Design, synthesis and evaluation of diphenyl ether analogues as antitubercular agents

Article abstract of DOI:10.1039/c6ra19821j

We herein report the investigation of new diphenyl ethers as Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (InhA) inhibitors by structure-based drug design approach. The virtual library of diphenyl ethers was designed and molecules with

Full text of DOI:10.1039/c6ra19821j

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Design, synthesis and evaluation of diphenyl ether analogues as antitubercular agents
Bharathkumar Inturi1, Gurubasavaraj V Pujar1*, Madhusudhan N. Purohit1, Viswanathan B.Iyer1,
Sowmya G.S2 and Madhuri Kulkarni2
1Department of Pharmaceutical Chemistry, JSS College of Pharmacy, Jagadguru Sri
Shivarathreeshwara University, Mysoreꢀ570015, Karnataka, India.
2Department of Microbiology, JSS Medical College and Hospital, Jagadguru Sri
Shivarathreeshwara University, Mysoreꢀ570015, Karnataka, India.
*Corresponding author:
Dr. Gurubasavaraj V. Pujar, Professor, Department of Pharmaceutical Chemistry, JSS College of
Pharmacy, Jagadguru Sri Shivarathreeshwara University, Mysore 570015, Karnataka, India,
Email: gvpujar@jssuni.edu.in, Phone: +918088532266; Fax: +918212548359
ABSTRACT
We here report the investigation of new diphenyl ethers as Mycobacterium tuberculosis enoylꢀ
acyl carrier protein reductase (InhA) inhibitors by structureꢀbased drug design approach. The
virtual library of diphenyl ethers was designed and molecules with appreciable physicochemical
and ADMET properties were docked. The best ranked molecules based on docking studies were
synthesized and characterized by spectral studies. Synthesized compounds were evaluated for in
vitro antitubercular activity against Mycobacterium tuberculosis H37Rv strain by Microplate
Alamar Blue Assay. Among the tested compounds, DE3 and DE2 exhibited substantial
antitubercular potential at 3.125 and 6.25 µg/mL concentrations respectively. The most active
compounds were further evaluated for cytotoxicity studies against Vero and HepG2 normal cell
lines by Microculture Tetrazolium assay and, ascertained to be safe against normal cell. The
molecular dynamic study reveals that the best active compounds have shown better binding free
energy than the reference compounds TCl and JPL at Mtb InhA binding site.
Keywords: Diphenyl ethers; Docking; InhA; Molecular Dynamics; Mycobacterium tuberculosis;
Triclosan.
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1. Introduction
Mycobacterium tuberculosis (Mtb) infection has been marked for mortality over two million
people worldwide every year. The World Health Organization reports indicated that at the end of
year 20201 more than one billion populations worldwide may be infected with tuberculosis (TB).
Clinical studies indicate that TB is more prevalent susceptible in AIDS patients and, there is an
increase in TB epidemics over the last five years may be associated to HIV coꢀinfection. The
current status of tuberculosis is magnified due to increase in multidrug resistant to the existing
drug therapy2–4.
Currently, the three major challenges that hinders our ability to eradicate TB effectively are drug
resistance strain infection, HIV coꢀinfection and regimen nonꢀcompliance. Consequently, there is
a need of addressing the issues of multidrug resistance and persistent TB infection. In view of
these facts, the development of drugs with novel modes of action has been the crucial of the
investigators. The pursuit for inhibitors targeting enzymes that are deemed specific, essential for
the replication and persistence of Mtb has been the core of antitubercular research. The targetꢀ
based screening approach for the discovery of new drugs was rendered possible with the
advances in proteomics, genomics and molecular genetics of mycobacterium. The targetꢀbased
screening approach yielded remarkable results in the field of cancer drug discovery and hope that
the same would results for TB drug discovery too.
The mycobacterial fatty acid synthase I (FAS I) and FAS II contributes in the biosynthesis of
mycolic acids, which are components of the mycobacterial cell wall. Whilst, FAS II has a
predominant role in the elongation of fatty acids derived from the end product of FAS I5. The
terminal step of FAS II, transꢀenoyl reduction is catalyzed by InhA and is marked for this
significant role. InhA catalyze the elongation of C16 fatty acid and longer, which is different
from Enoyl ꢀACP reductase (ENR) of other bacterial species. InhA has been validated as a
promising target for antitubercular drug discovery. Isoniazid (INH) and ethionamide (ETH) the
first line agents, and most prescribed drug to treat tuberculosis (TB), inhibits a NADHꢀdependent
InhA that provides precursors of mycolic acids. They are proꢀdrug that needs activation to form
the inhibitory INH/ETHꢀNAD adduct by KatG/ EthA encoding enzyme. These adducts are
marked as tight binding inhibitors of Mtb InhA6,7.The mutations of KatG and EthA have been
identified and are associated with the development of Mtb resistance to the INH and ETH,
respectively8,9. The inhibitors which can directly inhibit InhA without activation by KatG/ EthA
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can able to circumvent INH/ETH resistance mechanism. This approach is useful in the rational
design of potential antitubercular agents against MDRꢀTB and XDRꢀTB strains.
The pursuit for small molecules as potential Mtb InhA inhibitors is an effective strategy to
eradicate MDRꢀTB. Clinical studies highlight the importance of direct InhA inhibitors and
investigation of new small molecules for the same is on worldwide. The new small molecules
like pyrazoles10, indoleꢀ5ꢀamides11, diphenyl ethers12,13
,
pyrrolidine carboxamides14,
arylamides15, imidazopiperidines16 and 4ꢀhydroxyꢀ2ꢀpyridones17 have been reported as direct
InhA inhibitors. These molecules can inhibit InhA without prior activation by KatG and have
shown potential activity against MDR/INHꢀresistant TB strains. The Triclosan (TCl), a diphenyl
ether derivative was found to be a potent inhibitor of Mtb InhA with Ki value of 0.22 mM and
MIC value 12.5 µg/mL18. However, TCl use as antitubercular agent is limited due to its poor
bioavailability. The biochemistry involved in bacterial inhibition of TCl with the InhA is
possible through the πꢀπ ring stacking interactions between the aromatic ring of TCl and pyridine
of NAD+ cofactor. TCl also exhibits strong hydrogen bonding with Tyr 158 residue at the
catalytic site and NAD+ cofactor19.
In order to improve the bioavailability, TCl modified derivatives were synthesized with
improved pharmacokinetic parameters in the last decade, and these new compounds have shown
significant activity against both susceptible and resistant Mtb strains. In continuation and
exploring the structureꢀactivity relationship (SAR), the alkyl substituted diphenyl ethers were
synthesized with an improved affinity towards InhA inhibition. In this regard, 5ꢀoctylꢀ2ꢀ
phenoxyphenol has shown a potential activity (MIC:6ꢀ9 µM) against both drugꢀsensitive and
drug resistant strains of Mtb and, more importantly the mechanism of inhibition of InhA is KatG
independent5. The SAR highlights the InhA inhibitory potency also depends on the length of the
alkyl chain at 4th position of the Aꢀring. The optimal activity was observed with alkyl chain
length between 3ꢀ5 carbons. The removal of the two chlorine atoms on the diphenyl ether Bꢀring
moiety and also replacement of the chlorine function with an ethyl group in Aꢀring resulted in a
2ꢀfold increase in the IC50 value in comparison to TCl (Figureꢀ1). In spite of these structural
modifications in improvising the antitubercular efficiency, the limited bioavailability was the
foremost drawback of these alkyl chain substituted diphenyl ether derivatives may be attributed
to the higher log P values (>5). Hence, the investigation was focused to develop druggable
diphenyl ethers with improved antitubercular activity with appreciable ADMET properties using
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structureꢀbased drug design approach. We report herein the molecular docking, synthesis,
antimycobacterial evaluation and molecular dynamics study of new diphenyl ether derivatives.
