Synthesis and evaluation of novel coumarin-oxime ethers as potential anti-tubercular agents: Their DNA cleavage ability and BSA interaction study

Article abstract of DOI:10.1016/j.ejmech.2018.03.042

As a contribution to the development of novel coumarin-oxime ether conjugates with therapeutically interesting properties, a series of coumarin-oxime ether (1a–1j) was synthesised using S_N2 reaction of bromomethyl coumarins with butane-2,3-dione monoxime. Invitro anti-tuberculosis activityagainstMTBH₃₇Rv strain was established for the coumarin-oxime ether (1a–1j). Most of the compounds exhibited significant activity with minimum inhibitory concentration (MIC)in the range of 0.04–3.12 μg mL^?1. Compound (1h) was identified as a hit candidate exhibiting MIC of 0.04 μg mL^?1, closer to the MIC value of Isoniazid (0.02 μg mL^?1), a commercially available drug for the treatment of tuberculosis. Compound 1h also displayed a low level of toxicity in Vero cells along with a good safety profile in vitro. Compounds that showed potent anti-tubercular activity were also found to cleave DNA more efficiently and thereby exhibit nuclease activity. The most active compound (1h) was further studied to deduce the mode of interaction with model serum protein, bovine serum albumin (BSA).

Full text of DOI:10.1016/j.ejmech.2018.03.042

Accepted Manuscript
Synthesis and evaluation of novel coumarin-oxime ethers as potential anti-tubercular
agents: Their DNA cleavage ability and BSA interaction study
Dinesh S. Reddy, Manasa Kongot, Sandeep P. Netalkar, Mahantesh M. Kurjogi,
Rakesh Kumar, Fernando Avecilla, Amit Kumar
PII:
S0223-5234(18)30285-X
10.1016/j.ejmech.2018.03.042
EJMECH 10307
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 November 2017
Revised Date: 14 February 2018
Accepted Date: 15 March 2018
Please cite this article as: D.S. Reddy, M. Kongot, S.P. Netalkar, M.M. Kurjogi, R. Kumar, F. Avecilla,
A. Kumar, Synthesis and evaluation of novel coumarin-oxime ethers as potential anti-tubercular agents:
Their DNA cleavage ability and BSA interaction study, European Journal of Medicinal Chemistry (2018),
doi: 10.1016/j.ejmech.2018.03.042.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Synthesis and Evaluation of Novel Coumarin-Oxime Ethers as Potential
Anti-tubercular Agents: Their DNA Cleavage Ability and BSA
Interaction Study
a
a
b
c
Dinesh S. Reddy, Manasa Kongot, Sandeep P. Netalkar, Mahantesh M. Kurjogi, Rakesh
a
d
a
Kumar, Fernando Avecilla, Amit Kumar *
a
Centre for Nano and Material Sciences, Jain University, Jain Global Campus, Jakkasandra Post,
Bangalore 562112, India
b
P. G. Department of Studies in Chemistry, Karnatak University, Dharwad-580003, India
c
College of Horticulture, Nanjing AgriculturalUniversity, Nanjing, China
d
Grupo Xenomar, Centro de Investigacións Científicas Avanzadas (CICA), Departamento de
Química, Facultade de Ciencias, Universidade da Coruña, Campus de A Coruña, 15071 A
Coruña, Spain
*
To whom correspondence should be addressed.
E-mail: amit.kumar@jainuniversity.ac.inPhone: +91-80-27577251, Fax: +91-80-2757 7211
ABSTRACT
As a contribution to the development of novel coumarin-oxime ether conjugates with
therapeutically interesting properties, a series of coumarin-oxime ether (1a–1j) was synthesised
using S 2 reaction of bromomethyl coumarins with butane-2,3-dione monoxime. Invitro anti-
N
tuberculosis activityagainstMTBH37Rv strain was established for the coumarin-oxime ether (1a–
1
j).Most of the compounds exhibited significant activity with minimum inhibitory concentration
-1
(
MIC)in the range of 0.04–3.12µg mL .Compound (1h) was identified as a hit candidate
-1
-1
exhibiting MIC of 0.04µg mL , closer to the MIC value of Isoniazid (0.02 µg mL ), a
commercially available drug for the treatment of tuberculosis. Compound 1h also displayed a
low level of toxicity in Vero cells along with a good safety profile in vitro. Compounds that
showed potent anti-tubercular activity were also found to cleave DNA more efficiently and
thereby exhibit nuclease activity. The most active compound (1h) was further studied to deduce
the mode of interaction with model serum protein, bovine serum albumin (BSA).
Keywords: Coumarin-oxime ether, Anti-tubercular, DNA cleavage, Cytotoxicity, BSA
interaction.
1

