Radical scavenging and antibacterial activity of caffemides against gram positive, gram negative and clinical drug resistance bacteria

Article abstract of DOI:10.1016/j.bmcl.2016.10.089

A new series of caffemide were synthesized and their antioxidant and antibacterial activities were explored. Antioxidant and antibacterial activities were measured of different structures of caffemide containing different functional groups. Anti-oxidative caffemides 1b and 1g showed significantly higher activity against different bacteria with MIC values less than 50 μg/ml. These anti-oxidative and antibacterial properties of caffemides might be helpful for the treatment of secondary infections and discovery of new antibiotics.

Full text of DOI:10.1016/j.bmcl.2016.10.089

Bioorganic & Medicinal Chemistry Letters 26 (2016) 5943–5946
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Radical scavenging and antibacterial activity of caffemides against gram
positive, gram negative and clinical drug resistance bacteria
b
Kaushik Misra a, Himadri Sekhar Maity a, Ahindra Nag a, , Avinash Sonawane
⇑
a Department of Chemistry, IIT-Kharagpur 721302, West Bengal, India
b School of Biotechnology, KIIT University, Bhubaneswar 751024, Odisha, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2016
Revised 25 October 2016
Accepted 29 October 2016
Available online 31 October 2016
A new series of caffemide were synthesized and their antioxidant and antibacterial activities were
explored. Antioxidant and antibacterial activities were measured of different structures of caffemide con-
taining different functional groups. Anti-oxidative caffemides 1b and 1g showed significantly higher
activity against different bacteria with MIC values less than 50 lg/ml. These anti-oxidative and antibac-
terial properties of caffemides might be helpful for the treatment of secondary infections and discovery of
new antibiotics.
Keywords:
Caffemide
DPPH
Ó 2016 Elsevier Ltd. All rights reserved.
M. smegmatis
P. aeruginosa
S. aureus
Caffeic acid and its derivatives widely distributed in fruits, veg-
etables, grains, coffee, barks and roots have dietary significances.1
Caffeic acid containing catecholic moiety has anti-inflammatory,
anti-cancer, antibacterial, antiviral and variety of pharmacological
effects.2 Density functional theory calculations reveal that the
presence of at least two adjacent hydroxy groups (caffeic acid)
on a phenyl ring containing electron withdrawing moiety have rel-
atively weak bond dissociation energy of the phenolic–OH bond. It
enables the production of phenoxy radicals which are stabilized by
delocalization of their unpaired electron through extended conju-
gation and acting as potent antioxidant.3 Over the last decades,
many biological activities of ester and amide derivatives of caffeic
acid have been explored both in vivo and in vitro study by many
research groups. It was reported that the ester linkage of caffeic
acid ester was metabolically labile and the half life of such ester
was lower in presence of hydrolytic enzyme.4 Caffeic acid amide
(caffemide) is popular antioxidant agent and has many bioactive
properties due to their structural stability both in vivo and
in vitro.4,5 Amide derivatives of caffeic acid have many pharmaco-
logical activities such as antioxidant, anti-hyperglycemic, anti-pla-
telet, inhibitory effects on PG synthetase, MMP-2, MMP-9 and
arachidonate 5-lipoxygenase.6 In 2012 Jia et al. published that
Danshensue-cysteine analog connected with caffeic acid by sul-
phur linkage had protective activity on cardiovascular system.7
Moreover, caffemide had antiviral effects against influenza virus
and showed inhibitory activities on neuraminidases with MIC
value 7.2 l
M.8 The docking analysis indicated that the 3,4-dihy-
droxyphenyl group of caffemide could penetrate deeply into the
active site of neuraminidases and showed hydrogen bonding inter-
actions with Glu119 and Arg156 residues.8 Recently, Hai-liang Zhu
et al. reported that, a series of caffemides had considerable antibac-
terial activities against B. subtilis.9 Some hydroxycinnamic acid
amides and its analogues were synthesized as well as studied
against S. aureus and HIV-1 integrase inhibitors.10 Moreover, reac-
tive oxygen species alter cellular and molecular signaling which
results pathogenesis in gut mucosa of gastrointestinal tract.11
The presence and position of different functional groups
responsible for antioxidant as well as antibacterial activities of caf-
femides have discussed in this paper. It would be helpful for inex-
pensive synthesis of novel caffemides having antioxidant and
antibacterial properties.
Caffemides were synthesized by coupling reaction where caffeic
acid and different substituted amines were taken as starting mate-
rials in dry tetrahydrofuran (THF). N,N0-Dicyclohexyl carbodiimide
(DCC) was added as coupling reagent (Scheme 1).
The synthesized amides 1a to 1m were tested for radical scav-
enging activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.12
From all UV spectra, the percentage of inhibition with various con-
centrations of test samples was calculated using following
equation
⇑
Corresponding author.
E-mail address: ahinnag@chem.iitkgp.ernet.in (A. Nag).
% Inhibition ¼ ½fðA control ꢀ A testÞ=A controlg ꢁ 100ꢂ
http://dx.doi.org/10.1016/j.bmcl.2016.10.089
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

