Enabling the (3 + 2) cycloaddition reaction in assembling newer anti-tubercular lead acting through the inhibition of the gyrase ATPase domain: Lead optimization and structure activity profiling

Article abstract of DOI:10.1039/c4ob02049a

DNA gyrase, the sole type II topoisomerase present in Mycobacterium tuberculosis, is absent in humans and is a well validated target for anti-tubercular drug discovery. In this study, a moderately active inhibitor of Mycobacterium tuberculosis GyrB, the pharmaceutically unexploited domain of DNA gyrase, was reengineered using a combination of molecular docking and medicinal chemistry strategies to obtain a lead series displaying considerable in vitro enzyme efficacy and bacterial kill against the Mycobacterium tuberculosis H₃₇Rv strain. Biophysical investigations using differential scanning fluorimetry experiments re-ascertained the affinity of these molecules towards the GyrB domain. Furthermore, the molecules were completely devoid of hERG toxicity up to 30 μM, as evaluated in a zebra fish model with a good selectivity index, and from a pharmaceutical point of view, turned out as potential candidates against TB. This journal is

Full text of DOI:10.1039/c4ob02049a

View Article Online
View Journal
Organic &
Biomol ec ular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: J. U. Variam, R.
R. Srilakshmi, R. Janupally, B. Medapi, S. Saxena, J. Padma Sridevi, P. Kulkarni, P. Yogeeswari and D.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/obc

Page 1 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C4OB02049A
Enabling the (3+2) cycloaddition reaction in assembling newer antiꢀ
tubercular lead acting through the inhibition of Gyrase ATPase domain:
Lead optimization and structure activity profiling
a
a
a
Variam Ullas Jeankumar , Rudraraju Srilakshmi Reshma , Renuka Janupally , Shalini
a
a
a
b,c
Saxena , Jonnalagadda Padma Sridevi , Brahmam Medapi , Pushkar Kulkarni , Perumal
a
a
Yogeeswari , Dharmarajan Sriram *
a
Department of Pharmacy, Birla Institute of Technology & ScienceꢀPilani, Hyderabad
Campus, Shameerpet, R.R. District, Hyderabadꢀ500078, Andhra Pradesh, India.
b
Dr Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli,
Hyderabad 500046, India.
c.
Zephase Therapeutics (an incubated company at the Dr Reddy’s Institute of Life Sciences),
University of Hyderabad Campus, Gachibowli, Hyderabad 500046, India.
Corresponding Author*
D. Sriram
Chair Professor,
Department of Pharmacy,
Birla Institute of Technology & ScienceꢀPilani, Hyderabad Campus,
Jawahar Nagar, R.R. Dist., Hyderabadꢀ 500 078.
INDIA
Telephone: +91ꢀ40663030506
Fax: +91ꢀ4066303998
Email: dsriram@hyderabad.bitsꢀpilani.ac.in; drdsriram@yahoo.com
1

Organic & Biomolecular Chemistry
Page 2 of 24
View Article Online
DOI: 10.1039/C4OB02049A
Abstract
DNA Gyrase, the sole type II topoisomerase present in Mycobacterium tuberculosis is absent
in humans and is a well validated target for antiꢀtubercular drug discovery. In this study a
moderately active inhibitor of Mycobacterium tuberculosis GyrB ꢀ the pharmaceutically
unexploited domain of DNA gyrase was reengineered using a combination of molecular
docking and medicinal chemistry strategies to obtain a lead series displaying considerable in
vitro enzyme efficacy and bactericidal property against Mycobacterium tuberculosis H37Rv
strain. A biophysical investigation using differential scanning flourimetry experiments reꢀ
ascertained the affinity of these molecules towards the GyrB domain. Furthermore the
molecules were completely devoid of hERG toxicity until 30µM, evaluated in a zebra fish
model with a good selectivity index to be worked out from a pharmaceutical point of view as
potential candidate against TB.
2

Page 3 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C4OB02049A
Introduction:
Tuberculosis (TB) according to the recent world health organization (WHO) report claimed
about 1.3 million lives in 2012, with an overwhelming majority of these from developing
1
,2
portions of the world Despite the effectiveness of WHO initiated directly observed
treatment short course (DOTS) therapy with approximately 90% cure rate in human
immunodeficiency virus (HIV)–negative drugꢀsusceptible, active tuberculosis patients; the
disease has however resurged in a more deadly drug resistant form causing substantial
mortality and human suffering. Cofounding these issues is the association of TB with HIV,
which compromises the host defence mechanism and renders individuals more susceptible to
TB; necessitating further research into development of novel agents that lacks cross
3
ꢀ8
resistance mediated by mutation in bacterial target and shortens the duration of therapy.
The current TB drug discovery pipeline has many promising leads at the various stages of
8
clinical investigation including six new compounds specifically developed for tuberculosis.
The most promising among them is Moxifloxacin, a fluoroquinolone drug that acts through
the inhibition of DNA gyrase subunit A (GyrA) and has demonstrated promising activity
against both drug sensitive and drug resistant strains of Mycobacterium tuberculosis (M.tb).
In Mycobacterium tuberculosis, DNA gyrase is the sole type II topoisomerase that ensures
the regulation of DNA topology and has been genetically demonstrated to be a bactericidal
drug target. It is a heterodimeric enzyme with A2B2 complex, the A subunit aids in breakage
and reunion of the double stranded DNA, while the B subunit acts via ATPase activity,
9
ꢀ13
providing a sufficient amount of energy for the DNA supercoiling. The GyrA subunit, the
target of quinolone class of antibiotics has been clinically quite successful; however, the
emergence of quinolone resistance strain and the occurrence of some serious side effects;
10, 14ꢀ17
calls for novel research in this field.
3

