Design and Biological Evaluation of Furan/Pyrrole/Thiophene-2-carboxamide Derivatives as Efficient DNA GyraseB Inhibitors of Staphylococcus aureus

Article abstract of DOI:10.1111/cbdd.12529

DNA topoisomerases are well-validated targets in micro-organisms. DNA gyraseB is one of the most important enzymes among them as per their clinical importance. In earlier study, a novel lead 4-((4-(furan-2-carboxamido)phenyl)amino)-4-oxobutanoic acid was identified as inhibitor against DNA gyraseB with an IC₅₀ of 12.88 ± 1.39 μm. Subsequently, analogues of this lead were developed and evaluated through in vitro assays and in vivo studies. Among the 24 analogues, compound 22 was found to be the top hit with an improved DNA gyraseB activity of 5.35 ± 0.61 μm, and the binding affinity of this compound was further ascertained biophysically through differential scanning fluorimetry. The most potent ligand did not show any signs of cardiotoxicity in zebra fish ether-ago-go-related gene, ascertaining the safety profile of this series a breakthrough among the previously reported cardiotoxic gyraseB inhibitors.

Full text of DOI:10.1111/cbdd.12529

Received Date : 15-Oct-2014
Revised Date : 22-Dec-2014
Accepted Date : 06-Jan-2015
Article type : Research Letter
Design and biological evaluation of furan/pyrrole/thiophene-2-carboxamide
derivatives as efficient DNA gyraseB inhibitors of Staphylococcus aureus
Renuka Janupallya, Bhramam Medepia, Parthiban Brindha Devia, Priyanka Suryadevaraa, Variam
Ullas Jeankumara, Pushkar Kulkarnib,c, Perumal Yogeeswaria, Dharmarajan Srirama*
aDepartment of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Shameerpet, Jawahar Nagar, Ranga Reddy District, Hyderabad 500078,
Andhra Pradesh, India.
bDr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad
500046, India.
cZephase Therapeutics (An Incubated Company at the Dr. Reddy’s Institute of Life Sciences),
University of Hyderabad Campus, Gachibowli, Hyderabad 500046, India.
DNA topoisomerases are well validated targets in microorganisms. DNA gyraseB is one of the
most important enzyme among them as per their clinical importance. In earlier study, a novel lead
4-((4-(furan-2-carboxamido)phenyl)amino)-4-oxobutanoic acid, was identified as inhibitor
aganist DNA gyraseB with an IC50 of 12.88 1.39 µM. Subsequently, analogues of this lead were
developed and evaluated through in vitro assays and in vivo studies. Among the 24 analogues,
compound 22 was found to be the top hit with an improved DNA gyraseB activity of 5.35 0.61
µM, the binding affinity of this compound was further ascertained biophysically through
differential scanning fluorimetry. The most potent ligand did not show any signs of cardiotoxicity
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
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'Accepted Article', doi: 10.1111/cbdd.12529
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in zebra fish ether-ago-go-related gene (zERG), ascertaining the safety profile of this series a
breakthrough among the previously reported cardiotoxic gyraseB inhibitors.
Key words: Staphylococcus aureus, DNA gyrase, cytotoxicity, biofilm.
Abbreviations: e-pharmacophore, energy based pharmacophore; MRSA, methicillin resistant
Staphylococcus aureus; zERG, Zebra fish ether-ago-go-related gene.
