Triazole-diindolylmethane conjugates as new antitubercular agents: synthesis, bioevaluation, and molecular docking

Article abstract of DOI:10.1039/c8md00055g

We describe the synthesis of novel triazole-incorporated diindolylmethanes (DIMs) using a molecular hybridization approach. The in vitro antitubercular activity of the DIMs against Mycobacterium tuberculosis H37Ra (ATCC 25177) was tested in the active and

Full text of DOI:10.1039/c8md00055g

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DOI: 10.1039/C8MD00055G
TriazoleꢀDiindolylmethane Conjugates as New Antitubercular Agents: Synthesis,
Bioevaluation and Molecular Docking
Ashruba B. Dannea, Amit S. Choudharib, Shakti Chakrabortyb, Dhiman Sarkarb, Vijay M.
Khedkarc, Bapurao B. Shingate*a
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431
004, India
bCombi-Chem Bio-Resource Center, Organic Chemistry Division, CSIR-National Chemical
Laboratory, Pune 411 008, India
cDepartment of Pharmaceutical Chemistry, Shri Vile Parle Kelavani Mandal's Institute of
Pharmacy, Dhule, Maharashtra 424 001, India.
E-mail:* bapushingate@gmail.com (B. B. Shingate)
*Corresponding author. Tel.: (91)-240-2403312; Fax: (91)-240-2403113
Abstract
We have described the synthesis of novel triazole incorporated diindolylmethanes (DIMs) using
molecular hybridization approach and their in vitro antitubercular activity against
Mycobacterium tuberculosis H37Ra (ATCC 25177) both in active and dormant state. Among all
the synthesized conjugates, the compounds 6b, 6f, 6l, 6n, 6q, 6r and 6s displayed good
antitubercular activity against both active and dormant Mtb H37Ra strain. The compound 6l
exhibited good antitubercular activity against Mtb H37Ra dormant with IC50 value 1 ꢁg/mL and
IC90 (MIC) value 3 ꢁg/mL. The compounds 6b, 6l and 6r displayed good antitubercular activity
against Mtb H37Ra active with IC50 values 2.19, 1.52 and 0.22 µg/mL respectively. The
compounds 6b, 6h, 6l and 6s displayed more than 70% inhibition towards B. subtilus strain
against Gramꢀpositive bacteria at 3µg/mL. The molecular docking study shows the binding
modes of the titled compounds in active site of DprE1 enzyme and was helped to launch a
structural basis for the inhibition of Mycobacteria.
Keywords: Antitubercular activity; Diindolylmethanes; 1,2,3ꢀTriazoles; Molecular docking;
Molecular hybridization
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TriazoleꢀDiindolylmethane Conjugates as New Antitubercular Agents: Synthesis,
Bioevaluation and Molecular Docking
1. Introduction
Tuberculosis (TB) is one of the most perilous antique scourges, airborne infectious fatal disease
caused by the pathogen Mycobacterium tuberculosis (Mtb).1 In the WHO report, predictably 1.5
million people died and approximately 9 million peoples were diagnosed by TB.1 The current TB
treatment (isoniazid, rifampicin, pyrazinamide, streptomycin and ethambutol) lead to the poor
patient obedience due to the long therapy duration about 6ꢀ12 month. Moreover, presently used
first line medicaments are not responding to multiꢀdrugꢀresistant (MDR) TB and extensively
drugꢀresistant (XDR) TB,2 making obligatory use of secondꢀline drugs in combination with
bedaquiline and delamanid, recently approved drugs (Figure 1). The increasingly delaying for
the treatment of MDR and XDR TB, complex (combination of first line drugs) and prolonged
treatment is accompanying with a higher prevalence of adverse effects.1 Therefore, there is a
strong need to discover and develop a novel antitubercular drug having new mode of action.
Figure 1. Recentlyapproved drugs for the treatment of MDR TB.
The term, “click chemistry” first time coined by Sharpless and coworkers, describes the
development of a set of powerful, selective and modular building blocks, such as azide and
alkyne that works for both small and large scale.3 The Cu(I) catalyzed 1,3ꢀdipolar cycloaddition
between alkynes and azides became a reference click chemistry reaction.4 In recent years, some
drugs i.e.; 5ꢀaminoꢀ[4ꢀ(4ꢀchlorobenzoyl)ꢀ3,5ꢀdichlorobenzyl]ꢀ1,2,ꢀtriazoleꢀ4ꢀcarboxamide (CAI)
(calcium channel blocker), cefatrizine (broad spectrum antibiotic) and tazobactum (antibiotic)
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possesses 1,2,3ꢀtriazole pharmacophore unit in their structures are available in the market
(Figure 2).
Figure 2. The structures of 1,2,3ꢀtriazole containing drugs.
In the context of medicinal chemistry, 1,2,3ꢀtriazole moiety used as pharmacophore
because of the hydrogenꢀbonding and dipole interactions, triazole moiety readily associate with
biological target. The 1,2,3ꢀtriazole is an bioisostere of amide5 having noticeable importance of
biological applications including antibacterial, antiallergic, antiꢀHIV,4d antifungal6 and αꢀ
glycosidase inhibitor7 activities. In addition to this, there are several reports on the synthesis of
1,2,3ꢀtriazole containing compounds AꢀD possess good antitubercular activity8 (Figure 3).
Figure 3. The structures of 1,2,3ꢀtriazoles possess good antitubercular activity.
There are several reports on the synthesis of diindolylmethanes (DIMs) using
homogeneous, heterogeneous catalyst and also use of microwave, ultrasound sonication.9 Many
researchers throughout the world have paid their attention towards the synthesis of DIMs10a and
indole based molecules which exhibit various biological activities, including βꢀglucuronidase,10bꢀ
e carbonic anhydrase II inhibitors,10f,10g αꢀglucosidase,10h anticancer,10i antibacterial, antifungal,10j
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antimicrobial,10k antiꢀinflammatory,10l antioxidant,10m antiamoebic,10n antimetastatic,10o
antiangiogenic,10p cytotoxic,10q radical scavenging activity10r and used as dietary supplements for
humans.10s In addition to this, indole pharmacophore unit containing derivatives were reported to
displays good antitubercular activity profile11 (Figure 5).
Very recently, Sashidhara and coworkers have reported12 the synthesis of coumarinꢀ
diindolylmethane (E, F) and benzofuranꢀdiindolylmethane (G) hybrids and evaluated for
antihyperlipidemic activity (Figure 4). Perumal et. al reported13a the synthesis of DIMs
derivatised bistriazoles (H) as antiꢀinfective agents and indoleꢀ2ꢀcarboxylic acid derived bisꢀ
1,2,3ꢀtriazoles (I) as anticancer agents by Rao and coworkers13b (Figure 4).
Figure 4. Structures of coumarinꢀdiindolylmethane (E, F), benzofuranꢀdiindolylmethane (G),
diindolylmethaneꢀbistriazole (H) and indoleꢀbistriazole (I).
2. Results and discussion
2.1. Chemistry
Molecular hybridization is a new concept in drug design and development based on the
combination of different pharmacophoric moieties in a single molecular scaffold with improved
affinity and efficacy compared to the parent drugs.14 In view of the biological significance of
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DIMs, triazoles, indoleꢀheterocycle conjugates and continuation of our earlier work on the
synthesis and bioevaluation of various heterocyclic hybrids,8d,15 herein we would like to report,
the synthesis of new triazoleꢀindole conjugates via molecular hybridization approach and their
antitubercular activity evaluation for the first time. We have designed the new hybrids of triazole
incorporated DIMs, which looks like the shape of butterfly (Figure 5). It is the onus of medicinal
chemists to improve not only the pharmacological properties, but also the drugꢀlike properties of
the molecules. In addition to this, the computational study was carried out for the better
understanding of the drugꢀreceptor interaction for antitubercular activity.
Figure 5. Molecular design strategy of triazoleꢀindole conjugates.