Organic & Biomolecular Chemistry
Page 4 of 24
View Article Online
DOI: 10.1039/C4OB02049A
Later, novel (nonꢀfluoroquinolone) bacterial type II topoisomerase inhibitors (NBTIs) that
displayed broad spectrum antibacterial potency and were structurally and mechanistically
distinct from fluoroquinolones emerged as a probable replacement to quinolone class.
However their translation into a future clinical candidate was hampered by their significant
cardio toxic side effects, arising from their potent hERG inhibition and QTc prolongation inꢀ
18,19
vivo.
Researchers now focus on the pharmaceutically unexploited subunit of DNA Gyrase; the
GyrB subunit. Inhibitors of the GyrB subunit competitively block the ATPase activity
conferred by the DNA GyrB subunit and thereby abolish the energyꢀdependent reactions
catalyzed by DNA gyrase and hence offers tremendous opportunity to develop novel anti
tubercular agents displaying broad spectra efficacy, evading existing bacterial resistance with
minimal side effects.
In the light of these finding and in continuation of our group research efforts in designing
novel anti tubercular drugs; we report the development of an improved class of GyrB
2
0
inhibitor derived from a previously reported class ; that displays significant specificity
towards the mycobacterial GyrB domain and exhibits considerable inꢀvitro enzyme efficacy
3
and bacterial kill against Mycobacterium tuberculosis H Rv strain.
Results and Discussion:
The design strategy adopted here is based on the concept of molecular hybridisation through
reꢀalignment and positioning of two or more pharmacophoric units previously demonstrated
to have promising activity against the targeted protein / disease. The success of this strategy
in developing new substances which are therapeutically attractive has observed a significant
growth in distinct therapeutic classes, being amply used by the pharmaceutical industry to
discover new analogues of therapeutic innovations commercially attractive and also as a tool
4

Page 5 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C4OB02049A
2
1ꢀ23
useful in the molecular modification.
In the present protocol we integrated in one
molecular platform, the pharmacophoric units of two previously reported DNA GyraseB
inhibitors (1, 2 and 3 in Figure 1) to give the designed ligand 4 bearing a thiazole core on
the left hand side that runs through a triazole linker to the right handed coumarin nucleus as
shown in Figure 1.The left hand thiazole unit was derived from our previously reported
21
GyrB inhibitor ; that displayed moderate GyrB inhibitory potency and mycobacterial MIC.
Though Nꢀaminopiperdine nucleus has served as the linking unit in many of the previously
reported DNA Gyrase inhibitors, the significant cardiovascular safety risk associated with
18,19
this class
encouraged us to explore a novel traizole core as the linking unit. Additionally,
these 1,2,3ꢀtriazoles possess remarkable metabolic stability and prove to be amide surrogates
in various bioactive compounds. The presence of the coumarin core in many of the
9
previously reported GyrB inhibitor from natural source made it the right hand choice. Thus a
library of 36 hybrids analogues (Table 1) were designed and taken for synthesis as steps
towards the derivation of structureꢀactivity relationships (SAR) and lead optimization.
The synthetic pathway used to achieve the target compounds has been delineated in
Scheme1. We utilised the Huisgen’s (3+2) cycloaddition reaction as a useful strategy to
generate the designed library. Azido coumarins, required to engineer the right hand core of
24
the designed ligand were constructed as previously described by Sivakumar et al. Synthesis
of the left hand thiazole core began by condensing commercially available aryl (phenyl, 2ꢀ
pyrdiyl, 3ꢀpyrdiyl and 4ꢀpyridyl) thioamides (5aꢀd) with diethyl bromo malonate in refluxing
25
toluene as previously reported by Kerdesky et al , yielding the corresponding 4ꢀhydroxy
thiazole derivatives 6aꢀd. The acetylene tail required to fuse the thaizole core to right hand
nucleus was achieved by mistnobu reaction of hydroxyl group present in the thiazole moiety
(
6aꢀd) with propargyl alcohol, affording compounds 7aꢀd in good yields. These analogues
7aꢀd) were further hydrolysed into their corresponding acid derivatives (8aꢀd) and later
(
5

Organic & Biomolecular Chemistry
Page 6 of 24
View Article Online
DOI: 10.1039/C4OB02049A
converted to their amide derivatives counterparts (9aꢀd) as well, in an effort to understand the
ideal site for introducing chemical diversity. Finally Huisgen’s (3+2) cycloaddition of the
above obtained acetylenes (7aꢀd, 8aꢀd, 9aꢀd) with corresponding azido coumarins gave the
titled compounds (10ꢀ45 in Table 1) in excellent yields. An overview of the various
modification/substituent attempted in synthesis has been outlined in Figure 2.
In the preliminary screening, synthesized compounds were evaluated for their inꢀvitro
Mycobacterium smegmatis GyrB ATPase assay adapted to 96ꢀwell plate format as described
2
6,27
previously
and detailed extensively in the experimental section. The use of
Mycobacterium smegmatis GyrB protein as a surrogate for GyrB protein from
10,26ꢀ27
Mycobacterium tuberculosis has been well demonstrated in literature
as the activity of
GyrB protein from Mycobacterium tuberculosis was found be very low when compared to
that of Mycobacterium smegmatis. Novobiocin which has been previously demonstrated to be
a potent inhibitor of DNA GyrB was used as a positive control in the study. Negative controls
(0% inhibition) did not contain any inhibitory compounds. Compounds were also tested in the
presence of Brijꢀ35, a nonꢀanionic detergent to ascertain whether these inhibitions were an
artifact due to sequestration of the enzyme by drug aggregates. The other artifacts like auto
absorbance of the drug were also ruled out by nullifying its absorbance during the reaction.
In the GyrB assay, out of the 36 compounds tested, 34 compounds displayed GyrB inhibitory
IC < 50 µM, out of which 13 compounds demonstrated IC < 15 µM, 9 compounds showed
5
0
50
IC50 < 5 µM and 3 compounds were found to inhibit GyrB activity even at sub micromolar
concentration (IC50 < less 1 µM). Compound 27 emerged as the most promising leads with
and GyrB inhibitory IC of 0.44±1.1 µM.
5
0
With respect to structureꢀactivity relationship study we explored various modifications at
three different sites as shown in Figure 2. The site 1 of the thiazole core was derived from
6

