Design and synthesis of 9H-fluorenone based 1,2,3-triazole analogues as Mycobacterium tuberculosis InhA inhibitors

Article abstract of DOI:10.1111/cbdd.13127

We prepared fifty various 9H-fluorenone based 1,2,3-triazole analogues varied with NH, –S–, and –SO₂– groups using click chemistry. The target compounds were characterized by routine analytical techniques, ¹H, ¹³CNMR, mass, elemental, single-crystal XRD (8a) and screened for in vitro antitubercular activity against Mycobacterium tuberculosis (MTB) H37Rv strain and two “wild” strains Spec. 210 and Spec. 192 and MIC₅₀ was determined. Further, the compounds were evaluated for MTB InhA inhibition study as well. The final analogues exhibited minimum inhibitory concentration (MIC) ranging from 52.35 to >295?μm. Among the –NH– analogues, one compound 5p (MIC 58.34?μm), among –S– containing analogues four compounds 8e (MIC 66.94?μm), 8f (MIC 74.20?μm), 8g (MIC 57.55?μm), and 8q (MIC 56.11?μm), among –SO₂– containing compounds one compound 10p (MIC 52.35?μm) showed less than MTB MIC 74.20?μm: Compound 4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (10p) was found to be the most active compound with 73% InhA inhibition at 50?μm; it inhibited MTB with MIC 52.35?μm. Further, 10f and 10p were docked to crystal structure of InhA to know binding interaction pattern. Most active compounds were found to be non-cytotoxic against HEK 293 cell lines at 50?μm.

Full text of DOI:10.1111/cbdd.13127

DR MALLIKA ALVALA (Orcid ID : 0000-0003-3770-4725)
PROFESSOR KVG CHANDRASEKHAR (Orcid ID : 0000-0001-9833-353X)
Article type : Research Article
Design, synthesis of 9H-fluorenone based 1,2,3-triazole analogues as
Mycobacterium tuberculosis InhA inhibitors
[a]
[a]
[b]
Amaroju Suresh, Singireddi Srinivasarao, Napiórkowska Agnieszka, Augustynowicz-
[b]
[c]
[d]
Kopeć Ewa, Mallika Alvala, Christian Lherbet, Kondapalli Venkata Gowri Chandra
[a]∗
Sekhar,
[a]
Department of Chemistry, Birla Institute of Technology and Science, Pilani, Hyderabad
Campus, Jawahar Nagar, Hyderabad – 500 078, Telangana State, India.
[b]
Microbiology Department, National Tuberculosis and Lung Diseases Research Institute,
01-
1
38 Warsaw, Poland.
[
c]
National Institute of Pharmaceutical Education and Research-Hyderabad, Hyderabad,
00037, Telangana, India
5
[d]
Laboratoire SPCMIB (UMR CNRS 5068), Université Paul Sabatier, Université de
Toulouse, 118 route de Narbonne, 31062 Toulouse cedex, France; ITAV-USR3505,
Université de Toulouse, CNRS, UPS, F-31106 Toulouse, France.
∗
Corresponding author
Tel.:+91 40 66303527 E-mail: kvgc@hyderabad.bits-pilani.ac.in; kvgcs.bits@gmail.com
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13127
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Short Title: 9H-fluorenone analogues as MTB InhA inhibitors
Keywords: 1,2,3-triazole, InhA, 9H-Fluorenone, Hybridization, Mycobacterium
tuberculosis.
Abstract:
We prepared fifty various 9H-fluorenone based 1,2,3-triazole analogues varied with NH, -S-
and -SO2- groups using click chemistry. The target compounds were characterized by routine
1
13
analytical techniques, H, CNMR, Mass, Elemental, single crystal XRD (8a) and screened
for in vitro antitubercular activity against Mycobacterium tuberculosis (MTB) H37Rv strain
and two ‘wild’ strains Spec. 210 and Spec. 192 and MIC50 was determined. Further, the
compounds were evaluated for MTB InhA inhibition study as well. The final analogues
exhibited minimum inhibitory concentration (MIC) ranging from 52.35 - > 295 μM. Among
the -NH- analogues one compound 5p (MIC 58.34 µM), amongst -S- containing analogues
four compounds 8e (MIC 66.94 µM), 8f (MIC 74.20 µM), 8g (MIC 57.55 µM) & 8q (MIC
5
6.11 µM), among -SO
showed less than MTB MIC 74.20 µM: Compound 4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-
3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (10p), was found to be the most active compound
with 73% InhA inhibition at 50 µM; it inhibited MTB with MIC 52.35 µM. Further, 10f &
0p were docked to crystal structure of InhA to know binding interaction pattern. Most active
2
- containing compounds one compound 10p (MIC 52.35 µM)
(
1
compounds were found to be non cytotoxic against HEK 293 cell lines at 50 µM.