The synthesis of triazole incorporated diindolylmethanes in a convergent manner from
two fundamental building blocks; 1,2,3ꢀtriazole aldehydes 4a-j and indoles 5 were depicted in
Scheme 2. The arylꢀ(1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methanols 3a-j which were in turn prepared by
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Huisgen 1,3ꢀdipolar cycloaddition reaction between an aromatic azides 2a-j (prepared by the
diazotization of anilines followed by substitution reaction with sodium azide) and propargyl
alcohol catalyzed by Cu(I) in tꢀbutanol:H2O provided only the 1,4ꢀregioisomers in 63ꢀ82% yields
(Scheme 1). The structures of the arylꢀ(1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methanol 3a-j were confirmed by
1
FTIR and H NMR spectral analysis. FTIR showed the absence of stretching vibration of the
azide group and the presence of the broad OH stretching vibration at 3500ꢀ3600 cmꢀ1. In the 1Hꢀ
NMR spectra, the triazole proton were observed as a singlet at δ 8.06ꢀ8.15 ppm, whereas the CH2
group observed as a singlet at δ 4.66ꢀ4.89 ppm. The arylꢀ(1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methanols 3a-j
were further oxidized to the corresponding aldehydes 4a-j using Collins reagent (CrO3:2Py) in
dichloromethane in 56ꢀ68% yields. Structures of the desired arylꢀ1Hꢀ1,2,3ꢀtriazoleꢀ4ꢀ
1
carbaldehydes were confirmed by FTIR and H NMR spectral analysis. In the FTIR spectrum,
1
the stretching frequency was observed at 1690ꢀ1710 cmꢀ1 for aldehyde group. The H NMR
spectra specifies singlet at δ 10.05ꢀ9.95 ppm, corresponding to the aldehyde hydrogen. The
singlet signal at δ 8.06ꢀ8.19 ppm, were observed to the triazole hydrogen.
Scheme 1. Synthetic route for the preparation of substituted 1ꢀphenylꢀ1Hꢀ1,2,3ꢀtriazoleꢀ4ꢀ
carbaldehyde.
Reagents and conditions: (i) NaNO2, HCl, 0 °C; NaN3, 2ꢀ4 h, rt; (ii) propargyl alcohol, CuSO4,
sodium ascorbate, tꢀbutanol:H2O (1:1), 48ꢀ72 h, rt; (iii) Collins Reagent (CrO3:2Py), CH2Cl2, 2ꢀ6
h, rt.
The designed triazole incorporated DIMs 6a-s were synthesized from corresponding
1,2,3ꢀtriazole aldehydes 4a-j and indoles 5a-b in ethanolꢀacetic acid at 85 °C for 3ꢀ6 h in 85ꢀ90%
yields (Scheme 2). The structures of all the new triazoleꢀindole conjugates 6a-s were confirmed
by physical data and spectral analysis. The FTIR showed the presence of the broad NꢀH
1
stretching vibration at 3350ꢀ3450 cmꢀ1. In the H NMR spectra, the singlet signal observed at δ
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8.00ꢀ8.10 ppm, assigned to the triazole proton. The singlet signal observed at δ 5.50ꢀ5.95 ppm,
were assigned to methine proton. In the 13C NMR spectra, the peak observed at δ 30ꢀ32 ppm
assigned to the methine carbon.
Scheme 2: Synthesis of new 1,2,3ꢀtriazoleꢀdiindolylmethane conjugates.
2.2. Biological activity
2.2.1. Antibacterial activity
All the newly synthesized triazoleꢀdiindolylmethane conjugates 6a-s were screened for their
antibacterial activity against four bacterial strains: E. coli, P. aeruginosa as Gramꢀnegative and
B. subtilus, S. aureus as Gramꢀpositive bacteria at 3 µg/mL concentration, to assess their
selectivity towards Mtb. The obtained result of antibacterial activity is summarized in Table S2
(Supporting Information). The conjugates 6a-s showed higher specificity toward Mtb H37Ra and
some of the compounds 6b, 6h, 6l and 6s displayed more than 70 % inhibition towards B.
subtilus strain; also the compound 6r shows 71.77 % inhibition against S. aureus strain.
2.2.2. Antitubercular activity
In a standard primary screening, all the newly synthesized triazoleꢀdiindolylmethane conjugates
6a-s were tested for their in vitro antitubercular activity against M. tuberculosis H37Ra (ATCC
25177) at three different concentrations i.e. 30, 10 and 3 µg/mL using an established XTT
Reduction Menadione Assay (XRMA) antitubercular screening protocol16 and the results are
given in the Table S1 (Supporting Information). Rifampicin was used as a reference drug.
Among the synthesized compounds, 6b, 6f, 6l, 6n, 6q, 6r and 6s displayed inhibition more than
87.18 % towards M. tuberculosis H37Ra, which strengthens the fact of the antimicrobial nature
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of triazoleꢀdiindolylmethane conjugates. These compounds were further evaluated in secondary
screening at six different concentrations i.e. 30, 10, 3, 1, 0.3 and 0.1 in order to determine the
actual minimum inhibitory concentration (MIC). The obtained results of the screening are given
in Table 1. Also the compound 6h and 6j shows more than 88.22% inhibition (Table S1).
Table 1: In vitro antitubercular activity of compounds 6a-s against Mtb H37Ra strain.
M. Tuberculosis H37Ra (µg/mL)
Active Dormant
IC50 IC90
clogp
Entry
IC50
IC90
ꢀ
ꢀ
ꢀ
ꢀ
4.944
6a
2.19
30
1.55
9.39
7.236
5.443
6b
6c
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
7.505
5.055
6d
6e
ꢀ
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14.81
>30
2.96
>30
7.117
5.828
7.890
5.172
6f
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
6g
6h
6i
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
7.234
4.339
6.401
6j
6k
6l
1.52
6.16
1
3
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NO2
N
N
N
ꢀ
ꢀ
ꢀ
ꢀ
5.055
6m
NH
HN
2.27
>30
1
ꢀ
30
7.117
5.172
6n
6o
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
7.234
6p
2.04
0.22
>30
0.37
0.12
>30
5.828
7.890
6q
6r
13.5
9.84
6.25
>30
2.56
>30
7.117
3.710
6s
RP
ꢀ
0.020
0.53
0.0019
0.80
RPꢀ Rifampicin, clogpꢀ partition coefficient calculator (hydrophilicity), (ꢀ)ꢀ Not
calculated
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The presence of a ꢀNO2, ꢀNHCOCH3 and ꢀCl groups on the phenyl ring and ꢀBr group on
indole was found to be significantly increases the antiꢀTB activity in dormant and active state of
Mtb H37Ra. It is also observed that the nature of electron withdrawing substituents and position
of the substituents attached to the phenyl ring and indole are largely influenced the activity.
2.2.3. Structure Activity Relationship
The hybrid molecules derived from triazole aldehyde and indole has been used as basic scaffold
for the development of structural analogues, which produces the triazoleꢀdiindolylmethane
conjugates 6a-s. The different substituents on the phenyl ring and indole shows moderate to good
activity. From the results of the biological evaluation, the activity was significantly affected by
introducing a various substituents on phenyl ring (Table 1). From the conjugates 6a-s,
compound 6b (R1 = H and R2 = Br) showed good antitubercular activity with IC50 value 1.55
ꢁg/mL against dormant Mtb H37Ra strain. Compounds 6f (R1 = NO2 and R2 = Br) and 6l (R1 =
NHCOCH3 and R2 = Br) showed good antitubercular activity with IC50 value 2.96 and 1 ꢁg/mL
against dormant Mtb H37Ra respectively.
