Synthesis and antimycobacterial activity of 1-(β-D-Ribofuranosyl)-4-coumarinyloxymethyl- / -coumarinyl-1,2,3-triazole

Article abstract of DOI:10.1016/j.ejmech.2018.02.067

A series of β-D-ribofuranosyl coumarinyl-1,2,3-triazoles have been synthesized by Cu-catalyzed cycloaddition reaction between azidosugar and 7-O-/7-alkynylated coumarins in 62–70% overall yields. The in vitro antimycobacterial activity evaluation of the s

Full text of DOI:10.1016/j.ejmech.2018.02.067

Accepted Manuscript
Synthesis and antimycobacterial activity of 1-(β-D-Ribofuranosyl)-4-
coumarinyloxymethyl- / -coumarinyl-1,2,3-triazole
Smriti Srivastava, Devla Bimal, Kapil Bohra, Balram Singh, Prija Ponnan, Ruchi Jain,
Mandira Varma-Basil, Jyotirmoy Maity, M. Thirumal, Ashok K. Prasad
PII:
S0223-5234(18)30204-6
10.1016/j.ejmech.2018.02.067
EJMECH 10244
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 July 2017
Revised Date: 17 February 2018
Accepted Date: 21 February 2018
Please cite this article as: S. Srivastava, D. Bimal, K. Bohra, B. Singh, P. Ponnan, R. Jain, M.
Varma-Basil, J. Maity, M. Thirumal, A.K. Prasad, Synthesis and antimycobacterial activity of 1-(β-D-
Ribofuranosyl)-4-coumarinyloxymethyl- / -coumarinyl-1,2,3-triazole, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.067.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and Antimycobacterial Activity of 1-(β- -Ribofuranosyl)-4-
D
coumarinyloxymethyl- / -coumarinyl-1,2,3-triazole
Smriti Srivastava,#a Devla Bimal,#a Kapil Bohra,a,b Balram Singh,a Prija Ponnan,a Ruchi Jain,a
Mandira Varma-Basil,c Jyotirmoy Maity,a M. Thirumala and Ashok K. Prasada*
a Department of Chemistry, University of Delhi, Delhi-110007, India
b Department of Chemistry, Deen Dayal Upadhyaya College, University of Delhi, Delhi-110078, India
c Department of Microbiology, VP Chest Institute, University of Delhi, Delhi-110007, India
# First and second author contributed equally to this work and should be regarded as joint First authors.

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Synthesis and Antimycobacterial Activity of 1-(β- -Ribofuranosyl)-4-
D
coumarinyloxymethyl- / -coumarinyl-1,2,3-triazole
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Smriti Srivastava,#a Devla Bimal,#a Kapil Bohra,a,b Balram Singh,a Prija Ponnan,a Ruchi Jain,a
Mandira Varma Basil,c Jyotirmoy Maity,a M. Thirumala and Ashok K. Prasada*
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aDepartment of Chemistry, University of Delhi, Delhi-110007, India
bDepartment of Chemistry, Deen Dayal Upadhyaya College, University of Delhi, Delhi-110078, India
cDepartment of Microbiology, VP Chest Institute, University of Delhi, Delhi-110007, India
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Abstract
A series of β-D-ribofuranosyl coumarinyl-1,2,3-triazoles have been synthesized by Cu-
catalyzed cycloaddition reaction between azidosugar and 7-O- / 7-alkynylated coumarins in
62 to 70 % overall yields. The in vitro antimycobacterial activity evaluation of the
synthesized triazolo-conjugates against Mycobacterium tuberculosis revealed that compounds
were bactericidal in nature and some of them were found to be more active than one of the
first line antimycobacterial drug ethambutol against sensitive reference strain H37Rv, and 7
to 420 times more active than all four first line antimycobacterial drugs (isoniazid,
rifampicin, ethambutol and streptomycin) against multidrug resistant clinical isolate 591.
Study of in silico pharmacokinetic profile indicated the drug like characters for the test
molecules. Further, transmission electron microscopic experiments revealed that these
compounds interfere with the constitution of bacterial cell wall possibly by targeting
mycobacterial InhA and DNA gyrase enzymes. Study conducted on the activities of the test
compounds on bacterial InhA and DNA gyrase revealed that the most bactericidal test
compound, N1-(β-D-ribofuranosyl)-C4-(4-methylcoumarin-7-oxymethyl)-1,2,3-triazole (6b)
and its corresponding directly linked conjugate N1-(β-D-ribofuranosyl)-C4-(4-
methylcoumarin-7-yl)-1,2,3-triazole (11b) significantly inhibited the activity of both the
enzymes. The results were further supported by molecular docking studies of the compound
6b and 11b with bacterial InhA and DNA gyrase B enzymes. Further, the cytotoxicity study
of some of the better active compounds on THP-1 macrophage cell line using MTT assay
showed that the synthesised compounds were non-cytotoxic.
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Keywords: Antimycobacterial, Click chemistry, 1,2,3-Triazole, Cytotoxicity, In silico
pharmacokinetics, Docking study.
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*Corresponding Author
Ashok K. Prasad, Bioorganic Laboratory, Department of Chemistry, University of Delhi,
Delhi-110 007, India; Phone: 00-91-11-27662486; E-mail: ashokenzyme@gmail.com.
# First and second author contributed equally to this work and should be regarded as joint
First authors.
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Introduction
There is a great need for the development of newer molecular entities to act as
antimycobacterial agents due to the limitations of the existing drugs as well as the increasing
resistance against causative bacteria, Mycobacterium tuberculosis (M. tuberculosis) [1-4]. In
HIV-prevalent regions, infection by drug-resistant tuberculosis (TB) almost always has fatal
consequences [5-6]. Drugs that are effective in reducing the length of treatments providing
effective cure against multi-drug resistant TB (MDR), extremely drug resistant TB (XDR)
and latent TB is the requirement of the time [2-3].
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Carbohydrates are among the most ample raw materials provided by nature, which play a
significant role at the interface of chemistry and biology [7-8]. On the other hand coumarins
and their derivatives have shown their potential as antitubercular [1], anti-cancer [9-12], anti-
HIV [13], anti-inflammatory [14-15], anti-fungal [16], anti-oxidant, [14-15] and anti-bacterial
agents [17a]. Several coumarin derivatives, such as warfarin, carbochromen and
acenocoumarol have been approved by FDA for their clinical therapeutic uses [17a-c]. Both,
the sugar and the coumarin moieties are excellent biomolecules and have wide variety of
applications as well as good biocompatibility [18-19]. There are few reports of synthesis of
conjugates of sugar-coumarin or sugar-quinoline linked through a triazole ring that have
shown interesting biological activities (Figure 1) [20-25]. We report herein the synthesis of
small libraries of N1-(β-D-ribofuranosyl)-C4-(coumarin-7-oxymethyl)-1,2,3-triazole and N1-
(β-D-ribofuranosyl)-C4-(coumarin-7-yl)-1,2,3-triazole using Cu(I) catalyzed azide-alkyne 1,3-
dipolar cycloaddition ‘click’ reaction (CuAAC) and study of their in vitro antimycobacterial
activities against M. tuberculosis sensitive reference strain H37Rv and multidrug resistant
clinical isolate 591.
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OAc
OH
N
N
N
O
N
N
N
O
O
O
O
O
AcO
AcO
O
O
O
HO
HO
HO
HO
O
N
N
OAc
N
N
OH
OH
Anti-tubercular agent
Glycosidase enzyme inhibitor
Glycosylamine priming activity
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Figure 1: Biologically active sugar -quinoline / -coumarin triazoloconjugates
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Results and Discussion
Chemistry
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The synthesis of N1-(β-D-ribofuranosyl)-C4-(coumarin-7-oxymethyl)-1,2,3-triazoles has been
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achieved by Cu(I) catalyzed Huisgen-Sharpless-Meldal [3+2] dipolar cycloaddition reaction
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of 1-azido-2,3,5-tri-O-benzoyl-β- -ribofuranose (2) with 7-propargyloxycoumarins 4a-4d,
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followed by debenzoylation of the resultant conjugates in 62 to 68 % overall yields. The
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azido-sugar 2 was synthesised by the azidation of commercially available 1-O-acetyl-2,3,5-
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tri-O-benzoyl-β- -ribofuranose (1) with azido trimethylsilane in the presence of tin (IV)
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chloride in 88 % yield (Scheme 1) [26]. The coumarin precursors, 7-hydroxy-4-
methylcoumarin (3b) & 7-hydroxy-4-phenylcoumarin (3c) were synthesised by the
condensation of resorcinol with ethyl acetoacetate and ethyl benzoacetate in 78 and 80 %
yields, respectively and 6-chloro-4-methyl-7-hydroxycoumarin (3d) was synthesized by
condensation of 4-chloro-3-hydroxyphenol with ethyl acetoacetate in 75 % yield using
Pechmann condensation protocol [27]. However, 7-hydoxycoumarin (umbelliferone, 3a) was
obtained commercially from Sigma Aldrich Chemical Company, USA. The 7-
hydroxycoumarins 3a-3d were propargylated using propargyl bromide in acetone in the
presence of potassium carbonate as base to afford 7-propargyloxycoumarins (4a), 4-methyl-
7-propargyloxycoumarins (4b), 4-phenyl-7-propargyloxycoumarins (4c) and 6-chloro-4-
methyl-7-propargyloxycoumarin (4d) in 80-85 % yields (Scheme 2) [28-30].
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BzO
BzO
OAc
N3
O
O
Azido trimethylsilane, SnCl4
OBz OBz
OBz OBz
Dichloromethane, 4-5 h
88 %
β-Anomer
β-Anomer
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Scheme 1: Synthesis of 1-azido-2,3,5-tri-O-benzoyl-β- -ribofuranose
D
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The Cu(I) catalyzed cycloaddition reaction of 1-azido-2,3,5-tri-O-benzoyl-β- -ribofuranose
D
(2) with 7-propargyloxycoumarins 4a-4d in the presence of sodium ascorbate-CuSO4 in
THF:tert-BuOH:H2O (1:1:1, v/v/v) resulted in the formation of N1-(2,3,5-tri-O-benzoyl-β-D-
ribofuranosyl)-C4-(coumarin-7-oxymethyl)-1,2,3-triazoles 5a-5d in 75-80 % yields. The
debenzoylation of the resulted triazole derivatives 5a-5d with sodium methoxide in methanol
led to the formation of targeted compounds, N1-(β-D-ribofuranosyl)-C4-(coumarin-7-
oxymethyl)-1,2,3-triazoles 6a-6d in 80-85 % yields (Scheme 2).