Introduction
Tuberculosis (TB), which infects ill health in millions of people every year, is a major
universal health problem. It has been ranked above HIV/AIDS in causing death in the year
2015 [1]. If identified early and on proper treatment TB can be cured but irregular and
irrational usage of drugs causes drug resistance. Patients with multidrug-resistant TB (MDR-
TB) do not respond to isoniazid (INH) and rifampicin (RMP), the most prevailing, first-line
anti-TB drugs. Extensively drug-resistant TB (XDR-TB) is a more serious form of MDR-TB
[
2]
caused by bacteria that do not respond to the most effective second-line anti-TB drugs.
According to WHO, it is estimated that there were 1.4 million TB deaths in 2015, and an
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additional 0.4 million deaths resulted from amongst HIV-positive TB people. In terms of
cases, for 2015 there were 10.4 million new TB cases (including 1.2 million among HIV-
positive people), of which 5.9 million were among men, 3.5 million among women and 1.0
[
1]
million among children. Overall, 90% of cases were adults and 10% children. In view of
these facts and to overcome the issue of resistance there is a challenging need to develop
novel drugs using novel targets of MTB.
The fatty acid synthase arrangement of MTB contains unique signature fatty acid, the
mycolic acid (MA), which is a chief component of the mycobacterial cell wall. MA
biosynthesis is conceded by numerous sequential enzymatic cycles equivalent to two related
[3]
but different Fatty Acid Synthase (FAS) systems, FAS I and II. FAS II system is existent in
bacteria but is absent in humans. InhA protein (ENR, EC number: 1.3.1.9) is a key enzyme
of FAS II and has already been authenticated as the key molecular target of the frontline anti-
[4]
TB drug INH. It is a prodrug which upon activation by KatG, the mycobacterial catalase-
[
5]
peroxidase, forms NAD-INH. The X-ray structure of the complex InhA isonicotinoyl
moiety of this adduct displays that it dwells in a hydrophobic pocket. A number of research
groups documented this cavity as a suitable site to augment inhibitory potency. Several
[6]
methodologies exist towards the design of new inhibitors for InhA.
Genz-10850 (also called GEQ) (A) has been identified as a very promising inhibitor of InhA,
[
7]
after in vitro screening of a library of five million compounds. Later, He et al., synthesized
[
8]
a series of GEQ derivatives with InhA inhibitory activities ranging from 0.09 to 2.04 μM.
Amongst these, (4-(9H-fluoren-9-yl)piperazin-1-yl)(phenyl)methanone (B) was the most
active compound with IC50 0.09 μM. Unfortunately these molecules have poor in vitro
[
8]
activity against MTB (MIC ≥ 125 µM) because of their low absorptivity. Matviiuk et al.
reported 3-heteryl substituted pyrrolidine-2,5-diones derivatives with InhA inhibition at 50
μM ranging from 9 to 56% and its exhibited MTB MIC ranged from 2.5 to 40 µg/mL.
Amongst these, 1-benzyl-3-[4-(9H-fluoren-9-yl)-1-piperazinyl]-2,5-pyrrolidinedione (C) was
the most active compound with InhA inhibition of 25% at 50 μM and MTB MIC 45.3 μM
[9]
against MTB H37Rv. Chollet et al., incorporated modifications in GEQ skeleton;
piperazine was replaced with piperidine and other modifications include replacement of
amide with sulfonyl, phosphonyl and phosphinamide groups. They also introduced 2- and 3-
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alkyl-substituted fluorenone derivatives as inhibitors of InhA with IC50 102 to 2690 nM and
these derivatives exhibited MTB MIC ranging from 11 to 88.2 µM against MTB H37Rv.
Among this series, an enantiomer of (4-(3-(hexyloxy)-9H-fluoren-9-yl)piperazin-1-
yl)(phenyl) methanone (D) bearing an additional hexyloxy chain on the fluorenone moiety
demonstrated enhanced activity against InhA enzyme (IC50 up to 102 nM) as well as MTB
[10]
growth (MIC 22 μM). Described InhA active compounds are depicted in Figure 1.
Additionally, 1,2,3-triazole and its derivatives have enticed curiosity in the medicinal field
[11]
[12]
owing to their diverse biological activities such as anti-fungal, anti-bacterial, anti-
[
13]
[14]
[15]
[16]
allergic, anti-HIV, antiviral, anti-inflammatory, anesthetic, anti-cancer and β -
[17]
lactamase inhibition properties. It is quite evident that the encouraging properties of 1,2,3-
triazole ring viz., moderate dipole character, hydrogen bonding ability, and π stacking
interactions, rigidity and stability under in vivo conditions are responsible for their efficient
[
18]
binding to biological targets and also improve their solubility in living systems.