Page 7 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C4OB02049A
the previously reported GyrB inhibitor (1) and had phenyl, 2ꢀpyridyl, 3ꢀpyridyl, and 4ꢀpyridyl
substitutions. At site 2, we introduced a carboxylic acid and a carboxamide side chain in
addition to the ethyl carboxylate chain present in the previous inhibitor/lead. The site 3 had a
remarkable difference from the previous lead and had a coumarin nucleus with a hydroxyl
th
and methoxy substitution attempted at the 7 position, in addition to the unꢀsubstituted
analogue. Another notable difference is the newly introduced triazole linker for reasons
mentioned earlier. In the inꢀvitro assay, out of the various explorations attempted in synthesis,
the carboxamide analogues (16ꢀ18, 25ꢀ27, 34ꢀ36, 43ꢀ45) turned out to be the most promising
leads showing excellent GyrB inhibition even in the lower micromolar range. The most
promising leads compound 27 also belonged to this class. A general trend derived from the
GyrB assay has been summarized in Figure 2.
Information on the common properties of the binding groups is essential for resolving the
type of inhibitor binding to the target protein. In order to analyse the interaction profile of the
molecules reported from the assay, compounds were docked to the GyrB ATPase domain of
26
Mycobacterium smegmatis retrieved from protein data bank (PDB ID–4B6C). An initial
validation of the active site pocket was performed by redocking the crystal ligand (6ꢀ(3,4ꢀ
dimethylphenyl)ꢀ3ꢀ[[4ꢀ[3ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)propoxy]phenyl]amino]pyrazineꢀ2ꢀ
carboxamide), with the active site residues of the Mycobacterium smegmatis GyrB protein.
Redocking results showed that the ligand exhibited similar interactions as that of the original
0
crystal structure which was further confirmed with RMSD of 0.86A . Later compounds 10ꢀ45
28
were docked into the active site pocket using extra precision mode (XP) of Glide module.
All the molecules oriented nicely into the active pocket in the vicinity of the amino acids
similar to the crystal ligand. The docking results further validated the in vitro GyrB findings
with carboxamide derivatives (16ꢀ18, 25ꢀ27, 34ꢀ36, 43ꢀ45) giving the best docking scores in
ꢀ1
the range of ꢀ7.87 to ꢀ6.02 kcal mol . A closer look at the interaction profile of these
7

Organic & Biomolecular Chemistry
Page 8 of 24
View Article Online
DOI: 10.1039/C4OB02049A
2
molecules revealed the carboxamide –NH at site 2 to be involved in an important hydrogen
bonding interaction with Asp 79. This interaction is believed to be critical in improving the
bioactivity. The carboxylic acid (13ꢀ15, 22ꢀ24, 31ꢀ33, 40ꢀ42) and the ester analogues (10ꢀ12,
19ꢀ21, 28ꢀ30, 37ꢀ39) however failed to retain this interaction thus accounting for their
reduced activity. A cationꢀπ interaction was also observed between the πꢀelectron
+
environment of core thiazole ring and the ammonium ion (NH
3
) present in the guanidine
group of Arg82 in the case of active hits. Effect of hydrophobicity on activity determination
was also evident from the docking studies. The aryl/heteroaryl ring at site 1 was found to be
stabilised by hydrophobic interaction with Ala59, Val128, Val50, Val49, Met100, Ile171, or
Val199 amino acid residues and accounted for the good to moderate activity shown by these
molecules. A 2D interaction profile of few active and inactive compounds has been provided
in the supplementary information (Figure S4ꢀS9) for a better understanding.
Furthermore, the binding affinity of the most potent analogue was evaluated by measuring the
thermal stability of the protein–ligand complex using biophysical differential scanning
fluorimetry experiments. by measuring the fluorescence of the native protein and proteinꢀ
ligand complexes in presence of a fluorescent dye whose fluorescence increases when
exposed to non polar residues of the protein and reach the maximum when the protein
29
M
denature. A higher or positive shift of melting temperature (T ) of proteinꢀligand complex
compared to the native protein T signify a better stabilization of the proteinꢀligand complex,
M
which in turn would reflect on the inhibitor binding. The most potent analogue compound 27
displayed a T shift of 3.2°C compared with the native protein, a repercussion of strong
m
binding of the ligand to the protein and highly correlated with its GyrB IC50 of 0.44±1.1 µM.
The curves obtained are depicted in Figure 4.
8

Page 9 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C4OB02049A
2
7
Further the compounds were also subjected the DNA supercoiling assay using DNA gyrase
from Mycobacterium tuberculosis (Inspiralis, Norwich) to reconfirm their activity; as any
inhibition of ATPase activity conferred by the DNA GyrB subunit should also inhibit the
super coiling activity performed by GyrA domain. Moxifloxacin was used as a positive
control in the study. In the supercoiling assay out of the 36 compounds tested, 33 compounds
displayed supercoiling inhibitory IC50 < 25 µM, out of which 28 compounds demonstrated
IC50 < 15 µM, 11 compounds showed IC50 < 5 µM and 8 compounds were found to inhibit
supercoiling activity even at sub micromolar concentration (IC50 < less 1 µM). The most
potent compound from the GyrB assay compound 27 with a GyrB IC50 of 0.44±1.1 µM
showed an equally well correlating supercoiling IC of10.5±0.24 µM.
50
The compounds were also screened for their inꢀvitro antimycobacterial potency against
30
Mycobacterium tuberculosis H37Rv by the MABA assay. In general; a good correlation was
observed between the Mycobacterium smegmatis GyrB inhibitory IC50, Mycobacterium
tuberculosis DNA Gyrase supercoiling assay IC50 value and the inꢀvitro Mycobacterium
tuberculosis minimum inhibitory concentration (MIC). Eleven compounds inhibited
Mycobacterium tuberculosis with MIC less than 15 µM; Compounds 25, and 27 were
promising analogues, with MICs less than 10 µM. These compounds were found to be more
active than the firstꢀline antiꢀtubercular drug ethambutol (MIC = 15.31 µM) but were less
active compared with isoniazid (MIC = 0.66 µM) and rifampicin (MIC = 0.23 µM)
The most potent compounds were further examined for hERG channel inhibition by assessing
arrhythmogenic potential in a zebrafish model. Zebrafish (Danio rerio) is an attractive
preliminary inꢀvivo model for screening compounds with potential proꢀarrhythmic, hERG
31
blockade & QT prolongation. Compounds 18, 25 and 27 were exposed from 1µM to 30µM
3
2ꢀ33
concentration with 0.1% DMSO as a vehicle by using a previously reported protocol.
9

Organic & Biomolecular Chemistry
Page 10 of 24
View Article Online
DOI: 10.1039/C4OB02049A
Terfenadine (20µM) was used as a positive control. All the compounds were analyzed for
heart rate variations and AV ratio (Figure 4). The tested compounds though completely safe
at lower concentrations tested showed mild ventricular bradycardia, as compared to control
group at 30 µM concentration, which can be considered to be biologically insignificant from
an overall perspective. Encouragingly, there was no effect on atrioꢀventricular ratio in any
concentration of the test compounds. This data is of significant importance considering the
intrinsic challenges that some of the previously reported GyrB inhibitors (especially the
NBTIs class) had in achieving a acceptable cardiovascular safety profile.
Finally the safety profile of these molecules were also evaluated by checking the in vitro
cytotoxicity against RAW 264.7 celline (mouse macrophage) at 100 µM concentration by the
34
(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) assay.
All the
compounds tested were relatively safe and showed a selectivity profile.
A preliminary view of ADMET parameters predicted using QikProp3.5 module of
28
Schrodinger software depicted that all the hits preserved the basic criterion of drugꢀlikeness
showing characteristics (Table S1 in supplementary information) of a promising drug leads
which can be worked for rational drug design against Mycobacterium tuberculosis gyraseB
from pharmaceutical point of view.
Conclusion:
In the present study, using the concept of molecular hybridization, a library of 36 molecules
were designed and synthesized by fusing previously reported gyrase analogues through a
newly introduced triazole linker, incorporated primarily to reduce the hERG toxicity that is
believed to arise from the aminopiperidine nucleus ꢀ the linking unit in many of the previous
gyrase inhibitors. The synthesized molecules displayed good GyrB inhibitory potency with
1
0