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Scheme 2: Synthesis of N1-(β-D-ribofuranosyl)-C4-(coumarin-7-oxymethyl)-1,2,3-
triazoles. Reagents and conditions: (i) propargyl bromide, K2CO3, acetone, 8-10 h,
reflux, 80-85 %; (ii) sodium ascorbate, CuSO4, THF:tert-BuOH:H2O (1:1:1), 10-
15 h, rt, 75-80 %; (iii) NaOMe, MeOH, 5-6 h, rt, 80-85 %.
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Similarly, synthesis of N1-(β-D-ribofuranosyl)-C4-(coumarin-7-yl)-1,2,3-triazoles has been
achieved by Cu(I) catalyzed [3+2] cycloaddition reaction of azidosugar 2 with 7-
acetynylcoumarins 9a-9d followed by debenzoylation of the resulted N1-(2,3,5-tri-O-benzoyl-
β-D-ribofuranosyl)-C4-(coumarin-7-yl)-1,2,3-triazoles 10a-10d with sodium methoxide in
methanol in an overall yields of 62 to 70 % (Scheme 3). In turn, the synthesis of 7-
acetynylcoumarins 9a-9d was achieved in three steps starting with 7-hydroxycoumarins 3a-
3d. Thus the hydroxycoumarins 3a-3d were converted into their corresponding triflates 7a-7d
by treatment with trifluoromethane sulfonic anhydride (triflic anhydride) in dichloromethane-
o
pyridine at 0 C; alkynylation of the triflated coumarins 7a-7d with trimethylsilylacetylene
using Sonogashira coupling reaction followed by removal of the trimethylsilyl protection
from the resulted 7-trimethylsilylacetynylcoumarins 8a-8d led to the formation of 7-
acetynylcoumarins 9a-9d in an overall yields of 42 to 51 % (Scheme 3) [31-33].
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Scheme 3: Synthesis of N1-(β-D-ribofuranosyl)-C4-(coumarin-7-yl)-1,2,3-triazoles. Reagents
and conditions: (i) triflic anhydride, pyridine, 1-2 h, rt, 80-85 %; (ii) Et3N, Pd(PPh3)4, CuI,
acetonitrile, trimethylsilylacetylene, 5-6 h, 70-75 %; (iii) K2CO3, MeOH, 1-2 h, rt, 75-80 %;
(iv) CuSO4, sodium ascorbate, THF:tert-BuOH:H2O (1:1:1), 12-15 h, rt, 77-82 %; (v)
NaOMe-MeOH, 5-6 h, rt, 80-85 %.
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The structures of all the synthesized compounds 2, 4a-11a, 3b-11b, 3c-11c and 3d-11d were
unambiguously established on the basis of their spectral (IR, 1H-, 13C-, 1H-1H COSY, 1H-13C
HETCOR NMR and HRMS) data analysis. Three hydroxyl groups present in the sugar
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moiety of the targeted compounds 6a-6d and 11a-11d was confirmed by H NMR spectra
using D2O exchange experiments. The structures of known compounds 2, 3b-3d, 4a-4d, 7a-
7d, 8a, 8b, 8d, 9a, 9b and 9d was further confirmed by comparing their physical and spectral
data with those reported in the literature [26-33]. The structures, stereochemistry and
connectivities of the sugar and the coumarin moiety with triazole ring in N1-(β-
ribofuranosyl)-C4-(coumarin-7-oxymethyl)-1,2,3-triazoles 6a-6d and N1-(β-
-ribofuranosyl)-
C4-(coumarin-7-yl)-1,2,3-triazoles 11a-11d were unambiguously confirmed by X-ray
diffraction studies on the single crystals of N1-(β- -ribofuranosyl)-C4-(4-methylcoumarin-7-
oxymethyl)-1,2,3-triazole (6b) and N1-(β- -ribofuranosyl)-C4-(4-methylcoumarin-7-yl)-
D-
D
D
D
1,2,3-triazole (11b). The ORTEP diagrams of crystal structures of compounds 6b and 11b
have been given in Figure 2. The detailed crystallographic data of compounds 6b and 11b
was deposited in the Cambridge Crystallographic Data Centre with CCDC no. 1535099 and
1419805, respectively.
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Figure 2: ORTEP diagram of compounds 6b and 11b drawn in 50 % thermal probability
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ellipsoids showing atomic numbering scheme.
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Antimycobacterial Activity and Structure-Activity Relationship
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All the synthesized benzoyl and hydroxy conjugates, i.e. N1-(2,3,5-tri-O-benzoyl-β-
-
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ribofuranosyl)-C4-(coumarin-7-oxymethyl)-1,2,3-triazoles 5a-5d and N1-(β-
C4-(coumarin-7-oxymethyl)-1,2,3-triazoles 6a-6d, respectively with oxymethylene linker
between triazole and coumarin, and N1-(2,3,5-tri-O-benzoyl-β- -ribofuranosyl)-C4-
D-ribofuranosyl)-
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D
(coumarin-7-yl)-1,2,3-triazoles 10a-10d and N1-(β-D-ribofuranosyl)-C4-(coumarin-7-yl)-
1,2,3-triazoles 11a-11d with direct triazole-coumarin linkage were screened for their activity
against M. tuberculosis sensitive reference strain H37Rv and multidrug resistant clinical
isolate 591 obtained from Department of Microbiology, V. P. Chest Institute, University of
Delhi, India. The minimum inhibitory concentration (MIC) values of above mentioned
sixteen test compounds and reference compounds, i.e. isoniazid, rifampicin, ethambutol and
streptomycin were determined using the micro-plate alamar blue assay (MABA) (Table 1) [3,
34, 35]. The results revealed that the antimycobacterial activity of conjugates with
oxymethylene linker between triazole and coumarin moieties, i.e. 5a-5d and 6a-6d had
higher activity than the conjugates with direct triazole-coumarin linkage, i.e. 10a-10d and
11a-11d. The synthesized candidate drugs were active against both the sensitive reference
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strain as well as MDR clinical isolate 591. However, the antimycobacterial activity of almost
all synthesized compounds against MDR clinical isolates 591 was many times higher than
any of the four screened first line antitubercular drugs. The MIC of all sixteen tested
compounds against sensitive reference strain H37Rv and multidrug resistant clinical isolate
591 ranged from 4.4-30.5 µM. The compounds 5c, 6b and 6c were most active against both
sensitive reference strain H37Rv and multidrug resistant clinical isolate 591 having MIC
values 5.2, 5.1, 4.4 µM and 5.2, 10.3, 8.9 µM, respectively. The activity of these three
compounds against reference strain was found to be better than the activity of one of the first
line antitubercular drug, ethambutol. Importantly, the activity of compounds 5c, 6b and 6c
against MDR clinical isolate 591 was many folds higher (7-420 times) than the activity of
first line antitubercular drugs isoniazid, rifampicin, ethambutol and streptomycin,
respectively (Table 1).
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It was envisioned that respectively high level of activity of conjugates with -OCH2- bridge
between triazole and coumarin moieties may be due to the provision of greater flexibility to
the molecule to bind to the required target. The minimum bactericidal concentration (MBC)
of the synthesized conjugates and reference drug isoniazid was also determined which
revealed that hydroxyl conjugate 6b has the lowest MBC, i.e. 6.4 µM against reference strain
and 11.5 µM against MDR clinical isolate, respectively (Table 1). A compound is considered
to be bactericidal if the ratio of MBC/MIC is ≤ 4 [3, 34, 35]. The calculated MBC/MIC ratio
was 1.25 for reference strain and 1.13 for MDR clinical isolate for compounds 6b showing its
bactericidal nature. The MBC/MIC ratios of all the sixteen test compounds clearly revealed
their bactericidal nature (Table 1).
Serial
No.
First line
drug and
synthesized
compound
MIC against
H37Rv
MIC against
multidrug
resistant
clinical isolate
591
MBC (µM)
H37Rv
MBC/MIC ratio
H37Rv 591
591
sensitive
strain
(µM)
(µM)
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2
3
4
5
6
0.2
0.02
9.7
2187.5
151.9
73.4
69
0.3
-
-
-
-
-
-
1.6
-
-
-
-
-
-
Isoniazid
Rifampicin
Ethambutol
Streptomycin
5a
-
-
-
-
-
0.43
11.6
8.5
-
14.5
11.4
26.2
17.1
2.25
2
5b
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5.2
5.2
10.5
27.2
26.6
6.4
-
2
2
-
5c
5d
8
13.5
10.6
5.1
20.4
15.9
10.3
8.9
-
-
9
-
2.5
1.25
4
-
6a
10
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13
14
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20
11.5
1.13
6b
4.4
17.7
37.7
30.4
26.8
24.5
25.5
46.3
16.7
28.5
-
-
-
-
6c
18.9
15.2
11.9
10.9
14.2
28.9
16.6
14.2
30.5
18.9
30.4
29.8
13.6
14.2
28.9
22.2
18.9
25.4
-
2
6d
-
2
-
-
10a
10b
10c
10d
11a
11b
11c
11d
-
2.25
2.25
1.8
1.6
1
-
-
-
-
-
-
22.2
1
-
-
-
2
-
-
1
2
3
4
5
Table 1: MIC, MBC and (MBC/MIC ratio) results of test compounds and first line drugs against M.
tuberculosis sensitive reference strain H37Rv and multidrug resistant clinical isolate 591.
- Not assessed.
a Values are the results of three independent experiments.