Kumar and co-workers described the synthesis of triazole-isoniazid conjugates (E) and their
in vitro evaluation as possible anti-TB agents against MTB H37Rv. The compounds
exhibited potent activity against MTB strain with MIC values ranging from 0.195 to 1.56
[
19]
μM. Goverdhan et al., reported novel-substituted 1,2,3-triazolylmethyl carbazoles (F) for
in vitro antimycobacterial activity against MTB H37Rv with MIC values ranging from 6.25
[
20]
to 50 μg/mL. Menendez et al., reported 1,4-disubstituted triazoles and α-ketotriazole
derivatives (J). These derivatives with MIC values from 2 to 100 μg/mL against MTB
[
21, 22]
H37Rv also exhibited InhA inhibition from 10 to 58% at 50 μM.
triazole derivatives are given in Figure 2.
The representative
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Experimental Section
Materials and methods
Chemicals and solvents were used as commercially supplied. The solvents and reagents were
of LR grade and if necessary purified before use. Thin-layer chromatography (TLC) was
carried out on aluminium-supported silica gel plates (Merck 60 F254) with visualization of
components by UV light (254 nm). Column chromatography was carried out on silica gel
1
13
(
Merck 100-200 mesh). H NMR and C NMR spectra were recorded at 400 MHz and 101
MHz respectively using a Bruker AV 400 spectrometer (Bruker CO., Switzerland) in CDCl
3
and DMSO-d solution with tetramethylsilane as the internal standard and chemical shift
6
values (δ) were given in ppm. Melting points were determined on an electro thermal melting
point apparatus (Stuart-SMP30) in open capillary tubes and are uncorrected. Elemental
analyses were performed by Elementar Analysensysteme GmbH vario MICRO cube CHN
Analyzer. Mass spectra (ESI-MS) were recorded on Schimadzu MS/ESI mass spectrometer.
Single crystal XRD data collected on Rigaku Oxford diffraction Xta LAB. Purity of all tested
compounds were determined by LC-MS/MS on Schimadzu and was greater than 95%.
Chemistry
[
23]
Synthetic protocols of the intermediates (2, 3 , 4, 6, 7 and 9) and spectral data of all the
final compounds (5a-p, 8a-r, 10a-p) is given in supplementary information.
Synthesis of N-((1-substituted phenyl-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine
(5a-p)
A solution of N-(prop-2-yn-1-yl)-9H-fluoren-9-amine (4) (0.30 g, 1.0 equiv.) is reacted with
substituted phenyl azides (1.2 equiv.) in the presence of sodium ascorbate (0.01 equiv.),
t-
CuSO
4
.5H
2
O (0.02 equiv.) and BuOH:H
2
O (2:1), at room temperature for 4 h. On
completion of the reaction as indicated by TLC, the reaction was quenched with cold water
and extracted with DCM. The DCM layers were collected, washed with saturated brine
solution, dried over anhydrous Na
2 4
SO and concentrated in vacuo. The resultant crude
product was purified by column chromatography [MeOH / DCM (1 -3%)] to yield the title
[
24]
compounds 5a-p
.
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Synthesis of 4-(((9H-fluoren-9-yl)thio)methyl)-1-substituted phenyl-1H-1,2,3-triazole (8a-
r)
To a stirred solution of compound 7 (1.0 mmol) and substituted phenyl azide (1.2 mmol) in
t
4 2
butanol:water (1:1) (4 mL), CuSO .5H O (10 mol %) (0.2 mmol) and sodium ascorbate (5
mol %) (0.2 mmol) were added and the reaction mixture was stirred at RT for 16 h. After
completion of the reaction, as indicated by TLC, butanol was removed under reduced
pressure. The residue was extracted with ethyl acetate (3 x10 mL) and combined organic
2 4
layers were collected and washed with saturated brine solution, dried over anhydrous Na SO
and concentrated in vacuo to get the crude product. The product was further purified by
column chromatography [ethyl acetate / hexane (35 - 40%)] to afford the title compounds 8a-
[
24]
r
.
Synthesis of 4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-substituted phenyl-1H-1,2,3-triazole
10a-p)
(
To a stirred solution of compound 9 (1.0 mmol) and substituted phenyl azide (1.2 mmol) in
t
4 2
butanol:water (1:1) (4 mL), CuSO .5H O (1 mol %) (0.2 mmol) and sodium ascorbate (5 mol
%) (0.2 mmol) were added and the reaction mixture was stirred at room temperature for 16 h.
After completion of the reaction, as indicated by TLC, butanol was removed under reduced
pressure. The residue was extracted with ethyl acetate (3 x10 mL) and combined organic
layers were collected and washed with saturated brine solution, dried over anhydrous Na SO
2
4
and concentrated in vacuo to get the crude product. The product was further purified by
column chromatography [ethyl acetate / hexane (40 - 45%)] to afford the title compounds
[24]
10a-p
.
