Amino acid amides of piperic acid (PA) and 4-ethylpiperic acid (EPA) as NorA efflux pump inhibitors of Staphylococcus aureus

Article abstract of DOI:10.1016/j.bmcl.2016.07.062

A total of eighteen piperic acid (PA) and 4-ethylpiperic acid (EPA) amides (C1–C18) with α-, β- and γ-amino acids were synthesized, characterized and evaluated for their efflux pump inhibitory activity against ciprofloxacin resistant Staphylococcus aureus. The amides were screened against NorA overexpressing S. aureus SA-1199B and wild type S. aureus SA-1199 using ethidium bromide as NorA efflux pump substrate. EPI C6 was found to be most potent and reduced the MIC of ciprofloxacin by 16 fold followed by C18 which showed 4 fold reduction of MIC. Ethidium bromide efflux inhibition and accumulation assay proved these compounds as NorA inhibitors.

Full text of DOI:10.1016/j.bmcl.2016.07.062

Accepted Manuscript
Amino acid amides of piperic acid (PA) and 4-ethylpiperic acid (EPA) as NorA
efflux pump inhibitors of Staphylococcus aureus
Naiem Ahmad Wani, Samsher Singh, Saleem Farooq, Sudha Shankar, Surrinder
Koul, Inshad Ali Khan, Rajkishor Rai
PII:
S0960-894X(16)30791-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.07.062
BMCL 24106
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
23 May 2016
21 July 2016
27 July 2016
Please cite this article as: Wani, N.A., Singh, S., Farooq, S., Shankar, S., Koul, S., Khan, I.A., Rai, R., Amino acid
amides of piperic acid (PA) and 4-ethylpiperic acid (EPA) as NorA efflux pump inhibitors of Staphylococcus
aureus, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.07.062
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Amino acid amides of piperic acid (PA) and
4-ethylpiperic acid (EPA) as NorA efflux pump
inhibitors of Staphylococcus aureus
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Naiem Ahmad Wani a,†, Samsher Singh b, d, †, Saleem Farooq c,†
Sudha Shankar a, d Surrinder Koul c , Inshad Ali Khan b, d, * ,
,
Rajkishor Raia,d,
*

Bioorganic & Medicinal Chemistry Letters
Amino acid amides of piperic acid (PA) and 4-ethylpiperic acid (EPA) as NorA
efflux pump inhibitors of Staphylococcus aureus
Naiem Ahmad Wani a,†, Samsher Singh b, d, †, Saleem Farooq c,†, Sudha Shankar a, d , Surrinder Koul c,
Inshad Ali Khan b, d, *, Rajkishor Rai a, d,
*
a Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,Jammu-180001 India.
b Clinical Microbiology Division, CSIR-Indian Institute of Integrative Medicine, Canal road Jammu -180001 India
c Bioorganic Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal road Jammu -180001 India
dAcademy of Scientific and Innovative Research, CSIR-IIIM, Canal Road, Jammu-180 001, India
† Equal contribution
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A total of eighteen piperic acid (PA) and 4-ethylpiperic acid (EPA) amides (C1-C18) with -, -
and -amino acids were synthesized, characterized and evaluated for their efflux pump inhibitory
activity against ciprofloxacin resistant Staphylococcus aureus. The amides were screened
against NorA overexpressing S. aureus SA-1199B and wild type S. aureus SA-1199 using
ethidium bromide as NorA efflux pump substrate. EPI C6 was found to be most potent and
reduced the MIC of ciprofloxacin by 16 fold followed by C18 which showed 4 fold reduction of
MIC. Ethidium bromide efflux inhibition and accumulation assay proved these compounds as
NorA inhibitors.
Keywords:
Piperic acid
β-amino acid
γ-amino acid
NorA efflux pump
Staphylococcus aureus.
Multidrug resistance (MDR) pathogenic bacteria represents one
of the main causes for the failure of antimicrobial drugs.1-2 These
pathogens often emerge after the long-term use of antimicrobial
drugs. It is commonly characterized by the resistance to a series
of unrelated compounds.3 Reduced permeability of the bacterial
envelope and/or extrusion of the drug from the cell through
membrane-based efflux proteins are one of the major
mechanisms of MDR. NorA is a Staphylococcus aureus efflux
pump that gives reduced susceptibility to many structurally
unrelated compounds i.e. fluoroquinolones, biocides and dyes
resulting in a multidrug resistant (MDR) phenotype. Efflux
pumps are involved in 60% of global nosocomial infectious
(WHO press release, WHO, Geneva 2000). These are responsible
for the extrusion of toxic compounds including antibacterials
encountered in the bacterial environment.4 Efflux Pump
Inhibitors (EPI) have been proved to enhance the activity of
several antibiotics identified as the substrate of these efflux
pumps by blocking their removal from the core of bacterial cells.
possess several efflux pumps, notably NorA, TetK, MsrA and
MdeA, which transports fluoroquinolones, tetracyclines,
macrolides and mupirocin drugs, respectively.9-11
Most of the efflux pump inhibitors have been explored from the
plant sources.12 including piperine, an alkaloid, present in Piper
nigrum-L (black pepper) known for its different biological and
pharmacological activities13-19 as well as for its EPI activity.20 It
has been reported that piperoyl-amino conjugates, exhibited
biological activity better than piperine.21 Takao et al reported that
piperic acid amide as radical scavengers and -glucosidase
inhibitors.22 Piperine increases the bioavailability of many drugs
through a number of mechanisms.23,24 Several structural analogs
of piperine have been reported as inhibitor of bacterial NorA
efflux pump againt S. aureus.25 It has been reported that the
piperoyl amides display NorA efflux pump inhibitory activities.26
Recently, we have reported that the conjugate of 4-ethylpiperic
acid (EPA) with -disubstituted -amino acid inhibits the
invasion of metastasis in pancreatic Cancer Cells via
PI3K/Akt/NF-kB Pathway.27 Based on the previous studies with
piperine and its analogs, we envisaged to synthesise the amides
of piperic acid (PA) and 4-ethylpiperic acid (EPA) with -, -
and -amino acid esters (C1-C18). L-Ala, L-Val, L-Leu, L-Phe,
L-Pro, L-Trp and L-tert-Leu were used as -amino acid, while
3,3-Ac6c, 4-Ethyl-3,3-Ac6c, 4-tert-butyl-3,3-Ac6c and 3,3-Pip-
5,6
It has been reported that efflux is an important mechanism of
resistance in many clinically relevant pathogens, notably,
Streptococcus pneumoniae, Pseudomonas aeruginosa and
Neisseria gonorrhoeae7,8 Staphylococcus aureus (S. aureus) has
shown ability to acquired resistance to several antibacterial drugs
currently available in the market. The pathogen is known to