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In silico Pharmacokinetic Study
The benzoylated β-D-ribofuranosyl coumarinyl-1,2,3-triazoles 5a-5d & 10a-10d and β- -
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ribofuranosyl coumarinyl-1,2,3-triazoles 6a-6d & 11a-11d were further subjected to in silico
pharmacokinetic studies. Isoniazid (INH) was considered as the reference standard to
compare certain important pharmacokinetic parameters, such as molecular weight,
lipophilicity, hydrogen-bonding capabilities, etc. These properties were predicted using
molecular property prediction tool, Molinspiration and the results were analyzed on the basis
of Lipinski rule of five [36-38]. According to Lipinski’s rule an orally active drug should not
violate more than one of the following criteria-number of hydrogen bond donor and acceptor
that mostly determine membrane permeability should be ≤ 5 and ≤ 10, respectively;
molecular mass should be ≤ 500, Log P and Log S values corresponding to lipophilicity and
solubility and helpful in predicting the intestinal absorption and consequent efficiency of the
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trans-cellular transport of the drug should be ≤ 5 [36]. All the eight hydroxylated β- -
D
ribofuranosyl coumarinyl-1,2,3-triazoles 6a-6d and 11a-11d exhibited good drug likeness
properties and did not violated any of the pharmacokinetic parameters as proposed in
Lipinski’s rule (Table 2). None of the benzoylated conjugates passed all four criteria of
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Lipinski’s filter possibly due to the presence of benzoyl groups instead of hydroxyls, which
make them too lipophilic to behave like a drug.
#Log P
¥Log S
+HBD
Drug
Likeness
No. of
Violations
*HBA
Compound
No.
Molecular
weight
687.65
701.67
763.74
736.12
375. 33
389.36
451.43
423.80
657.62
671.65
733.72
706.09
345.30
359.33
421.40
393.77
137.14
5.69
6.04
7.37
6.64
-0.74
-0.39
0.93
0.20
5.22
6.27
7.59
6.87
-0.52
-0.17
1.16
0.11
0.82
-9.51
-9.61
-10.75
-10.25
-3.28
-3.38
-4.52
-4.02
-9.93
-10.03
-11.57
-10.40
-3.69
-3.80
-4.94
0.43
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12
12
12
9
0
0
0
0
3
3
3
3
0
0
0
0
3
3
3
3
1
-0.49
-0.44
-0.50
-0.30
-0.37
-0.44
-0.37
-0.04
-0.69
-0.61
-0.67
-0.42
-0.57
-0.62
-0.62
-0.24
0.67
3
3
3
3
0
0
0
0
3
3
3
3
0
0
0
0
0
5a
5b
5c
5d
6a
9
6b
9
6c
9
6d
11
11
11
11
8
10a
10b
10c
10d
11a
11b
11c
11d
INH
8
8
8
-0.86
4
4
5
6
7
8
Table 2: Some important pharmacokinetic parameters of test compounds; INH reference anti-TB
drug, #log P octanol/water partition coefficient (log P ≤ 5), ¥log S water solubility (log S ≤ 5), *HBA
hydrogen bond acceptor (HBA ≤ 10), +HBD hydrogen bond donor (HBD ≤ 5).
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Transmission Electron Microscopic Study
On the basis of MIC, MBC and in silico pharmacokinetic study results, one of the most active
compound 6b and its directly linked conjugate 11b was selected for transmission electron
microscopy. The untreated and treated cells of M. tuberculosis sensitive reference strain
H37Rv with test compounds 6b at 1.38 µM, 11b at 4.15 µM and first line antitubercular drug
isoniazid at 0.05 µM concentrations (1/4th concentration of their MICs) were subjected to
transmission electron microscopy (TEM). The TEM images of the treated cells with 6b, 11b
and isoniazid revealed cell-wall disintegrating nature of the test compounds as well as the
drug (Figure 3). It has been observed that the untreated / control cells retained their shape
with a clearly visible cell wall, however cell-wall disruption at quite a few places was
observed in the treated cells of the bacilli (Figure 3a-d) [3, 39]. The cell-wall in some treated
cells was found to be completely diminished with cell remnants being seen while some of the
disintegrating cells showed vacuole formation (Figure 3c-f).
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The TEM study indicated the change in cell morphology of the treated H37Rv strain of M.
tuberculosis, which encouraged us to explore the possibility of finding drug targets present in
the cell wall of M. tuberculosis. The other reason for the investigation of the targets in the
cell wall is the presence of coumarin moiety in the synthesized candidate drugs which has
been previously reported to target InhA and DNA gyrase bacterial enzymes [3, 40, 41]. Thus,
the interaction of the synthesized drug candidates with both the bacterial enzymes, i.e. InhA
and DNA gyrase was studied.
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11
12
13
Figure 3: Transmission electron micrographs of untreated and compound-treated M. tuberculosis
H37Rv cells. (a) untreated cells; (b) Isoniazid treated; (c) & (d) 6b treated and (e) & (f) 11b treated.
Arrows indicate changes in cell wall morphology, ranging from initiation of cell wall disappearance to
complete disintegration and show empty cell-like appearance.
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Assay for InhA Enzyme Inhibition
InhA, the enoyl acyl carrier protein reductase (EACPR) from M. tuberculosis is one of the
essential enzymes involved in the mycobacterial fatty acid elongation cycle and is important
for cell survival [41-44]. It is an effective antimicrobial drug target [44]. InhA–drug
interaction experiment was carried out to validate InhA as drug target for our test compounds
as indicated by TEM studies [41]. To identify whether the test compounds 6b and 11b inhibit
InhA activity of M. tuberculosis; the enzyme activity was observed in the presence and the
absence of compound 6b and 11b at single concentration, i.e. 5 µM (Figure 4). In the
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experiment, a visible decrease in the absorbance at 340 nm was reported in the reaction
mixture containing enzyme InhA, cofactor NADH and substrate crotonoyl-CoA. However,
on addition of the test compounds 6b and 11b to the reaction mixture, a slight decrease in
absorbance was observed initially and there after it remained constant. This indicated the
inhibition of InhA enzyme activity in the presence of compounds 6b and 11b (Figure 4). The
results were statistically found to be significant by one way ANOVA test showing p value ≤
0.0019.
InhA enzyme activity
0.4
enzyme -substrate
enzyme-subtrate-inhibitor 6b
0.3
0.2
0.1
0.0
enzyme-subtrate-inhibitor 11b
0
5
10
Time (min.)
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Figure 4: Assay for the activity of bacterial enzyme InhA in presence of test compounds 6b and 11b.
10
Values are the results of three independent experiments.
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DNA Gyrase Inhibition Assay
DNA gyrase-drug interaction experiment was carried out by DNA gyrase inhibition assay to
validate DNA gyrase as drug target [40, 45-47]. Bacterial DNA gyrase, a type II DNA
topoisomerase found exclusively in all bacteria and is essential for cell survival. It is a proven
target for antibacterial chemotherapy and is a common target of many drugs which includes
coumarin and quinolones derived drugs such as ciprofloxacin, novobiocin, chlorobiocin etc
[40]. The DNA gyrase B protein from E. coli and mycobacterium species such as M.
tuberculosis and Mycobacterium smegmatis (M. smegmatis) share ~ 80 % amino acid
sequence identity (supporting information S23 Figure 1). Hence, the two active test
compounds 6b and 11b were studied for their effect on E. coli DNA gyrase enzyme at single
concentration of 0.66 ꢀM and 100 ꢀM (Figure 5). The inhibition of DNA gyrase activity was
checked by agarose gel electrophoresis and visualized in GELDOC. Results showed
inhibition of DNA gyrase supercoiling activity in the presence of ciprofloxacin (Figure 5,
Lane R5 and R8 taken as positive control), test compounds 6b (Figure 5, Lane R4 and R7)
and 11b (Figure 5, Lane R3 and R6) where as supercoiling was observed in negative control
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(relaxed DNA and DNA gyrase enzyme only) (Figure 5, Lane R2). Result shows
ciprofloxacin and the test compounds 6b and 11b significantly inhibited the bacterial DNA
gyrase activity at 0.66 ꢀM and showed complete inhibition at 100 ꢀM concentration.
Preliminary assay results showed inhibition of DNA gyrase activity by our test compounds
6b and 11b having IC50 0.8 ꢀM and 3.2 ꢀM respectively (supporting information S25 Figure
3 and S26 Figure 4). The DNA gyrase enzyme inhibition assay results at single point
concentration as well and IC50 determination for the DNA gyrase enzyme inhibition indicated
DNA gyrase as one of the probable drug target for our test compounds.
R0
R1
R2
R3
R4
R5
R6
R7
R8
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Lane
R0
Lane
R1
Lane
R2
Lane
R3
Lane
R4
Lane
Lane
R6
Lane
R7
Lane
R8
R5
Supercoiled DNA
Relaxed DNA
DNA Gyrase
DMSO (µl)
Ciprofloxacin
6b
+
-
-
+
-
-
+
+
-
-
-
-
-
-
+
-
+
+
-
+
+
-
+
+
-
+
+
+
-
-
-
0.2
0.2
-
0.2
0.2
-
-
-
-
-
-
0.66µM
-
100 µM
-
-
-
-
-
-
-
0.66µM
-
100µM
-
11b
-
-
-
0.66µM
-
100µM
-
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Figure 5: 6b, 11b and Ciprofloxacin (0.66 µM and 100 µM) showed inhibition of supercoiling
activity of DNA gyrase enzyme; Lane R0: Supercoiled Plasmid control, Lane R1: Linear relaxed
plasmid, Lane R2: Super coiling of linear plasmid in presence of DNA-Gyrase, Lane R3: 11b (0.66
µM), Lane R4: 6b (0.66 µM), Lane R5: ciprofloxacin (0.66 µM), Lane R6: 11b (100 µM), Lane R7:
6b (100 µM), Lane R8: Ciprofloxacin (100 µM).
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Molecular Docking
The molecular docking studies were performed to understand the interaction of active β- -
D
ribofuranosyl coumarinyl-1,2,3-triazoles 6b & its directly linked conjugate 11b and the
docking protocol was validated using the prediction for the interaction of co-crystallized
ligands (carboximde, chlorobiocin and aminopyrazinamide derivatives) with the binding site
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of M. tuberculosis InhA , E.coli DNA gyrase B and M. smegmatis DNA gyrase B, and the
results of the docked conformations of best scored compounds are given in Table 3.