2. Results and Discussion
2.1. Strategies of drug design
In view of facts discussed earlier, the present work was aimed to develop diphenyl ethers with
the optimal lipophilicity (log P values between 3 and 5) as favorable for drug likeness properties.
Prior to the drug design strategy, the reported diphenyl ethers physicochemical properties were
studied. The results highlights that most of the compounds have significantly higher log P values
(>5), while other physicochemical parameters were well within the acceptable range. These
compounds have exhibited potent in vitro antitubercular activity and their Clog P values ranged
between 4 and 7. Despite their promising in vitro activity, some of compounds exhibits poor in
vivo efficacy, which may attributed to high Clog P values. The SAR of diphenyl ethers indicates
that the dichloro substitutions on ringꢀB of TCl can be attributed for the higher log P values. The
studies also indicated the compounds were to be involved in unfavorable steric interactions with
the enzyme and the removal of chlorine functionality from the scaffold would increase the
affinity of inhibitors by seven folds towards enzyme20. Consequently, in the present investigation
diphenyl ethers were designed with hydrophilic substitutions at the 4th position of ringꢀA and
exclusion of chlorine substitutions on ringꢀB. The hydrophilic linkers were employed to achieve
desired lipophilicity. The substitutions on aryl moiety with both electron donating and electron
withdrawing functions were made towards experimenting and understanding the possible
interaction to effect the inhibition of the enzyme21. Over all modifications of diphenyl ethers
were aimed to reduce the inherent lipophilicity of TCl derivatives without compromising its
orientations and catalytic interactions at the InhA binding site. The drug design strategy is
depicted in Figureꢀ1.
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Design Stratergy
Essential for Binding
Mode
OH
OH
O
O
Cl
B
OH
Removal of Bꢁring
chlorine groups
2
O
1
3
A
Alkyl
X
groups
6
4
Cl
Cl
5
Imroved Activity
6ꢁ9 ꢀM
Triclosan
12.5 ꢀg/mL
Liphophilic
modifier
OH
O
O
N
NH
R
X
Design outcome
logP: 3ꢁ5
Figureꢀ1: Design strategy of diphenyl ether analogues
2.2. Molecular modelling studies
2.2.1. In silico ADMET Studies
The designed compounds were screened for their appreciable ADMET properties using
PreADME online tool22. All the compounds have appropriate values towards the evaluated in
silico parameters. The Lipinski parameters of the molecules towards their biological efficacy
were encouraging with zero Lipinski violation. The in silico ADMET results shows that the
selected compounds have appreciable oral bioavailability and protein binding efficiency. The
predicted oral availability was excellent as the molecules exhibited a calculated percentage of
absorption (HIA) values ranging from 92 to 96%. The predicted plasma protein binding (PPB)
exhibits parentage binding values ranging from 89 to 100 %. The compounds also shows
moderate Caco2 cell permeability effect, which is in the range of 15ꢀ32 nm/sec. The compounds
which have <2 blood brain barrier penetration values indicate poor penetration. The ADMET
properties data is provided as supplementary material. Compounds that satisfied ADMET and
Lipinski’s parameters of drug likeness were docked against Mtb InhA.
2.2.2. Molecular docking study
The molecular docking technique was used to explore, predict and understand the
protein/enzyme interactions with designed diphenyl ethers at Mtb InhA binding site. The docking
study was performed on Mtb InhA pdb protein (PDB ID: 3FNG) using the SYBYLꢀX 2.1
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molecular modelling software. The molecular docking study was executed initially by the
analysis of binding site structural features, followed by docking and interpretation of the results
and finally the validation of the docking protocol.
Mtb InhA binding site analysis: The structural features of InhA reveals that it has four
monomer units and each consists of 269 amino acid residues. The molecular weight of each
monomer unit is ~29000 Da. The monomer unit builds with Rossmann fold structure and the
core of the binding site contains eight α helices and seven β sheets, representing the dinucleotide
binding for the cofactor, NADH. The substrate binding loop is consisting of 15 amino acid
residues (194–208) and further categorized into three regions, upper, middle and lower substrate
binding loop (USL, MSL, and LSL) (Figure 2). The top USL cover the substrate binding pocket
(SBP) by forming interꢀloop interactions. The dinucleotide binding site is close to LSL and
forms interactions with NADH.
Figureꢀ2: Substrate binding pocket of InhA and structural components. (USL: Upper substrate
binding loop, MSL: Middle substrate binding loop and LSL: Lower substrate binding loop.)
The InhA complex of fatty acyl substrate and NAD+ cofactor crystal structure reveals that the
fatty acyl substrate binds in Uꢀshaped conformation. The residues Tyr158 and Lys165 are very
much essential for the transꢀenoyl reduction. They promote the removal of a proton from the 2'ꢀ
nicotinamide ꢀOH group. The InhA enzyme plays a crucial role in the reduction of C2–C3
double bond by transfer of hydrate ion to C3 carbon. The Tyr 158 hydroxyl group donates a
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proton to the carbonyl oxygen of (C1) fatty acid results in an enolate anion. The substrate, fatty
acyl chain is present in the core of lipophilic residues and most of the lipophilic residues are
abreast to the SBL (Met103, Phe149, Tyr158, Lys165, Thr196, Met199, Leu207 and Ile215).
The SBL of InhA is larger compared to enoylꢀACP reductases of other organisms and facilitates
a deeper substrate binding area. The Tyr 158 interaction with fatty acyl substrate is the crucial
feature in all of enoylꢀACP reductases
2.2.3. Docking Results:
Molecular docking results revealed that the basic scaffolds of DE 1ꢀ10 (5ꢀ((Substituted
imino)methyl)ꢀ2ꢀphenoxyphenol) fits in the binding pocket of InhA similar to TCl and 5ꢀ
(cyclohexylmethyl)ꢀ2ꢀ(2,4ꢀdichlorophenoxy)phenol (coꢀcrystallized ligand of 3FNG; JPL)
(Figure. 3a & 3b). Furthermore, docking study reveal that the formation of hydrogen bonding
interactions between phenolic –OH and ether oxygen of the designed compounds with Tyrꢀ158
and NAD+ of Mtb InhA, which is vital as per the docking literature. The hydrogen bonding
network is consistent in all the designed compounds, and this feature is reported to be a
conserved one among all the InhAꢀinhibitor complexes including TCl. Hence, the core structural
features of TCl (diphenyl ether nucleus) and the hydroxyl group in ringꢀA were kept intact in all
the designed analogues. Based on docking scores (>7), interactions and binding with above said
crucial residues; the best ranked molecules were considered for synthesis. The docking score
data of the synthesized compounds is presented in Table 1.
Figureꢀ3: (a): Binding poses of TCl and JPL at InhA binding site, Green (TYR 158), Purple
(NAD+), Yellow lines (Hydrogen Bonding). (b). Binding pose of compounds DE3 and JPL at
InhA binding site, Green (TYR 158), Purple (NAD+), Yellow lines (Hydrogen Bonding).
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Tableꢀ1: Molecular docking scores of synthesized compounds DE 1ꢀ10.