5944
K. Misra et al. / Bioorganic & Medicinal Chemistry Letters 26 (2016) 5943–5946
Table 1
EC50 values of caffemides.
O
OH
HO
HO
R
HO
HO
N
H
DCC, Amine
10-16h, r.t
O
Compound
EC50 S.D (
l
M)
Compound
EC50 S.D (lM)
1a
1b
1c
1d
1e
1f
5.51 0.9
6.57 1.12
7.21 1.64
10.62 1.71
11.90 1.38
13.18 1.75
14.67 1.38
1h
1i
CA
1j
1k
1l
1m
24.06 1.42
27.04 1.39
30.88 1.09
36.01 2.28
47.30 2.15
52.63 1.55
58.81 2.32
Scheme 1.
where A control was absorbance of the control (DPPH without test
sample) and A test was absorbance of DPPH solution with caf-
femides. The radical scavenging activity of caffemides was
1g
Data are shown as
DPPH ethanolic solution was 100
l
M
S.D of three independent trials. The final concentration of
M.
expressed in terms of EC50 which defined as concentration of
lM
l
required for 50% decreased in absorbance of DPPH radical at
517 nm. A plot% of inhibition against concentration was drawn to
establish the standard curve and calculated EC50 value of synthe-
sized caffemides (Fig. 1).From Fig. 1, the observed EC50 values of caf-
femides were presented in tabular form (Table 1).
It was observed from Table 1, electron withdrawing group
attached with caffemide had lower EC50 value than caffeic acid
i.e. they behaved more potent antioxidant with respect to caffeic
acid whereas electron donating moiety attached with caffemide
had higher EC50 value compared to caffeic acid.
whereas exposure to 50 lg/ml of compound 1g was found to kill
51.9% and 78.1% of the M. smegmatis population after 6 h and
24 h respectively (Fig. 2a and b). Compounds 1b and 1g could com-
pletely eliminate M. smegmatis colonies beyond concentration
200
negative bacteria, approximately 50% of P. aeruginosa population
was killed between 10–50 g/ml and 50–100 g/ml by compound
1b after 1 h and 3 h respectively (Fig. 2c). But exposure to 50 g/ml
lg/ml after 6 h and 24 h treatment periods. In case of Gram-
l
l
l
of compound 1g was found to kill 58.9% and 62.1% of the P. aerug-
inosa population after 1 h and 3 h incubation period (Fig. 2d). It
was found that both 1b and 1g could completely eliminate P.
aeruginosa colonies beyond the concentration 300 lg/ml after 1 h
and 3 h treatment period.
In vitro antibacterial activity of synthesized caffemides was
done against a panel of human pathogens belonging to Gram-pos-
itive (S. aureus), Gram negative (P. aeruginosa), acid fast (M. smeg-
matis) and multidrug S. aureus MRSA with different time interval
by colony forming unit (CFU assay).13 Among all of the tasted caf-
femides (1a–1m), compounds 1b and 1g were exhibited significant
antibacterial activity against tested microorganisms in a dose-
dependent manner. Caffemides which had minimum inhibitory
Among all tested bacteria, S. aureus WT was found to be more
susceptible to compound 1b such that 60.9% of bacterial popula-
tion was killed between 1 and 10