*Corresponding author: D. Sriram (dsriram@hyderabad.bits-pilani.ac.in)
Occurrence of increasingly serious threat to global public health is a consequence of antimicrobial
resistance, which threaten the effective prevention and treatment of an increasing range of
infections caused by the bacteria (1). Staphylococcus aureus, a gram positive bacteria a normal
flora, inhabiting 30% of human population, had emerged as a causative agent of many pathogenic
infections globally, a major cause of morbidity and mortality in community associated
environments and hospital environment (2-3). As expected, resistance to first-line drugs to treat
infections caused by Staphlylococcus aureus is also increasing alarmingly. However development
of methicillin resistant Staphylococcus aureus (MRSA- multidrug-resistant) strain in the early
1960s, re-urged the necessity of novel drugs against this dreadful bacteria (4). Presently,
vancomycin is the only available source to treat S. aureus infections to certain extent. One of the
important target clinically validated as bactericidal in S. aureus was DNA topoisomerase II and
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IV. DNA gyrase belongs to DNA topoisomerase II that is required to maintain the DNA topology
during replication, transcription in bacteria. It consists of two A subunits and two B subunits
attached to form a holoenzyme (5). For the bacterial cell to survive, the DNA as to exist in a
negative supercoiled form. The functional enzyme performs the negative supercoiling of the
DNA. The dimeric subunit A involves in the cleavage and religation of the DNA while the B
subunit aids in the catalytic ATP hydrolysis. As fluoroquinolones have almost become ineffective
due to fast emerging Staphylococcal strains, caused due to alteration in the interaction of the
fluoroquinolones with their target sites and also due to alterations that affect drug (6). The other
drawback of fluoroquinolones is that they are more potent against the gram-negative bacteria
when compared with gram-positive ones like S. aureus due to the presence of another
homologous target topoisomerase IV (7). Furthermore, research groups from both industry and
academia focused on the development of antibacterial DNA gyraseB inhibitors against various
organisms have identified many potent inhibitors belonging to the chemical class of indolinones
(8), pyrimidines (9), indazoles (10), rhodanines (11), pyrazolothiazoles (12), pyrrolopyrimidines
(13), pyrrolamides (14) as potential hits, though clinically very little has been achieved when it
comes to Staphylococcal gyrase B inhibitors. Keeping this in view, we designed novel class of
furan/pyrrole/thiophene-2-carboxamide derivatives molecules, targeting DNA gyraseB domain. In
the present study, we explored furan/pyrrole/thio-phene-2-carboxamide derivatives and their
inhibitory profiles against DNA gyrase, cell cytotoxities and zERG activity as well to know the
overall effect of these compounds biologically.
As reported in our earlier publication (2), twelve compounds were procured from Asinex
database through e-pharmacophore based screening approach using PDB Id: (3TTZ). Among the
promising in silico results of the twelve virtual screening hits obtained, compound 5 (considered
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as lead in this series) was one among them with S. aureus DNA gyraseB activity of 12.88 1.39
µM and an MIC of 79.03 µM. Figure 1 depicts the structure of the lead compound 5. Considering
this compound as a lead in this series, twenty three analogues were synthesized with the goal of
obtaining a potent compound with tractable SAR and potencies better than the identified initial
screening hit. Based on the potency and synthetic feasibility; a series of twenty three analogues
were designed and synthesized around the lead molecule 5 in the ligand expansion step, to trace
the structure activity relationship. Considering the important interaction that the p-
phenylenediamine linker retained with Asp 81, it was decided to retain the linker and explore the
right and left hand core as shown in Table 1. The synthetic pathway used to assemble library has
been depicted in Scheme 1. The first step involved the selective protection of p-
phenylenediamine using Boc anhydride under controlled conditions by a previously reported
protocol. The monoprotected linker was then functionalized with various substituted heterocyclic
acids to give the corresponding amide derivatives in excellent yields. Further de-protection and
treatment with various anhydrides gave the corresponding acid analogues in moderate yield. The
corresponding ester and hydrazide derivative of the acid analogue were also attempted in
synthesis, to understand the role of this group in activity determination.
The DNA gyraseB assay was performed as per the protocol (14-15). Absence of highly
active DNA gyraseB ATPase activity of the purified S. aureus gyraseB in vitro like other bacteria
made us use the E. coli DNA gyraseA for enhancing the activity of S. aureus gyraseB (2).