Compounds
Docking results with
M. tuberculosis InhA
Docking results with
E. coli DNA gyrase B
Docking results with M.
smegmatis DNA gyrase B
XP
Glide
XP
Glide
XP
Glide
GScore
Energy
GScore
Energy
GScore
Energy
-9.88
-9.34
-66.18
-63.45
-9.21
-9.96
-60.34
-73.05
-8.28
-8.22
-59.30
-54.33
Compound 6b
Compound 11b
-9.98
-73.10
-
-
-
-
(3S)-1-Cyclohexyl -
n-(3,5-dichloro
phenyl)- 5-oxo
pyrrolidine-3-
carboxamide
-
-
-
-
-10.32
-
-76.28
-
-
-
Chlorobiocin
-9.72
-64.59
6-(3,4-Dimethyl
phenyl)-3-[[4-[3-(4-
methylpiperazin-1-
yl) propoxy]
phenyl] amino]
pyrazine-2-carb
oxamide
3
4
5
6
Table 3: Glide XP docking scores (kcal mol-1) and docking energies of best bioactive molecules along
with the reference inhibitors: pyrrolidine carboxamide, chlorobiocin and aminopyrazinamide bound to
M. tuberculosis InhA, E. coli DNA gyrase and M. smegmatis DNA gyrase respectively.
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These docking results clearly indicated that compounds 6b and 11b exhibited significant
binding affinities towards the M. tuberculosis InhA protein (Glide energy range -66.19
kcalmol-1 to -52.14 kcalmol-1 and Glide score in range -9.88 to -7.11 kcalmol-1). The
predicted binding pose of compounds 6b and 11b with hydrogen bonding and hydrophobic
interactions in the binding site of M. tuberculosis InhA protein has been shown in supporting
material (S27 Figure 5). The lowest binding energy pose (-66.19 kcalmol-1) and high Glide
XP score (-9.88 kcalmol-1) of compound 6b, when docked in the ligand binding site of M.
tuberculosis InhA has been shown in S27 Figure 5A. Compound 6b binds deep in the InhA
binding site forming hydrogen bond between nitrogen of triazole ring of the compound and
side chain OH of Tyr158 (S27 Figure 5A). Further, a π-π interaction between aromatic ring
of Tyr158 and the triazole ring of the compound was observed. These binding patterns are
similar to that observed for the co-crystallized pyrrolidine carboxamide in M. tuberculosis
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InhA protein [44]. However, the binding pose for compound 11b having Glide energy: -
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63.45 kcalmol-1 and Glide XP score: -9.34 kcalmol-1 is predicted to form hydrogen bond
interactions between the OH group of hydroxymethyl substituent at tetrahydrofuran moiety of
the compound and main chain carbonyl group of Met98 residue of M. tuberculosis InhA (S27
Figure 5B).
5
6
One of the frontline drugs for treating TB is isonicotinic acid hydrazide (INH), which is an
inhibitor of NADH-dependent enoyl-ACP reductase (InhA). In fact, INH is a prodrug which
is first activated to acyl-radical by another enzyme, catalase-peroxidase (KatG). NADH
bound as a co-factor in InhA forms a covalent adducts with acyl-radical of INH, i.e. INH acyl
radical-NADH-InhA. Mutations associated with KatG accounts for 50 % of the INH-resistant
TB clinical isolates. Therefore, efforts were made to develop potent InhA inhibitors that are
capable of directly binding to InhA active site, avoiding activation by KatG. So far, none of
the direct inhibitors of bacterial InhA has been approved for the treatment of TB. This
signifies the importance of presently synthesized candidate drugs. Similarly, the interaction
of test compounds 6b and 11b with the active site of E.coli DNA gyrase B (S28 Figure 6)
and M. smegmatis DNA gyrase B (S29 Figure 7) was studied using molecular docking to
understand hydrogen bonding and hydrophobic interactions of the candidates [45-47]. The
interaction of compounds 6b and 11b was studied with the DNA gyrase B protein from E.
coli (S28 Figure 6). Both the active test compounds showed H-bond and hydrophobic
interactions similar to that of the co-crystallized ligand chlorobiocin in the active site of E.
coli DNA gyrase B protein [40(d)]. Compound 6b with Glide energy -60.34 kcal mol-1 and
Glide XPscore -9.21 kcal mol-1 showed H-bond interaction between the carbonyl oxygen
atom of pyran ring and side chain nitrogen atoms of Arg136 residue. Another H-bond
interaction between the two OH groups of furano sugar moiety and side chain oxygen atom
of Asp73 is observed in supporting information S28 Figure 6A. Compound 11b with Glide
energy -73.05 kcal mol-1 and Glide XPscore -9.96 kcal mol-1 shows H-bond interaction
between heteroatom oxygen of pyran ring and side chain nitrogen atom of Arg136 and
another H-bond interaction between the two OH groups of furano sugar moiety and side
chain oxygen atom of Asp73 residue. Further, a cation-π interaction is observed between side
chain of Arg76 residue of the protein and pyran ring of the compound 11b shown in
supporting information S28 Figure 6B. Such interaction is evident in the binding of
chlorobiocin (a coumarin analogue) in the active site of DNA gyrase B crystal structure.
Further the interaction of compounds 6b and 11b was predicted in the binding site of M.
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smegmatis DNA gyrase B active site. The binding pose of 6b having the best binding scores
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with Glide energy -59.30 kcal mol-1 and Glide XPscore -8.28 kcal mol-1 indicates hydrogen
bonding interaction between one of the secondary OH group of furano-sugar moiety of the
compound and oxygen atom of Asp79 side chain in supporting information S29 Figure 7A.
The interaction pattern of compound 11b with the Glide energy -54.33 kcal mol-1 and Glide
XPscore -8.22 kcal mol-1 is shown in supporting information S29 Figure 7B. In the case of
compound 11b, the OH group of hydroxymethyl substituent at tetrahydrofuran of 11b forms
hydrogen bond with oxygen atom of Asp79 side chain. Further, two hydrogen bonds were
formed with the side chain NH atoms of the conserved Arg141 and ethereal oxygen &
carbonyl oxygen of coumarin ring. Such interaction is evident in the binding of
aminopyrazinamide analogue in the active site of DNA gyrase B crystal structure [47].
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Cytotoxicity Study against THP-1 Macrophage Cell Line using MTT Assay
Out of sixteen tested compounds, N1-(β-
triazoles (5a-5d & 6a-6d) and N1-(β-
D
-ribofuranosyl)-C4-(coumarin-7-oxymethyl)-1,2,3-
-ribofuranosyl)-C4-(coumarin-7-yl)-1,2,3-triazoles
D
(10a-10d & 11a-11d); the four active compounds 5c, 6a, 6b and 6c having MIC ≤ 10 µM
were screened for their cytotoxicity on THP-1 macrophage cell line using MTT assay [3,47].
The cytotoxicity study was also carried out for the corresponding directly linked hydroxyl
conjugates 11b and 11c of the most active compounds 6b and 6c (Table 4). The stock
solutions of the test compounds were prepared with maximum concentration 150 µg/mL and
were used in serial double dilution for MTT assay. Cells were incubated with different
concentration of test compounds (4.68, 9.36, 18.72, 37.48, 75.00 and 150 µg/mL) and cell
viability was checked after 24 and 48 h (supporting information, S30, Table 1). All the six
tested compounds were found to be non-toxic to THP-1 cells even after the incubation of 48 h
at 150 µg/mL concentration; compounds 6a, 6b, 6c, 11b and 11c were almost as safe as
isoniazid and the other screened standard compound streptomycin under the same condition.
Among six tested compounds, THP-1 cells viability was found to be more than 90 % for
compounds 6b, 6c and 11b after 24 h of incubation and up to the concentration 48.1 µM, 41.5
µM and 52.1 µM, respectively (37.48 µg/mL). The two most active compounds, 6b (IC50
>385 µM) and 6c (IC50 >332 µM) were found to be safer than streptomycin at the highest
studied concentration i.e., 385 µM (150 µg/mL) and 332 µM (150 µg/mL), respectively even
after 48 h of incubation (supporting information, S30, Table 1). Antimycobacterial activity is
considered to be specific when the selectivity index is >10. The selectivity index indicates the
range of concentration of the compound on which it is active against mycobacteria but not
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toxic towards host cells [3]. Although the therapeutic index of isoniazid is very high, it is
important to consider the significance of this study with respect to the developing resistance
among microorganisms against available antibiotics. In the current work all the compounds
exhibited good selectivity index i.e. SI >10, indicating their potential as an antimycobacterial
agent, and can be explored further for drug development.
Compound No.
IC50
Selectivity Index (SI)
(IC50/MIC)
(µM)
547
2735
600
37.6
37.7
75.4
75.4
25
Isoniazid
>258
>196
>400
>385
>332
>417
>355
Streptomycin
5c
6a
6b
6c
11b
11c
25
7
8
9
Table 4: IC50 , Selectivity index and Cell viability (%) of human THP1 cell line after 48 h treatment
with test compounds and first line drug isoniazid and streptomycin at different concentrations.
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Conclusion
Sixteen novel β- -ribofuranosyl coumarinyl 1,2,3-triazole conjugates have been synthesized
D
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using Cu-catalyzed 1,3-dipolar cycloaddition reaction of azidosugar with 7-O- / 7-
alkynylated coumarins in good yields. These compounds possess moderate to high
antimycobacterial activity against M. tuberculosis sensitive reference strain H37Rv as well as
multidrug resistant clinical isolate 591. Compounds 5c, 6b and 6c were most active having
MICs ≤ 5.2 µM against sensitive reference strain H37Rv and MICs ≤ 10.3 µM in case of
multidrug resistant clinical isolate 591. Notably, the activity of compounds 5c, 6b and 6c
against MDR clinical isolate 591 was many folds higher than the activity of first line
antitubercular drugs. The most bactericidal compound N1-(β-
D
-ribofuranosyl)-C4-(4-
methylcoumarin-7-oxymethyl)-1,2,3-triazole (6b) and its directly linked conjugate N1-(β-
D-
ribofuranosyl)-C4-(4-methylcoumarin-7-yl)-1,2,3-triazole (11b) were direct inhibitors of
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bacterial InhA and DNA gyrase B bacterial enzymes and interferes with the constitution of
call wall to exhibit its antimycobacterial activity. Isonicotinic acid hydrazide (INH) is the first
line drug for the treatment of TB, which acts as an inhibitor of InhA. However, INH acts as
prodrug and has to be activated by another enzyme KatG to act as an active inhibitor of InhA.