lg/ml after 6 h incubation period.
Treatment with doses 200 g/ml and 50
l
l
g/ml of compound 1b
concentration (MIC) value greater than 50
lg/ml were not consid-
was killed approximately 93.8% and 89.6% S. aureus WT bacterial
colonies after 6 h and 24 h exposure period respectively (Fig. 3a).
In case of compound 1g, 86.8% and 68.2% of bacterial population
ered in this study. Exposure to 50 g/ml of compound 1b was
l
found to kill 63.8% and 45.6% of the M. smegmatis population,
was killed with dose 50
24 h treatment period. Moreover, compound 1g with dose
100 g/ml, 92.3% and 84.9% bacterial colonies were eliminated
within 6 h and 24 h respectively (Fig. 3b). It was also observed that
caffemides 1b and 1g were effective against S. aureus drug resistant
bacteria and were found to kill 57.5% and 52.8% of S. aureus MRSA
lg/ml against S. aureus WT after 6 h and
R=
Cl
l
NO2
NO2
OH
Cl
O
N
H
N
H
N
H
N
H
C
N
H
N
H
OH
OH
1b
1e
1f
1c
1d
1a
at concentration 50 lg/ml after 6 h, whereas exposure to 50 lg/ml
NH2
of compound 1b and 1g were found to kill 64.8% and 61.2% of the S.
aureus MRSA population after 24 h incubation period
(Fig. 3c and d). No viable colonies were found beyond the concen-
F
N
H
N
H
N
H
N
H
N
H
N
H
S
HN
1l
1h
1j
1k
1g
1i
tration 100 lg/ml by compounds 1b and 1g after 6 h and 24 h
treatment period.
N
H
This experimental study was designed in three different ways to
understand the role of functional groups of caffemides were
responsible for antioxidant and antibacterial properties. Ring A
was fixed but the ring B contained electron donating, electron
withdrawing and hydroxyl group present along with electron
donating and withdrawing moiety (Fig. 4). Electron withdrawing
group attached with caffemides (1a, 1c, 1d and 1f) had lower
N
H
1m
100
80
60
40
20
0
1a
1b
1c
1d
1e
1f
EC50 values compared to caffeic acid (30.88
lM) and vitamin E
(50.85 M) i.e. they exhibited more potent antioxidant whereas
l
1g
1h
1i
electron donating moiety attached with caffemides (1j–1m)
showed less antioxidant property with respect to caffeic acid. Com-
pounds 1b (R1 = OH, R4 = CH3) and 1e (R1 = OH, R4 = t-Bu) showed
less EC50 values due to presence of an extra hydroxyl group
(R1 = OH) at ring B which probably took part to scavenge the free
radical (Fig. 4). Compound 1a (R3 = OH, R2 and R4 = Cl) containing
electron withdrawing moiety along with hydroxyl group
(R3 = OH) in ring B displayed lowest EC50 value.
CA
1j
1k
1l
1m
2.0x10-5 4.0x10-5 6.0x10-5 8.0x10-5 1.0x10-4
0.0
The observed EC50 value of caffemide 1g (R1 = NH-Ph) was les-
ser than 1i (R1 = S-Ph) and lesser than caffeic acid. Compounds hav-
ing radical scavenging activity would help to reduce the
unnecessary stress inside the cell. It was well accepted that various
Concentration (M)
Fig. 1. % of inhibition of synthesized caffemides. Final concentration of DPPH was
100 M.
l