Subsequently, E. coli DNA gyraseA in combination with S. aureus DNA gyraseB was used to do
the ATPase activity. As the reference ligand 5 from Asinex database (BAS04380545) showed an
IC50 of 12.88 1.39 µM search was made to obtain a potent ligand with better inhibitory profile,
this compound was found to be in hydrogen bonding with residues Gly85 and Arg144 as shown
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in Figure 2. Also a cation-pi interaction was present with Arg81 and strong hydrophobic
interactions by the furan ring with Ile51, Val79, Ile86 and Ile175. Hence, based on these
interactions, the compound was further modified and derivatized placing the substituents majorly
at three positions – substituents over five membered ring (R1), substituents at oxygen in furan ring
(X) and final substitution at the terminal hydroxyl group (R2). The synthesized compounds were
docked with the crystal structure of gyraseB of S. aureus (PDB code–3TTZ) so as to observe the
mode of interaction of the compounds at the active pocket and to determine its orientation in
silico, subsequently the biological activity too was performed with different drug concentrations
starting from a range of 100 µM to 1.56 µM. Fourteen compounds showed greater than 50%
inhibitions at 100 and 50 µM concentration of the drug concentration. Further, all these molecules
were tested at lower drug concentrations of 25, 12.5, 6.25, 3.12 and 1.56 µM. Initially,
substitutions were made at R2 position replacing hydroxyl group with methoxy 13, and hydrazine
21. These compounds were found to be inactive with docking score of -3.9 and -4.1 kcal/mol
respectively, with gyraseB assay values ranging from 27-36 μM suggesting the favorability of
hydroxyl group for the activity as in 5. Hydroxyl group was involved in hydrogen bonding with
Arg144, an important active site residue, deletion of which was found to deplete the activity. The
next substitution was done at both R1 and R2 positions, where R1 is occupied by 2-methyl moiety
and the R2 position with hydroxyl 6, methoxy 14 and hydrazine 22 groups. Of these, compound
22, N-(4-(4-hydrazinyl-4-oxobutanamido) phenyl)-5-methylfuran-2-carboxamide emerged as the
top hit molecule with a docking score of -8.2 with a fitness value of 1.9, a DNA gyraseB IC50 of
5.35 0.44 µM almost one half of the lead compound 5, calculated by GraphPad Prism software
and an MIC of 18.92 μM. The dose response curve for this compound is shown in Figure 3. The
binding profile of this compound revealed the presence of four hydrogen bonds at active site with
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Val79, Asp81, Gly85 and Thr173 as shown in Figure 4. It was found that the presence of
hydrazine group at R2 position contributed for three hydrogen bonds and made the compound
orient towards the hydrophobic pocket adding up to non-polar interactions. The compounds were
re-screened for the final time in triplicates to ensure their activity results. The other two molecules
were found to be orienting totally in opposite direction shown as shown in supporting information
Figure S1.
The substitutions were further carried out using nitro at R1 position and the same hydroxyl in 7
with docking score of -4.6, methoxy group in 15 with docking score of -4.3 and hydrazine 23
group at R2 position with docking score of -4.9. These compounds were found to be inactive
against gyraseB (IC50 around 27 – 40 μM). The docking studies of these compounds revealed
their orientation with nitro group facing the hydrophobic pocket making it impossible to involve
in any hydrophobic contacts resulting the compounds inactivity.
The further substitutions were done by replacing the furan ring with thiophene in 8, 16 and 24 and
2-methyl thiophene in 9, 17 and 25 with the same three substitutions at R2 position. All these
compounds were found to be inactive (IC50 ranging 21 – >40 μM) except compound 25 with a
docking score of -7.3 and a fitness value of 1.9. Potency of this compound (IC50 – 9.711 μM and
MIC – 18.04 μM) may be attributed to the presence of hydrazine which was found to be
participating in hydrogen bonds with Val79, Asp81 and Thr173 and the carbonyl oxygen next to
thiophene ring was found to be in polar contact with Arg144 which were lacking in the remaining
compounds as shown in supporting information Figure S2. The ethylene linker was found to be
involved in strong hydrophobic contacts with Ile51 and Ile175 and thiophene ring with Pro87.
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The replacement of hydrazine in compound Compounds 9 and 17 resulted in loss of activity up to
five folds showing up the importance of hydrazine for S. aureus gyraseB.
Substitution of furan with 2-nitro thiophene and same three R2 substitutions in 10, 18 and 26,
yielded compound 18 as active one with IC50 of 12.49 μM in gyraseB assay and a docking of -7.8
and a fitness value of 1.7. This may be reasoned for the for the hydrogen bond interactions with
Asp81 and Arg144. The main reason can be given by non-polar interactions with thiophene ring
at the hydrophobic pocket with Ile51 and Ile175. The other two compounds were not involved in
hydrogen bonding with Asp81, an important active site residue, as the substitutions at R2 position
oriented the molecules away from Asp81.