Page 11 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C4OB02049A
equally well correlating supercoiling inhibitory activity. Compound 27 emerged as the most
potent lead with a Mycobacterium semgmatis GyrB inhibitory IC50 of 0.44 ± 1.1 µM,
Mycobacterium tuberculosis supercoiling IC50 of 0.5 ± 0.24 µM and Mycobacterium
tuberculosis MIC of 6.5 µM. Structural handles critical for retaining the bioactivity were
identified through inꢀsilico investigations which revealed the interaction with Asp 79 along
with the hydrophobic contacts that are the key attributes to activity. Compounds in the series
demonstrated excellent inꢀvitro mycobacterial kill and showed no signs of cardiotoxicity
unlike some of the previously reported gyrase analogues.
Experimental section:
Chemistry:
Representative experimental procedures
General procedure utilised for developing the designed analogues (10ꢀ45): To a mixture
2
of the corresponding acetylene (1 equiv) and corresponding azide (1.1equiv) in THF:H O
o
(
1:1) system was added CuSO .5H O (0.05 equiv), sodium ascorbate (0.1equiv) at 27 C. The
4
2
o
reaction was then stirred at 27 C for 12ꢀ15 hours (monitored by TLC & LCMS for
completion). The residue was further diluted with water (2 mL) and dichloromethane (4 mL)
and the layers separated. The aqueous layer was reꢀextracted with dichloromethane (2 x 4
mL) and the combined organic layer was washed with brine (3 mL), dried over anhydrous
sodium sulphate and evaporated under reduced pressure and the residue reꢀcrystallized form
diethyether/ethanol to afford the desired product in good yield as described below.
4ꢀ((1ꢀ(2ꢀoxoꢀ2Hꢀchromenꢀ3ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ5ꢀyl)methoxy)ꢀ2ꢀphenylthiazoleꢀ5ꢀ
carboxamide (16): The compound was synthesized according to the above general procedure
using 4ꢀ(Propꢀ2ꢀynꢀ1ꢀyloxy)ꢀ2ꢀphenylthiazoleꢀ5ꢀcarboxamide (9a, 0.1 g, 0.39 mmol), 3ꢀ
azidoꢀ2Hꢀchromenꢀ2ꢀone (0.08 g, 0.43 mmol), CuSO .5H O (0.005 g, 0.02 mmol), sodium
4
2
ascorbate (0.008 g, 0.04 mmol) to afford 16 (0.13 g, 75.6 %) as buff coloured solid. M.p: 190
1
1

Organic & Biomolecular Chemistry
Page 12 of 24
View Article Online
DOI: 10.1039/C4OB02049A
o
1
13
–
3 3
192 C. H NMR (CDCl ): δH. 5.79 (s, 2H), 6.96 – 8.79 (m, 11H). C NMR (CDCl ):
δc.165.8, 163.3, 161.1, 153.4, 148.6, 144.1, 143.5, 141.1, 131, 129.3, 128.8, 128.5, 128.3,
+
1
27.8, 125.3, 123.5, 123.3, 122.2, 115.9, 63.4. ESIꢀMS m/z 446.3 (M+H) . Anal Calcd for
22 15 5 4
C H N O S; C, 59.32; H, 3.39; N, 15.72; Found: C, 59.31; H, 3.4; N, 15.74.
4ꢀ((1ꢀ(7ꢀhydroxyꢀ2ꢀoxoꢀ2Hꢀchromenꢀ3ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ5ꢀyl)methoxy)ꢀ2ꢀ
phenylthiazoleꢀ5ꢀcarboxamide (17): The compound was synthesized according to the above
general procedure using 4ꢀ(Propꢀ2ꢀynꢀ1ꢀyloxy)ꢀ2ꢀphenylthiazoleꢀ5ꢀcarboxamide (9a, 0.1 g,
0
.39 mmol), 3ꢀazidoꢀ6ꢀhydroxyꢀ2Hꢀchromenꢀ2ꢀone (0.087 g, 0.43 mmol), CuSO
4 2
.5H O
(0.005 g, 0.02 mmol), sodium ascorbate (0.008 g, 0.04 mmol) to afford 17 (0.14 g, 78.2 %)
o
1
as buff coloured solid. M.p: 262 ꢀ 264 C. H NMR (CDCl
3
): δH. 5.78 (s, 2H), 6.86 – 8.80 (m,
1
3
1
1
0H). C NMR (CDCl ): δc.165.4, 163.2, 161.3, 156.4, 154.2, 148.4, 143.8, 143.2, 141.3,
3
30.9, 130.2, 129.2, 128.4, 128.3, 123.1, 122.9, 114.6, 112.7, 102.6, 63.3. ESIꢀMS m/z 462.1
+
(
M+H) . Anal Calcd for C22
H
15
N
5
O
5
S; C, 57.26; H, 3.28; N, 15.18; Found: C, 57.24; H, 3.29;
N, 15.2.
4ꢀ((1ꢀ(7ꢀmethoxyꢀ2ꢀoxoꢀ2Hꢀchromenꢀ3ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ5ꢀyl)methoxy)ꢀ2ꢀ
phenylthiazoleꢀ5ꢀcarboxamide (18): The compound was synthesized according to the above
general procedure using 4ꢀ(Propꢀ2ꢀynꢀ1ꢀyloxy)ꢀ2ꢀphenylthiazoleꢀ5ꢀcarboxamide (9a, 0.1 g,
0.39 mmol), 3ꢀazidoꢀ6ꢀmethoxyꢀ2Hꢀchromenꢀ2ꢀone (0.094 g, 0.43 mmol), CuSO
4 2
.5H O
(0.005 g, 0.02 mmol), sodium ascorbate (0.008 g, 0.04 mmol) to afford 18 (0.16 g, 88.9 %)
o
1
as buff coloured solid. M.p: 240 ꢀ 242 C. H NMR (DMSOꢀd
6
): δH. 3.89 (s, 3H), 5.78 (s, 2H),
1
3
6
1
1
3
.97 – 7.15 (m, 3H), 7.54 – 8.81 (m, 9H). C NMR (DMSOꢀd ): δc. 165.6, 163.4, 161.3,
6
59.4, 156.1, 154.5, 142.5, 135.7, 132.2, 131.3, 130.6, 129.3, 125.9, 120.1, 113.5, 111.4, 109,
+
00.6, 63.4, 56.1. ESIꢀMS m/z 476.1 (M+H) . Anal Calcd for C23
H
17
N
5
O
5
S; C, 58.10; H,
.60; N, 14.73; Found: C, 58.09; H, 3.59; N, 14.74.
1
2