The compounds 6n (R1 = NO2 and R2 = Br) and 6q (R1 = Cl and R2 = H) showed good
antitubercular activity with IC50 value 1 and 0.37 ꢁg/mL against dormant Mtb H37Ra
respectively. Compound 6r (R1 = Cl and R2 = Br) showed good antitubercular activity with IC50
value 0.12 ꢁg/mL against dormant Mtb H37Ra. The compound 6s (R1 = NO2 and R2 = Br)
showed promising antitubercular activity with IC50 value 2.56 ꢁg/mL against dormant Mtb
H37Ra. The other compounds from the series 6a-s do not displays significant antitubercular
activity against dormant Mtb H37Ra. Hence, among all the synthesized compounds 6a-s, the
derivatives 6b, 6f, 6l, 6n, 6q, 6r and 6s showed moderate to good antitubercular activity against
dormant Mtb H37Ra (Table 1).
The compound 6b showed good antitubercular activity with IC50 value 2.19 ꢁg/mL
against active Mtb H37Ra (Table 1). Compounds 6f and 6l showed good antitubercular activity
with IC50 value 14.81 and 1.52 ꢁg/mL against active Mtb H37Ra respectively. The compounds
6n and 6q showed good antitubercular activity with IC50 value 2.27 and 2.04 ꢁg/mL against
active Mtb H37Ra, respectively. The compounds 6r and 6s showed good antitubercular activity
with IC50 value 0.22 and 6.25 ꢁg/mL against active Mtb H37Ra, respectively. The remaining
compound does not display significant antitubercular activity against active Mtb H37Ra. Hence,
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among all the synthesized compounds, the hybrids 6b, 6f, 6l, 6n, 6q, 6r and 6s showed moderate
to good antitubercular activity against active Mtb H37Ra (Table 1).
Electron withdrawing groups, such as nitro and halogens enhanced the antitubercular
activity against dormant/active Mtb H37Ra. On the contrary, electron donating groups such as
methyl and methoxy decreased the antitubercular activity. Exceptionally, compound 6l (R1 =
NHCOCH3) with electron donating group showed high activity. Among all the synthesized
compounds, 6l showed good antitubercular activity with IC90 (MIC) value 3 ꢁg/mL and IC50
value 1 ꢁg/mL against dormant Mtb H37Ra. The above results shows that, the presence of bromo
group on indole nucleus in compounds 6b, 6f, 6l, 6n, 6r and 6s displays better activity than the
unsubstituted indole based conjugates 6a, 6e, 6k and 6m (Figure 6). In addition to this, the
compound 6q doesn’t have bromo group on indole nucleus also shows better activity. The
compounds 6f, 6n and 6s having same group i.e nitro on phenyl ring showed different activity,
the activity increased in the order 2ꢀNO2 >3ꢀNO2 >4ꢀNO2 against both dormant and active Mtb
H37Ra strain.
Figure 6. Structure activity relationship for triazoleꢀdiindolylmethane conjugates.
2.3. Computational study
2.3.1. Molecular docking
Promising level of antitubercular potential demonstrated by some of the triazoleꢀindole
conjugates investigated herein prompted us to perform molecular docking studies to identify the
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potential target for these compounds and understand the key molecular mechanisms governing
the inhibitory activity against Mycobacterium tuberculosis. Scanning through the Protein Data
Bank for crystal structures available for mycobacterium targets resulted in a fairly good
agreement between experimental antitubercular data and the docking results against DprE1
enzyme. Furthermore, the high "druggability" of DprE1 proven by the number of unrelated
inhibitors discovered in the last five years motivated us to evaluate the potential of these
molecules to bind to this target.17 Therefore, in an effort to obtain more insight into the binding
mode and to obtain additional validations for experimental results, molecular docking study was
performed for the most active triazoleꢀdiindolylmethane conjugates 6b, 6f, 6l, 6n, 6q, 6r and 6s.
DecaprenylphosphorylꢀDꢀribose oxidase (DprE1), an oxidase enzyme involved in the
arabinogalactan biosynthesis, has been shown to be a critical target for the survival of
Mycobacterium tuberculosis. It is a key enzyme involved in the biosynthesis of the basic
precursor for the mycobacterial cell wall core decaprenylphosphorylꢀDꢀarabinose (DPA), the
only known donor of Dꢀarabinofuranosyl residues for the synthesis of arabinogalactan. DprE1 in
association with decaprenylphosphorylꢀ2ꢀketoꢀribose reductase (DprE2) catalyses the
epimerization of decaprenylphosphorylꢀDꢀribose (DPR) to decaprenylphosphorylꢀDꢀarabinose
(DPA) via the formation of the intermediate decaprenylphosphoryl 2ꢀketoꢀribose (DPX).17b,18 In
this context DprE1 is shown to be essential for cell growth and survival, thus making it a
potential target for antimycobacerial drug design strategy. In silico tools viz. molecular docking
has become very beneficial to identify the targets for different ligands, especially in the absence
of available resources to carry out the enzymatic assays.
Results from the ensuing docking simulation revealed that the triazoleꢀdiindolylmethane
conjugates could snugly fit into the active site of DprE1 occupying positions very close to that of
the native ligand with varying magnitude of affinity. All the conjugates adopted a very similar
topology of binding and were stabilized within the active pocket by formation of several steric
and electrostatic interactions. Their binding affinity has been analyzed and discussed on the basis
of four main parametersꢀ Glide score (docking score), Glide energy, bonded (Hꢀbonds and pi
stacking) and nonꢀbonded interactions (van der Waals and Coulombic). Their docking scores
varied from ꢀ8.491 for the most active analogue 6l to ꢀ7.812 for the least active 6f. The
theoretical predictions from docking calculations corroborated well with the experimental
antitubercular activity wherein the most active compounds showed higher docking scores while
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those with relatively low inhibition were predicted to have a lower docking score. A detailed perꢀ
residue interaction analysis to understand the thermodynamic components involved in the
binding event of these molecules through which we can speculate regarding the binding patterns
in the cavity. However, for the sake of brevity these results have been elaborated for the most
active analogues 6l in the next section.
The minimum energy docked conformation (Figure 7) of the most active molecule 6l
revealed that the compound binds at the same coꢀordinates as the native ligand with a
significantly higher binding affinity. Though it showed multiple interactions with the residues in
the active site, however for visibility and clarity only selected interacting residues are shown in
the figure. The compound binds with a docking score of ꢀ8.491 with an overall binding energy of
ꢀ63.731 kcal/mol. The binding energy signifies the energy required to cover the entire protein by
a ligand molecule and the putative interaction of the molecules at the active site of the enzyme.
Figure 7: Binding mode of 6l into the active site of DprE1 (on right side: green lines signify πꢀπ
stacking interactions while the pink lines represent the hydrogen bonding interactions).
Lower its value the tighter will be the binding affinity towards the target. The higher binding
affinity is attributed to the specific bonded and nonꢀbonded interactions with the residues lining
the active site of DprE1. A detailed analysis of the perꢀresidual interactions revealed that the van
der Waals interactions are more prevalent over the electrostatic contribution in its affinity
towards DprE1.
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The most significant contribution to the binding of 6l within the active site of DprE1
resulted from an extensive network of favorable van der Waals interactions with two indole
pharmacophores via Ala417 (ꢀ1.982 kcal/mol), Cys387 (ꢀ2.98 kcal/mol), Val365 (ꢀ4.835
kcal/mol), Leu363 (ꢀ1.985 kcal/mol), Gln336 (ꢀ1.987 kcal/mol), Gly321 (ꢀ1.822 kcal/mol),
Leu317 (ꢀ2.965 kcal/mol), Pro316 (ꢀ1.878 kcal/mol), Tyr314 (ꢀ1.836 kcal/mol), Lys134 (ꢀ1.914
kcal/mol), Gly133 (ꢀ1.926 kcal/mol), His132 (ꢀ3.127 kcal/mol), Gly117 (ꢀ3.155 kcal/mol)
residues lining the active site. Furthermore van der Waals interactions through the triazolyl
nucleus with Lys418 (ꢀ2.977 kcal/mol), Tyr415 (ꢀ1.951 kcal/mol), His132 (ꢀ3.127 kcal/mol),
Ile131 (ꢀ1.794 kcal/mol), Thr118 (ꢀ2.222 kcal/mol), Tyr60 (ꢀ1.939 kcal/mol) and through the
phenyl acetamide side chain with Val121 (ꢀ1.875 kcal/mol), Pro116 (ꢀ3.517 kcal/mol), Ser59 (ꢀ
1.894 kcal/mol), Arg58 (ꢀ2.145 kcal/mol) also stabilized the molecule in the active site of DprE1.