The final substitutions were done with tetra hydro furan in 11, 20 and 28 and pyrrole ring in 12,
19 and 27 at R1 position with the same R2 substitution compounds. Compounds 11 and 20 were
found to be active with docking scores of -7.3 and -7.7, with two hydrogen bonds Asp81 and
Arg144 and the hydrophobic interactions with tetra hydro furan ring. Compound 28 was inactive
with a docking score of -3.9 which can be explained by the reversal of binding pattern with the
addition of hydrazine as the R1 position was towards solvent which decreases the activity. All the
pyrrole substituted compounds were found to be highly inactive suggesting the un-favorableness
of pyrrole ring as it was weakly involved in hydrophobic interactions. All the pyrrole substituted
compounds were found to be inactive suggesting the moiety unfavourable for gyraseB protein.
To sum up, it was observed that not all substitutions were favourable for S. aureus gyraseB
activity. Some of the key features of compounds that are crucial for activity may be – (i) a
hydrophobic ring (like furan, thiophene, pyrrole) at one terminal of compound is important for the
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activity and (ii) a combination of substitutions like methoxy over furan or thiophene at one
terminal and hydrazine (-NH-NH2) at the other terminal of the compound is highly fruitful as
observed in compounds 22 and 25. Compounds with such above features can be desirable in drug
development process for S. aureus targeting gyraseB. Novobiocin, an aminocoumarin was
considered as a standard compound in this assay with an IC50 of 0.125 0.24 µM.
Subsequently, all the molecules were screened for the DNA supercoiling assay as well to
confirm the inhibitory profiles of the gyraseB inhibitors (2, 16) S. aureus DNA supercoiling kit
was procured (Inspiralis Pvt. Limited, Norwich) and the different range of drug concentrations
were used from 100 to 1 µM. Compounds 9, 6, 7, 25, 10, 20 and 11 showed better inhibitory
profiles with an IC50 of less than 10 µM. While the reference compound 5 had an IC50 of 3.12 1.6
µM, the synthesized compound 22 had a better IC50 of 2.79 0.59 µM. In this assay too
novobiocin was considered as a standard inhibitor with an IC50 of 0.030 0.01 µM. The dose
dependent inhibitor profile is shown in Figure 5.
Twenty four synthesized compounds were further screened for their in vitro antibacterial
activity against two bacterial strains of Staphylococcus aureus, namely S. aureus MTCC 3160 and
MRSA 96. The assay was performed according to the Clinical and Laboratory Standards Institute
(17) using the protocol as described in supporting information. The concentration of the drugs
used was in a range of 256-0.75 µg/mL. While almost all the compounds showed an MIC less
than 100 µM, in MTCC 3160 strain, they showed two-three fold higher MIC values in MRSA 96
strain elucidating the drugs efficiency to inhibit the multi-resistant strain as well (2). As per the
table, compounds 22 and 17 showed better MIC values at 18.92 µM and 16.56 µM almost one-
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fourth less concentration when compared to the lead 5. Ofloxacin and ciprofloxacin were included
as reference compounds in the assay. The overall results of the antibacterial screening are
presented in Table 1. Few compounds 6, 10, 27, 5, 24, 9, 19, 12 and 28 exhibited weak
antimicrobial activities which could be attributed to their permeability or efflux pump
transportation hindrances.
All the compounds were also tested for their biofilm inhibition studies too. Being an
extracellular polysaccharide, produced by S. aureus species, biofilm aids in attachment and matrix
formation around the bacteria that result in the alteration in the phenotype of the organism,
resulting in the development of resistance towards the drugs. According to the recent statistics, it
was estimated that 65% of all the human bacterial infections were due to biofilm formation (18)
moreover people with MRSA infections are estimated to be 64% more likely to die than people
with a non-resistant form (1). While the emergence of greater resistance of S. aureus biofilms
against the antibacterial agents was attributed to failure of antibiotics to penetrate the biofilm, the
other important parameter being the existence of different metabolic states of the cells in the
biofilm aggregates associated with differential expression of genes by the bacteria concerned in
the same biofilm aggregates (19). All the synthesized compounds were screened for inhibition of
S. aureus biofilm formation in a strong biofilm producer MRSA 96 strain. The extent of inhibition
and the efficiency of the drugs were monitored quantitatively and qualitatively by the Congo red
method (20). Assays were performed in a 96 flat-bottomed well plates at different drug
concentration range starting from a high 250 to 0.75 µM. While the potent ligand 22 showed an
IC50 of 18.92 µM, all the compounds except 21, showed an IC50 less than 100 µM indicating the
efficiency of these series of compounds towards inhibition of biofilm formation.