Docking scores
Crash
ꢀ2.75
Compounds
Total
7.74
8.07
7.06
8.25
8.56
8.07
8.71
8.81
8.04
8.05
8.86
5.07
Polar
2.25
2.11
2.12
1.02
2.18
1.13
2.02
2.21
1.26
1.84
2.24
2.41
DE1
DE2
DE3
DE4
DE5
DE6
DE7
DE8
DE9
DE10
JPL
ꢀ3.15
ꢀ1.96
ꢀ1.44
ꢀ1.11
ꢀ1.02
ꢀ2.61
ꢀ1.58
ꢀ1.21
ꢀ2.08
ꢀ0.58
ꢀ0.29
TCl
Total Score = The total SurflexꢀDock score expressed as ꢀlog(Kd).
Crash = The degree of inappropriate penetration by the ligand into the protein.
Polar = Contribution of the polar interactions to the total score.
2.2.4. Docking Validation:
The accuracy of the docking protocol detailed under method was extensively validated by
reproducing the ligandꢀreceptor (Mtb InhA) complex (3FNG) deposited in the RCSB PDB. The
Root Mean Squire Deviation (RMSD) value and poses were used for validation of docking
protocol. The coꢀcrystallized ligand (JPL) of the PDB structure was extracted and docked along
with the designed ligands. The docking poses of designed ligands were compared with the
binding pose of JPL and TCl (Figureꢀ3a). The RMSD value was calculated between the coꢀ
crystallized and docked ligand. The RMSD value for the coꢀcrystallized and docked ligand was
0.948 Å and is lower than the acceptable limit (< 1.5 Å), accordingly indicates that the docking
protocol is validated.
2.3. Chemistry:
The synthetic protocol for new series of diphenyl ether scaffold is outlined in Schemeꢀ1. The
starting material, 3ꢀmethoxyꢀ4ꢀphenoxybenzaldehyde (1a) was prepared according to LamꢀCham
Oꢀarylation with vanillin and phenyl boronic acid. Demethylation of methoxy aldehyde
o
derivative 1 was carried out in the presence of Boron tribromide (BBr3) at ꢀ78 C (dry ice and
acetone) to afford 3ꢀhydroxyꢀ4ꢀphenoxybenzaldehyde (2a) which was further condensed with
various acid hydrazides to afford title compounds DE 1ꢀ10 (Tableꢀ2). The synthesized
compounds were characterized by IR, 1H NMR, 13C NMR, mass spectral and elemental analysis
data. In IR spectra, the absorption band around ~1590 cmꢀ1 were observed in all the synthesized
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1
derivatives may be attributed for –C=N stretching. The spectral characterization by H NMR
spectra of the derivatives indicates the chemical shift signals in the range of ꢀ 6.6ꢀ7.9 ppm (Arꢀ
H) as multiplet for aromatic protons. The NH proton of the amide group resonated as a singlet
around ꢀ 11.5 ppm attributing for the amide bond formation. Mass spectrum of the compounds
exhibits molecular ion peak (M+) corresponding to their respective molecular weights.
1
The H NMR spectrum of the compound 1a exhibited a singlet at ꢀ 9.9 ppm value corresponds to
aldehyde functionality and also exhibits 8 protons in the aromatic region (ꢀ 7.6ꢀ7.0 ppm), it
1
confirms the formation of diphenyl ether nucleus. In the H NMR spectra of compound 1b, the
singlet at ꢀ 10.1 and ꢀ 9.9 ppm values were attributed to the proton of aldehyde functional group
1
and the phenolic OH of diphenyl ether, respectively. The H NMR spectral data concludes the
formation of diphenyl ether and its demethylation product. The IR spectrum of DE3 exhibited
absorption bands at 3435 cm−1 attributing for NꢀH stretching and 1732 cm−1 accounting for
1
stretching of C=O functionality. The H NMR spectrum of the compound DE3 exhibited singlet
protons at ꢀ 11.8 and 9.8 ppm values corresponding to NH of hydrazide functionality and phenolic
OH group of diphenyl ether respectively. The spectral characterization data of all the synthesized
compounds are in accordance with the proposed structures as depicted in the scheme. The
experimental log P values of all the synthesized compounds were determined by RPꢀHPLC method
to affirm and towards a comparative study with respect to the calculated log P values. The
physicochemical properties data of DE 1ꢀ10 is listed in Tableꢀ2.
Schemeꢀ1: Synthesis of designed compounds
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Tableꢀ2: Physicochemical properties data of diphenyl ether derivatives.
Lipinski Rule of Parameters
R
log P
(Experimental)
Compound
Acceptor Donor
Clog P
M.W*
333.3
(Aꢀring C4)
Count
count
(Calculated)
O
N
DE1
6
2
3.29
3.34
HN
O
DE2
DE3
5
5
2
2
332.3
366.7
4.40
5.02
4.25
4.8
HN
O
Cl
HN
Cl
O
DE4
DE5
DE6
5
5
8
2
1
2
366.7
318.3
377.3
4.38
2.44
4.36
4.44
2.56
4.43
HN
O
N
O
NO2
HN
HO
O
DE7
6
2
362.3
4.49
4.35
HN
H3C
O
DE8
DE9
5
5
5
2
2
2
346.3
346.3
346.3
4.56
4.90
4.53
4.42
4.85
4.55
HN
O
CH3
HN
O
DE10
NH
* Molecular Weight
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2.4. Biological Activity:
2.4.1. Antitubercular Activity:
The synthesized compounds were evaluated for in vitro antitubercular activity by Microplate
Alamar Blue assay (MABA) against Mtb H37Rv strain. The antitubercular activities of DE 1ꢀ10
are expressed as minimum inhibitory concentration (MIC) values using INH and TCl as the
reference drugs towards a comparative study. The in vitro antitubercular evaluation of diphenyl
ethers indicates that few of the tested compounds exhibit significant activity (Tableꢀ3). The
compounds, DE3 was found to be potent antitubercular among all tested compounds with a MIC
value of 3.125 µg/mL. The results indicate that the better activity of compound DE3 may be
attributed to the electron withdrawing function chlorine at the para position and a comparatively
higher log P value (4.8). In support of the above view, compound DE2, the phenyl derivative
exhibited a MIC value of 6.25 µg/mL. Whilst, the methyl group substituted derivatives DE8 and
DE9, examples for electron donating groups were exhibiting a trivial antitubercular activity with
MIC values >100 µg/mL. The oꢀchloro derivative (DE4) exhibited a higher MIC value (12.5
µg/mL) in comparison to the pꢀchlorophenyl derivative (DE3; MIC3.125 µg/mL), but
comparatively better than that of the pꢀnitrophenyl (DE6 MIC=50 µg/mL). The better activity of
compound DE3 in comparison to DE4 and DE6 may be attributed to its log P value. The
compound DE3 log P value (4.80) is higher than that of compound DE6 and DE4, which have
log P value of 4.43 and 4.44, respectively. The efficiency of the drug to produce an
antitubercular effect depends on the accumulation of the same in the cell and resulting in cell
death. The higher lipophilic nature of compound DE3 may be correlated to its potential to cross
the phospholipid membrane of Mtb and leading to a significant accumulation of it in the cell and
resulting in considerable antitubercular activity. The compounds with pyridinyl (DE1 & DE5)
substitutions demonstrated poor activity (MIC >100 µg/mL). The activity data reveals that the
antitubercular activity of the derivatives drastically improved with the replacement of pyridinyl
moiety (DE1 and DE5 MIC>100 µg/mL) by phenyl/ substituted phenyl moiety (DE2; MIC=6.25
µg/mL). Moreover, the low log P values of DE1 (3.34) and DE5 (2.56) may also be correlated
with the lower antitubercular activity of pyridinyl derivatives. The investigation also indicated
that the antitubercular activity decreases with increase in carbon chain length between the amide
function and the phenyl moiety, compound DE10; benzyl derivative exhibited a MIC value of
12.5 µg/mL in contrast to DE2 (MIC=6.25 µg/mL). The considerable antitubercular activity of
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DE3 may be attributed to the electronegative functional group substitutions on the phenyl
hydrazide moiety and a significantly higher log P (4ꢀ5) values.