The enhanced binding affinity of 6l is also attributed to the favorable electrostatic interactions
observed with Lys418 (ꢀ7.602 kcal/mol), Asp389 (ꢀ1.878 kcal/mol), Asn385 (ꢀ1.113 kcal/mol),
Lys134 (ꢀ1.561 kcal/mol), Cys129 (ꢀ1.41 kcal/mol) and Arg119 (ꢀ1.121 kcal/mol) residues. Two
very prominent hydrogen bonding interaction were also observed first between the triazole
nitrogen and Lys418 and second between the indole nitrogen and Tyr60 residues with a bonding
distance of 2.27 Å and 1.83 Å respectively. Furthermore, the enzymeꢀinhibitor complex is
observed to be stabilized by close πꢀπ stacking interactions observed between the triazole and the
indole ring with His132 residue. Such type of hydrogenꢀbonding and the πꢀπ stacking
interactions serve as an "anchor" and guide the 3D orientation of the ligand into the active site
facilitating the steric and electrostatic interactions.
A similar binding mode and network of interactions was observed for 6b (Figure S1), 6f
(Figure S2), 6n (Figure S3), 6q (Figure S4), 6r (Figure S5) and 6s (Figure S6) as well but
decreasing gradually with their observed antitubercular activity. However, for the sake of brevity
the details are summarized in supporting information Table S3. The perꢀresidue ligand
interaction analysis suggest that the mechanical interlocking of these molecules is governed by
the steric complementarity with the active site residues of DprE1 which is reflected in the
relatively higher contribution of favorable van der Waals interactions than the other components
contributing to the overall binding affinity. Overall, it is evident from these docking simulations
and more specifically from the perꢀresidue interaction energy profile that these molecules have
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excellent affinity for the mycobacterial DprE1 enzyme qualifying them as pertinent starting
points for structureꢀbased lead optimization.
2.3.2. In silico ADMET predictions
In silico ADMET predictions approach gives a cost effective alternative to filter their
pharmacokinetic, safety and drugꢀlikeness profile and optimize the lead compounds in the early
stage of drug discovery. In the present study, we calculated molecular weight (MW), molecular
volume (MV), logarithm of partition coefficient (miLog P), number of hydrogen bond donors (nꢀ
OHNH), number of hydrogen bond acceptors (nꢀON), topological polar surface area (TPSA),
number of rotatable bonds (nꢀROTB) and Lipinski’s rule of five19 using a Molinspiration online
property calculation toolkit.20 A computational study of active compounds 6b, 6f, 6l, 6n, 6q, 6r
and 6s was performed for the prediction of ADMET properties and the obtained values are
presented in Table 3. The Drugꢀlikeness model score was computed by Molsoft software21 and is
a combined property of physicalꢀchemical properties, pharmacokinetics and pharmacodynamics
of a compound represented by a numerical value. The drug likeness model score is larger, then
the probability of activity is higher. The % absorption (% ABS) was calculated by % ABS =
109ꢀ(0.345×TPSA).22 It is observed that, conjugates exhibited a good % ABS ranging from
71.69 to 87.50 %. Furthermore, the compounds 6b, 6c, 6d, 6f, 6g, 6h, 6j, 6l, 6n, 6o, 6p, 6q, 6r
and 6s violated Lipinski’s rule of five. Remaining all other compounds did not violated
Lipinski’s rule of five. Orally active drug candidate should not show more than one violation of
the following four criteria: miLog P (octanol–water partition coefficient) ≤5, molecular weight
≤500, number of hydrogen bond donors ≤5, and number of hydrogen bond acceptors ≤10.23 All
the synthesized compounds followed the criteria for orally active drug, and therefore, these
compounds can be further developed as oral drug candidates.
Table 3. Pharmacokinetic parameters important for good oral bioavailability.
Entry %ABS
nꢀ
atoms
TPSA
(A2)
nꢀ
ROTB
MV
MW
milog
p
nꢀ
nꢀ
Lipinski's
Drugꢀ
ON OHNH violations likeness
model
score
Rule
6a
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
<500
≤5
<10
5
<5
2
≤1
0
ꢀ
87.506
87.506
30
32
62.30
62.30
4
4
350.11 389.46
385.88 547.25
4.83
6.40
ꢀ1.28
ꢀ0.97
5
2
2
6b
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87.506
87.506
71.698
71.698
87.506
87.506
84.322
84.322
77.467
77.467
71.698
71.698
84.322
84.322
87.506
87.506
71.698
31
33
33
35
31
33
32
34
34
36
33
35
32
34
31
33
35
62.30
62.30
108.12
108.12
62.30
62.30
71.53
71.53
91.40
91.40
108.12
108.12
71.53
71.53
62.30
62.30
108.12
4
4
5
5
4
4
5
5
5
5
5
5
5
5
4
4
5
366.67 403.49
402.44 561.28
373.44 434.46
409.21 592.25
363.64 423.91
399.41 581.70
375.65 419.49
411.42 577.28
398.05 446.51
433.82 604.31
373.44 434.46
409.21 592.25
375.65 419.49
411.42 577.28
363.64 423.91
399.41 581.70
409.21 592.25
5.28
6.84
4.79
6.36
5.50
7.08
4.88
6.45
4.04
5.62
4.95
6.52
5.05
6.62
5.69
7.26
6.54
5
5
8
8
5
5
6
6
7
7
8
8
6
6
5
5
8
2
2
2
2
2
2
2
2
3
3
2
2
2
2
2
2
2
1
2
0
2
1
2
0
2
0
2
0
2
1
2
1
2
2
ꢀ1.25
ꢀ1.11
ꢀ1.11
ꢀ1.01
ꢀ0.99
ꢀ1.15
ꢀ1.05
ꢀ0.92
ꢀ0.60
ꢀ0.50
ꢀ1.18
ꢀ0.96
ꢀ1.14
ꢀ0.93
ꢀ1.03
ꢀ0.98
ꢀ0.88
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
3. Conclusion
In summary, we have synthesized a novel triazoleꢀindole conjugates and evaluated for their in
vitro antitubercular activity against Mtb H37Ra both in active and dormant state. Among all the
screened compounds, 6b, 6f, 6l, 6n, 6q, 6r and 6s were recognized as the active compounds with
IC50 values ranging from 0.12 to 14.81 ꢁg/mL and MIC ranging from 3 to >30 ꢁg/mL against
both active and dormant Mtb H37Ra strain. The most active compound 6l exhibited good
antitubercular activity against Mtb H37Ra dormant with IC50 value 1 ꢁg/ml and MIC value 3
ꢁg/ml. Compounds 6b, 6h, 6l and 6s showed potential inhibition i.e more than 70 % towards B.
subtilus strain as Gramꢀpositive bacteria at 3 µg/mL. The predictions of molecular docking study
were found to be in agreement with the experimental data of antitubercular activity, thereby
signifying that the molecules may act as inhibitors of DprE1. Diverse structural modifications are
underway through iterative synthesis in conjugation with in silico methodologies and the results
will be communicated in future.
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4. Experimental section
4.1. Chemistry
Commercially available starting materials and reagents were purchased from SigmaꢀAldrich,
Spectrochem, Alfa Aesar and used without further purification. TLC was performed on 300ꢀ400
mesh gel plates. Melting points were recorded using open capillary tube on electrothermal
apparatus and were uncorrected. Infrared (IR) spectra were measured on a bruker alpha FTꢀIR
spectrophotometer. 1H and 13C NMR spectra were recorded on Bruker Avance 400 spectrometer
using tetramethylsilane (TMS) as an internal standard and CDCl3 and DMSOꢀd6 as solvents. All
the chemical shift values (δ) were recorded in ppm and coupling constants J in hertz (Hz).