Results and discussion
Design and chemistry
In our design, two key elements for InhA inhibition, i.e. the fluorenyl and the triazole
[10]
moieties, were considered. The fluorenyl moiety that could act as an anchor
will occupy
the same hydrophobic position than the long alkyl chain of the substrate. The triazole moiety
could interact by hydrogen bonds with the hydroxyl group of the key residue Tyr158,
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essential for a good recognition. Furthermore, triazoles, synthesized in one step by “click”
[
7]
chemistry reaction could effectively mimic amide bonds as found in molecule A and could
[
22]
bring diversity. With this collective information and emphasizing on molecular
hybridization approach we drew a synthetic stratagem to fit these imperative pharmacophoric
groups into one distinct scaffold and synthesized N-((1-substituted phenyl-1H-1,2,3-triazol-4-
yl)methyl)-9H-fluoren-9-amine, 4-(((9H-fluoren-9-yl)thio)methyl)-1-substituted phenyl-1H-
1,2,3-triazole, and 4-(((9H-fluoren-9-yl)sulfonyl)methyl)-1-substituted phenyl-1H-1,2,3-
triazole analogues (Figure 3). Of note, only aryl groups were introduced on the triazole
moiety of our designed target (Figure 3) because molecules bearing alkyl groups at this
[10]
position were found to be less efficient as inhibitors against InhA enzyme.
We synthesized N-((1-substituted phenyl-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amine,
-(((9H-fluoren-9-yl)thio)methyl)-1-substituted phenyl-1H-1,2,3-triazole, and 4-(((9H-
4
fluoren-9-yl)sulfonyl)methyl)-1-substituted phenyl-1H-1,2,3-triazole analogues as sketched
in scheme 1.1 and scheme 1.2.
In the scheme 1.1 we adopted reported procedure with slight modification to prepare 9H-
fluoren-9-ol (2), then the compound 2 was brominated with phosphorus tribromide to form
[23]
compound (3). Compound 3 on reacting with propargyl amine in the presence of K
2 3
CO
formed N-alkyl product (4). The free acetylene group was converted to various N-((1-
substituted phenyl-1H-1,2,3-triazol-4-yl)methyl)-9H-fluoren-9-amines (5a-p) using different
[
24]
aromatic azides via click chemistry method.
In the scheme 1.2, 9H-fluorene-9-thiol (6) was obtained from 2 using Lawesson reagent in
[25]
toluene at 110 °C for 16 h. 6 on reacting with propargyl bromide in the presence of TEA
formed (9H-fluoren-9-yl)(prop-2-yn-1-yl)sulfane (7). The compound 7 was converted to
various 4-(((9H-fluoren-9-yl)thio)methyl)-1-substituted phenyl-1H-1,2,3-triazoles (8a-r)
[24]
using different aromatic azides via click chemistry method; further compound 7 on
oxidation with meta-chloroperbenzoic acid in the presence of DCM at room temperature
formed 9-(prop-2-yn-1-ylsulfonyl)-9H-fluorene (9). Subsequently, 9 was converted to various
4-(((9H-fluoren-9-yl) sulfonyl) methyl)-1-substituted phenyl-1H-1,2,3-triazole (10a-p) using
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[
24]
an array of aromatic azides. The purity of compounds synthesized was checked by LC-MS
1
and elemental analyses. Structures of the compounds were confirmed by spectral data. In H
13
NMR and C NMR, the signals of the respective protons and carbons were verified on the
basis of their chemical shifts, multiplicities, and coupling constants. The results of elemental
analysis were within ± 0.05 of the theoretical values.
In-vitro screening
All the synthesized derivatives were tested for their capacity to inhibit the growth of MTB. In
assay three different MTB strains were used. One of them was reference strain MTB H37Rv
[25]
ATTC 25618 and the others were ‘wild’ strains isolated from TB patients. MTB strain
spec. 210 was resistant to p-aminosalicylic acid (PAS), INH, ethambutol (ETB) and RMP and
[26]
another Spec. 192 was fully sensitive to the administrated tuberculostatics. Three different
strains were used for screening as we wanted to explore the kind of activity synthesized
compounds showed against the reference strain as well as against the strains isolated from TB
patients. The influence of the compound on the growth of mycobacteria at concentrations 3.1,
6
.2, 12.5, 25, 50 and 100 μg/mL were evaluated. INH was used as reference drug. The in
vitro anti-TB results of title compounds are given in Table 1 as MIC in μM and the activity
ranged from 52.35 ->250 μM.
Among the three series of 9H-fluorenone analogues, a total of fifty compounds were
screened. Fourteen compounds (5b, 5c, 5d, 5h, 8a, 8d, 8p, 10b, 10c, 10d, 10f, 10h, & 10l)
showed moderate activity with MIC ranging from 141.87 to 109.78 μM. Five compounds
(
5p, 8e, 8f, 8g, & 8q) showed good activity with MIC 74.20 to 56.11 μM. Compound 10p (4-
((9H-fluoren-9-yl)sulfonyl)methyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole) was
(
found to be the most active compound with in vitro MIC 52.35 μM.