The compounds exhibiting significant antitubercular activity (DE2, DE3, DE4, DE7 and DE10)
with a MIC values less than 25 µg/mL were further screened for cell viability assay by
Microculture Tetrazolium Assay (MTT) against Vero (epithelial cells) and HepG2 (hepatocytes)
cell lines to ascertain their antitubercular activity is not due to cytotoxicity and also to highlight
their safety profile on normal cell. The approximate CC50 values and selectivity index (SI) are
tabulated in Table 3. These findings indicate that the active derivatives target Mtb to a greater
extent compared to macrophage cell lines.
Tableꢀ3: In vitro antitubercular, antibacterial activity (MIC) and cytotoxicity (CC50) of diphenyl
ether derivatives
MICa (µg/mL)
CCb50 (µg/mL)
Compounds
SIc
Mtb H37Rv
S.aureas
6.25
3.125
3.125
3.125
25
E.coli
12.5
3.125
6.25
12.5
12.5
25
Vero
nc
HepG2
nc
DE1
DE2
DE3
DE4
DE5
DE6
DE7
DE8
DE9
>100
6.25
3.125
12.5
>100
50
ꢀ
>300
>300
>300
nc
>300
>300
>300
nc
>10
>10
>10
ꢀ
12.5
12.5
25
25
12.5
ꢀ
nc
nc
ꢀ
25
50
>300
nc
>300
nc
>10
ꢀ
ꢀ
>100
>100
12.5
<3.125
12.5
ꢀ
12.5
100
25
ꢀ
ꢀ
nc
nc
DE10
INH
TCl
>300
nc
262
nc
>10
ꢀ
ꢀ
ꢀ
ꢀ
nc
nc
Ampicillin
nc: not carried out
<3.125
<3.25
nc
nc
a MIC = minimal drug concentration required to stop the growth of Mycobacterium tuberculosis H37Rv.
b CC50 = minimal drug concentration required for 50% death of viable cells.
c SI (Selective index) = CC50/MIC.
2.4.2. Antibacterial Activity:
The synthesized compounds were also evaluated for in vitro antibacterial activity against Gramꢀ
positive Staphylococcus aureus (ATCCꢀ25923) and Gramꢀnegative Escherichia coli (ATCCꢀ
25922) by tube dilution method. The antibacterial data (Table 3) reveals that few of the diphenyl
ether derivatives exhibited significant activity against the tested strains. Among the tested,
compounds DE2, DE3 and DE4 have shown potential antibacterial activity with a MIC value of
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3.125 µg/mL against S.aureus. While, compound DE3 was also effective against the E.coli with
a MIC value of 6.25 µg/mL.
2.5. Molecular dynamic studies
The synthesized compounds were subjected to molecular dynamics (MD) simulation studies in
comparison to the wellꢀknown inhibitor, TCl and JPL (3FNG coꢀcrystalized ligand). The MD
trajectories from the end of the simulation time were used to apply the MMꢀPB/SA method. The
absolute free energy of a system is estimated from a combination of molecular mechanics
following the Poisson–Boltzmann protocol. This protocol involves the estimation of electrostatic
free energy determined from the exposed surface area and estimate of entropy of the system
derived from normal mode calculations. RMSD plots of the simulation studies are presented in
Figureꢀ4. The RMSD plot shows that all the molecular dynamic simulation systems were
stabilized and the results highlighting that designed ligands are stable in InhA core. The MMꢀ
PB/SA results and the average of the RMSD values (0ꢀ5 ns) for all the evaluated molecules are
presented in Table 4. The studies indicate that few of the compounds have shown better binding
free energy (ꢁG binding>ꢀ29) than that of the TCl (ꢁG binding=ꢀ14.70) and JPL (ꢁG binding=ꢀ
28.96). The results highlight that the best active compounds would have a better inhibitory
efficacy than TCl and comparable to that of JPL at Mtb InhA binding site. The video files of
molecular dynamic trajectories and detailed report files of MMꢀPB/SA are given as
supplementary data.
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Figureꢀ4: MD simulations RMSD plots of TCl (green), JPL (blue) and DE3 (red).
Tableꢀ4: Calculated MMPBSA binding free energies of the compound DE3, TCl and JPL
MMꢀPB/SA Results
Cpd Code Avg. RMSD (Å)
ꢁVDW
ꢁEEL
ꢁEPB
ꢁ G
DE1
DE2
DE3
DE4
DE5
DE6
DE7
DE8
DE9
DE10
TCl
2.05
1.73
1.84
1.86
1.88
1.85
1.95
2.01
2.26
2.29
1.87
1.96
ꢀ49.73
ꢀ49.80
ꢀ54.54
ꢀ49.24
ꢀ43.39
ꢀ44.09
ꢀ49.28
ꢀ40.12
ꢀ47.13
ꢀ54.26
ꢀ36.41
ꢀ49.97
ꢀ20.08
ꢀ11.39
ꢀ35.90
ꢀ9.26
49.03
39.96
65.15
36.86
34.66
43.91
41.70
45.14
36.35
51.43
34.90
28.69
ꢀ21.25
ꢀ25.46
ꢀ29.56
ꢀ26.04
ꢀ20.73
ꢀ24.29
ꢀ28.01
ꢀ22.62
ꢀ20.49
ꢀ29.34
ꢀ14.70
ꢀ28.96
ꢀ7.95
ꢀ27.89
ꢀ15.84
ꢀ16.45
ꢀ5.14
ꢀ22.06
ꢀ10.10
ꢀ3.36
JPL
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3. Conclusion
In the present investigation, a library of diphenyl ethers was designed by structureꢀbased drug
design approach. The ADMET studies indicate that designed compounds have drugꢀlikeness
properties. The docking study indicates that the best ranked diphenyl ether molecules exhibit
major interactions with 2ꢀhydroxyl moiety of the nicotinamide ribose and the hydroxyl group of
Tyr158 residue at InhA, which are similar to TCl and JPL. Best ranked diphenyl ether
derivatives were synthesized and evaluated for antitubercular and antibacterial activities. The
antitubercular activity of tested compounds was encouraging. Wherein, compounds DE2 and
DE3 were found to be the potent molecules with MIC value 3.125 and 6.25 µg/mL respectively.
The cytotoxicity results of the most active compounds indicate that the tested compounds are
nonꢀtoxic. The selectivity index values were found to be >10,. The antibacterial activity of test
compounds were promising and compound DE3 was found to be an effective antibacterial agent
against the tested bacterial strains. The molecular dynamics study supports that best active
molecules have stable proteinꢀligand complex and exhibits better binding free energy.