This signifies the importance of development of direct inhibitors of InhA as compounds 6b
and 11b. The cytotoxicity study revealed that the most active synthesised compounds 5c, 6a,
6b and 6c, as well as the compounds 11b and 11c, which are directly linked conjugates of
compounds 6b and 6c, respectively are safe against human THP1 cell line.
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Experimental Section
General
Reactions were conducted under nitrogen atmosphere, when anhydrous solvents were used.
Melting points were determined on Buchi M-560 instrument and are uncorrected. The IR
spectra were recorded on a Perkin-Elmer model 2000 FT-IR spectrometer by making KBr disc
for solid samples and thin film for oils. The 1H- and 13C NMR spectra were recorded on a Jeol
alpha-400 spectrometer at 400 and 100.6 MHz, respectively, using TMS as internal standard.
The chemical shift values are on δ scale and the coupling constants (J) are in Hz. Signals from
OH groups in 1H NMR spectra were verified by removing them by shaking the NMR solution
with D2O. Mass spectra were recorded on Agilent-G6530AA high-resolution mass
spectrometer in positive ion mode. HRMS-ESI-TOF analyses were carried out on a microTOF
instrument from Bruker Daltonics, Bremen on ESI positive mode. Analytical TLCs were
performed on precoated Merck silica-gel 60F254 plates; the spots were detected under UV light
and carbohydrate compounds were detected by charring using 4 % H2SO4 in ethanol solution.
Silica gel (100-200 mesh) was used for column chromatography. Chemicals were obtained
from commercial suppliers and were used without any further purification unless otherwise
noted. The single crystal X-ray diffraction data was collected on an Oxford Diffraction
X’Calibur single crystal X-ray instrument having CCD camera [Cu Kα radiation (λ =
0.71073)] at USIC, University of Delhi, Delhi.
General procedure for the synthesis of N1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-C4-
(methylenoxy-coumarin-7-yl)-1,2,3-triazoles (5a-5d)
To a stirred solution of 7-propargyloxycoumarin (4a-4d, 0.20 g, 0.06 mmol) and 1-azido sugar
(2, 0.066 mmol) in a solvent mixture of THF:tert-BuOH:Water (1:1:1, 30 mL) were added
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copper sulphate (0.024 mmol) and sodium ascorbate (0.048 mmol). Reaction mixture was
stirred for 10-15 h at room temperature. On completion of reaction (checked by analytical
TLC), the reaction mixture was concentrated on rotavapour and coevaporated with toluene (2 x
20 mL) to remove water completely from reaction mixture. The crude thus obtained was
purified by silica gel column chromatography using methanol in chloroform as gradient solvent
system.
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8
N1-(2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl)-C4-(coumarin-7-oxymethyl)-1,2,3-triazole
9
(5a)
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It was obtained as off-white solid (0.53 g, 78 % yield); Rf = 0.5 (10 % methanol in
o
chloroform). M.Pt.: 120-122 C; IR (KBr)νmax: 1719, 1610, 1261, 1118, 1022, 835 and 754
1
cm-1; H NMR (CDCl3, 400 MHz): δ 8.04-7.94 (6H, m), 7.88 (1H, s), 7.64-7.36 (11H, m),
6.87 (2H, s), 6.45 (1H, d, J = 4.0 Hz), 6.28 (1H, s), 6.26 (1H, t, J = 4.0 Hz), 6.14 (1H, t, J =
5.2 Hz), 5.15 (2H, q, J = 10.0 Hz), 4.92-4.82 (2H, m) and 4.60 (1H, dd, J = 12.0, 4.0 Hz); 13C
NMR (CDCl3, 100.6 MHz): δ 166.05, 165.12, 165.04, 161.13, 161.09, 155.66, 143.51,
143.31, 133.93, 133.75, 133.46, 129.86, 129.78, 129.72, 128.89, 128.60, 128.58, 128.52,
128.33, 122.27, 113.50, 113.01, 112.63, 102.01, 90.43, 81.19, 75.23, 71.41, 63.46 and 61.98.
HRMS: m/z calculated for [C38H29N3O10+H+] 688.1931, observed 688.1924.
N1-(2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl)-C4-(4-methylcoumarin-7-oxymethyl)-1,2,3-
triazole (5b)
It was obtained as white solid (0.52 g, 80 % yield); Rf = 0.5 (10 % methanol in chloroform).
o
1
M.Pt.: 230-234 C; IR (KBr)νmax: 1723, 1612, 1267, 1119, 753 and 710 cm-1; H NMR
(CDCl3, 400 MHz): δ 8.04-7.94 (6H, m), 7.90 (1H, s), 7.60-7.36 (10H, m), 6.90-6.86 (2H,
m), 6.46 (1H, d, J = 2.8 Hz), 6.27 (1H, t, J = 8.0 Hz), 6.16-6.13 (2H, m), 5.14 (2H, q, J = 8.8
13
Hz), 4.91-4.82 (2H, m), 4.59 (1H, dd, J = 12.0, 4.4 Hz) and 2.39 (3H, s); C NMR (CDCl3,
100.6 MHz): δ 165.99, 165.07, 164.97, 161.14, 160.90, 154.97, 152.45, 133.85, 133.69,
133.38, 129.78, 129.70, 129.65, 129.11, 128.53, 128.49, 128.45, 128.28, 125.64, 122.42,
114.00, 112.24, 112.18, 101.96, 90.37, 81.08, 75.16, 71.37, 63.41, 61.85 and 18.60. HRMS:
m/z calculated for [C39H31N3O10+H+] 702.2088, observed 702.2084.
N1-(2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl)-C4-(4-phenylcoumarin-7-oxymethyl)-1,2,3-
triazole (5c)
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It was obtained as white fluffy solid (0.41 g, 75 % yield); Rf = 0.5 (10 % methanol in
o
chloroform). M.Pt.: 151-153 C; IR (KBr)νmax: 1721, 1607, 1265, 1150, 1119 and 755 cm-1;
3
1H NMR (CDCl3, 400 MHz): δ 8.04-7.89 (7H, m), 7.61-7.36 (15H, m), 6.95 (1H, s), 6.83
(1H, dd, J = 9.2, 2.0 Hz), 6.46 (1H, d, J = 3.2 Hz), 6.27-6.24 (2H, m), 6.14 (1H, t, J = 5.2
Hz), 5.16 (2H, dd, J = 22.0, 12.0 Hz), 4.92-4.83 (2H, m) and 4.60 (1H, dd, J = 12.4, 4.8 Hz);
13C NMR (CDCl3, 100.6 MHz): δ 166.04, 165.11, 165.03, 161.10, 155.77, 155.69, 143.50,
135.42, 133.92, 133.74, 133.45, 129.86, 129.78, 129.71, 129.61, 128.83, 128.56, 128.51,
128.36, 128.14, 122.29, 113.02, 112.32, 112.20, 102.27, 90.44, 81.19, 75.24, 71.42, 63.45
and 61.96. HRMS: m/z calculated for [C44H33N3O10+H+] 764.2244, observed 764.2223.
4
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N1-(2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl)-C4-(6-chloro-4-methylcoumarin-7-
oxymethyl)-1,2,3-triazole (5d)
It was obtained as light yellow fluffy solid (0.45 g, 77 % yield); Rf = 0.5 (10 % methanol in
o
chloroform). M. Pt.: 155-157 C; IR (KBr)νmax: 1720, 1606, 1259, 1158, 1098, 754 and 709
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cm-1; H NMR (CDCl3, 400 MHz): δ 8.00-7.88 (6H, m), 7.89 (1H, s), 7.57-7.33 (10H, m),
6.98 (1H, s), 6.44 (1H, d, J = 2.8 Hz), 6.26 (1H, t, J = 8 Hz), 6.15 (1H, s), 6.13 (1H, t, J = 5.2
Hz), 5.17 (2H, q, J = 12.4 Hz), 4.89-4.79 (2H, m), 4.57 (1H, dd, J = 12.0, 4.8 Hz) and 2.34
13
(3H, s); C NMR (CDCl3, 100.6 MHz): δ 166.02, 165.08, 165.00, 160.49, 156.02, 153.33,
151.47, 143.02, 133.87, 133.70, 133.38, 129.83, 129.75, 129.69, 129.13, 128.56, 128.48,
128.33, 125.49, 122.56, 119.22, 114.29, 113.17, 101.69, 90.38, 81.17, 75.18, 71.40, 63.42,
63.00 and 18.61. HRMS: m/z calculated for [C39H3035ClN3O10+H+] 736.1698, observed
736.1687.
General procedure for the synthesis of N1-(β-D-ribofuranosyl)-C4-(coumarin-7-
oxymethyl)-1,2,3-triazoles (6a-6d)
A solution of compound 5a-5d (0.2 g, 0.05 mmol) was taken in a round bottom flask in
methanol solvent (20 mL) and sodium methoxide (0.1mmol) was added into it, reaction
mixture was stirred at room temperature upto the completion of the reaction, checked on
analytical TLC. After the completion of reaction, it was neutralized using seralite (H+) resin and
filtered and solvent was evaporated under vaccum. The crude thus obtained was purified by
silica gel column chromatography using methanol in chloroform as gradient solvent system.