Structure activity relationship (SAR) of N-((1-substituted phenyl-1H-1,2,3-triazol-4-
yl)methyl)-9H-fluoren-9-amines derivatives (5a-p)
In this series, we screened sixteen compounds against the three different strains (MTB
H37Rv, MTB spec. 192 & MTB spec. 210). We noticed that among electron withdrawing and
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electron donating substituents on the triazoles, electron donating substituent show major
impact in exhibiting anti-TB activity. SAR is explained based on activity of 5a. Compound
5
a was inhibiting 99% growth of MTB strains at 295.49 μM. Introduction of electron
donating group on the phenyl ring increased activity. Compounds 5b (MIC 141.87 μM), 5c
MIC 136.44 μM) and 5d (MTB 135.71 μM) with methyl, ethyl and methoxy groups
increased the activity by two folds compared to 5a. Introduction of electron withdrawing
groups viz., F, Cl, Br and NO either second or fourth position in phenyl resulted in either
(
2
decrease in activity or the activity remained unaltered. Exception being electron withdrawing
th
CF at 4 position with which the activity increased by two fold (5h, MIC 123.30 μM).
3
Compound 5p with three electron donating methoxy groups emerged to be the most active
compound among these sixteen derivatives with MIC 58.34 μM exhibiting fivefold increase
in activity compared to 5a.
SAR of sulfide derivatives (8a-r)
Among the eighteen sulfide derivates, SAR is explained with respect to compound 8a
th
(
140.66 μM). Presence of electron donating groups at the 4 position impacted the activity.
th
Interestingly, presence of electron withdrawing halogens like F, Cl, Br at the 4 position
resulted in increase in activity by two folds. But the presence of electron withdrawing group
at the ortho position decreased the activity by two folds. Among the dichloro substituted
compounds, 8p was most active with MIC 117.82 μM similar to that of 8a. In this series,
trimethoxy derivative 8q was most active with MIC 56.11 μM.
SAR of sulfonyl derivatives (10a-p)
Eighteen compounds based on sulfonyl were synthesized and screened for MTB. SAR is
explained with respect to 10a which showed MIC 258.09 μM. Activity increased by more
than two folds with electron donating 4-methyl, 4-ethyl and 4-methoxy (MIC 124.53, 120.33
and 119.76 μM respectively). Among the halo derivatives, activity remained unaltered with
bromo where as it increased by two folds with 4-flouro and 4-chloro derivatives. Electron
nd
th
withdrawing NO at 2 & 4 positions did not impact the activity; with electron withdrawing
2
disubstituted derivatives activity remained unaltered (10m, 10n & 10o). Presence of electron
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donating dimethyl increased the activity by two folds (10l, MIC 120.33). Among this series,
0p with three methoxy groups emerged to be the most active compound (MIC 52.35 μM).
1
Over all, we notice that sulfide derivatives exhibited better anti-TB activity followed by
sulfonyl derivatives and amines. Electron donating 3,4,5-trimethoxy derivatives emerged to
be the most active compounds in all the series of compounds.
InhA enzyme Inhibition studies
The synthesized molecules were tested, in the presence of InhA, for their ability to inhibit the
reduction of the substrate double bond by NADH. The experiments were done in triplicate in
the presence of the substrate analogue 2-trans-dodecenoyl-CoA and the InhA percentage
inhibition was measured by using the conversion of the NADH cofactor to its oxidized form
+
[28]
NAD by determining the absorbance at 340 nm. The molecules were tested at 50 μM,
GEQ was used as reference and results are reported in Table 2.
Fifteen compounds whose activities were ≤ 50 μg/mL were selected for screening the InhA
inhibitory activity. Among the amine derivatives (5a-p) the most active compound 5p (MIC
25 μg/mL) was selected for screening; among the sulfide derivatives except 8d all other
compounds with MIC ≤ 50 μg/mL were selected. Among the sulfonyl derivatives except 10b
and 10d, remaining all other compounds with MIC ≤ 50 μg/mL were selected for screening.
Investigations on these fifteen compounds indicated that among the -NH- group containing
9
H-fluorenone derivative, 5p exhibited only 7% inhibition. Presence of sulpur in the 9H-
fluorenone increased the InhA activity, as the results were moderate in this series of
2
compounds. 8p showed the maximum InhA inhibition of 31%. The series of sulfonyl (-SO -)
compounds showed relatively good inhibition. Compounds 10f and 10p were the most active
with 74% and 73% inhibition. They showed that the triazole group could be an attractive
alternative to the amide group found in GEQ.
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On the whole we notice that compounds with (O=S=O) exhibited the highest InhA inhibition
[10]
which is in agreement with the already reported literature. The most active compounds 10f
and 10p were docked into the crystal structure of InhA (PDB ID: 1BVR) in complex with
+
NAD and the C16-fatty-acyl-substrate to know the exact binding pattern with the receptor.