Page 13 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C4OB02049A
4ꢀ((1ꢀ(2ꢀoxoꢀ2Hꢀchromenꢀ3ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ5ꢀyl)methoxy)ꢀ2ꢀ(pyridinꢀ2ꢀyl)thiazoleꢀ
5ꢀcarboxamide (25): The compound was synthesized according to the above general
procedure using 4ꢀ(Propꢀ2ꢀynꢀ1ꢀyloxy)ꢀ2ꢀ(pyridinꢀ2ꢀyl)thiazoleꢀ5ꢀcarboxamide (9b, 0.1 g,
.39 mmol), 3ꢀazidoꢀ2Hꢀchromenꢀ2ꢀone (0.08 g, 0.43 mmol), CuSO .5H O (0.005 g, 0.02
0
4
2
mmol), sodium ascorbate (0.008 g, 0.04 mmol) to afford 25 (0.13 g, 75.6 %) as buff coloured
o
1
13
3
solid. M.p: 184 – 186 C. H NMR (CDCl ): δH. 5.77 (s, 2H), 7.01 ꢀ 8.82 (m, 10H), C NMR
(CDCl
3
): δc. 164.2, 163, 160.7, 154.6, 152.9, 148.9, 147.6, 142.8, 141.2, 137.4, 128.7, 128.5,
+
1
27.9, 125.2, 123.9, 123.4, 123.1, 122.9, 122, 115.9, 63.2. ESIꢀMS m/z 447.1 (M+H) . Anal
14 6 4
Calcd for C21H N O S; C, 56.50; H, 3.16; N, 18.82; Found: C, 56.49; H, 3.14; N, 18.8.
4ꢀ((1ꢀ(7ꢀhydroxyꢀ2ꢀoxoꢀ2Hꢀchromenꢀ3ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ5ꢀyl)methoxy)ꢀ2ꢀ(pyridinꢀ2ꢀ
yl)thiazoleꢀ5ꢀcarboxamide (26): The compound was synthesized according to the above
general procedure using 4ꢀ(Propꢀ2ꢀynꢀ1ꢀyloxy)ꢀ2ꢀ(pyridinꢀ2ꢀyl)thiazoleꢀ5ꢀcarboxamide (9b,
0.1 g, 0.39 mmol), 3ꢀazidoꢀ6ꢀhydroxyꢀ2Hꢀchromenꢀ2ꢀone (0.08 g, 0.43 mmol), CuSO
4 2
.5H O
(0.005 g, 0.02 mmol), sodium ascorbate (0.008 g, 0.04 mmol) to afford 26 (0.15 g, 84.3 %)
o
1
as buff coloured solid. M.p: 237 – 239 C. H NMR (CDCl
3
): δH. 5.75 (s, 2H), 6.89 – 8.81 (m,
1
3
9
1
H). C NMR (CDCl ): δc. 164, 162.8, 160.5, 156.1, 154.6, 154.3, 148.7, 147.6, 143, 141.1,
3
37.2, 130.2, 128.5, 124, 123.5, 123.3, 123.1, 114.6, 112.6, 102.4, 63. ESIꢀMS m/z 463.2
+
(
M+H) . Anal Calcd for C21
H
14
N
6
O
5
S; C, 54.54; H, 3.05; N, 18.17; Found: C, 54.52; H,
3.06; N, 18.14.
4ꢀ((1ꢀ(7ꢀmethoxyꢀ2ꢀoxoꢀ2Hꢀchromenꢀ3ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ5ꢀyl)methoxy)ꢀ2ꢀ(pyridinꢀ2ꢀ
yl)thiazoleꢀ5ꢀcarboxamide (27): The compound was synthesized according to the above
general procedure using 4ꢀ(Propꢀ2ꢀynꢀ1ꢀyloxy)ꢀ2ꢀ(pyridinꢀ2ꢀyl)thiazoleꢀ5ꢀcarboxamide (9b,
0
.1 g, 0.39 mmol), 3ꢀazidoꢀ6ꢀmethoxyꢀ2Hꢀchromenꢀ2ꢀone (0.08 g, 0.43 mmol), CuSO
4 2
.5H O
(0.005 g, 0.02 mmol), sodium ascorbate (0.008 g, 0.04 mmol) to afford 27 (0.16 g, 87 %) as
o
1
6
buff coloured solid. M.p: 224 – 226 C. H NMR (DMSOꢀd ): δH. 3.89 (s, 3H), 5.78 (s, 2H),
1
3