Splitting patterns are designated as s, singlet; d, doublet; t, triplet; m, multiplet. The mass
spectrometry experiments of the compound were recorded with electrospray ionization ESIꢀMS
spectrometry.
4.1.1. General Procedure for the Preparation of compounds 6a-s
In a roundꢀbottom flask equipped with a magnetic stirring bar, a mixture of 1,2,3ꢀtriazole
aldehydes 4a-j (0.5 mmol) and indoles 5 (1 mmol) was stirred in ethanol and acetic acid at 85 °C
for 3ꢀ6 h. The progress of reaction was monitored by TLC and after completion, the reaction
mixture was poured in to 50 mL ice water, neutralized and the obtained solid was filtered, dried
and recrystallized from aqueous ethanol.
4.1.2. 3,3'-((1-phenyl-1H-1,2,3-triazol-4-yl)methylene)bis(1H-indole) (6a)
The compound 6a was prepared according to general procedure from aldehyde 4a and indole 5a,
as a white solid in 86% yield. m.p. 196ꢀ198 °C. IR ν max/cmꢀ1 3410 (NH), 1596 (C=C, Ar), 1221
1
(CꢀN), 1039, 794, 742, 679. H NMR (400 MHz, DMSOꢀd6) δ 10.88 (s, 2H), 8.59 (s, 1H), 7.89
(d, J = 7.5 Hz, 2H), 7.55 (t, J = 7.9 Hz, 2H), 7.49ꢀ7.44 (m, 3H), 7.35 (d, J = 8.1 Hz, 2H), 7.14 (d,
J = 2.0 Hz, 2H), 7.04 (t, J = 7.1 Hz, 2H), 6.90 (t, J = 7.1 Hz, 2H), 6.07 (s, 1H). 13C NMR (100
MHz, DMSOꢀd6) δ 151.8, 136.8, 136.5, 129.8, 128.3, 126.3, 123.5, 120.9, 120.4, 119.9, 118.9,
118.3, 116.6, 111.5, 31.5. MALDIꢀTOF (ms): Calcd for C25H19N5, 389.1640: found: 412.6925
[M+Na]+.
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4.1.3. 3,3'-((1-phenyl-1H-1,2,3-triazol-4-yl)methylene)bis(5-bromo-1H-indole) (6b)
The compound 6b was prepared according to general procedure from aldehyde 4a and indole 5b,
as a white solid in 85% yield. m.p. 147ꢀ150 °C. IR ν max/cmꢀ1 3420 (NH), 1500 (C=C, Ar),
1
1455, 1330, 1225 (CꢀN), 1098, 1047, 877, 792 (CꢀBr), 681. H NMR (400 MHz, DMSOꢀd6) δ
11.13 (s, 2H), 8.61 (s, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.62 (d, J = 1.8 Hz, 2H), 7.56 (t, J = 7.9 Hz,
2H), 7.45 (t, J = 7.4 Hz, 1H), 7.34 (s, 1H), 7.32 (s, 1H), 7.22 (d, J = 2.2 Hz, 2H), 7.16 (d, J = 1.9
Hz, 1H), 7.14 (d, J = 1.9 Hz, 1H), 6.06 (s, 1H). 13C NMR (100 MHz, DMSOꢀd6) δ 151.0, 136.8,
135.2, 129.8, 128.4, 128.1, 125.2, 123.5, 121.2, 120.5, 119.9, 116.0, 113.6, 111.0, 31.0. MALDIꢀ
TOF (ms): Calcd for C25H17Br2N5, 544.9851: found: 583.0309 [M+K]+.
4.1.4. 3,3'-((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methylene)bis(1H-indole) (6c)
The compound 6c was prepared according to general procedure from aldehyde 4b and indole 5a,
as a white solid in 88% yield. m.p. 214ꢀ216 °C. IR ν max/cmꢀ1 3412 (NH), 1508 (C=C, Ar),
1
1450, 1413, 1340, 1222 (CꢀN), 1089, 1038, 787, 736, 663. H NMR (400 MHz, CDCl3) δ 8.06
(s, 2H), 7.61 (s, 1H), 7.53 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 7.9 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H),
7.23 (d, J = 8.3 Hz, 2H), 7.17 (t, J = 7.5 Hz, 2H), 7.02 (t, J = 7.4 Hz, 2H), 6.92 (d, J = 2.0 Hz,
2H), 6.18 (s, 1H), 2.38 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 138.4, 136.7, 130.0, 129.3, 127.7,
126.7, 123.3, 122.0, 120.3, 120.0, 119.7, 119.4, 117.7, 111.2, 32.1, 21.0. MALDIꢀTOF (ms):
Calcd for C26H21N5, 403.1797: found: 442.6175 [M+K]+.
4.1.5. 3,3'-((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methylene)bis(5-bromo-1H-indole) (6d)
The compound 6d was prepared according to general procedure from aldehyde 4b & indole 5b,
as a white solid in 87% yield. m.p. 260ꢀ262 °C. IR ν max/cmꢀ1 3433 (NH), 1510 (C=C, Ar), 1447
(C=C, Ar), 1333, 1213 (CꢀN), 1045, 873, 793 (CꢀBr). 1H NMR (400 MHz, DMSOꢀd6) δ 11.14 (s,
2H), 8.56 (s, 1H), 7.77 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 1.9 Hz, 2H), 7.37 (s, 1H), 7.35 (s, 2H),
7.33 (s, 1H), 7.24 (d, J = 2.2 Hz, 2H), 7.17 (d, J = 1.9 Hz, 1H), 7.15 (d, J = 1.9 Hz, 1H), 6.07 (s,
13
1H), 2.36 (s, 3H). C NMR (100 MHz, DMSOꢀd6) δ 150.9, 137.9, 135.2, 134.6, 130.2, 128.1,
125.2, 123.5, 121.2, 120.4, 119.8, 116.1, 113.6, 111.0, 31.0, 20.6. MALDIꢀTOF (ms): Calcd for
C26H19Br2N5, 559.0007: found: 560.8829 [M+H]+.
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4.1.6. 3,3'-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(1H-indole) (6e)
The compound 6e was prepared according to general procedure from aldehyde 4c and indole 5a,
as a white solid in 89% yield. m.p. 214ꢀ216 °C. IR ν max/cmꢀ1 3407 (NH), 1601 (C=C, Ar), 1519
1
(NO2 asymmetric), 1342 (NO2 symmetric), 1229 (CꢀN), 1102, 1039, 852, 789, 745. H NMR
(400 MHz, DMSOꢀd6) δ 10.89 (s, 2H), 8.78 (s, 1H), 8.39 (d, J = 9.1 Hz, 2H), 8.20 (d, J = 9.1
Hz, 2H), 7.46 (d, J = 7.9 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 1.8 Hz, 2H), 7.04 (t, J =
7.5 Hz, 2H), 6.89 (t, J = 7.4 Hz, 2H), 6.08 (s, 1H). MALDIꢀTOF (ms): Calcd for C25H18N6O2,
434.1491: found: 457.70 [M+Na]+.