4. Materials and Methods:
4.1. Molecular docking study
The 3D structures of the designed compounds were generated using SYBYLꢀX 2.1 molecular
modelling software (Tripos Associates, St. Louis, MO, USA)23. The molecules were subjected to
energy minimization with MMFF94s force field using a distance dependentꢀdielectric function,
energy gradient of 0.001 kcal/mol and electrostatics. The SurflexꢀDock tool was used to dock the
designed molecules against InhA binding site to identify the binding mode and structural
optimization. SurflexꢀDock adopted is an empirical scoring function and a patented searching
engine was employed for molecular docking. The crystal structure of Mtb InhA complexed with
5ꢀ(cyclohexylmethyl)ꢀ2ꢀ(2,4ꢀdichlorophenoxy)phenol (PDB ID: 3FNG, 1.97 Å Xꢀray resolution)
was taken from the protein databank ( http://www.rcsb.org/pdb)24. The protein structure was
prepared for the molecular docking study by removing all water molecules. The missing
hydrogen atoms were assigned to the InhA crystal structure. The coꢀcrystalized ligand (JPL) was
extracted from the protein and used as reference molecule for the validation study. The protomol
was generated by keeping the parameters of coꢀcrystalized ligand (JPL), threshold and bloat
parameters unchanged from the default values of 0.50 and 0 Å. The mode of interaction of the
coꢀcrystalized ligand against 3FNG (InhA crystal structure) was used as a standard docking
15

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model. The molecular docking was performed by analyzing 20 poses per ligand without any
constraints. The docked complex assumed to represent the proteinꢀligand interactions, which was
selected based on docking score, the orientation of the ligands at the active site in a similar to
reference ligands and preservation of the two key interactions (Hꢀ bonds with Tyr158 and
NAD+).
4.2. Synthesis and Characterization of designed molecules
All the chemicals and solvents used in this study were procured from Aldrich Chemical Co.,
Spectrochem Ltd., and Sd fine chemicals. All commercially available reagents procured were
used without further purification. Column chromatography was carried out on 100ꢀ200 mesh
Silica Gel. The progress of the reactions was monitored by TLC using Aluminum backed sheets
of Silica Gelꢀ60 F24 (Merck). Melting points were recorded using laboratory melting point
1
apparatus and are uncorrected. H NMR spectra were recorded on a NMR Spectrometer
(VNMRS400 ꢀ 400 MHz) using DMSOꢀd6 as the solvent. Mass spectroscopy was performed
using LCꢀMS using methanol as solvent. IR spectrum was obtained using FTIR
Spectrophotometer (Shimadzu, Japan) using KBr pellets.
3ꢀMethoxyꢀ4ꢀphenoxybenzaldehyde (Ia)
To the stirred solution of vanillin (1 mmol) in anhydrous dichloromethane (60 mL), activated
molecular sieves (4 A˚, 2.5 g), phenylboronic acid (1.5 mmol), copper (II) acetate (1.5 mmol)
and anhydrous pyridine (2 mmol) were added successively. The resulting suspension was stirred
o
at 25–27 C for 72 h25. The progress of the reaction was monitored by TLC, using hexane:ethyl
acetate (4:1) as the mobile phase. After the completion of reaction (72 h), the reaction mixture
was diluted with dichloromethane and filtered under vacuum. The filtrate was washed with dilute
aqueous hydrochloric acid solution (2M), followed by water, dried over anhydrous MgSO4 and
evaporated under vacuum. The crude compound obtained was purified by column
chromatography over silica 100–200 with hexane:ethyl acetate (4:1) as the mobile phase to
afford the target compound.
o
3ꢀmethoxyꢀ4ꢀphenoxybenzaldehyde (1a): Yield = 75%; mp = 40–42 C; Anal.Calcd. For
C14H12O3: C, 73.67; H, 5.30; Found: C, 73.62; H, 5.36 %; IR (KBr, νmax, cmꢀ1): 3051 (ArꢀH),
2928 (CꢀH), 1674 (C=O), 1583, 1489, 1413 (ArꢀC=C), 1276 (Asym. CꢀOꢀC), 1134 (Sym.CꢀOꢀ
1
C); H NMR (400 MHz, DMSOꢀd6): 9.94 (s, 1H, CHO), 7.62 (s, 1H, ArꢀH), 7.54ꢀ7.56 (d, 1H,
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ArꢀH), 7.38ꢀ7.42 (t, 2H, ArꢀH), 7.15ꢀ7.18 (t, 1H, ArꢀH), 7.05ꢀ7.07 (d, 1H, ArꢀH), 7.00ꢀ7.01 (d,
2H, ArꢀH), 3.87 (s, 3H, CH3); LCꢀMS (m/z): 229.2 [M+].
3ꢀHydroxyꢀ4ꢀphenoxybenzaldehyde (2a):
To the solution of compound 1a (3 g, 13.16 mmol) in dicloromethane (50 mL), BBr3 (1 mmol in
o
o
dicloromethane 26.32 mmol) was added at ꢀ78 C, and the reaction continued for 6 h at 0ꢀ10 C.
The progress of the reaction was monitored by TLC, using hexane:ethyl acetate (4:1) as mobile
phase. The resulting reaction mixture was poured into water and extracted with ethyl acetate
(3x50 mL). The organic layers were combined and washed with saturated the sodium
bicarbonate solution, dried over anhydrous magnesium sulphate and evaporated under vacuum.
The crude product obtained was purified by column chromatography over silica 100–200 with
hexane:ethyl acetate (4:1) as the mobile phase to afford the target compound.
Yield = 55%; mp = 82–84 oC; Anal.Calcd. For C13H10O3: C, 72.89; H, 4.71; Found: C, 72.92; H,
4.65 %; IR (KBr, νmax, cmꢀ1): 3051 (ArꢀH), 2928 (CꢀH), 1674 (C=O), 1583, 1489, 1413 (Arꢀ
C=C), 1276 (Asym. CꢀOꢀC), 1134 (Sym.CꢀOꢀC); 1H NMR (400 MHz, DMSOꢀd6): 10.08 (s, 1H,
CHO), 9.85 (s, 1H, OH), 7.54 (d, 1H, ArꢀH), 7.42–7.32 (m, 3H, ArꢀH), 7.32–7.20 (m, 1H, ArꢀH),
7.20–7.07 (m, 2H, ArꢀH), 6.86–6.84 (d, 1H, ArꢀH).; LCꢀMS (m/z): 215.1 [M+].
Preparation of acid hydrazides : The acid hydrazides(3aꢀh) were prepared as per the reported
procedure26.
Synthesis of (E)ꢀN'ꢀ(3ꢀhydroxyꢀ4ꢀphenoxybenzylidene) isonicotinohydrazide (DE1):
To the solution of 3ꢀHydroxyꢀ4ꢀphenoxybenzaldehyde (1 mmol) in absolute ethanol (10 mL),
Isoniazid (1 mmol) was added and refluxed for 4 h, allow the solution to cool it for 12 h. The
progress of the reaction was monitored by TLC, using hexane:ethyl acetate (1:1) as a mobile
phase. The reaction mixture was cooled, poured into water and the precipitate was filtered, dried.
The crude compound obtained was purified by column chromatography over silica 100–200 with
hexane:ethyl acetate (1:1) as the mobile phase to afford the target compound. Similar procedure
was adopted to synthesize title compounds DE2ꢀ10 using appropriate acid hydrazide.