Organic & Biomolecular Chemistry
Page 14 of 24
View Article Online
DOI: 10.1039/C4OB02049A
1
3
6
1
5
6
.94 – 8.84 (m, 9H). C NMR (DMSOꢀd ): δc. 164.3, 163.1, 160.6, 159.9, 154.6, 153.9, 149,
47.8, 143.1, 141.2, 137.4, 130, 128.6, 124, 123.7, 123.4, 123.2, 114.8, 110.9, 100.5, 63.1,
+
6.2. ESIꢀMS m/z 477.1 (M+H) . Anal Calcd for C22
H
16
N
6
O
5
S; C, 55.46; H, 3.38; N, 17.64;
Found: C, 55.43; H, 3.35; N, 17.62. For details regarding the experimental section of other
compounds synthesised eg. 6aꢀd, 7aꢀd, 8aꢀd, 9aꢀd, 10ꢀ15, 19ꢀ24 and 28ꢀ45 refer ESI.
Biological evaluation
MS GyrB ATPase assay:
Being the gyrase enzyme catalytic site the gyraseB domain performs the ATPase assay with
the sole GyrB subunit. The assay was performed in 30 ꢁL reaction volume for 120 min at
25°C in reaction buffer containing 60 mM HEPESꢀKOH pH 7.7, 250 mM potassium
glutamate, 200 mM KCl, 2 mM magnesium chloride, 1 mM DTT, 2% Glycerol, 4% DMSO,
0
.001% BriJ, 0.65 mM ATP, 40 nM GyrB as previously published method (Pravin etal,
012). All the test compounds were diluted in 4% DMSO to about eight concentrations for
2
the determination of IC . ATPase assay was performed in Vꢀshaped 96ꢀwell plates
50
(
Polystyrene untreated). Initially 15 µL of 2x assay buffer containing purified GyrB enzyme
and substrate mix were placed in the assay well followed by 1 µL of test compound,
subsequently the enzyme reaction was initiated by adding 14 µL of MgCl solution, as metal
2
ion triggers the enzyme. The reaction was allowed to proceed for 120 min at room
temperature. At the end, 20µL malachite green reagent (Bioassay systems) was added to
quench the reaction and incubated for 20 min to determine the inorganic phosphates (Pi)
released when measured at 635 nm wavelength against the blank absorbance. In this assay,
novobiocin was considered as positive control and moxifloxacin as the negative control.
Inꢀvitro Mycobacterium tuberculosis (M.tb) MABA assay:
1
4

Page 15 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C4OB02049A
The compounds were further screened for their in vitro antimycobacterial activity against M.
S7
tuberculosis H37Rv by microplate Alamar blue assay method . Briefly, the inoculum was
prepared from fresh LJ medium reꢀsuspended in 7H9ꢀS medium (7H9 broth, 0.1% casitone,
0.5% glycerol, supplemented oleic acid, albumin, dextrose, and catalase [OADC]), adjusted
to a McFarland tube No. 1, and diluted 1:20; 100 µl was used as inoculum. Each drug stock
solution was thawed and diluted in 7H9ꢀS at fourꢀfold the final highest concentration tested.
Serial twoꢀfold dilutions of each drug were prepared directly in a sterile 96ꢀwell microtiter
plate using 100 µl 7H9ꢀS. A growth control containing no antibiotic and a sterile control were
also prepared on each plate. Sterile water was added to all perimetre wells to avoid
evaporation during the incubation. The plate was covered, sealed in plastic bags and
o
incubated at 37 C in normal atmosphere. After 7 days incubation, 30 ml of alamar blue
solution was added to each well, and the plate was reꢀincubated overnight. A change in
colour from blue (oxidised state) to pink (reduced) indicated the growth of bacteria, and the
MIC was defined as the lowest concentration of drug that prevented this change in colour.
For details regarding the protocol utilised for cloning and purification of protien, supercoiling
assay, docking, toxicity evaluation, DSF experiments and ADMET calculations refer ESI.
References:
1
. http://www.who.int/mediacentre/factsheets/fs104/en/ 28/04/2014.
. WHO Global Tuberculosis Report 2013
2
http://www.who.int/tb/publications/global_report/en/
3
4
5
. C. Dye and B.G. Williams, Science, 2010, 328, 856ꢀ861.
. M.C. Raviglione, Bull World Health Organ., 2007, 85, 327ꢀ.
. E.L. Corbett, C.J. Watt, J. Catherine, N. Walker, D. Maher, B.G. Williams, M.C.
Raviglione and C. Dye, Arch. Intern. Med., 2003, 163, 1009 ꢀ 1021.
. A. Mahmoudi, and M.D. Iseman, J. Am. Med. Assoc., 1993, 270, 65−68.
6
1
5

Organic & Biomolecular Chemistry
Page 16 of 24
View Article Online
DOI: 10.1039/C4OB02049A
7
8
9
. C. Lienhardt, M. Raviglione, M. Spigelman, R. Hafner, E. Jaramillo, M. Hoelscher,
A. Zumla and J. Gheuens, J. Infect. Dis. 2012, 205, Suppl 2:S241ꢀ 249.
. Z. Ma, C. Lienhardt, H. McIlleron, A. Nunn and X. Wang, Lancet, 2010, 375, 2100ꢀ
2109.
. S. Chopra, K. Matsuyama, T. Tran, J.P. Malerich, B. Wan, S.G. Franzblau, S. Lun, H.
Guo, M.C. Maiga, W.R. Bishai and P.B. Madrid, J. Antimicrob. Chemother., 2012,
67, 415 ꢀ 421.
1
0. M. G. Kale, A. Raichurkar, P. S. H, D. Waterson, D. McKinney, M. R. Manjunatha,
U. Kranthi, K. Koushik, L. K. Jena, V. Shinde, S. Rudrapatna, S. Barde, V.
Humnabadkar, P. Madhavapeddi, H. Basavarajappa, A. Ghosh, V. Ramya, S. Guptha,
S. Sharma, P. Vachaspati, K.N. Kumar, J. Giridhar, J. Reddy, V. Panduga, S.
Ganguly, V. Ahuja, S. Gaonkar, C. N. Kumar, D. Ogg, J. A. Tucker, P. A. Boriackꢀ
Sjodin, S. M. de Sousa, V. K. Sambandamurthy and S. R. Ghorpade, J. Med.
Chem. 2013, 56, 8834– 8848.
1
1
1
1. A. Maxwell, Trends Microbiol., 1997, 5, 102ꢀ109.
2. A. Maxwell and D. M. Lawson, Curr. Top. Med. Chem., 2003, 3, 283ꢀ303.
3. A. Zumla, P. Nahid and S. T. Cole, Nat. Rev. Drug Discov. 2013, 12, 388ꢀ404.
1
4. W.J. Burman, S. Goldberg, J. L. Johnson, G. Muzanye, M. Engle, A. W. Mosher, S.
Choudhari, C. L. Daley, S. S. Munsiff, Z. Zhao, A. Vernon and R. E. Chaisson, Am.
J. Respir. Crit. Care. Med., 2006, 174, 331ꢀ338.
1
5. M.B. Conde, A. Efron, C. Loredo, G.R. De Souza, N.P. Graca, M.C. Cezar, M. Ram,
M. A. Chaudhary, W. R. Bishai, A. L. Kritski and R. E. Chaisson, Lancet, 2009, 373,
1183ꢀ1189.
1
6