4.1.7. 3,3'-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(5-bromo-1H-indole) (6f)
The compound 6f was prepared according to general procedure from aldehyde 4c and indole 5b,
as a white solid in 85% yield. m.p. 264ꢀ266 °C. IR ν max/cmꢀ1 3420 (NH), 2921 (CꢀH), 1597
(C=C, Ar), 1515 (NO2 asymmetric), 1455, 1339 (NO2 symmetric), 1222 (CꢀN), 1028, 848, 781
1
(CꢀBr). H NMR (400 MHz, DMSOꢀd6) δ 11.16 (s, 2H), 8.82 (s, 1H), 8.42 (d, J = 9.2 Hz, 2H),
8.24 (d, J = 9.2 Hz, 2H), 7.63 (d, J = 1.8 Hz, 2H), 7.35 (s, 1H), 7.33 (s, 1H), 7.24 (d, J = 2.2 Hz,
2H), 7.17 (d, J = 1.9 Hz, 1H), 7.15 (d, J = 1.9 Hz, 1H), 6.11 (s, 1H). 13C NMR (100 MHz,
DMSOꢀd6) δ 152.2, 146.9, 141.5, 135.7, 128.5, 125.9, 125.7, 123.9, 121.6, 121.5, 120.9, 116.3,
114.0, 111.5, 31.4.
4.1.8. 3,3'-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(1H-indole) (6g)
The compound 6g was prepared according to general procedure from aldehyde 4d and indole 5a,
as a white solid in 88% yield. m.p. 230ꢀ232 °C. IR ν max/cmꢀ1 3410 (NH), 1696 (C=C, Ar),
1
1490, 1414, 1339, 1224 (CꢀN), 1088, 1039, 996, 827, 785, 734 (CꢀCl), 658. H NMR (400 MHz,
DMSOꢀd6) δ 10.88 (s, 2H), 8.62 (s, 1H), 7.94 (d, J = 8.9 Hz, 2H), 7.62 (d, J = 8.9 Hz, 2H), 7.47
(d, J = 7.9 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 2.0 Hz, 2H), 7.04 (t, J = 8.0 Hz, 2H),
13
6.90 (t, J = 7.9 Hz, 2H), 6.07 (s, 1H). C NMR (100 MHz, DMSOꢀd6) δ 151.9, 136.5, 135.6,
132.5, 129.8, 126.3, 123.5, 121.5, 120.9, 120.5, 118.9, 118.3, 116.5, 111.5, 31.5.
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4.1.9.
(6h)
3,3'-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(5-bromo-1H-indole)
The compound 6h was prepared according to general procedure from aldehyde 4d and indole 5b,
as a white solid in 85% yield. m.p. 256ꢀ258 °C. IR ν max/cmꢀ1 3436 (NH), 2917 (CꢀH), 1559
(C=C, Ar), 1494, 1448, 1222 (CꢀN), 1093, 1043, 851 (CꢀCl), 788 (CꢀBr). MALDIꢀTOF (ms):
Calcd for C25H16Br2ClN5, 578.9461: found: 601.43 [M+Na]+.
4.1.10. 3,3'-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(1H-indole) (6i)
The compound 6i was prepared according to general procedure from aldehyde 4e and indole 5a,
as a white solid in 86% yield. m.p. 228ꢀ230 °C. IR ν max/cmꢀ1 3393 (NH), 1596 (C=C, Ar),
1
1506, 1446, 1416, 1248 (CꢀN), 1035 (CꢀOꢀC), 825, 789, 739. H NMR (400 MHz, DMSOꢀd6) δ
10.86 (s, 2H), 8.47 (s, 1H), 7.79 (s, 1H), 7.77 (s, 1H), 7.47 (d, J = 7.9 Hz, 2H), 7.34 (d, J = 8.1
Hz, 2H), 7.13 (d, J = 2.0 Hz, 2H), 7.09 (s, 1H), 7.07 (s, 1H), 7.03 (t, J = 7.6 Hz, 2H), 6.89 (t, J =
7.5 Hz, 2H), 6.04 (s, 1H), 3.80 (s, 3H). 13C NMR (100 MHz, DMSOꢀd6) δ 159.0, 151.5, 136.5,
130.3, 126.4, 123.4, 121.5, 120.9, 120.4, 119.0, 118.3, 116.6, 114.8, 111.5, 55.5, 31.5. MALDIꢀ
TOF (ms): Calcd for C26H21N5O, 419.1746: found: 442.0596 [M+Na]+.
4.1.11. 3,3'-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(5-bromo-1H-indole)
(6j)
The compound 6j was prepared according to general procedure from aldehyde 4e and indole 5b,
as a white solid in 89% yield. m.p. 240ꢀ242 °C. IR ν max/cmꢀ1 3435 (NH), 3150, 1603 (C=C,
1
Ar), 1510, 1445, 1330, 1250, 1211 (CꢀN), 1100, 1041 (CꢀOꢀC), 870, 784 (CꢀBr). H NMR (400
MHz, DMSOꢀd6) δ 11.13 (s, 2H), 8.51 (s, 1H), 7.81 (s, 1H), 7.79 (s, 1H), 7.64 (d, J = 1.9 Hz,
2H), 7.35 (s, 1H), 7.33 (s, 1H), 7.24 (d, J = 2.2 Hz, 2H), 7.17 (d, J = 1.9 Hz, 1H), 7.15 (d, J = 1.9
Hz, 1H), 7.11 (s, 1H), 7.09 (s, 1H), 6.06 (s, 1H), 3.81 (s, 3H). 13C NMR (100 MHz, DMSOꢀd6) δ
159.0, 150.8, 135.2, 130.3, 128.1, 125.2, 123.5, 121.6, 121.3, 120.5, 116.2, 114.8, 113.6, 111.0,
55.6, 31.0. MALDIꢀTOF (ms): Calcd for C26H19Br2N5O, 574.9956: found: 574.3549 [M+].
4.1.12. N-(4-(4-(di(1H-indol-3-yl)methyl)-1H-1,2,3-triazol-1-yl)phenyl)acetamide (6k)
The compound 6k was prepared according to general procedure from aldehyde 4f and indole 5a,
as a white solid in 85% yield. m.p. above 270 °C. IR ν max/cmꢀ1 3404 (NH), 3250 (NH), 1663
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1
(C=O), 1608 (C=C, Ar), 1528, 1446, 1354, 1321, 1226 (CꢀN), 1047, 831, 741, 680. H NMR
(400 MHz, DMSOꢀd6) δ 10.87 (s, 2H), 10.17 (s, 1H), 8.48 (s, 1H), 7.80 (d, J = 9.0 Hz, 2H), 7.74
(d, J = 9.0 Hz, 2H), 7.47 (d, J = 7.9 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 1.8 Hz, 2H),
7.04 (t, J = 7.4 Hz, 2H), 6.90 (t, J = 7.4 Hz, 2H), 6.05 (s, 1H), 2.07 (s, 3H). 13C NMR (100 MHz,
DMSOꢀd6) δ 168.6, 151.6, 139.3, 136.5, 131.9, 126.3, 123.4, 120.9, 120.5, 120.2, 119.6, 118.9,
118.3, 116.6, 111.5, 31.6, 24.0. MALDIꢀTOF (ms): Calcd for C27H22N6O, 446.1855: found:
469.5413 [M+Na]+.
4.1.13. N-(4-(4-(bis(5-bromo-1H-indol-3-yl)methyl)-1H-1,2,3-triazol-1-yl)phenyl)acetamide
(6l)
The compound 6l was prepared according to general procedure from aldehyde 4f and indole 5b,
as a white solid in 86% yield. m.p. above 270 °C. IR ν max/cmꢀ1 3416 (NH), 3200 (NH), 1666
(C=O), 1605 (C=C, Ar), 1522, 1452, 1310, 1223 (CꢀN), 1102, 1044, 878, 831, 792 (CꢀBr), 718.
1H NMR (400 MHz, DMSOꢀd6) δ 11.13 (s, 2H), 10.19 (s, 1H), 8.51 (s, 1H), 7.81 (d, J = 9.1 Hz,
2H), 7.75 (d, J = 9.0 Hz, 2H), 7.63 (d, J = 1.7 Hz, 2H), 7.35 (s, 1H), 7.33 (s, 1H), 7.23 (d, J = 2.1
Hz, 2H), 7.17 (d, J = 1.9 Hz, 1H), 7.15 (d, J = 1.9 Hz, 1H), 6.06 (s, 1H), 2.08 (s, 3H). 13C NMR
(100 MHz, DMSOꢀd6) δ 168.6, 150.9, 139.4, 135.2, 131.8, 128.1, 125.2, 123.5, 121.2, 120.5,
120.3, 119.6, 116.1, 113.6, 111.0, 31.0, 24.0. MALDIꢀTOF (ms): Calcd for C27H20Br2N6O,
602.0065: found: 641.9423 [M+K]+.