The binding pattern of 10f and 10p with InhA is shown in Figures 4 & 5.
Compound 10f with 74% of inhibition at 50 μM exhibits hydrophilic interaction with
THR196 and SER94 as shown in Figure 4. The active site in the hydrophobic pocket is
within the vicinity of MET103, ILE215, ALA157, LEU218, PRO193, MET199, PHE149,
ILE194, ALA191, ILE21, MET147, ILE95 and MET161. 10f showed π-π interactions with
PHE149 and TYR 158, but no hydrogen bond between triazole and Tyr158 residue could be
observed.
Compound 10p with 73% of inhibition at 50 μM showed a different binding pattern. The
active site in the hydrophobic pocket is within the vicinity of MET103, MET199, ALA198,
ILE194, ILE21, ALA22, PHE41, VAL65, ILE122, PHE97, LE63, ILE15, ILE16 and ILE95
as well as some polar amino acid residues THR196, SER20, SER94, GLN66, THR39 and
SER13 respectively. The ligand also exhibited π-π interactions with PHE41. No interaction of
compound 10p with Tyr158 could be observed.
In vitro cytotoxicity studies
Compounds with MTB MIC < 25 µg/mL were subjected to cytotoxicity studies against HEK
2
93 cell line. Cytotoxicity assay of 5p, 8e, 8f, 8g, 8q & 10p was determined. Cell viability
[29]
was measured by in vitro MTT assay. Cells were exposed to compounds for 24 hours at
three concentrations 50µM, 25 µM and 10 µM (n=2). Data represent mean values of
measurements ± s.d. (Figure 6). Data clearly indicate the active compounds were not toxic at
even 50 µM.
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Single Crystal X-ray Crystallographic Structure of Compound 8a:
Single crystal X-ray of compound 8a is recorded and crystallographic data for 8a is deposited
in the Cambridge Crystallographic Data Center and corresponding deposition (CCDC)
number is 1523811 (complete details are available in supplementary information).
Conclusion
In this communication, we designed novel 9H-fluorenone analogues with three different
series of substitutions. (N4-(((9H-fluoren-9-yl)thio)methyl)-1-substituted phenyl-1H-1,2,3-
triazole, N4-(((9H-fluoren-9-yl)thio)methyl)-1-substituted phenyl-1H-1,2,3-triazole and 4-
(
((9H-fluoren-9-yl) sulfonyl) methyl)-1-substituted phenyl-1H-1,2,3-triazole) by the
molecular hybridization approach using reported MTB InhA inhibitors and substituted 1H-
,2,3-triazole anti-tubercular compounds. Fifty compounds were synthesized and
1
characterized. One of the compounds 10p showed good MTB activity with MIC 52.35 μM.
Out of fifty compounds studied InhA activity was studied for fifteen compounds. Amongst
these compounds, 10f & 10p showed >73% of inhibition at 50 μM. Further, the most active
compounds did not exhibit cytotoxicity against HEK 293 cell line for the most active
compounds at 50 μM.
Acknowledgements
The financial assistance provided by Department of Science and Technology (DST),
(F.No.SB/S1/OC-60/2013) and DST FIST grant (SR/FST/CSI-240/2012) Government of
India, New Delhi is great fully acknowledged. AS thanks DST for providing senior research
fellowship. SS acknowledges CSIR for providing SRF.
Conflict of Interest
The authors declare no conflict of interest.
Supporting Information
1
13
H and C NMR spectra of synthesized compounds have been provided.
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Legends to the figures and schemes
Figure 1. Structures of literature reported InhA inhibitors
Figure 2. Heterocyclic hybrids of [1,2,3] triazole with antitubercular activity
Figure 3. Design strategy to achieve title compounds
Figure 4. Docked pose of compound 10f inside the 1BVR, showing two-dimensional
interactive diagram.
Figure 5. Docked pose of compound 10p inside the 1BVR, showing two-dimensional
interactive diagram.
Figure 6. Cytotoxicity assay of 5p, 8e, 8f, 8g, 8q, 10p on HEK-293 cells
Scheme 1.1. Synthetic protocol to achieve the compound (5a-p)
Reagents and conditions: (i) NaBH
ºC - rt, 4 h. (iii) Propargyl amine (1.2 eq), K
phenyl azides, CuSO .5H
4 3
(0.5 eq), MeOH, 0 ºC - rt, 4 h. (ii) PBr (1.2 eq), DCM, 0
2
CO
3
(2.0 eq), ACN, rt, 16 h. (iv) Substituted
t-
4
2
O (0.02 eq), Sodium ascorbate (0.01 eq), H
2
O: BuOH (1:2), rt, 4 h.
Scheme 1.2. Synthetic protocol to achieve the compounds (8a-r) & (10a-p).