K. Misra et al. / Bioorganic & Medicinal Chemistry Letters 26 (2016) 5943–5946
5945
10090
100
90
Control
Control
Control
Control
a
a
b
b
90
80
90
80
1µg/ml
1µg/ml
1µg/ml
1µg/ml
80
70
10µg/ml
10µg/ml
80
70
10µg/ml
10µg/ml
50µg/ml
50µg/ml
50µg/ml
50µg/ml
70
70
60
60
100µg/ml
100µg/ml
100µg/ml
100µg/ml
60
50
50
40
40
60
50
200µg/ml
200µg/ml
200µg/ml
50
40
40
30
30
30
30
20
20
20
20
10
10
10
10
0
0
0
0
24h24h
6h6h
6
6
h
h
24h
24h
80 c
100
160
160
Control
Control
Control
Control
c
d
d
90
70
140
140
1µg/ml
1µg/ml
1µg/ml
1µg/ml
80
120
70
10µg/ml
10µg/ml
10µg/ml
10µg/ml
60
120
50µg/ml
50µg/ml
50µg/ml
50µg/ml
100µg/ml
100µg/ml
50
100
100µg/ml
100µg/ml
100
60
200µg/ml
200µg/ml
200µg/ml
40
80
50
80
40
60
30
60
30
40
20
20
40
10
20
20
10
0
0
0
0
24h
3h
6h
1h
1h
6h
24h
3h
Fig. 3. (a) Activity of 1b against Staphylococcus aureus WT. (b) Activity of 1g against Staphylococcus aureus WT. (c) activity of 1b against Staphylococcus aureus MRSA. (d)
Activity of 1g against Staphylococcus aureus MRSA. Values were the means S.D of three independent trials. The results were found to be statistically significant p 6 0.05 (⁄),
p 6 0.001 (⁄⁄), p 6 0.0001 (⁄⁄⁄) compared to that of control group without treatment by 1b and 1g.
played an important role for the activity of caffemide 1b. The oppo-
R4
B
site pattern was observed for compound 1i (R1 = S-Ph) structurally
identical with compound 1g (R1 = NH-Ph) had higher MIC value
against P. aeruginosa and S. aureus MRSA and inactive on M. smeg-
matis and S. aureus WT, whereas caffemide 1g showed significant
activity on M. smegmatis, S. aureus WT, P. aeruginosa and S. aureus
R3
R2
O
HO
HO
N
H
A
R1
MRSA with MIC values less than 50 lg/ml.
In summary, a series of novel caffemide were synthesized and
their antioxidant and antibacterial properties were measured.
The experimental observation reveals that caffemides having more
antioxidant property may not be active against specified bacteria
but the presence of functional groups of caffemides at ring B plays
a crucial role for their antioxidant and antibacterial properties.
Compounds 1b and 1g had antioxidant property along with
antibacterial activity which could be helpful for the treatment of
oxidative stress and secondary infection for human. Finally, this
experimental study might be helpful for the discovery of new
antibiotics in future after detailed SAR analysis.
Fig. 4. Schematic representation of functional groups on caffemides.
bacteria could attack the cell in higher oxidative stress conditions.
On the basis of antioxidant property, antibacterial activities of caf-
femides were investigated. The experimental result revealed that,
caffemides 1c (R3 = NO2) and 1d (R2 = COCH3) had more radical
scavenging property but showed toxic effect towards tested bacte-
rial colonies due to presence of strong electron withdrawing group
(Fig. 4). Caffemides 1a, which had highest antioxidant activity was
also toxic at a dose 1 l
g/ml. However, compounds 1f (R1 = CH3,
R3 = NO2), 1h (R3 = F) and 1i (R1 = S-Ph) were activities against M.
smegmatis and S. aureus WT, but the MIC values were greater than
Acknowledgement
50
(6.57
l
g/ml. The observed EC50 values of caffemides 1b and 1e were
The author Kaushik Misra is indebted to UGC-Delhi, India, for
the financial support of this research.
l
M) and (11.90 M) respectively, which were less than caf-
l
feic acid and vitamin E. Compound 1e which had tertiary group
at the meta position (ring B) with respect to amide linkage almost
similar structure with compound 1b, exhibited antibacterial activ-
ity against M. smegmatis and S. aureus WT bacteria but the MIC
A. Supplementary data
value was greater than 50 lg/ml and inactive against on P. aerugi-
nosa and S. aureus MRSA whereas caffemide 1b was significantly
active towards all tested bacteria. This observation revealed that
one hydroxy and one methyl group at ring B (R1 = OH, R4 = CH3)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.10.
089.

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K. Misra et al. / Bioorganic & Medicinal Chemistry Letters 26 (2016) 5943–5946
Pain S, Guiseppe Pelicci P, Saccani A, Storto M, Thaler F, Varasi M, Villa M, Plyte
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