Page 17 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C4OB02049A
1
6. D. A. Duong, T. H. Nguyen, T. N. Nguyen, V. H. Dai, T. M. Dang, S. K.Vo, D. A.
Do, V. V. Nguyen, H. D. Nguyen, N. S. Dinh, J. Farrar and M. Caws, Antimicrob.
Agents. Chemother., 2009, 53, 4835ꢀ4839.
17. J. J. Ruiz. Antimicrob. Chemother. 2003, 51, 1107ꢀ1119.
1
8. (a). F. Reck, R. Alm, P. Brassil, J. Newman, B. Dejonge, C.J. Eyermann, G. Breault,
J. Breen, J. ComitaꢀPrevoir, M. Cronin, H. Davis, D. Ehmann, V. Galullo, B. Geng, T.
Grebe, M. Morningstar, P. Walker, B. Hayter and S. Fisher, J. Med. Chem. 2011, 54,
7834−47. (b). F. Reck, R. A. Alm, P. Brassil, J. V. Newman, P. Ciaccio, J. McNulty,
H. Barthlow, K. Goteti, J. Goteti, J. ComitaꢀPrevoir, M. Cronin, D. E. Ehmann, B.
Geng, A. A. Godfrey and S. L. Fisher, J. Med. Chem. 2012, 55, 6916−6933.
1
9. B.D. Bax, P.F. Chan, D.S. Eggleston, A. Fosberry, D.R. Gentry, F. Gorrec, I.
Giordano, M.M. Hann, A. Hennessy, M. Hibbs, J. Huang, E. Jones, J. Jones, K.K.
Brown, C.J. Lewis, E.W. May, M.R. Saunders, O. Singh, C.E. Spitzfaden, C. Shen, A.
Shillings, A.J. Theobald, A. Wohlkonig, N.D. Pearson and M.N. Gwynn, Nature,
2010, 466, 935−940.
2
0. V. U. Jeankumar, J. Renuka, P. Santosh, J. P. Sridevi, P. Suryadevara, P.
Yogeeeswari, D. Sriram, Eur. J. Med. Chem. 2013, 70, 140−153.
2
1. a). C. Lazar, A. Kluczyk, T. Kiyota and Y. Konishi, J. Med. Chem., 2004, 47,
6
1
973−6982. (b). R. Maia, C. Do and C. A. M. Fraga, Curr. Enzyme Inhib., 2010, 6,
71−182. (c). C. A. M. Fraga, Expert Opin. Drug Discovery, 2009, 4, 605−609.
2
2. (a). C. Viegas junior, A. Danuello, V. da Silva Bolzani, E.J. Barreiro and C. A. M.
Fraga, Chem. Med. Chem., 2007, 14, 1829ꢀ1850. (b). M. L. Lidia and E. J. Lidia
Barreiro, Curr. Med. Chem., 2005, 12, 23ꢀ49. (c). S. V. Frye, Nat. Chem. Biol., 2010,
6, 159−161. (d). C. ViegasꢀJunior, A. Danuello, V. da Silva Bolzani, E. J. Barreiro
and C. A. M. Fraga, Curr. Med. Chem., 2007, 14, 1829−1852.
1
7

Organic & Biomolecular Chemistry
Page 18 of 24
View Article Online
DOI: 10.1039/C4OB02049A
2
3. (a). S.R. Patpi, L. Pulipati, P. Yogeeswari, D. Sriram, N. Jain, B. Sridhar, R. Murthy,
T. Anjana Devi, S. V. Kalivendi and S. Kantevari, J. Med. Chem., 2012, 55,
3911−3922. (b). T. Yempala, D. Sriram, P. Yogeeswari and S. Kantevari, Bioorg.
Med. Chem. Lett., 2012, 22, 7426–7430.
2
2
2
4. K. Sivakumar, F. Xie, B. M. Cash, S. Long, H.N. Barnhill and Q. Wang. Org. Lett.,
2004, 6, 4603ꢀ4606.
5. F. A. J. Kerdesky, J.H. Holms, J.L. Moore, R.L. Bell, R.D. Dyer, G.W. Carter and
D.W. Brooks, J. Med. Chem., 1991, 34, 2158ꢀ2165.
6. P. S. Shirude, P. Madhavapeddi, J. A. Tucker, K. Murugan, V. Patil, H.
Basavarajappa, A.V. Raichurkar, V. Humnabadkar, S. Hussein, S. Sharma, V. K.
Ramya, C. B. Narayan, T. S. Balganesh and V. K. Sambandamurthy, ACS Chem.
Biol., 2013, 8, 519−523.
2
7. V. U. Jeankumar, R. Janupally, V. K. Pulla, V. Soni, J. P. Sridevi, P. Suryadevara, M.
Shravan, R. Medishetti, P. Kulkarni, P. Yogeeswari and D. Sriram. Int. J. Antimicrob.
Agents. 2013, 43, 269ꢀ278.
2
2
3
8. Glide, version 5.8, Schrödinger, LLC, New York, NY, 2012.
9. F.H. Niesen, H. Berglund, and M. Vedadi, Nat Protoc. 2007, 2, 2212ꢀ2221.
0. G. S. Franzbalu, S. R. Witzig, C. J. McLaughlin, P. Torres, G. Madico, A.
Hernandez, T. M. Degnan, B. M. Cook, K. V. Quenzer, M. R. Ferguson, and H. R.
Gilman, J.Clin.Microbiol., 1998, 36, 362ꢀ366.
3
1. G. H. Chaudhari, K.S. Chennubhotla, K. Chatti, and P. Kulkarni, J. Pharmacol.
Toxicol. Methods, 2013, 67, 115ꢀ120.
3
2. P. Kulkarni, G. H. Chaudhari, V. Sripuram, R. K. Banote, K. T. Kirla, R. Sultana, P.
Rao, S. Oruganti and K. Chatti. Pharmacol Rep., 2014, 66, 179ꢀ183.
1
8

Page 19 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C4OB02049A
3
3
3. V. U. Jeankumar, J. Renuka, S. Kotagiri, S. Saxena, S. S. Kakan, J. P. Sridevi, S.
Yellanki, P. Kulkarni, P. Yogeeswari and D. Sriram, ChemMedChem, 2014, 9,1850ꢀ
1859.
4. D. Gerlier and N. Thomasset, Immunol. Methods, 1986, 94, 57−63.
1
9

Organic & Biomolecular Chemistry
Page 20 of 24
View Article Online
DOI: 10.1039/C4OB02049A
Figure 1: Chemical structure of previously reported synthetic inhibitors of DNA GyrB
bearing thiazole nucleus (1), natural product inhibitor novobiocin (2) and quercetin (3)
bearing coumarin nucleus and the inhibitor designed through molecular hybridisation (4).
2
0