(E)ꢀN'ꢀ(3ꢀhydroxyꢀ4ꢀphenoxybenzylidene)isonicotinohydrazide (DE1):
o
Yield = 75%; mp = 186–188 C; Anal. Calcd. For C19H15N3O3: C, 68.46; H, 4.54; N, 12.61;
Found: C, 68.41; H, 4.60; N, 12.58 %; IR (KBr, νmax, cmꢀ1): 3556 (ꢀOH ), 3435 (ꢀNH ), 3055 (Ar,
1
ꢀCH ), 1722 (ꢀC=O ), 1592 (C=N), 1221 (C–O– C); H NMR (400 MHz, DMSOꢀd6, δ ppm):
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11.98 (s, 1H, CONH), 9.84 (s, 1H, OH), 8.77 (s, 2H, ArꢀH), 8.35 (s, 1H, N=CH), 7.80ꢀ7.78 (d,
2H, ArꢀH), 7.43ꢀ7.42 (d, 1H, ArꢀH), 7.31–7.29 (dd, 2H, ArꢀH), 7.12–6.88 (m, 5H, ArꢀH); 13C
NMR (400 MHz, DMSOꢀd6, δ ppm): 161.92, 157.74, 150.77, 149.88, 149.01, 145.49, 140.96,
131.40, 130.17, 122.98, 121.93, 121.73, 120.60, 117.32, 114.81; LCꢀMS (m/z): 334.14 [M+].
(E)ꢀN'ꢀ(3ꢀhydroxyꢀ4ꢀphenoxybenzylidene)benzohydrazide (DE2): Yield: 68 %; mp = 118–
o
120 C (ethanol); Anal. Calcd. for C20H16N2O3: C, 72.28; H, 4.85; N, 8.43; O; Found: C, 72.30;
H, 4.90; N, 8.40 %; IR (KBr, νmax, cmꢀ1): 3565 (ꢀOH ), 3435 (ꢀNH ), 3058 (Ar, ꢀCH ), 1714 (ꢀ
1
C=O ), 1590 (C=N), 1228 (C–O– C); H NMR (400 MHz, DMSOꢀd6, δ ppm): 11.77 (s, 1H,
CONH), 9.82 (s, 1H, OH), 8.35 (s, 1H, N=CH), 7.90ꢀ7.89 (d, 2H, ArꢀH), 7.52–7.49 (m, 3H, Arꢀ
H), 7.43ꢀ7.42 (d, 1H, ArꢀH), 7.34–6.89 (m, 7H, ArꢀH); 13C NMR (400 MHz, DMSOꢀd6, δ ppm):
163.47, 157.83, 149.89, 147.77, 145.26, 133.95, 132.12, 131.77, 130.14, 128.90, 128.02, 122.90,
121.79, 120.33, 117.24, 114.71, 114.71; LCꢀMS (m/z): 333.15 [M+].
(E)ꢀN'ꢀ(3ꢀhydroxyꢀ4ꢀphenoxybenzylidene)ꢀ4ꢀchlorobenzohydrazide (DE3): Yield: 70 %; mp
o
= 154ꢀ156 C (ethanol); Anal. Calcd. for C20H15ClN2O3: C, 65.49; H, 4.12; N, 7.64; Found: C,
65.55; H, 4.16; N, 7.66 %; IR (KBr, νmax, cmꢀ1): 3564 (ꢀOH ), 3435 (ꢀNH ), 3056 (Ar, ꢀCH ),
1
1732 (ꢀC=O ), 1592 (C=N), 1230 (C–O– C); H NMR (400 MHz, DMSOꢀd6, δ ppm): 11.83 (s,
1H, CONH), 9.82 (s, 1H, OH), 8.34 (s, 1H, N=CH), 7.93ꢀ7.91 (d, 2H, ArꢀH), 7.59ꢀ7.58 (d, 2H,
13
ArꢀH), 7.42 (s, 1H, ArꢀH), 7.34ꢀ7.29 (m, 2H, ArꢀH), 7.11–6.89 (m, 5H, ArꢀH); C NMR (400
MHz, DMSOꢀd6, δ ppm): 162.39, 157.80, 149.88, 148.12, 145.37, 136.98, 132.64, 131.65,
130.38, 130.14, 129.96, 129.01, 122.92, 121.77, 120.40, 118.31, 117.26, 114.75; LCꢀMS (m/z):
367.11 [M+].
(E)ꢀN'ꢀ(3ꢀhydroxyꢀ4ꢀphenoxybenzylidene)ꢀ2ꢀchlorobenzohydrazide (DE4): Yield: 65 %; mp
o
= 152ꢀ153 C (ethanol); Anal. Calcd. For C20H15ClN2O3: C, 65.49; H, 4.12; N, 7.64; Found: C,
65.6; H, 4.10; N, 7.61; IR (KBr, νmax, cmꢀ1): 3552 (ꢀOH ), 3425 (ꢀNH ), 3042 (Ar, ꢀCH ), 1734 (ꢀ
1
C=O ), 1594 (C=N), 1225 (C–O– C); H NMR (400 MHz, DMSOꢀd6, δ ppm): 11.87 (s, 1H,
CONH), 9.84 (s, 1H, OH), 8.37 (s, 1H, N=CH), 7.94ꢀ7.89 (d, 2H, ArꢀH), 7.59ꢀ7.58 (d, 2H, Arꢀ
H), 7.44 (s, 1H, ArꢀH), 7.41 (s, 1H, ArꢀH) 7.36ꢀ7.34 (d, 1H, ArꢀH), 7.13–6.92 (m, 5H, ArꢀH); 13C
NMR (400 MHz, DMSOꢀd6, δ ppm): 167.42, 159.78, 152.14, 149.82, 146.31, 139.26, 133.27,
131.93, 129.91, 129.64, 127.76, 126.41, 122.52, 119.73, 119.48, 117.39, 116.76; LCꢀMS (m/z):
367.11 [M+].
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(E)ꢀN'ꢀ(3ꢀhydroxyꢀ4ꢀphenoxybenzylidene)isonicotinamide (DE5): Yield: 54 %; mp = 135ꢀ
136 oC (ethanol); Anal. Calcd. For C19H14N2O3: C, 71.69; H, 4.43; N, 8.80; Found: C, 71.70; H,
4.48; N, 8.76; %; IR (KBr, νmax, cmꢀ1): 3568 (ꢀOH ), 3048 (Ar, ꢀCH ), 1715 (ꢀC=O ), 1602
1
(C=N), 1216 (C–O– C); H NMR (400 MHz, DMSOꢀd6, δ ppm): 9.87 (s, 1H, OH), 8.64 (s, 2H,
ArꢀH), 8.37 (s, 1H, N=CH), 7.81ꢀ7.79 (d, 2H, ArꢀH), 7.44ꢀ7.42 (d, 1H, ArꢀH), 7.33–7.32 (dd,
13
2H, ArꢀH), 7.08–6.82 (m, 5H, ArꢀH); C NMR (400 MHz, DMSOꢀd6, δ ppm): 167.92, 161.74,
154.77, 147.68, 147.14, 144.42, 141.16, 134.47, 130.87, 124.38, 123.23, 121.73, 121.61, 119.32,
114.81; LCꢀMS (m/z): 319.15 [M+].
(E)ꢀN'ꢀ(3ꢀhydroxyꢀ4ꢀphenoxybenzylidene)ꢀ4ꢀnitrobenzohydrazide (DE6): Yield: 62 %; mp =
o
154ꢀ156 C (ethanol); Anal. Calcd. For C20H15N3O5: C, 63.66; H, 4.01; N, 11.14; Found: C,
63.70; H, 4.15; N, 11.20 %; IR (KBr, νmax, cmꢀ1): 3560 (ꢀOH ), 3441 (ꢀNH ), 3065 ( ꢀCH ), 1708
1
(ꢀC=O ), 1590 (C=N), 1225 (C–O– C); H NMR (400 MHz, DMSOꢀd6, δ ppm): 12.04 (s, 1H,
CONH), 9.83 (s, 1H, OH), 8.34ꢀ8.32 (m, 3H, N=CH and ArꢀH), 8.14ꢀ8.12 (d, 2H, ArꢀH), 7.45 (s,
1H, ArꢀH), 7.34ꢀ7.30 (m, 2H, ArꢀH), 7.13–6.89 (m, 5H, ArꢀH); 13C NMR (400 MHz, DMSOꢀd6,
δ ppm): 162.39, 157.80, 149.88, 148.12, 145.37, 136.98, 132.64, 131.65, 130.38, 130.14, 129.96,
129.01, 122.92, 121.77, 120.40, 118.31, 117.26, 114.75; LCꢀMS (m/z): 378.5 [M+].