OH as -amino acids. -amino acids have been reported as an
important element of biologically active natural products such as
taxol, bleomycin and cytotoxic microcystin.28 Gabapentin, an
anti-epileptic drug used in neuropathic pain29, was used as -
amino acid. The chemical structures of the synthesized amides
are shown in Fig 1. All the compounds were evaluated for EPI
activity against wild type strain S. aureus SA-1199 having
normal expression level of NorA as well as NorA over
expressing strain S. aureus SA-1199B.
All the amides, C1-C18 were evaluated for their efflux pump
inhibitory activity in combination with ciprofloxacin drug on a
NorA overexpressing strain of S. aureus having overexpression
level of NorA (Table 1) and further tested for reduction in MIC
of ethidium bromide using SA-1199B (NorA overexpressing;
Table 2) and SA-1199 (wild type S. aureus). Minimum inhibitory
concentration (MIC) potentiation studies of ethidium bromide in
combination with compounds C1-C18 against S. aureus SA-
1199, which is a wild type strain having normal expression level
of NorA, shows the activity as EPIs (Table 3).
Table 1
Activity of ciprofloxacin in combination with piperic acid (PA)
and 4-ethylpiperic acid (EPA) amides, C1-C18.
Compound MIC of MEC4 MIC of ciprofloxacin against
EPI
SA-1199B at 12.5 µg/ml of
respective EPI
(µg/ml)
Without With
Fold
Reduction
0
EPI
8
EPI
8
C1
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
12.5
25
C2
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
2
4
0
0
4
2
16
4
2
0
0
2
4
0
0
0
4
2
4
0
0
4
0
C3
C4
C5
C6
3.12
6.25
50
0.5
C7
2
4
8
8
4
2
8
8
8
2
4
2
8
8
2
8
C8
C9
>50
>50
25
C10
C11
C12
C13
C14
C15
C16
C17
C18
PA
12.5
>50
50
>50
12.5
25
Figure 1. Chemical structure of piperine; piperic acid (PA); 4-
ethylpiperic acid (EPA) and their amides with -, - and -amino
acids, C1-C18.
Piperine was extracted from the ripened dried seeds of Piper
nigrum using methanol and converted into piperic acid (PA). 4-
ethylpiperic acid (EPA), was synthesized using the procedure
reported by us earlier.30 β,β-disubstituted-β-amino acids were
synthesized by a procedure reported in the literature.31 The
amides of piperic acid (PA) and 4-ethylpiperic acid (EPA), C1-
C16 were synthesized by the reaction of piperic acid (PA) and
4-ethylpiperic acid (EPA) with amino ester hydrochloride in
presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDCI.HCl) and 4-methylmorpholine (NMM) in
dry dichloromethane. Ac--Pip(EPA)-Aib-OMe, C17 was
synthesized by coupling of Ac--Pip(EPA)-OH with Aib-
OMe.HCl using EDCI.HCl/HOBt and NMM in dry
dichloromethane and synthesis of C18 was achieved by coupling
6.25
>50
>50
12.5
>50
EPA
SK-20
Piperine
No antibacterial activity of EPIs was observed at 50µg/ml that was the
highest concentration tested.
For determining potentiation of ciprofloxacin, EPI were tested at
concentration range of 50–0.8µg/ml.
a MEC4, minimum effective concentration at which MIC is four fold reduced.
b MIC, minimum inhibitory concentration.
of
Valeryl--Pip(NH)-NH-NH-Ph
with
EPA
using
Among all the compounds, the EPA amide with L-Proline, C6
was found to be the most potent compound with 16 fold decrease
in MIC of ciprofloxacin with minimum effective concentration
(MEC4; four fold reduction in MIC) at 3.12 µg/mL. In addition,
EPA derived amides C7 and C18 exhibited MEC4 at 6.25 µg/mL
EDCI.HCl/HOBt and NMM in dry dichloromethane. All the
amides were characterised by ESI-HRMS, H-NMR and 13C
1
NMR spectroscopy.