Reagents and conditions: (i) Lawesson reagent (2.0 eq), toluene, 110 ºC, 16 h. (ii) Propargyl
bromide (80% in toluene) (2.0 eq), TEA (3.0 eq), DCM, 16 h. (iii) Substituted aromatic
t-
azides, CuSO
4
.5H
2
O (10 mole %), Sodium ascorbate (5 mole %), H
2
O: BuOH (1:2), rt, 16 h.
(iv) meta-Chloroperoxybenzoic acid (2.0 eq), DCM, rt, 2h. (v) Substituted aromatic azides,
t-
4 2 2
CuSO .5H O (10 mol %), Sodium ascorbate (5 mole %), H O: BuOH (1:2), rt, 16 h.
Table 1 Result of Antimycobacterial screening of title compounds
Table 2 Inhibition of MTB InhA activity
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Legends to the supplementary information
S1 Procedures of intermediates and spectral data of final compounds
S2 Biological activity
S3 Molecular Docking Study
S4 Single Crystal X-ray Crystallographic Structure
1
13
S5 HNMR & CNMR spectra of representative synthesized compounds
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This article is protected by copyright. All rights reserved.

Table 1 Result of Antimycobacterial screening of title compounds
MIC
*
*
*
MIC
MIC
Entry
Ar
against MTB
against MTB
H37Rv
against MTB
Spec. 192
Spec. 210
>
295.49
5a
Phenyl
>295.49 (>100)
>295.49 (>100)
(
>100)
5
b
4-Methylphenyl
4-Ethylphenyl
141.87 (50)
136.44 (50)
135.71 (50)
280.58 (100)
268.22 (100)
239.63 (100)
141.87 (50)
136.44 (50)
135.71 (50)
141.87 (50)
136.44 (50)
5
c
5d
4-Methoxyphenyl
4-Fluorophenyl
4-Chlorophenyl
4-Bromophenyl
>271.42 (>100)
5
e
280.58 (100) >280.58 (>100)
5
f
268.22 (100)
239.63 (100)
268.22 (100)
239.63 (100)
5
g
4
-
5h
123.30 (50)
123.30 (50)
123.30 (50)
Trifluoromethylphenyl
>
260.82
5
i
4-Nitrophenyl
>260.82 (>100)
280.58 (100)
>260.82 (>100)
280.58 (100)
(
>100)
5j
2-Fluorophenyl
2-Nitrophenyl
280.58 (100)
>260.82
5k
260.82 (>100)
>260.82 (>100)
(
>100)
>
272.88
5l
3,4-dimethylphenyl
>272.88 (>100)
>272.88 (>100)
(
>100)
3
-Chloro,4-
>255.85
(>100)
5
m
>255.85 (>100)
>245.51 (>100)
>255.85 (>100)
>245.51 (>100)
fluorophenyl
5
n
2,4-dichlorophenyl
>245.51
This article is protected by copyright. All rights reserved.

*
*
*
MIC
MIC
MIC
Entry
Ar
against MTB
against MTB
H37Rv
against MTB
Spec. 192
Spec. 210
(>100)
>
245.51
5o
3,5-dichlorophenyl
>245.51 (>100)
>245.51 (>100)
(
>100)
5
p
3,4,5-trimethoxyphenyl
Phenyl
58.34 (25)
140.66 (50)
135.32 (50)
260.74 (100)
129.70 (50)
66.94 (25)
74.20 (25)
57.55 (25)
58.34 (25)
140.66 (50)
135.32 (50)
58.34 (25)
140.66 (50)
135.32 (50)
8
a
8b
4-Methylphenyl
4-Ethylphenyl
8
c
260.74 (100) >260.74 (>100)
8d
4-Methoxyphenyl
4-Fluorophenyl
4-Chlorophenyl
4-Bromophenyl
129.70 (50)
66.94 (25)
74.20 (25)
57.55 (25)
>259.41 (>100)
>267.77 (>100)
>256.80 (>100)
115.1 (50)
8
e
8
f
8
g
4
-
>236.21
(>100)
8
h
>236.21 (>100)
>249.71 (>100)
>256.47 (>100)
>249.71 (>100)
>260.74 (>100)
>236.21 (>100)
>249.71 (>100)
>256.47 (>100)
>249.71 (>100)
>260.74 (>100)
Trifluoromethylphenyl
>249.71
8
i
4-Nitrophenyl
(>100)
>256.47
8
j
2-Chlorophenyl
2-Nitrophenyl
(>100)
>249.71
8k
(>100)
>260.74
8
l
3,4-dimethylphenyl
(>100)
This article is protected by copyright. All rights reserved.