(E)ꢀN'ꢀ(3ꢀhydroxyꢀ4ꢀphenoxybenzylidene)ꢀ2ꢀhydroxybenzohydrazide (DE7): Yield: 54 %;
mp = 154ꢀ156 oC (ethanol); Anal. Calcd. For C20H16N2O4: C, 68.96; H, 4.63; N, 8.04; Found: C,
68.90; H, 4.68; N, 8.07 %; IR (KBr, νmax, cmꢀ1): 3563 (ꢀOH ), 3430 (ꢀNH ), 3058 (ꢀCH ), 1726 (ꢀ
1
C=O ), 1590 (C=N), 1225 (C–O– C); H NMR (400 MHz, DMSOꢀd6, δ ppm): 11.88 (s, 1H,
CONH), 11.79 (s, 1H, OH), 9.85 (s, 1H, OH), 8.37 (s, 1H, N=CH), 7.89ꢀ7.87 (d, 1H, ArꢀH),
7.45ꢀ7.40 (d, 2H, ArꢀH), 7.33ꢀ7.29 (m, 2H, ArꢀH), 7.13ꢀ7.02 (m, 2H, ArꢀH), 6.98ꢀ6.89 (m, 5H,
ArꢀH); 13C NMR (400 MHz, DMSOꢀd6, δ ppm): 165.22, 159.59, 157.77, 149.89, 148.76,
145.51, 134.26, 131.50, 130.16, 128.91, 122.95, 121.74, 120.56, 119.38, 117.74, 117.30, 116.26,
114.87; LCꢀMS (m/z): 349.13 [M+].
(E)ꢀN'ꢀ(3ꢀhydroxyꢀ4ꢀphenoxybenzylidene)ꢀ4ꢀmethylbenzohydrazide (DE8): Yield: 60 %; mp
o
= 150ꢀ151 C (ethanol); Anal. Calcd. For C21H18N2O3: C, 72.82; H, 5.24; N, 8.09; Found: C,
72.85; H, 5.19; N, 8.12 %; IR (KBr, νmax, cmꢀ1): 3558 (ꢀOH ), 3438 (ꢀNH ), 3051(Ar, ꢀCH ), 1728
1
(ꢀC=O ), 1585(C=N), 1238 (C–O– C); H NMR (400 MHz, DMSOꢀd6, δ ppm): 11.62 (s, 1H,
CONH), 9.79 (s, 1H, OH), 8.34 (s, 1H, N=CH), 7.89ꢀ7.87 (d, 2H, ArꢀH), 7.51–7.49 (m, 3H, Arꢀ
H), 7.42 (s, 1H, ArꢀH), 7.33–7.27 (m, 6H, ArꢀH), 1.17 (s, 3H, CH3); 13C NMR (400 MHz,
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DMSOꢀd6, δ ppm): 161.34, 158.11, 148.32, 146.27, 143.26, 131.96, 131.12, 129.77, 129.14,
127.92, 126.22, 121.96, 121.19, 119.73, 116.24, 114.21, 24.71; LCꢀMS (m/z): 347.2 [M+].
(E)ꢀN'ꢀ(3ꢀhydroxyꢀ4ꢀphenoxybenzylidene)ꢀ2ꢀmethylbenzohydrazide (DE9): Yield: 60 %; mp
o
= 146ꢀ148 C (ethanol); Anal. Calcd. For C21H18N2O3: C, 72.82; H, 5.24; N, 8.09; Found: C,
72.80; H, 5.22; N, 8.10 %; IR (KBr, νmax, cmꢀ1): 3562 (ꢀOH ), 3435 (ꢀNH ), 3048 (Ar, ꢀCH ),
1
1736 (ꢀC=O ), 1590 (C=N), 1233 (C–O– C); H NMR (400 MHz, DMSOꢀd6, δ ppm): 11.57 (s,
1H, CONH), 9.77 (s, 1H, OH), 8.35 (s, 1H, N=CH), 7.87ꢀ7.84 (d, 2H, ArꢀH), 7.53–7.51 (m, 3H,
13
ArꢀH), 7.41 (s, 1H, ArꢀH), 7.29–7.22 (m, 6H, ArꢀH), 1.19 (s, 3H, CH3); C NMR (400 MHz,
DMSOꢀd6, δ ppm): 163.11, 159.26, 148.12, 146.74, 144.16, 131.27, 130.82, 129.93, 128.74,
126.92, 126.31, 121.56, 119.84, 119.13, 117.44, 113.20, 22.64; LCꢀMS (m/z): 347.3 [M+].
N'ꢀ[(E)ꢀ(3ꢀhydroxyꢀ4ꢀphenoxyphenyl)methylidene]ꢀ2ꢀphenylacetohydrazide (DE10): Yield:
o
65 %; mp = 146ꢀ148 C (ethanol); Anal. Calcd. For C21H18N2O3: C, 72.82; H, 5.24; N, 8.09;
Found: C, 72.90; H, 5.20; N, 8.12 %; IR (KBr, νmax, cmꢀ1): 3556 (ꢀOH ), 3441 (ꢀNH ), 3052 (Ar,
1
ꢀCH ), 1726 (ꢀC=O ), 1594(C=N), 1236 (C–O– C); H NMR (400 MHz, DMSOꢀd6, δ ppm):
11.71 (s, 1H, CONH), 9.86 (s, 1H, OH), 8.33 (s, 1H, N=CH), 7.89ꢀ7.87 (d, 2H, ArꢀH), 7.57–7.54
13
(m, 3H, ArꢀH), 7.47 (s, 1H, ArꢀH), 7.41–7.10 (m, 7H, ArꢀH), 3.02 (s, 2H, CH2C6H5); C NMR
(400 MHz, DMSOꢀd6, δ ppm): 162.64, 159.71, 149.22, 147.43, 144.26, 133.95, 132.12, 131.77,
130.14, 128.90, 128.02, 122.90, 121.79, 120.33, 117.24, 114.71, 114.71, 32.47; LCꢀMS (m/z):
347.4 [M+].
4.3. Antitubercular activity
Synthesized compounds were screened against M. tuberculosis H37Rv using MABA27,28 and
Middlebrook 7H9ꢀS broth was used as media. Frozen stock culture suspension of M. tuberculosis
H37Rv (ATTCC 27294) from Lowenstein–Jensen slants in complete 7H9 broth was vortexed,
adjusted to a turbidity equivalent to that of a 1 McFarland standard (3x108 CFU/mL). It was
further diluted to a concentration of 2x105 CFU/mL and used as inoculum in the MABA assay.