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Furthermore, all the compounds were tested for their eukaryotic safety profile information.
The compounds were screened for their in vitro cytotoxicity against mammalian RAW 264.7 cell
line at 100 µM concentration in triplicates by utilizing (4,5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide (MTT) assay (21-22). It was observed that all the twenty four
compounds inhibitory range was below 25 µM. The most potent compound 22 showed only
9.35% cytotoxicity at the highest concentration tested which was on par with novobiocin standard
which had 11.4%.
We tried to test the potent ligand 22 in an in vivo mouse septicaemia model too. In brief,
S. aureus inoculum of 3*10^7 cfu/mouse was administered to CD-1 female mice (10 per group)
via intraperitoneal route (2). The infected mice were then treated with compound 6 in varying
doses of 5, 10, and 25 mg/kg weight at time of and 4 h of post infection. Standard vancomycin
was used as a control and dosed at 5 mg/kg weight. The mice survival was assessed 24 h post
infection. Compound 22 was found to protect 40% of animals at 5 mg/kg, 60% and 100%
protection at 10 and 25 mg/kg respectively as shown in Figure 6.
The fluoroquinolones, considered as the only clinically approved DNA gyrase inhibitors
till date suffer from hERG toxicity mainly the potent moxifloxacin (23-24). As per literature,
previously reported antibacterial C- and N-linked aminopiperidine DNA gyrase inhibitors
suffered from serious hERG toxicity, we tested the top hits to ensure that the newly designed
molecules did not suffer from such drawbacks. Compound 22 and 25 were subjected for hERG
channel inhibition studies by assessing the arrhythmogenic potential on zebra fish ether-a-go-go-
related gene (zERG) which was orthologous to the human ether-a-go-go-related gene (hERG),
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due to their conserved homology. This method of assessment, possessed significant advantage
over the current conventional animal models which include ethical issues, low compound
requirement, manually less tedious and low cost. Furthermore, Milan et al.(25) and Mittelstadt et
al. (26) had reported that bradycardia and atrio-ventricular dissociation in zebrafish larvae can be
used as a surrogate marker for hERG channel inhibition thereby affecting the rapid component of
the repolarizing potassium current and inducing arrhythmia in the model. In our study, compound
22 and 25 were subjected to hERG channel inhibition in concentrations ranging from 1 µM to 30
µM with 0.1% DMSO as vehicle, the heart rate variations and AV ratio were analysed by using a
protocol described in more detail in supporting information. Both the compounds 22 and 25 were
found to be safe when compared to the positive control (20 µM terfenadine), by not showing any
significant cardiotoxicity until 30 µM concentration of the drug. Furthermore, there was no
significant change in the heart rate or AV ratio, in comparison to control group making them
relatively safe, a significant breakthrough when compared to otherwise cardiotoxic terfenadine
and amiodarones as illustrated in Figure 7a-7b. The hERG figure (S3a and S3b) for compound
25 is shown in supporting information.
To know the thermal stability of the DNA gyraseB protein when associated with the
ligand differential scanning fluorimetry (DSF) studies were performed (22, 27). The most active
compound 22 from this furan/thio-phene-2-carboxamide series was subjected to DSF. Compound
22 enhanced the thermal stability of the catalytic domain of the gyraseB protein when performed
o
in the presence and absence of the inhibitor. A significant positive shift of 2.9 C was observed
with respect to its Tm of 44 oC confirming the stability of the protein–ligand complex as shown in
Figure 8.
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A structure based e-pharmacophore model was built and based on the built hypothesis
twelve molecules were shortlisted. Among them, BAS04380545, 4-((4-(furan-2-
carboxamido)phenyl)amino)-4-oxobutanoic acid a compound which has been synthetically
derivitized to obtain another twenty three molecules for which the in vitro screening and in vivo
screening were performed. On hit expansion of this lead compound 5, a more potent inhibitor 22
with better biological properties was obtained. Further, structural elements were identified that
undoubtedly provide the basis for further optimization of the new inhibitors.