4.1.14. 3,3'-((1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(1H-indole) (6m)
The compound 6m was prepared according to general procedure from aldehyde 4g and indole
5a, as a white solid in 88% yield. m.p. 148ꢀ150 °C. IR ν max/cmꢀ1 3406 (NH), 1606 (C=C, Ar),
1
1533 (NO2 asymmetric), 1349 (NO2 symmetric), 1229 (CꢀN), 1094, 1043, 788, 745. H NMR
(400 MHz, DMSOꢀd6) δ 10.89 (s, 2H), 8.43 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.92 – 7.88 (m,
1H), 7.85 – 7.78 (m, 2H), 7.45 (d, J = 7.9 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 7.07ꢀ7.03 (m, 4H),
13
6.90 (t, J = 7.5 Hz, 2H), 6.07 (s, 1H). C NMR (100 MHz, DMSOꢀd6) δ 151.3, 144.2, 136.5,
134.3, 130.9, 129.4, 127.6, 126.3, 125.4, 123.9, 123.4, 120.9, 119.0, 118.3, 116.6, 111.5, 31.5.
MALDIꢀTOF (ms): Calcd for C25H18N6O2, 434.1491: found: 457.0612 [M+Na]+.
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4.1.15. 3,3'-((1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(5-bromo-1H-indole) (6n)
The compound 6n was prepared according to general procedure from aldehyde 4g and indole 5b,
as a white solid in 85% yield. m.p. 140ꢀ142 °C. IR ν max/cmꢀ1 3414 (NH), 1602 (C=C, Ar), 1527
(NO2 asymmetric), 1450, 1344 (NO2 symmetric), 1222 (CꢀN), 1093, 1037, 871, 782 (CꢀBr), 744.
1H NMR (400 MHz, DMSOꢀd6) δ 11.15 (s, 2H), 8.46 (s, 1H), 8.19 (d, J = 8.1 Hz, 1H), 7.92 –
7.77 (m, 3H), 7.61 (d, J = 1.7 Hz, 2H), 7.34 (d, J = 8.6 Hz, 2H), 7.17 (s, 3H), 7.15 (d, J = 1.9 Hz,
13
1H), 6.10 (s, 1H). C NMR (100 MHz, DMSOꢀd6) δ 150.6, 144.2, 135.3, 134.3, 130.9, 129.4,
128.0, 127.6, 125.4, 125.2, 123.9, 123.6, 121.4, 116.1, 113.6, 111.0, 30.9. MALDIꢀTOF (ms):
Calcd for C25H16Br2N6O2, 589.9701: found: 628 [M+K]+.
4.1.16. 3,3'-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(1H-indole) (6o)
The compound 6o was prepared according to general procedure from aldehyde 4h and indole 5a,
as a white solid in 86% yield. m.p. 142ꢀ144 °C. IR ν max/cmꢀ1 3404 (NH), 1604 (C=C, Ar),
1
1504, 1458, 1343, 1246 (CꢀN), 1043 (CꢀOꢀC), 792, 746. H NMR (400 MHz, CDCl3) δ 8.12 (s,
2H), 7.79 (s, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 7.9 Hz, 2H), 7.35 (d, J = 8.0 Hz, 3H),
13
7.15 (t, J = 7.3 Hz, 2H), 7.07ꢀ7.01 (m, 4H), 6.93 (s, 2H), 6.17 (s, 1H), 3.74 (s, 3H). C NMR
(100 MHz, CDCl3) δ 151.2, 151.2, 136.7, 129.8, 126.7, 125.5, 124.5, 123.4, 121.9, 121.9, 121.1,
119.8, 119.2, 117.8, 112.1, 111.2, 55.9, 32.2.
4.1.17. 3,3'-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(5-bromo-1H-indole)
(6p)
The compound 6p was prepared according to general procedure from aldehyde 4h and indole 5b,
as a white solid in 85% yield. m.p. 218ꢀ220 °C. IR ν max/cmꢀ1 3418 (NH), 1604 (C=C, Ar),
1
1506, 1457, 1247 (CꢀN), 1104, 1046 (CꢀOꢀC), 878, 792 (CꢀBr). H NMR (400 MHz, CDCl3) δ
8.65 (s, 2H), 7.77 (s, 1H), 7.72 (dd, J = 7.9, 1.5 Hz, 1H), 7.51 (d, J = 1.2 Hz, 2H), 7.39 – 7.35
13
(m, 1H), 7.17 – 7.12 (m, 4H), 7.07 – 6.99 (m, 2H), 6.73 (s, 2H), 5.95 (s, 1H), 3.79 (s, 3H). C
NMR (100 MHz, CDCl3) δ 151.4, 149.6, 135.4, 130.2, 128.1, 126.3, 125.5, 124.9, 124.7, 124.6,
121.9, 121.0, 116.3, 112.9, 112.4, 112.2, 56.0, 31.9. MALDIꢀTOF (ms): Calcd for
C26H19Br2N5O, 574.9956: found: 597.42 [M+Na]+.
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4.1.18. 3,3'-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(1H-indole) (6q)
The compound 6q was prepared according to general procedure from aldehyde 4i and indole 5a,
as a white solid in 86% yield. m.p. 188ꢀ190 °C. IR ν max/cmꢀ1 3409 (NH), 2919 (CꢀH), 1587
(C=C, Ar), 1222 (CꢀN), 1038, 735 (CꢀCl). 1H NMR (400 MHz, DMSOꢀd6) δ 10.91 (s, 2H), 8.70
(s, 1H), 8.06 (s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.55 (t, J = 8.1 Hz, 1H), 7.49 (d, J = 7.5 Hz, 3H),
7.37 (d, J = 8.0 Hz, 2H), 7.16 (s, 2H), 7.06 (t, J = 7.5 Hz, 2H), 6.92 (t, J = 7.5 Hz, 2H), 6.09 (s,
13
1H). C NMR (100 MHz, DMSOꢀd6) δ 152.5, 138.3, 136.9, 134.6, 131.9, 128.5, 126.8, 123.9,
121.4, 121.1, 120.0, 119.4, 118.8, 118.8, 116.9, 111.9, 32.0.
4.1.19.
(6r)
3,3'-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(5-bromo-1H-indole)
The compound 6r was prepared according to general procedure from aldehyde 4i and indole 5b,
as a white solid in 85% yield. m.p. 145ꢀ148 °C. IR ν max/cmꢀ1 3411 (NH), 1586 (C=C, Ar),
1446, 1220 (CꢀN), 1090, 1037, 870 (CꢀCl), 776 (CꢀBr). 1H NMR (400 MHz, DMSOꢀd6) δ 11.14
(s, 2H), 8.69 (s, 1H), 8.03 (t, J = 1.7 Hz, 1H), 7.90 (d, J = 8.9 Hz, 1H), 7.61 (d, J = 1.6 Hz, 2H),
7.57 (t, J = 8.1 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.34 (s, 1H), 7.32 (s, 1H), 7.22 (d, J = 2.1 Hz,
2H), 7.16 (d, J = 1.7 Hz, 1H), 7.14 (d, J = 1.8 Hz, 1H), 6.06 (s, 1H). 13C NMR (100 MHz,
DMSOꢀd6) δ 151.8, 138.3, 135.7, 134.7, 132.0, 128.7, 128.5, 125.7, 124.0, 121.7, 121.3, 120.2,
118.9, 116.4, 114.1, 111.5, 31.5. MALDIꢀTOF (ms): Calcd for C25H16Br2ClN5, 578.9461: found:
601.42 [M+Na]+.