Test samples and standard compounds (Isoniazid and TCl) were uniformly dissolved in DMSO
and sterilized by filtering through syringe is driven filters (0.22 µm) to prepare stock solutions of
concentration 20,000 µg/mL. The stock solutions (4X) were diluted serially with media in a 96
deep well plate to afford working solutions. Dehydration of perimeter wells of the 96 well plates
was prevented by filling with sterile deionized water during an incubation period. Twofold serial
dilutions of the test compounds were done directly on the plate by using a multichannel
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micropipette by adding Media (100 µL) was added to each well of the 96 well plate and 100 µL
of inoculum (2 x105 CFU/mL) was added to each well to obtain 200 µL and the final drug
concentrations tests were 3.125–100 µg/mL. Isoniazid and TCl were used as standard and
DMSO as blank. The error was minimized by applying positive control (inoculum) and negative
o
control (media) in the plate. Then the plates were incubated at 37 C under aeration. On the
seventh day of incubation, 20 µL of Alamar blue reagent solution added to each well of the plate
o
and incubated for another 24 h at 37 C. A change in color from blue to pink was considered as
the growth of the Mycobacterium at that concentration of the drug. For better interpretation of
the results, the color was compared to the color present in the growth control wells. The MIC
was defined as the lowest concentration of drug that inhibited bacterial growth.
4.4. Cytotoxicity Screening
The cytotoxicity of the best active compounds was assessed by MTT29 and against Vero and
HepG2 cells (NCCS, Pune, India). The stock solutions of the test compounds were diluted in a
96 deep well plate aseptically with MEM (without FBS) to achieve concentrations 300, 250, 200,
150, 100 and 50 ꢂg/mL. The 96 well plates containing the cells were taken and kept inverted on
filter paper to remove the supernatant media and washed gently with PBS and decanted. 100ꢂL
of sterile water were added to outer perimeter wells. Then 100 ꢂL of each test compound
dilutions were added to the wells. DMSO was used as a control and the plates were incubated in
an incubator (5% CO2) at 37 °C 72 h and 24 h for Vero cells and HepG2 respectively. After the
incubation, plates were inverted on filter paper to remove the supernatant media followed by
PBS washing. To this, 50 ꢂL of MTT solution was added to each well in dark place and
incubated for 3 h. After the incubation, the MTT solution was removed from the well by
inverting gently on filter paper and 50 ꢂL of DMSO was added to each well and kept in dark
place for 2 h. Then the optical density readings of the plates were taken using Elisa reader at 540
nm. The safety profile (CC50) was carried out by following equations 1&2.
Determination of safety profile (CC50)
(1)
Percentage cell Inhibition = 100 ꢀ % Cell Viability
(2)
CC50 was calculated by extrapolating a graph with % cell inhibition on Yꢀaxis against the
concentration of test compound on Xꢀaxis.
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4.5. Determination of log P
Reverse phase HPLC method was applied to determine the log P (lipophilicity) of the
compounds30,31. All the chromatographic runs were conducted on HPLC (Shimadzu, Japan) at
room temperature using ODSꢀ4 (Intersil ODSꢀ4, 5 ꢂm, 4.6, 150 mm, GL Science Inc., Japan)
column and PDA detector. Numerical analysis and data processing were done using Lab
solutionꢀ2013 software. 3ꢀMorpholinopropaneꢀ1ꢀsulfonic acid (MOPS, 4.18 g) was added to 900
mL octanol saturated MilliQ water, and the volume was made up to 1 L. pH of the buffer was
adjusted to 7.4. A mixture of methanol (0.25%v/v octanol) and buffer at the ratio of 60:40, 65:45
and 70:30, was used to elute the test sample. 5 ꢂL of the sample was injected, and the flow rate
was kept at 1 mL/min. Signal was detected at λmax 254 nm. Sample runtime was kept in
between 20 min. Capacity factor (k0) was calculated for each run by using the equationꢀ332.
k'=tRꢀt0/t0
(3)
Where tR is the retention time of the sample, t0 is the retention time of blank (methanol). A graph
was plotted by taking log k' (yꢀaxis) and % methanol (3–4% concentrations) (xꢀaxis). The
logarithm of k' was extrapolated to a 0% concentration of methanol in the graph. log k' at 0%
methanol was calculated from the regression equation (R2 = 0.99) generated from the graph to
determine log P of the compounds. The log P calculation of compound DEꢀ3 is given in the
supplementary file.
4.6. Molecular dynamics
The molecular dynamic simulations were carried out by AmberTools 15.0 software. The
topology and coordinate files were prepared for the selected Mtb InhA crystal structure
complexes. The initial structure of the Mtb InhAꢀligand complex was taken from the molecular
docking study. The AMBER (Assisted Model Building with Energy Refinement) LeaP
module33,34 was used to setup FF99SB force field parameters for protein and the ligand force
fields parameters were taken from the General Amber Force Field (GAFF) and AM1 RSP atomic
partial charges were assigned. The parameters missing for the NADH were taken from the amber
parameter database, university of Manchester35,36. The prepared complexes were solvated with
TIP3P water model by creating an isometric water box, where the distance of the box was set to
10Å from a periphery of protein37. Molecular complexes were neutralized through the AMBER
tleaP module by the addition of a necessary amount of counter ions (Na+) to construct the system
in electrostatically preferred positions. The whole assembly was then saved as per the
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requirement of free energy calculation. This involved the preparation of parameter and
coordinate files for the complex, protein and the ligand without salvation. Further, the prepared
topology and coordinate files of solvated complexes were used as input for sander module of the
AMBER38. The optimization and relaxation of solvent and ions were performed by means of two
energy minimization cycles using 1500 and 2000 steps. The initial 1500 steps of each
minimization cycle were performed using steepest descent followed by conjugate gradient
minimization for rest of the steps. In the first part of minimization, InhAꢀligand complex was
kept fixed to allow water and ion molecules to move, followed by the minimization of the whole
system (water, ions and complex) in the second part. Heating was performed by six steps using a
NVT ensemble for 500 ps where InhAꢀligand complex was restrained with a very small force
constant of 5kcal/mol/Å. The temperature was allowed to rise slowly in 0ꢀ300K in first 50 ps.
Then the system was further equilibrated under constant pressure at 300K for the period of 200
ps without restraining on the complex. Final simulations (production phase)was performed for 5
ns on NPT ensemble at 300K temperature and 1atm pressure. The step size of 1fs was kept for
the entire simulation study. Langevin thermostat and barostat were used for temperature and
pressure coupling. SHAKE algorithm was applied to constrain all bonds containing hydrogen
atoms. Trajectory snapshots were taken at each 2 ps of the production phase, which were used
for final analysis. The minimization, equilibration and production were performed by sander
module of Ambertools 15. The production run was considered for the analysis, which was
carried out using the cpptraj module of the Ambertools 1539 and Chimera40.
4.6.1. MMꢀPB/SA Calculations
The MMꢀPBSA method41 was used to calculate the binding free energy of the proteinꢀligand
complex. The binding free energy is estimated by the equationꢀ4:
∆G = ∆H – T∆S
(4)
T is the temperature of the system at 300 K. The binding free energy (∆G) of the proteinꢀligand
complex is computed as per equationꢀ5.
∆G = Gcomplex – [ Gprotein + Gligand
]
(5)
Detailed calculations of MMꢀPB/SA were given in supplementary data file.
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5. Acknowledgments
Authors are thankful to The Principal, JSS College of Pharmacy, JSS University, Mysuru, India
for providing necessary facilities. Authors are beholden to DBT for the financial assistance (Ref.
No: BT/PR5594/MED/29/540/20l2). Authors express gratitude to The Director, University of
Mysore, Central Instrumentation and Research Facility for spectral data.
Authors declare no conflict of interest
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