*
*
*
MIC
MIC
MIC
Entry
Ar
against MTB
against MTB
H37Rv
against MTB
Spec. 192
Spec. 210
>
238.98
8
m
4-Fluoro, 2-nitrophenyl >238.98 (>100)
>238.98 (>100)
>235.65 (>100)
>235.65 (>100)
(
>100)
>
235.65
8
n
2,4-dichlorophenyl
3,4-dichlorophenyl
>235.65 (>100)
>235.65 (>100)
(
>100)
>
235.65
8
o
(
>100)
8
p
3,5-dichlorophenyl
117.82 (50)
56.11 (25)
117.82 (50)
56.11 (25)
>235.65 (>100)
56.11 (25)
8q
3,4,5-trimethoxyphenyl
3
-Chloro,4-
8r
245.61 (100)
245.61 (100) >245.61 (>100)
fluorophenyl
>
258.09
10a
Phenyl
>258.09 (>100)
258.09 (>100)
(
>100)
1
0b
0c
0d
0e
0f
4-Methylphenyl
4-Ethylphenyl
124.53 (50)
120.33 (50)
119.76 (50)
123.32 (50)
118.51 (50)
124.53 (50)
120.33 (50)
119.76 (50)
123.32 (50)
118.51 (50)
124.53 (50)
240.66 (>100)
119.76 (50)
123.32 (50)
118.51 (50)
1
1
4-Methoxyphenyl
4-Fluorophenyl
4-Chlorophenyl
1
1
>
214.43
1
0g
4-Bromophenyl
>214.43 (>100)
109.78 (50)
>214.43 (>100)
109.78 (50)
(
>100)
4-
10h
109.78 (50)
Trifluoromethylphenyl
This article is protected by copyright. All rights reserved.

*
*
*
MIC
MIC
MIC
Entry
Ar
against MTB
against MTB
H37Rv
against MTB
Spec. 192
Spec. 210
>
231.24
1
0i
4-Nitrophenyl
2-Nitrophenyl
>231.24 (>100)
>231.24 (>100)
>231.24 (>100)
>231.24 (>100)
(
>100)
>231.24
10j
(
>100)
1
0k
2-Chlorophenyl
237.02 (100)
120.33 (50)
237.02 (100)
120.33 (50)
237.02 (100)
1
0l
3,4-dimethylphenyl
120.33 (>100)
>
222.00
1
0m 4-Fluoro, 2-nitrophenyl >222.00 (>100)
>222.00 (>100)
>219.13 (>100)
>219.13 (>100)
(
>100)
>219.13
1
0n
2,4-dichlorophenyl
3,4-dichlorophenyl
>219.13 (>100)
>219.13 (>100)
(
>100)
>219.13
1
0o
(
>100)
1
0p
3,4,5-trimethoxyphenyl
-
52.35 (25)
0.66
52.35 (25)
0.66
52.35 (25)
12.5
INH
*
The values indicated in parenthesis correspond to MIC in μg/mL
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Table 2: Inhibition of MTB InhA activity
%
of inhibition
Compound
Code
S. No
at 50 μM of inhibitor
1
2
3
4
5
6
7
8
9
5p
8a
7
23
24
6
8b
8e
8f
< 5
NI
21
31
30
27
NI
74
27
NI
73
88
8g
8h
8p
8q
1
1
1
1
1
1
1
0
1
2
3
4
5
6
10c
10e
10f
10h
10l
10p
GEQ (A)
NI = No inhibition
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This article is protected by copyright. All rights reserved.

This article is protected by copyright All rights reserved

Scheme 1.1. Synthetic protocol to achieve the compound (5a-p)
Reagents and conditions: (i) NaBH
ºC - rt, 4 h. (iii) Propargyl amine (1.2 eq), K
2
phenyl azides, CuSO .5H O (0.02 eq), Sodium ascorbate (0.01 eq), H O: BuOH (1:2), rt, 4 h.
4
(0.5 eq), MeOH, 0 ºC - rt, 4 h. (ii) PBr
3
(1.2 eq), DD CM,
0
2
CO (2.0 eq), ACN, rt, 16 h. (iv) Substituted
3
t-
4
2
This article is protected by copyright All rights reserved

Scheme 1.2. Synthetic protocol to achieve the compounds (8a-r) & (10a-p).
Reagents and conditions: (i) Lawesson reagent (2.0 eq), toluene, 110 ºC, 16 h. (ii) Propargyl
bromide (80% in toluene) (2.0 eq), TEA (3.0 eq), DCM, 16 h. (iii) Substituted aromatic
t-
azides, CuSO .5H O (10 mole %), Sodium ascorbate (5 mole %), H O: BuOH (1:2), rt, 16 h.
4
2
2
(
iv) meta-Chloroperoxybenzoic acid (2.0 eq), DCM, rt, 2h. (v) Substituted aromatic azides,
t-
4 2 2
CuSO .5H O (10 mol %), Sodium ascorbate (5 mole %), H O: BuOH (1:2), rt, 16 h.
This article is protected by copyright. All rights reserved.