N1-(β-D-Ribofuranosyl)-C4-(coumarin-7-oxymethyl)-1,2,3-triazole (6a)
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It was obtained as off white solid (0.089 g, 82 % yield); Rf = 0.3 (10 % methanol in
chloroform). M.Pt.: 162-164 oC; IR (KBr)νmax: 3302, 2922, 1729, 1605, 1399, 1032, and 826
cm-1; 1H NMR (DMSO-d6, 400 MHz): δ 8.51 (1H, s), 7.98 (1H, d, J = 9.2 Hz), 7.63 (1H, d, J
= 8.0 Hz), 7.15 (1H, s), 7.03 (1H, dd, J = 4.4, 2.0 Hz), 6.31 (1H, d, J = 9.6 Hz), 5.96 (1H, d,
J = 4.4 Hz), 5.60 (1H, d, J = 6.0 Hz), 5.28 (1H, s), 5.26 (2H, d, J = 5.2 Hz), 5.00 (1H, t, J =
6.0 Hz), 4.39 (1H, q, J = 6.0 Hz), 4.13 (1H, q, J = 5.2 Hz), 3.98 (1H, q, J = 4.4 Hz) and 3.64-
3
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5
6
13
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3.48 (2H, m); C NMR (DMSO-d6, 100.6 MHz): δ 161.10, 160.11, 155.31, 144.30, 142.30,
8
129.54, 123.55, 112.85, 112.73, 112.64, 101.57, 92.09, 85.94, 75.15, 70.39, 61.62 and 61.30.
9
HRMS: m/z calculated for [C17H17N3O7+H+] 376.1145, observed 376.1135.
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N1-(β-D-Ribofuranosyl)-C4-(4-methylcoumarin-7-oxymethyl)-1,2,3-triazole (6b)
It was obtained as white solid (0.094 g, 85 % yield); Rf = 0.3 (10 % methanol in chloroform).
o
M.Pt.: 160-162 C; IR (KBr)νmax: 3409, 1699, 1613, 1395, 1280, 1113, 1023 and 826 cm-1;
1H NMR (DMSO-d6, 400 MHz): δ 8.51 (1H, s), 7.68 (1H, d, J = 8.8 Hz), 7.14 (1H, s), 7.03
(1H, d, J = 9.2 Hz), 6.21 (1H, s), 5.95 (1H, d, J = 3.6 Hz), 5.62 (1H, d, J = 3.6 Hz), 5.27 (3H,
s), 5.00 (1H, t, J = 5.2 Hz), 4.38-3.96 (3H, m), 3.61-3.48 (2H, m) and 2.38 (3H, s); 13C NMR
(DMSO-d6, 100.6 MHz): δ 161.02, 160.00, 154.68, 153.46, 142.34, 126.57, 123.55, 113.44,
112.58, 111.34, 101.60, 92.08, 85.95, 75.16, 70.40, 61.61, 61.31 and 18.19. HRMS: m/z
calculated for [C18H19N3O7+H+] 390.1296, observed 390.1287.
N1-(β-D-Ribofuranosyl)-C4-(4-phenylcoumarin-7-oxymethyl)-1,2,3-triazole (6c)
It was obtained as off white solid (0.10 g, 85 % yield); Rf = 0.3 (10 % methanol in
o
chloroform). M.Pt.: 191-192 C; IR (KBr)νmax: 3238, 1716, 1605, 1378, 1027 and 813 cm-1;
1H NMR (DMSO-d6, 400 MHz): δ 8.52 (1H, s), 7.55-7.51 (5H, m), 7.35 (1H, d, J = 9.2 Hz),
7.27 (1H, d, J = 2.0 Hz), 7.00 (1H, dd, J = 9.2, 2.4 Hz), 6.25 (1H, s), 5.95 (1H, d, J = 4.4 Hz),
5.63 (1H, s), 5.29 (3H, s), 5.01 (1H, s), 4.37 (1H, t, J = 4.4 Hz), 4.11 (1H, s), 3.96 (1H, d, J =
4.0 Hz) and 3.61-3.48 (2H, m); 13C NMR (DMSO-d6, 100.6 MHz): δ 161.12, 159.98, 155.36,
154.93, 142.21, 134.88, 129.64, 128.82, 128.39, 127.82, 123.60, 112.89, 112.13, 111.57,
102.03, 92.05, 85.88, 75.10, 70.36, 61.65 and 61.26. HRMS: m/z calculated for
[C23H21N3O7+Na+] 474.1277, observed 474.1267.
N1-(β-D-Ribofuranosyl)-C4-(6-chloro-4-methylcoumarin-7-oxymethyl)-1,2,3-triazole (6d)
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It was obtained as white solid (0.09 g, 80 % yield); Rf = 0.3 (10 % methanol in chloroform).
o
M.Pt.: 114-116 C; IR (KBr)νmax: 3358, 2930, 1724, 1609, 1387, 1280, 1207, 1044 and 848
3
cm-1; 1H NMR (DMSO-d6, 400 MHz): δ 8.54 (1H, s), 7.83 (1H, s), 7.48 (1H, s), 6.29 (1H, s),
5.97 (1H, d, J = 4.8 Hz), 5.61 (1H, d, J = 6.4 Hz), 5.37 (2H, s), 5.25 (1H, d, J = 5.2 Hz), 4.98
(1H, t, J = 5.2 Hz), 4.39 (1H, q, J = 4.4 Hz), 4.11 (1H, q, J = 4.4 Hz), 3.97 (1H, q, J = 4.8
Hz), 3.63-3.42 (2H, m) and 2.39 (3H, s); 13C NMR (DMSO-d6, 100.6 MHz): δ 159.75,
155.80, 153.24, 152.72, 141.82, 126.01, 123.65, 117.73, 114.03, 112.22, 102.16, 92.09,
85.96, 75.12, 70.39, 62.57, 61.22 and 18.24. HRMS: m/z calculated for
[C18H1835ClN3O7+Na+] 446.0731, observed 446.0739.
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4-Phenyl-7-trimethylsilylethynylcoumarin (8c)
4-Phenyl-7-triflouromethanesulfonyloxycoumarin (7c, 0.8 g, 0.13 mmol), copper iodide (0.02
g, 0.013 mmol) and tetrakis triphenylphosphine palladium (0) (0.03 g, 0.006 mmol) were taken
in round bottom flask under argon atmosphere. Triethyl amine (0.29 mL, 0.26 mmol) and
acetonitrile was added into the reaction mixture at 0 oC and the reaction mixture was stirred at
room temperature for 5-6 h. Reaction mixture was concentrated under reduced pressure and the
crude thus obtained was purified by silica gel column chromatography using ethyl acetate in
petroleum ether as gradient solvent system. It was obtained as light yellow solid (0.48 g, 70 %
yield); Rf = 0.5 (15 % EtOAc in Pet. ether); M.Pt. = 167-169 C; IR (cm-1, KBr) νmax: 3058,
o
1
2954, 1725, 1606 and 842cm-1; H NMR (400 MHz, CDCl3): δ 7.51-7.37 (8H, m), 6.34 (1H, s)
13
and 0.25 (9H, s); C NMR (100.6 MHz, CDCl3): δ 160.66, 155.29, 153.99, 135.09, 130.04,
129.17, 128.61, 127.86, 127.05, 126.97, 120.55, 119.17, 115.64, 103.37 and 98.87; HRMS: m/z
calculated for [C20H18O2Si+H]+ 319.1154, observed 319.1164.
7-Ethynyl-4-phenylcoumarins (9c)
4-Phenyl-7-trimethylsilylethynylcoumarin (8c, 0.4 g, 0.1 mmol) was dissolved in methanol (15
ml) and potassium carbonate (0.08 g, 0.44 mmol) was added to the reaction. After 1 hr stirring at
rt, the completion of the reaction was checked by TLC. Reaction mixture was filtered and washed
with methanol 2-3 times and the solvent obtained was removed on rotavapour. The crude thus
obtained was purified by silica gel column chromatography using ethyl acetate in petroleum ether
as gradient solvent system. It was obtained as light orange solid (0.23 g, 75 % yield); Rf = 0.4
(20 % EtOAc in Pet. ether); M.Pt. = 148-150 oC; IR (cm-1, KBr) νmax: 1724, 1607, 1491 and
1
759 cm-1; H NMR (400 MHz, CDCl3): δ 7.55-7.30 (8H, m), 6.39 (1H, s) and 3.28 (1H, s); 13C
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NMR (100.6 MHz, CDCl3): δ 160.19, 155.28, 153.65, 134.57, 129.86, 128.97, 128.37, 127.74,
126.92, 125.74, 120.65, 119.22, 115.64, 82.18 and 80.69; HRMS: m/z calculated for
[C17H10O2+H]+ 247.0749, observed 247.0754.
3
4
5
General procedure for the synthesis of N1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-C4-
(coumarin-7-yl)-1,2,3-triazoles (10a-10d)
6
7
To a stirred solution of 7-ethynylcoumarin (9a-9d, 0.2 g, 0.06 mmol) and 1-azido sugar (2,
0.066 mmol) in a solvent mixture of THF:tert-BuOH:Water (1:1:1, 30 mL) were added copper
sulphate (0.024 mmol) and sodium ascorbate (0.048 mmol). Reaction mixture was stirred for
10-15 h at room temperature. On completion, reaction mixture was concentrated on rotavapour
and coevaporated with toluene (2 x 20 mL) to remove water completely from reaction mixture.
The crude thus obtained was purified by silica gel column chromatography using methanol in
chloroform as gradient solvent system.
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N1-(2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl)-C4-(coumarin-7-yl)-1,2,3-triazole (10a)
It was obtained as white solid (0.59 g, 77 % yield); Rf = 0.5 (10 % methanol in chloroform).
M.Pt.: 124-126 oC; IR (KBr)νmax: 1728, 1618, 1267, 1108, 985, 757 and 711 cm-1; 1H NMR
(CDCl3, 400 MHz): δ 8.09-7.97 (7H, m), 7.71-7.39 (13H, m), 6.54 (1H, d, J = 3.6 Hz), 6.43
(1H, d, J = 9.6 Hz), 6.29 (1H, d, J = 5.2 Hz), 6.16 (1H, t, J = 5.6 Hz), 4.96-4.92 (2H, m) and
13
4.62 (1H, dd, J = 14.8, 4.4 Hz) ; C NMR (CDCl3, 100.6 MHz): δ 166.14, 165.15, 165.05,
160.61, 154.32, 146.60, 142.96, 133.93, 133.77, 133.66, 133.54, 129.86, 129.79, 129.65,
129.08, 128.75, 128.59, 128.54, 128.29, 121.74, 119.57, 118.59, 116.61, 113.52, 90.48,
81.40, 75.23, 71.48 and 63.34. HRMS: m/z calculated for [C37H27N3O9+Na+] 680.1645,
observed 680.1623.