and showed fourfold reduction in MIC of the drug. EPA derived
amide C4, and PA derived amides C12 and C16 showed 4 fold
reduction in the MIC of the ciprofloxacin at 12.5 µg/mL. The
above results corroborate our earlier findings25 about the higher
potent EPI activity profiles of EPA derived amides over piperine
and its amides.
Further to confirm the inhibitory mechanism of most potent
EPIs, ethidium bromide efflux and accumulation assay were used
as described in biological evaluation studies.32 The most potent
compounds, C6 and C18 were studied along with piperine and
reserpine as standard EPIs. These EPIs inhibited the efflux of
ethidium bromide effectively in a concentration dependent mode
with maximum inhibition at 50µg/mL resulting in significant
decrease in fluorescence over the time period of 30 min (Fig. 2).
Table 2
Potentiation of ethidium bromide MIC by active derivatives
using SA-1199B
Compound MIC
MEC4 MIC of EtBr against SA-
1199B
of
EPI
µg/ml
Without With
Fold
EPI
EPI at Reduction
12.5
µg/ml
C4
C5
C6
C7
C12
C16
C18
SK-20
Piperine
>50
>50
>50
>50
>50
>50
>50
>50
>50
25
16
16
16
16
16
16
16
16
16
8
4
4
4
8
8
2
4
8
2
4
4
4
2
2
8
4
2
6.25
3.12
3.12
50
50
3.12
12.5
25
Figure 2. Ethidium bromide efflux of C6 and C18 using NorA
over expressing SA-1199B
Table 3
Potentiation of ethidium bromide MIC by active derivatives
using SA-1199.
Similarly, both the compounds were assessed for ethidium
bromide accumulation in the presence and absence of EPI.
Introduction of EPI after 30 min resulted in the sharp increase in
the fluorescence (Fig. 3).
Compound MIC
MEC4 MIC of EtBr against SA-1199
of
Without With
Fold
EPI
µg/ml
EPI
EPI
12.5
Reduction
at
µg/ml
C4
C5
C6
C7
C12
C16
C18
SK-20
Piperine
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
25
50
50
25
12.5
25
4
4
4
4
4
4
4
4
4
2
2
2
2
4
4
1
4
4
2
2
2
2
0
0
4
0
0
>50
The compounds having MEC4 at 12.5 µg/mL or lower than it
were further tested for reduction in MIC of ethidium bromide
using SA-1199B (NorA overexpressing) and SA-1199 (wild type
S. aureus). Tables 2 and 3 show the activity of ethidium bromide
in combination with C4, C5, C6, C7, C12, C16, and C18 against
SA-1199B and SA-1199, respectively. As expected, NorA
overexpressing SA-1199B exhibited higher MIC against
ethidium bromide compared to the wild type SA-1199. Of these
compounds, C6 and C7 exhibited the MEC4 at 3.12 µg/mL in
ethidium bromide against SA-1199B, while MEC4 was 25 and 50
µg/mL for the same compounds against SA-1199. Further, C18
exhibited eight fold reduction in MIC at 3.12 µg/mL against SA-
1199B, while 4 fold reduction in MIC was observed at 12.5
µg/mL against SA-1199. In case of C5, 4 fold reduction in MIC
at 6.25 µg/mL was observed against SA-1199B.
Figure 3. Ethidium bromide accumulation of C6 and C18 using
NorA overexpreesing SA-1199B.
From EPI screening and ethidium bromide assay, it was
concluded that EPA amide with L-proline exhibited EPI activity
(MEC4 = 3.12 µg/mL) which is four fold better than of EPA
piperidine (SK-20) having MEC4 at 12.5 µg/ml.25 The compound
C18, in which -amino acid, 3,3-Pip-OH is conjugated with
EPA, also exhibited better EPI activity (MEC4 = 6.25µg/mL as
compared to SK-20. In addition, C16, an amide of piperic acid
with Gpn, shows similar EPI activity (MEC4 =12.5) µg/mL like
SK-20.