4.1.20. 3,3'-((1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl)methylene)bis(5-bromo-1H-indole) (6s)
The compound 6s was prepared according to general procedure from aldehyde 4j and indole 5b,
as a white solid in 89% yield. m.p. 160ꢀ162 °C. IR ν max/cmꢀ1 3410 (NH), 1612 (C=C, Ar), 1529
(NO2 asymmetric), 1451, 1345 (NO2 symmetric), 1223 (CꢀN), 1094, 1040, 876, 790 (CꢀBr), 733.
1H NMR (400 MHz, CDCl3) δ 8.49 (t, J = 2.1 Hz, 1H), 8.37 (s, 2H), 8.25– 8.22 (m, 1H), 8.11 –
8.08 (m, 1H), 7.73 (s, 1H), 7.67 (t, J = 8.2 Hz, 1H), 7.53 (s, 2H), 7.26 – 7.21 (m, 4H), 6.85 (d, J
13
= 2.0 Hz, 2H), 6.03 (s, 1H). C NMR (100 MHz, CDCl3) δ 152.1, 148.9, 137.8, 135.4, 130.9,
128.1, 125.9, 125.2, 124.7, 123.0, 121.8, 120.1, 116.3, 115.2, 113.0, 112.9, 31.9.
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4.2. Pharmacology
4.2.1. Anti-mycobacterial activity
All the chemicals such as sodium salt XTT and MTT, DMSO, Ampicillin and Rifampicin were
purchased from SigmaꢀAldrich, USA. Synthesized compounds were dissolved in DMSO and
was used as a stock solution (10 mg/mL) for further biological testing. Microbial strain such as
Mtb H37Ra (ATCC 25177) was obtained from Astra Zeneca, India. The stock culture was
o
maintained at ꢀ80 C and subcultured once in a liquid medium before inoculation into an
experimental culture. For the antimycobacterial assay, M. pheli medium (minimal essential
medium) was used. It contains 0.5 g KH2PO4, 0.25 g trisodium citrate, 60 mg MgSO4, 0.5 g
asparagine and 2 mL glycerol in distilled water (100 mL) followed by pH adjustment to 6.6. It
takes at least 8ꢀ10 days for OD 1 at 620 nm. The antimycobacterial assay was performed in 96 ꢀ
well plates for active as well as dormant stages. All the synthetic compounds were screened for
their in vitro activity against M. tuberculosis H37Ra using dilutions ranging from 30ꢀ0.1 µg/mL,
in order to determine the actual minimum inhibitory concentration (MIC). The screening of M.
Tuberculosis H37Ra has been done by XTT Reduction Menadione Assay (XRMA) by reading
absorbance at 470 nm. The optical density was read on a micro plate reader at 470 nm filter for
XTT against a blank prepared from cellꢀfree wells. Absorbance given by cells treated with the
vehicle (DMSO) alone was taken as 100% cell growth. Initially, primary screening was done at
30, 10 and 3 µg/mL. Compounds showing 75% inhibition of MTB at or lowers than 30µg/mL
were selected for further dose response curve. All experiments were performed in duplicates and
the quantitative value was expressed as the average ± standard deviation. MIC and IC50 values of
the selected compound were calculated from their dose response curves by using Origin 8
software. Percentage inhibition was calculated using the formula: % inhibition = [(control–CMP)
/ (control–blank)] x 100 where ‘control’ is the activity of Mycobacterium without compounds,
‘CMP’ is the activity of Mycobacterium in the presence of compounds and ‘blank’ is the activity
of the culture medium without Mycobacterium.
4.2.2. Antibacterial activity
Bacterial strains E. coli (NCIM 2688), P. aeruginosa (NCIM 2036) as Gramꢀnegative and B.
subtilus (NCIM 2079), S. aureus (NCIM 2010) as Gramꢀpositive were obtained from NCIM
(NCL, Pune) and were grown in Luria Burtony medium from Himedia, India. Once the culture
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reaches 1 O.D600, it was used for the antibacterial assay. Briefly, 0.1 OD600 bacterial cultures
were treated with synthesized compound at 3 µg/mL concentration and incubated for 8 h at 37
°C. Ampicillin served as positive control for antibacterial activity. The in vitro screening values
(% inhibition) against microorganisms tested and are summarized in supplementary information.
4.3. Molecular Docking
Molecular docking study was performed using the standard protocol implemented in the GLIDE
(Gridꢀbased Ligand Docking with Energetics) module incorporated in the Schrodinger Molecular
modeling package to predict the binding modes of triazoleꢀdiindolylmethane conjugates into the
active site of DprE1 enzyme.24 With this purpose, the Xꢀray crystal structures of DprE1
(decaprenylphosphorylꢀβꢀDꢀriboseꢀ2'ꢀepimerase) enzyme of mycobacterium tuberculosis in
complex with its inhibitor (4FDO.pdb) was retrieved from the RCSB protein data bank25 and
used as the primary model for the docking study. First, protein structure was refined for docking
simulation using the Protein Preparation Wizard incorporated in the Glide program. This
involved eliminating all crystallographically observed water as no water molecule was found to
be conserved in the interaction with the protein, addition of missing hydrogens/side chain atoms
and assigning the appropriate charge and protonation state to the protein structure corresponding
to pH 7.0 considering the appropriate ionization states for the acidic as well as basic amino acid
residues. Then the structure was subjected to energy minimization using OPLSꢀ2005 force field
with root mean square deviation (RMSD) cutꢀoff value of 0.30Å to relieve the steric clashes
among the residues due to addition of hydrogen atoms. The three dimensional structures of the
triazoleꢀdiindolylmethane conjugates were sketched with build panel in Maestro and optimized
using the ligprep module which performs addition of hydrogens, adjusting realistic bond lengths
and angles, correct chiralities, ionization states, tautomers, stereo chemistries and ring
conformations. Partial charges were ascribed to the structures using the OPLSꢀ2005 forceꢀfield
and the resulting structures were then subjected to energy minimization until their average
RMSD reached 0.001 Å. The active site of the DprE1 enzyme for docking was defined using the
Receptor Grid Generation panel which generates two cubical boxes having a common centroid
to organize the calculations: a larger enclosing and a smaller binding box. The binding region
was defined by a grid box of 12X12X12Å dimensions centered on the centroid of the native
ligand in the crystal complex which was big enough to explore a larger region of the enzyme
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structure. Using this setup automated docking was carried out to evaluate the binding affinities of
the aforementioned compounds within the macromolecule using the extra precision (XP) Glide
scoring function to rank the docking poses and to measure their binding affinities. GLIDE
searches for favorable interactions between the ligand and the active site of the enzyme using a
filtering an approach wherein each of the ligand pose pass through a series of hierarchical filters
that evaluate the ligand's interaction with the receptor. The output files i.e. the docking poses of
the ligands were visualized and analyzed using the Maestro’s Pose Viewer utility. The protocol
adopted for docking simulation was validated by extracting the native ligand from the crystal
structure and docking it into active site of the DprE1 using the above defined settings and
monitoring its ability to reproduce the experimentally observed binding mode. The RMSD
between the experimental conformation of the native ligand in the crystal structure and that
obtained from its docking was found to be less than 1Å, confirming the reliability of the docking
procedure in reproducing the experimentally observed binding mode for triazoleꢀ
diindolylmethane conjugates investigated herein.
Acknowledgments
The author A.B.D. is very much grateful to the University Grants Commission, New Delhi for
the award of research fellowship. A.C (File no: PDF/2016/003615) is grateful to Science and
Engineering Research Board (SERB), New Delhi for the award of NPDF. The authors are also
thankful to the Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada University,
Aurangabadꢀ431 004, India for providing laboratory facilities. Authors are also thankful to
Schrodinger Inc. for providing the Demo license of Schrodinger Molecular modeling Suite that
has significantly helped in this study.
Conflict Of Interest
The authors declare no competing interest.
Notes and References
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2016.
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