
Mukesh Pasupuleti, Department of
Microbiology, CSIR-Central Drug Research
Institute, B.S. 10/1 Sector-10, Janakipuram
Extension, Sitapur Road, Lucknow 226031,
Uttar Pradesh, India.
Email: mukeshp@cdri.res.in
Present address
National Institute of Pharmaceutical Education
and Research, Mohali, Sector 67, Phase
10, Sahibzada Ajit Singh Nagar 160062,
Punjab, India.
Funding information
All India Council for Technical Education,
Grant/Award Number: 06/AICTE/RIFD(Policy-
4th)/EF(01)/2012-13; Council for Scientific
and Industrial Research, Grant/Award Number:
09/384(159)/2013.EMR-I
K E Y W O R D S
amide coupling, antibacterial activity, antitubercular activity, piperamides
1
|
INTRODUCTION
Black pepper (fruits of Piper nigrum L.) has been in use in tradi-
tional medicine as stimulant, carminative, diuretic, and antiasthmatic.
Only four new small molecule antibacterials were approved for human
use from 2014 and bedaquiline is the only new antimycobacterial agent
since 2012 (Jackson, Czaplewski, & Piddock, 2018; Lohrasbi et al., 2018).
Multidrug resistance among the “ESKAPE” organisms, Enterococcus
faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa and Enterobacter spp. is of concern as
they are causing many serious infections in global public healthcare sys-
tems and hospitals (Tommasi, Brown, Walkup, Manchester, & Miller,
2015). Therefore, there is an urgent need to discover newer agents for
drug-resistant bacteria. Naturally occurring chemical compounds are a
good source of antibacterial agents (Wright, 2017).
It is also prescribed in fevers, dyspepsia, and used as gargle for sore
throat (Aumeeruddy & Mahomoodally, 2019; Khare, 2007). Piperine
and various amides of piperic acid (PA) were found to potentiate
antibacterial action of ciprofloxacin against S. aureus (Sangwan et al.,
2008). Piperic acid conjugates of curcumin were found to be active
(MIC: 2.5 μM/ml) on P. aeruginosa and P. pyocynin (Mishra, Narain,
Mishra, & Misra, 2005).
Substituted cinnamic acid amides of 7-Aminocephalosporanic
acid (ACA) were synthesized and tested against S. aureus.
3,4-Dihydroxycinnamic acid amide was found to have MIC of 4 μg/ml
(1; Figure 1) (López et al., 2004).
Drug Dev Res. 2019;1–8.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

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ACHANTA ET AL.
Methylenedioxycinnamic acid (MDCA) amides of isoniazid (INH)
and benzoic acid hydrazide were synthesized and tested along with
few other cinnamic acid derivatives for their antimycobacterial activ-
ity. Most of the isoniazid hybrids including MDCA-INH were reported
to be active at 3.12 μg/ml (2; Figure 2) (Carvalho, da Silva, de Souza,
Lourenço, & Vicente, 2008).
DMAP, HOBT were procured from Hymedia (Mumbai). The amine
portion of aztreonam was purchased from Ark Pharm Inc., USA.
7-ADCA, 7-ACA, 6-APA, amine of Cefixime, 5-methyl-2-amino thia-
zole, and 4-aminosalicylic acid were gift samples from Orchid Pharma,
Chennai, Ramdev Pharma, Mumbai and Biosynth, Hyderabad.
By using molecular hybridization strategy, amines which are
either drugs or part of biologically active molecules were conjugated
with PA, MDCA and Piperonylic Acid (PoA) and evaluated for their
antibacterial and antimycobacterial actions. Selected compounds were
tested for their cytotoxicity on normal (HaCaT) cell line.
2.2
|
Isolation of piperine (7)
Fresh fruits of black pepper (500 g) were purchased from the local
market and powdered. The powder was macerated with 1 L of chloro-
form for 72 hr in a conical flask. Chloroform extract was collected by
filtration and the marc was extracted three more times. Chloroform
was removed in vacuo and the residue was washed with diethyl ether
(5 × 100 ml). This yielded piperine (20 g) as a pale yellow powder
whose identity and homogeneity was established by comparing with a
standard sample. It was taken up for hydrolysis without further
purification.
The in silico evaluation of ADME properties of the compounds
was carried out on admetSAR (Miteva et al., 2006) and FAF-Drugs4
(Cheng et al., 2012).
2
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EXPERIMENTAL
| General
2.1
All the solvents and general chemicals were of laboratory grade, and
procured from SD Fine Chemicals (Mumbai) and Merck (Hyderabad).
TLC plates: Normal phase silica gel 60 F254 and Reversed phase silica
gel 60 RP-18F254s (both of E. Merck). Silica gel for open column
chromatography: Normal phase (E. Merck, #230–400) and reversed
phase (C-18, Sorbent technologies). Melting points were recorded on
Mel-Temp M4125 and were uncorrected. UV: Elico UV–visible spec-
trophotometer SL 159 and IR: Bruker Alpha FTIR spectrometer. NMR
spectra were recorded at 27ꢀC with a Bruker 400 or 300 MHz with a
5-mm triple-resonance probe equipped with puls-filed z-gradient; d in
ppm, J in Hz in DMSO-d6 or Methanol-d4. Mass spectra were
recorded on Agilent 5973i quadrupole mass spectrometer.
2.3
|
Procedures for syntheses and bioevaluation
All the methods and the NMR and mass spectral data of piper amides
and lower homologs is given as supplementary information.
3
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RESULTS AND DISCUSSION
Amide synthesis is usually achieved by converting the acid to acid
chloride using thionyl chloride and then treating the activated acid
with amine in the presence of a base. But amides of piperic acid,
methylenedioxycinnamic acid, and piperonylic acid could not be
obtained when this procedure was followed. DCC-DMAP and DCC-
HOBt-DMAP combinations were also tried but did not prove success-
ful. However, desired products could be obtained using HATU-DIPEA
combination (Scheme1) (Carpino, 1993; Carpino, El-Faham, & Albe-
ricio, 1995). Later, the title compounds were also synthesized by using
Piperonal and oxone were purchased from Sigma Aldrich
(Bengaluru). HATU was purchased from Alfa Aesar (Hyderabad). DCC,
FIGURE 1 Strategy to modify compound 1
FIGURE 2 Strategy to modify compound 2