N1-(2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl)-C4-(4-methylcoumarin-7-yl)-1,2,3-triazole
(10b)
It was obtained as white fluffy solid (0.59 g, 82 % yield); Rf = 0.5 (10 % methanol in
o
chloroform). M.Pt.: 170-172 C; IR (KBr)νmax: 1716, 1620, 1257, 1092, 1026, 859 and 754
cm-1; 1H NMR (CDCl3, 400 MHz): δ 8.08-7.96 (7H, m), 7.69-7.39 (12H, m), 6.54 (1H, d, J =
3.6 Hz), 6.31-6.29 (2H, m), 6.17 (1H, t, J = 5.6 Hz), 4.95-4.90 (2H, m), 4.62 (1H, dd, J =
13
12.0, 5.6 Hz) and 2.45 (3H, s); C NMR (CDCl3, 100.6 MHz): δ 166.05, 165.14, 165.05,
160.65, 153.77, 151.99, 146.56, 133.91, 133.75, 133.64, 133.44, 129.86, 129.78, 129.65,
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129.09, 128.72, 128.58, 128.53, 128.31, 125.08, 121.49, 119.72, 119.62, 115.05, 113.65,
90.47, 81.38, 75.23, 71.52, 63.37 and 18.60. HRMS: m/z calculated for [C38H29N3O9+Na+]
694.1796, observed 694.1790.
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4
5
N1-(2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl)-C4-(4-phenylcoumarin-7-yl)-1,2,3-triazole
(10c)
6
7
It was obtained as yellow fluffy solid (0.47 g, 80 % yield); Rf = 0.5 (10 % methanol in
chloroform). M.Pt.: 150-152 oC; IR (KBr)νmax: 1722, 1260, 1093, 1026, 804 and 754 cm-1; 1H
NMR (CDCl3, 400 MHz): δ 8.08-7.97 (7H, m), 7.65-7.38 (17H, m), 6.54 (1H, d, J = 3.2 Hz),
6.39 (1H, s), 6.29 (1H, q, J = 3.6 Hz), 6.17 (1H, t, J = 5.2 Hz), 4.95-4.91 (2H, m) and 4.62
(1H, dd, J = 13.2, 4.8 Hz); 13C NMR (CDCl3, 100.6 MHz): δ 166.16, 166.05, 165.20, 165.13,
165.05, 160.66, 155.27, 154.43, 146.44, 133.90, 133.75, 133.70, 133.63, 133.56, 133.24,
129.85, 129.77, 129.68, 129.65, 129.42, 129.08, 128.89, 128.71, 128.57, 128.52, 128.40,
128.29, 127.46, 121.45, 119.80, 118.66, 114.99, 113.87, 93.24, 90.46, 81.36, 79.78, 75.23,
71.47, 71.40, 63.70 and 63.35. HRMS: m/z calculated for [C43H31N3O9+H+] 734.2139,
observed 734.2140.
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N1-(2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl)-C4-(6-chloro-4-methylcoumarin-7-yl)-1,2,3-
triazole (10d)
It was obtained as yellow solid (0.5 g, 78% yield); Rf = 0.5 (10 % Methanol in Chloroform).
M.Pt.: 187-189 oC; IR (KBr)νmax: 1727, 1612, 1452, 1267, 1098, 809 cm-1; 1H NMR (CDCl3,
400 MHz): δ 8.44 (1H, s), 8.13 (1H, s), 7.98-7.90 (6H, m), 7.53-7.32 (10H, m), 6.47 (1H, d, J
= 3.6 Hz), 6.26 (2H, s), 6.14 (1H, t, J = 5.2 Hz), 4.87-4.79 (2H, m), 4.55 (1H, dd, J = 12.0,
3.6 Hz) and 2.32 (3H, s) ; 13C NMR (CDCl3, 100.6 MHz): δ 166.11, 165.15, 165.10, 160.14,
151.90, 150.71, 143.10, 133.93, 133.75, 133.44, 131.69, 129.87, 129.80, 129.72, 129.06,
128.54, 128.34, 127.71, 126.44, 125.87, 123.02, 120.53, 117.61, 116.31, 90.39, 81.41, 75.23,
71.58, 63.47 and 18.44. HRMS: m/z calculated for [C38H2835ClN3O9+Na+] 728.1412,
observed 728.1440.
General procedure for the synthesis of N1-(β-D-Ribofuranosyl)-C4-(coumarin-7-yl)-1,2,3-
triazoles (11a-11d)
A solution of compound 10a-10d (0.2 g, 0.05mmol) in methanol was taken in a round bottom
flask, and sodium methoxide (0.1mmol), was added into it, reaction mixture was stirred at rt
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upto the completion of the reaction, checked on analytical TLC. The solution was neutralized
using seralite (H+) resin and filtered and solvent was evaporated under vaccum. The crude thus
obtained was purified by silica gel column chromatography using methanol in chloroform as
gradient solvent system.
3
4
5
6
N1-(β-D-Ribofuranosyl)-C4-(coumarin-7-yl)-1,2,3-triazole (11a)
7
It was obtained as white solid (0.08 g, 80 % yield); Rf = 0.3 (10 % methanol in chloroform).
o
1
8
M.Pt.: 220-222 C; IR (KBr)νmax: 3410, 2924, 1690, 1616, 1110 and 852 cm-1.; H NMR
(DMSO-d6, 400 MHz): δ 8.96 (1H, s), 8.08 (1H, d, J = 10.0 Hz), 7.88-7.79 (3H, m), 6.49
(1H, d, J = 9.2 Hz), 5.97 (1H, d, J = 4.4 Hz), 5.68 (1H, d, J = 5.6 Hz), 5.30 (1H, d, J = 5.6
Hz), 5.01 (1H, t, J = 5.2 Hz), 4.42 (1H, q, J = 5.6 Hz), 4.15 (1H, q, J = 4.8 Hz), 3.99 (1H, q, J
= 3.6 Hz) and 3.65-3.50 (2H, m); 13C NMR (DMSO-d6, 100.6 MHz): δ 159.65, 145.01,
144.30, 129.09, 121.72, 118.51, 116.03, 112.23, 92.15, 86.14, 74.56, 69.98 and 61.22.
HRMS: m/z calculated for [C16H15N3O6+H+] 346.1034, observed 346.1035.
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N1-(β-D-Ribofuranosyl)-C4-(4-methylcoumarin-7-yl)-1,2,3-triazole (11b)
It was obtained as white solid (0.09 g, 85 % yield); Rf = 0.3 (10 % methanol in chloroform).
o
1
M.Pt.: 213-215 C; IR (KBr)νmax: 3427, 2939, 1669, 1617, 1057, 1014 and 852 cm-1.; H
NMR (DMSO-d6, 400 MHz): δ 8.96 (1H, s), 7.87-7.80 (3H, m), 6.38 (1H, d, J = 1.6 Hz),
5.98 (1H, d, J = 4.4 Hz), 5.69 (1H, d, J = 3.7 Hz), 5.31 (1H, d, J = 5.2 Hz), 5.00 (1H, t, J =
4.8 Hz), 4.43 (1H, q, J = 8.0, 4.4 Hz), 4.16 (1H, q, J = 12.0, 5.2 Hz), 4.00 (1H, q, J = 8.8, 4.4
Hz), 3.65-3.48 (2H, m) and 2.43 (3H, s); 13C NMR (DMSO-d6, 100.6 MHz): δ 159.79,
153.53, 153.07, 145.25, 133.98, 126.30, 121.40, 121.06, 119.25, 114.54, 112.46, 92.36,
86.01, 75.15, 70.33, 61.36 and 18.11. HRMS: m/z calculated for [C17H17N3O6+H+] 360.1191,
observed 360.1190.
N1-(β-D-Ribofuranosyl)-C4-(4-phenylcoumarin-7-yl)-1,2,3-triazole (11c)
It was obtained as light yellow solid (0.09 g, 82 % yield); Rf = 0.3 (10 % methanol in
o
chloroform). M.Pt.: 160-162 C; IR (KBr)νmax: 3281, 2926, 1681, 1622, 1387, 1134 and 839
cm-1; 1H NMR (DMSO-d6, 400 MHz): δ 8.99 (1H, s), 7.93 (1H, s), 7.86 (1H, d, J = 8.4 Hz),
7.58-7.53 (6H, m), 6.43 (1H, s), 5.98 (1H, d, J = 4.4 Hz), 5.70 (1H, d, J = 5.2 Hz), 5.35 (1H,
dd, J = 14.8, 3.6 Hz), 5.03 (1H, brs), 4.43 (1H, q, J = 4.4 Hz), 4.15 (1H, q, J = 4.4 Hz), 3.99
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(1H, q, J = 4.4 Hz), 3.67-3.52 (2H, m); 13C NMR (DMSO-d6, 100.6 MHz): δ 159.88, 154.28,
145.01, 134.35, 129.58, 128.76, 121.93, 121.38, 114.14, 113.05, 94.75, 92.02, 85.70, 84.66,
75.24, 74.82, 70.03, 62.12 and 60.99. HRMS: m/z calculated for [C22H19N3O6+H+] 422.1352,
observed 422.1336.