Acknowledgements
We are sincerely grateful to Prof. M. Meera and Gujjula Ravi Kumar (Sir Ronald Ross Institute of
Tropical and Communicable Diseases (Nallakunta, India), Osmania University for their help with
S. aureus MRSA 96 strain. DS acknowledges the University Grant Commission, India for a
U.G.C research award, JR is thankful for CSIR for providing the financial assistance.
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Supporting information
Additional supporting information may be found in the online version of this article.
Appendix S1: Methodology used for docking, spectral details of all the synthesised compounds
and the protocol used for biological evaluation.
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Table1. Biological activities of furan/pyrrole/thiophene-2-carboxamide derivatives
GyraseB
assay(IC50)a
(µM)
Supercoiling
assay(IC50)b
(µM)
MIC
ATCC
(µM)c
MRSA
MIC
Biofilm
Inhibi.
(IC50)e
Cytotoxic
ity (%
Comp
R1
R2
X
(µM)d
inhibi.)f
H
CH3
NO2
H
-OH
OCH3
OCH3
OCH3
OCH3
OCH3
0CH3
OCH3
OCH3
-OH
O
O
12.88 1.39
27.63 1.09
22.64 0.66
37.63 0.91
37.52 1.22
12.49 0.58
11.06 0.92
24.86 0.65
45.42 2.54
10.33 0.51
28.94 0.83
21.99 0.74
24.66 0.82
29.49 0.81
42.45 0.88
11.69 0.5
3.12 1.6
26.15 0.3
8.41 0.22
11.2 0.69
22.6 0.91
6.12 0.49
7.5 0.39
79.03
75.67
17.29
37.60
72.17
16.56
40.89
39.02
79.03
39.51
35.99
39.26
39.26
68.81
81.61
39.02
79.03
75.67
34.58
75.2
79.03
75.67
34.58
37.60
72.1
15.95
21.22
19.88
16.64
17.34
17.82
15.5
5 (Lead)
6
7
O
S
8
CH3
NO2
-H
S
72.17
16.56
40.89
78.04
158.06
79.02
71.98
78.52
78.52
68.81
81.61
78.04
9
S
33.12
81.78
39.02
79.03
39.51
35.99
39.26
39.26
39.26
81.61
39.02
10
11
12
13
14
15
16
17
18
19
20
THF
N
-H
13.7 0.77
28.18 0.6
6.12 0.7
12.54
17.66
18.33
13.26
24.62
18.35
18.55
15.22
17.06
H
O
CH3
NO2
H
O
-OH
O
25.8 0.35
12.5 0.51
12.5 0.83
26.8 1.25
13.7 0.45
6.12 0.28
-OH
S
CH3
NO2
-H
-OH
S
-OH
S
-OH
N
-H
-OH
THF
H
-NH-NH2
O
39.41 0.97
23.9 0.4
79.03
79.03
79.03
14.21
21
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CH3 -NH-NH2
NO2 -NH-NH2
O
O
S
5.35 0.44
35.49 1.54
32.56 2.41
9.71 0.69
2.79 0.59
25.1 0.44
28.1 0.67
6.12 0.22
18.92
34.59
75.23
18.04
18.92
34.59
75.23
36.08
18.92
34.59
75.23
36.08
68.05
9.35
22
23
24
25
22.53
10.22
19.31
13.54
H
-NH-NH2
CH3 -NH-NH2
NO2 -NH-NH2
S
S
N
26.44 0.77
54.87 0.84
41.2 0.83
19.3 0.88
43.1 0.68
23.1 0.45
0.03 0.01
ND
68.05
78.03
78.03
0.25
68.05
156.06
156.06
ND
26
27
-H
-H
-NH-NH2
NH-NH2
156.06 17.33
THF
78.03
ND
15.4
11.4
ND
28
0.125 0.24
0.81 0.15
Novobiocin
Moxifloxacin
ND
ND
ND
ND, indicates not determined.
a S. aureus gyraseB ATPase activity.
b S. aureus DNA gyrase supercoiling activity.
c In vitro (MTCC 3160) activity.
d In vitro (MTCC 96) MRSA MIC activity.
e Biofilm inhibition of MTCC 96.
f At 100 µM against RAW 264.7 cells.
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.