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ACHANTA ET AL.
3
oxalyl chloride to convert the acid to acid chloride and adding it to a
substituted amine in THF. The yields obtained by this method were
lower when compared to the yields obtained by the use of HATU-
DIPEA as coupling reagent.
from byproducts and purified by reversed phase (C-18) open column
chromatography as they did not resolve well on normal phase TLC.
The UV spectra of all the compounds gave two typical absorption
bands. The IR spectra gave peaks characteristic of the functional
groups present in the compounds but are highly overlapped due to
the presence of similar functionalities like C C alkenyl and C C aro-
matic, N H str (amide), and O H str (carboxylic acid), C O amide
and C O acid. Therefore, IR spectral analysis was not useful in con-
firming the formation of the products (File S1).
Amines such as 6-APA, 7-ACA, 5-methyl-2-aminothiazole,
3-carboxymethyl-4,5-dimethyl-2-aminothiophene, 2-aminopyridine,
2-aminopyrimidine, dapsone, sulphamethoxazole, amine of
cefipime were attempted for coupling with piperic acid. The reac-
tions were not successful and products were not obtained.
Sulafnilamide, ADCA and INH amides of piperic acid were obtained
in low yields (10–21%). The yields of the other amides were
between 38 and 65%.
The 1H NMR spectrum of compound 1a showed a signal at ~ δ
6.0 ppm, a singlet integrating for two protons, characteristic of met-
hylenedioxy (
O CH2 O ) group. A number of signals between δ
The three amides obtained by 3-desacetyl-7-aminocephalosporanic
acid with piperic acid, MDCA and piperonylic acid had to be separated
6.5 and 8 ppm with complex splitting pattern were observed. Large
J values of signals at δ 6.26 (H-2, d) and δ 7.36 (H-4, dd) (14.81 Hz
SCHEME 1 Synthesis of compounds of series 1–3

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ACHANTA ET AL.
and 14.79, 10.66 Hz, respectively) in 1a indicated the trans, trans con-
figuration in the diene in the side chain.
3.1
|
Antibacterial screening
The compounds of series-2 contain only two alkenic protons and so
they show two doublets in their 1H NMR spectra. In 2c, the J values of
signals at δ 6.7 ppm (H-2) and 7.54 ppm (H-3) due to alkenic protons
were found to be ~15 Hz confirming their trans orientation in MDCA.
1H NMR spectra of the compounds of series-3 show three char-
acteristic signals, a weak doublet (H-3) due to J4 (meta) coupling
(1–2 Hz), a weak doublet of doublet (dd, H-7) due to J3 and J4 cou-
pling (8, 2 Hz) and a doublet (H-6) due to J2 coupling (~8 Hz).
Initially, few piperamides were screened against a panel of bacteria,
E. coli, P. aeruginosa, S. aureus, B. subtilis, S. marcescens, K. pneumonia,
Aeromonas hydrophila and a fungus, Candida albicans. The minimum
inhibitory concentration (MIC) of all the synthesized compounds on
the above listed microbes was found to be more than the highest
tested concentration (100 μg/ml). Later, all the synthesized com-
pounds were subjected to screening against S. aureus and
P. aeruginosa by growth count assay. It can be observed (Figure 3) that
though the tested compounds showed concentration dependent
responses, they did not inhibit the growth of S. aureus at 50 μg/ml. But
some compounds inhibited the growth of P. aeruginosa at 50 μg/ml
and all the compounds inhibited the growth at 100 μg/ml (Figure 4).
Therefore, they were also tested on vancomycin resistant P. aeruginosa
(BAA-427) (Figure 5). Piperic acid amide of anthranilic acid showed
activity on all the three strains. The positive results were further con-
firmed by performing viable count assay. Radial diffusion assay of PA-
AA on S. aureus produced 10 mm zone of inhibition at 25 μg/ml
7-ADCA and the amine of aztreonam have free amino groups in their
structures. The possibility of racemization in 1, 2, 3: e–f was eliminated by
the use of HATU with DIPEA as a catalyst and base. This reagent was
reported to retain the chirality in the products during peptide synthesis
(Carpino, 1993; Carpino et al., 1995). Ene-formation, which is an essential
intermediate step during the racemization, is unlikely to take place
because of the ring constraint in these compounds. The coupling con-
stants in the reactants (amine) and the products did not change (J = ~4 Hz
in 1-3e and 2–3 Hz 1-3f) confirming that the racemization did not occur.
FIGURE 3 Antibacterial activity of synthesized compounds on S. aureus 25,923
FIGURE 4 Antibacterial activity of synthesized compounds on P. aeruginosa 25,668