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4
5
6
N1-(β-D-Ribofuranosyl)-C4-(6-chloro-4-methylcoumarin-7-yl)-1,2,3-triazole (11d)
7
It was obtained as light yellow solid (0.09 g, 80 % yield); Rf = 0.3 (10 % methanol in
o
8
chloroform). M.Pt.: 138-140 C; IR (KBr)νmax: 3484, 2914, 2116, 1665, 1606, 1388, 1244,
9
1056 and 753 cm-1; 1H NMR (DMSO-d6, 400 MHz): δ 9.14 (1H, s), 8.00 (2H, d, J = 5.4 Hz),
6.49 (1H, s), 6.08 (1H, d, J = 4.0 Hz), 5.74 (1H, d, J = 5.6 Hz), 5.31 (1H, d, J = 5.6 Hz), 5.15
(1H, t, J = 5.6 Hz), 4.45 (1H, q, J = 5.2 Hz), 4.21 (1H, q, J = 5.2 Hz), 4.03 (1H, q, J = 4.0
10
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12
13
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16
13
Hz), 3.72-3.54 (2H, m) and 2.46 (3H, s); C NMR (DMSO, 100.6 MHz): δ 159.55, 152.14,
151.95, 142.22, 132.10, 127.58, 125.44, 123.89, 120.77, 116.39, 115.71, 93.05, 85.99, 75.06,
69.73, 61.45, 17.74. HRMS: m/z calculated for [C17H1635ClN3O6+H+] 394.0806, observed
394.0808.
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Compound code
6b
11b
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C18 H19 N3 O7
389.36
293(2) K
0.71073 A°
C17 H17 N3 O6
359.33
293(2) K
0.71073 A°
Orthorhombic
P 21 21 21
Monoclinic
P 21
Unit cell dimensions
a = 7.107(5) A° alpha = 90 deg
b = 15.119(5) A° beta = 90 deg.
c = 16.055(5) A° gamma = 90 deg.
a = 6.7824(5) A° alpha = 90 deg.
b = 8.0573(5) A° beta = 90 deg.
c = 29.495(2) A° gamma = 90 deg.
1611.86(19) A°3
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Volume
Z
Calculated density
Absorption coefficient
F(000)
1716.8(14) A°3
4
1.506 Mg/m3
0.120 mm-1
704
1.427 Mg/m3
0.111 mm-1
724
Theta range for data collection
2.88 to 25.00 deg.
2.88 to 25.00 deg.
Limiting indices
- 8<=h<=8, -17<=k<=17, -19<=l<=19
20161/6024 [R(int) = 0.1248]
= 25.00 99.8 %
-8<=h<=8, -9<=k<=9, -35<=l<=35
18672 / 2844 [R(int) = 0.0777]
= 25.00 99.8 %
Reflections collected / unique
Completeness to theta
Refinement method
Data / restraints / parameters
Goodness-of-fit on F^2
Final R indices [I>2sigma (I)]
R indices (all data)
Absolute structure parameter
Largest diff. peak and hole
CCDC No.
Full-matrix least-squares on F2
6024/1/505
Full-matrix least-squares on F2
2844 /0/235
0.908
0.806
R1 = 0.0836, wR2 = 0.2001
R1 = 0.1858, wR2 = 0.2863
3(3)
R1 = 0.0473, wR2 = 0.1344
R1 = 0.0523, wR2 = 0.1394
0.7(15)
0.308 and -0.300 e.A°-3
1535099
0.343 and -0.354 e.A°-3
1419805
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Table 5. Single crystal X-ray diffraction data of compound 6b and 11b
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Experimental: Biology
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Antimycobacterial Activity (MIC & MBC study)
The strains and isolates used for antimycobacterial activity study comprised of the reference
strain M. tuberculosis H37Rv and the MDR clinical isolate 591. They were obtained from the
Department of Microbiology, V. P. Chest Institute, Delhi, India. The cultures were
maintained on Middlebrook 7H9 medium (Difco Laboratories, MI, USA). The MICs of the
series of test compounds and the first line drugs isoniazid, rifampicin, ethambutol and
streptomycin, taken as reference standards, were determined using the micro-plate alamar
blue assay (MABA) [3,34,35]. Sterile water (200 mL) was added to all the boundary wells of
a 96-well U-bottom plate to minimize evaporation and maintain humidity. A single-cell
suspension was prepared from a log-phase culture of M. tuberculosis by passing it through an
8 µm filter (Millipore). The Optical Density at 600 nm was measured using a Cary UV
spectrophotometer and the cell density was then calculated using McFarland standards. The
required amount of 7H9 medium, the calculated amount of the drug and 1x106 cells were
added to each well, so as to make up the volume to 200 mL. Positive (medium+inoculum)
and negative (medium only) controls were also included. The plates were then sealed with
parafilm and incubated aerobically at 37 °C. After 10 days of incubation, 30 mL of 0.02 %
resazurin solution was added to each well and the plates were again incubated overnight at
37 °C before being observed for colour change. A colour change from blue to pink was
considered as growth and the MIC value was recorded as the lowest drug concentration that
prevented visible growth/colour change. A similar procedure was also followed for MDR
clinical isolate 591 and the dye was added on the fifteenth day of incubation. Different range
of drug concentrations was checked to determine the exact MIC and the experiments were
done in triplicate to check the result reproducibility. MBCs were determined using the
method adapted from a previously reported protocol [3]. MBC study was carried out directly
from the 96-well plates used for MIC determination. Observations were taken on days 15, 21
and 28. The MBC was recorded as the minimum concentration of the drug that led to 99 %
inhibition of growth at the end of 4 weeks of incubation. A compound is considered to be
bactericidal if the ratio of MBC/MIC is ≤ 4 and if the MBC/MIC is > 4 it is considered to be
bacteriostatic in nature. MBC/MIC ratios were also calculated to determine whether the
compounds are bacteriostatic or bactericidal in nature.
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In silico Pharmacokinetic Property Predictions
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The active series of compounds were further subjected to in silico pharmacokinetic prediction
to gather information about certain important pharmacokinetic parameters, such as
lipophilicity, solubility, hydrogen bond accepters and hydrogen bond donors. Isoniazid was
taken as the reference standard for comparing the compound series. The said properties were
predicted considering parameters of Lipinski filter using online drug likeness predicting
software Molinspiration. This software uses certain mathematical models to quantitatively
predict properties by a set of rules/keys that specify threshold pharmacokinetic characteristics
for the chemical structure of the molecules, based on the available drug information.
Lipophilicity is estimated from log P values. The model is based on a genetic partial least
squares method on a training set of 784 compounds with experimentally measured
solubilities. This property explorer software enables the prediction of drug-relevant properties
for any valid structure [36-38]. Structures can be drawn online using this software and the
results can be seen immediately. The prediction results as obtained are valued.
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Transmission Electron Microscopy Studies
Transmission electron microscopy of the untreated as well as the drug-treated bacterial
samples was carried out in order to examine the changes in cell morphology. Sensitive
reference strain H37Rv was grown in the presence and absence of sub-lethal doses (1/4th
MIC) of the chosen test compounds 6b, 11b and first-line drug INH. The initial inoculum
was fixed at 106 cells / mL. After 30 days of incubation, cells were harvested and washed
thrice with 0.1 M phosphate buffer (PB). The cells were then fixed in 2.5 % glutaraldehyde at
48 °C for 18–24 h. The fixative was then removed from the cell pellet by washing with 0.1 M
phosphate buffer thrice [3, 39] The cell pellet was then resuspended in 0.1 M phosphate
buffer and the samples were submitted to the Sophisticated Analytical Instrumentation
Facility (SAIF), Department of Anatomy, All India Institute of Medical Sciences, New Delhi
for further analysis.
Assay for InhA Enzyme Activity
Cloning, expression and purification of InhA (Rv1484) was done to study the effect of test
molecules on the activity of the enzyme. InhA (Rv1484) was cloned in E. coli and purified
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InhA protein. Protein estimation was done by Bradford’s method. InhA-drug interaction
experiment was carried out to validate the drug target proposed through docking experiment.
Assay was performed in 96 well plates with 200 µL reaction volume in each well, reactions
were performed in triplicate. 2 µg purified enoyl-ACP reductase (InhA) was incubated with
test compounds (6b and 11b having concentration 5 µM from 100 µM stock solution
respectively, dissolved in DMSO such that the final concentration of this co-solvent was
constant at ≤ 1 % v/v) in 100 mM sodium phosphate buffer pH 7.5 for 30 minutes. Enzyme
activity was carried out using a plate reader spectrophotometer (model: Infinite f200 pro;
Tecan) by monitoring NADH absorbance at 340 nm (A340). Decrease in absorbance over
time indicates the enzyme activity which facilitates the conversion of NADH to NAD+ as the
reaction proceeds [41]. The assay was started by addition of 100 µM NADH and 100 µM
crotonoyl-CoA. The plate was incubated at room temperature and OD at 340 nm was
recorded over a time period of 15 minutes using spectrophotometer (model: Infinite f200 pro;
Tecan) [41-44].The chemicals and reagents, NADH and Crotonoyl CoA were purchased from
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Sigma-Aldrich Chemical Company (St. Louis, Missouri USA). The results were statistically
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checked for significance by one way ANOVA test using Graph Pad Prism software.
DNA gyrase Inhibition Assay
DNA gyrase-drug interaction experiment was performed by DNA gyrase inhibition assay
using protocol as recommended in manufacturer’s manual provided with New England Bio
Labs (NEB), E. coli DNA gyrase (Catalogue No. M0306S). Assays were performed with
relaxed pUC19 plasmid DNA. The reaction was carried in 30 µL volume containing DNA
gyrase assay buffer 1X, Relaxed pUC19 DNA 0.3 µg, 1 unit DNA gyrase enzyme. To this
reaction mix, 0.2 µL ciprofloxacin (taken as positive control from 100 µM stock), DMSO
(solvent control) and 0.2 µL of the test compounds 6b and 11b (from 100 µM stock) were
added respectively. The reaction mixtures were incubated at 37 ºC for one hour. The
reactions were terminated by addition of 50 % glycerol and 0.25 % bromophenol blue. The
samples were subjected to electrophoresis in 1 % agarose gel in 0.5X Tris-borate EDTA
(TBE) buffer pH 8.3, for 16 hours at 30 mA. The gel was stained in ethidium bromide (0.7µg
/ mL) and visualized in UV light. The plasmid and gyrase enzyme was purchased from New
England Bio Labs, USA (Lot no.0021609).
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