26. Sangwan, P. L.; Koul, J. L.; Koul, S.; Reddy, M. V.; Thota, N.;
Khan, I. A.; Kumar, A.; Kalia, N. P.; Qazi, G. N. Bioorg. Med.
Chem. 2008, 16, 9847.
27. Amin, H.; Wani, N. A.; Farooq, S.; Nayak, D.; Chakraborty, S.;
Shankar, S.; Rasool, R. U.; Koul, S.; Goswami, A.; Rai, R. ACS
Med. Chem. Lett. 2015, 6, 1071.
28. Kudo, F.; Miyanaga, A.; Eguchi, T. Nat. Prod. Rep. 2014, 31, 1056.
29. Rosenberg, J. M.; Harrell, C.; Ristic, H.; Werner, R. A.; De Rosayro,
A. M. Clin. J. Pain. 1997, 13, 251.
30. Koul, S.; Koul, J. L.; Taneja, S. C.; Gupta, P.; Khan, I. A.; Mirza, Z.
M.; Kumar, A.; Johri, R. K.; Pandita, M. A.; Khosa, U.S. Patent
Application US 20070004645 A1, 2013.
In conclusion, piperic acid and 4-ethylpiperic acid amides with
amino acid esters (i. e. -, - and - amino acids) when tested in
combination with ciprofloxacin have shown to exhibit efflux
pump inhibitory activity property against ciprofloxacin resistant
Staphylococcus aureus. Compounds C6, C7, C12, C16 & C18
were found to be the active compounds, and C6 displayed 16 fold
reduction in the MIC of ciprofloxacin against NorA
overexpressing strain of S. aureus (SA-1199B). Further, through
ethidium bromide efflux inhibition and accumulation assays,
these compounds have shown potent NorA inhibitors. The
present study can be extended to develop the potent EPIs based
on conjugation of piperic and 4-ethylpiperic acid with ,-
disubstituted-- and -amino acids.
31. Wani, N. A.; Gupta, V. K.; Kant, R.; Aravinda, S.; Rai, R.; Acta
Crystallogr., Sect. E: Struct. Rep. Online. 2013, 69, o888.
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42, 2032.
Acknowledgments
We acknowledge the Council of Scientific and Industrial
Research (CSIR) for financial assistance under Network Project
BSC-120. IIIM publication number is IIIM/1913/2016
Supplementary Material
Detailed procedure for the synthesis of piperic acid, 4-
ethylpiperic acid and compounds C1-C18, analytical data, and
biological assays are provided in the supporting information.
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Highlights
 A series of piperic acid (PA) and 4-
ethylpiperic acid (EPA) amides with --
and-amino acids were synthesised,
characterized and evaluated for their efflux
pump
inhibitory
activity
against
ciprofloxacin resistant
Staphylococcus
aureus.
 The potent compounds were tested against
NorA
overexpressing
and
Staphylococcus
aureus 1199
B
wild type
Staphylococcus aureus 1199 using ethidium
bromide as NorA efflux pump substrate.
 Compounds C6 and C18 were found most
potent and reduce the MIC of ciprofloxacin
by sixteen and four folds, respectively.
 The present study can be extended to
develop the potent EPIs based on
conjugation of piperic and 4-ethylpiperic
acid with ,-disubstituted-- and -amino
acids.