Page 21 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C4OB02049A
Figure 2: Structureꢀactivity profiling.
O
O
S
OH
S
Ar
Ar
NH2
N
R
N
Ar = Phenyl, 2-Pyridyl
-Pyridyl, 4-Pyridyl
N
N
O
3
O
5
a-d
1
3-15, 22-24, 31-33, 40-42
a
e
O
O
O
O
S
S
S
S
OH
NH2
O
O
Ar
Ar
Ar
Ar
d
f
b
N
N
N
O
N
O
O
OH
6a-d
7a-d
c
8a-d
9a-d
g
O
O
S
O
S
NH2
N
Ar
Ar
N
R
N
R
O
O
N
N
N
N
O
N
O
O
O
10-12, 19-21, 28-30, 37-39
16-18, 25-27, 34-36, 43-45
Scheme 1: Reagents and conditions. a. diethylbromomalonate, pyridine, toluene, reflux; b. propargyl
o
4 2
alscohol, DEAD, triphenylphosphine, THF,0 C to rt; c. Azido coumarin, CuSO , sodium ascorbate, THF:H O,
rt; d. LiOH, THF:CH OH::H O, rt; e. azido coumarin, CuSO , sodium ascorbate, THF:H O, rt; f. (i) CO Cl ,
3
2
4
2
2
2
DCM, DMF (cat), (ii) NH
4
OH; g. azido coumarin, CuSO
4
2
, Sodiuma ascorbate, THF:H O.
2
1

Organic & Biomolecular Chemistry
Page 22 of 24
View Article Online
DOI: 10.1039/C4OB02049A
o
Figure 3: DSF curve for compound 27 a T
M
shift of 3.2 C showing an increase in thermal
stability between the native protein G24 protein (red) and DNA gyraseB proteinꢀcompound
complex (blue).
Figure 4: Evaluation of cardiotoxicity of the compounds 18, 25 and 27 in zebra fish model.
2
2

Page 23 of 24
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C4OB02049A
Table 1: Inꢀvitro inhibitory potency of synthesised analogues 10 ꢀ 45.
GyrB
assay (IC50
Supercoiling MIC Cytotoxicity
Cmpd.
Ar
R
R’
b
a
c
e
)
assay (IC50
)
µM
(% inhib:)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
OC
2
2
2
H
H
H
5
5
5
H
OH
OCH
H
19.1±1.3
16.13±0.17
18.38±0.76
>50
7.3±0.62
11.31±0.13
13.5±0.81
>25
26.34
25.49
24.79
56.0
23.57
24.23
25.88
27.31
36.06
27.99
26.31
30.17
21.86
14.58
29.35
24.69
39.16
13.96
31.93
23.61
29.42
26.72
11.32
22.22
11.52
27.57
24.78
20.67
31.89
24.91
13.80
35.5
OC
OC
3
3
3
3
3
3
3
3
3
3
3
OH
OH
OH
OH
OCH
H
21.8±0.82
20.36±1.3
1.2±0.48
17.4±0.66
14.9±0.42
0.62±0.30
0.71±0.16
0.63±0.28
10.2±0.71
11.2±0.64
5.5±0.24
>25
27.03
26.23
14.03
27.08
13.15
26.29
12.72
12.72
111.75
26.97
26.18
7.00
NH
NH
NH
2
OH
OCH
H
1.8±0.66
2
0.92±0.28
21.45±0.97
14.66±0.33
5.7±0.18
2
2ꢀPyridyl OC
2ꢀPyridyl OC
2ꢀPyridyl OC
H
2
2
2
5
5
5
H
H
OH
OCH
H
2ꢀPyridyl
2ꢀPyridyl
2ꢀPyridyl
2ꢀPyridyl
2ꢀPyridyl
2ꢀPyridyl
OH
OH
OH
>50
OH
OCH
H
16.7±1.2
15.8±0.63
15.6±0.31
0.44±0.17
0.8±0.32
0.5 ±0.55
11.3±0.92
10.6±1.12
0.5±0.22
16.1±0.41
18.2±0.76
12.4±0.27
1.7±0.61
0.75±0.21
8.5±0.92
9.1±0.51
9.7±0.13
8.3±0.31
>25
18.93±0.77
0.71±0.72
1.6±0.72
NH
NH
NH
2
OH
OCH
H
13.5
2
0.44±1.1
6.5
2
3ꢀPyridyl OC
3ꢀPyridyl OC
3ꢀPyridyl OC
2
2
2
H
5
5
5
15.8±1.5
26.29
25.43
12.36
27.93
53.95
26.18
13.99
27.03
13.12
26.28
25.43
24.72
111.75
26.97
26.18
H
H
OH
OCH
H
14.3±0.91
2.24±0.22
23.6±0.82
27.54±1.1
22.7±2.1
3ꢀPyridyl
3ꢀPyridyl
3ꢀPyridyl
3ꢀPyridyl
3ꢀPyridyl
3ꢀPyridyl
OH
OH
OH
OH
OCH
H
NH
NH
NH
2
2.896±0.14
1.354±0.25
7.3±0.21
2
OH
OCH
H
2
4ꢀPyridyl OC
4ꢀPyridyl OC
4ꢀPyridyl OC
2
2
2
H
5
5
5
18.91±0.8
15.25±0.53
19.8±0.24
32.1±0.46
17.38±0.75
14.98±0.44
H
H
OH
OCH
H
56.5
41.5
4ꢀPyridyl
4ꢀPyridyl
4ꢀPyridyl
OH
OH
OH
15.8
OH
OCH
14.9±0.48
11.4±0.91
18.64
34.49
2
3

Organic & Biomolecular Chemistry
Page 24 of 24
View Article Online
DOI: 10.1039/C4OB02049A
4
4
4
3
4
5
4ꢀPyridyl
4ꢀPyridyl
4ꢀPyridyl
NH
NH
NH
2
2
2
H
OH
17.27±1.6
7.79±0.43
16.28±0.18
46±10 nM
nd
11.8±0.31
1.56±0.89
13.3±0.24
180±3.9 nM
nd
27.99
13.51
26.23
nd
30.41
26.88
29.69
81.67
nd
OCH
3
Novobiocin
Isoniazid
Rifampicin
Ofloxacin
0.66
0.23
2.16
nd
nd
nd
nd
nd
nd
a
b
c
Mycobacterium smegmatis GyrB ATPase activity, Mycobacterium tuberculosis DNA Gyrase supercoiling activity, in
vitro Mycobacterium tuberculosis activity, At 100µM against RAW 264.7 cells, nd: indicates not determined
d
2
4