ACCEPTED MANUSCRIPT
1
. INTRODUCTION
Tuberculosis (TB) is one of the most fatal infectious diseases caused by Mycobacterium
tuberculosis that may lead to tubercle lesions in the lungs, abnormalities in skin, lymph nodes,
and brain.[1]. It is the second leading cause of death after the human immunodeficiency virus
(
HIV), accounting for a mortality rate of 1945 deaths every day. World Health Organization
WHO) has estimated one-third of the world's population at risk due to the latent infection with
(
Mycobacterium tuberculosis [1, 2]. In year 2015, nearly 1 million deaths and 10.4 million new
cases were caused by this deadly infection [1a]. Despite the availability of a large variety of
drugs [3], tuberculosis remains a major threat for the humans being [1, 2]. The major obstacle in
eradication of tuberculosis is the emerging of resistance of Mycobacterium tuberculosis to the
existing drugs [4] (Figure 1), that has resulted in multidrug-resistant tuberculosis (MDR-TB) and
extensively drug-resistant tuberculosis (XDR-TB) [1, 2]. The co-morbidity of TB with HIV-
AIDS is another major concern and in some cases nearly two-third of the patients diagnosed with
TB are also HIV-1 seropositive [5]. The rapid emergence of multidrug resistant strains along
with co-infection with HIV, have created an alarming situation that necessitates an urgent
development of new and effective anti-tubercular drugs.
O
H N
2
HO
O
OH
NHNH2
OH
HO
N
O
O
HO
OH
H
N
AcO
HOOH OH
O
Isoniazid
O
OH OH
H N
2
OH
O
OH
NH2
Inhibits mycolic acid
synthesis
RS (ref: 4a, 4f)
NH
HO
MeO
O
O
OH
O
N
N
H
2
N
NH
2
O
NH
OH
2
OH
O
N
O
NH2
O
O
Kanamycin
NH2
HN
O
Inhibits protein synthesis
RS (ref: 4c)
OH
OH
NH2
Rifampicin
Inhibits transcription
RS (ref: 4d)
Amikacin
Inhibits protein synthesis
RS (ref: 4c)
Cycloserine
Inhibits peptidoglycan
synthesis
NH2
O
O
RS (ref: 4b)
H O
N
O
N
O
H N
2
NH
2
S
N
N
F
H
H
H N
N
O
NH2
2
NH
HN
H
H
N
N
H N
2
N
N
O
N
N
O
O
H
Ethionamide
Pyrazinamide
O
N
H
O
H N
2
Ofloxacin
Inhibits DNA supercoiling
RS (ref: 4h)
Inhibits mycolic acid
biosynthesis
Inhibit the fatty acid
synthetase I (FASI) enzyme
RS (ref: 4g)
Capreomycin IA
Inhibit protein synthesis
RS (ref: 4e)
N
RS (ref:4f)
Figure1. Commercially available anti-tubercular drugs and their modes of actions.[3] Drug
resistant strains for these drugs have been reported.[4] RS: resistance strains.
2

ACCEPTED MANUSCRIPT
Re-engineering of the known anti-tubercular scaffolds amenable to the improved anti-
mycobacterial activity against multidrug-resistant strains, and enhanced pharmacokinetic
properties, offers an attractive strategy for the effective control [6].
The coumarin moiety is well-regarded as a ‘privileged’ structural motif due to its broad
spectrum of pharmacological activities [7]. Moreover the unique structure of coumarin allows its
derivatives to readily interact with a diversity of biomacromolecules through weak interactions
and thereby exhibit wide potentiality in medicines [8]. Hence, coumarin-derived compounds
have been explored as promising lead targets in medicinal chemistry [9] and recently, they have
also been envisaged as potential candidates for the treatment of tuberculosis [10-12], for e.g. bis
aryloxy linked coumarin triazole (1) [10c], phenyl-substituted coumarin (2) [10d], 4-
aryl/alkylsulfonylmethylcoumarins (3) [10e], iodinated-4-aryloxymethyl-coumarin (4) [10f],
chalconated coumarin (5) [10g], coumarin-thiazoline conjugates (6) [12a] and benzocoumarin-
pyrimidine conjugates (7) [12b] exhibited potential antitubercular activity with MIC of 0.2, 0.24,
0
.78, 1.56, 3.0, 0.09 and 0.05 µg/mL respectively (Figure 2). Reports have also suggested that
coumarin class compounds target the fatty acyl-ACP synthetase activity of the FadD32 enzyme,
essential for survival of Mycobacterium tuberculosis [10d, 11b]. Further, from our previous
studies it was found that coumarin based compounds exhibited promising anti-tubercular
activities along with good safety profiles.[12]. Hence, looking into the biological significance of
coumarin particularly in the field of tubercular treatment, it was desirable for us to introduce
coumarin as the core structural unit of the compound.
O
O
H N
2
O
N
N
N
O
N
S
O
N
N
N
CH
3
N
O
(
3)
(
1)
O
O
(2)
H CO
O
O
3
O
O
H C
3
O
O
MIC (MTB) = 0.2 µg/ml
ref: 10c
OCH3
MIC (MTB) = 0.24 µg/ml
ref: 10d
OR
MIC (MTB) = 0.78 µg/ml
ref: 10e
I
S
N
F
S
Cl
(
4)
N
O
N
O
O
O
(6)
Cl
(5)
MIC (MTB) = 0.09 µg/mL
ref: 12a
(
7)
H CO
O
O
3
O
O
O
O
R = H, MIC (MTB) = 3.0 µg/ml
MIC (MTB) = 0.05 µg/mL
ref: 12b
MIC (MTB) = 1.56 µg/ml R = 2-ethylpiperidine, MIC (MTB) = 3.5 mg/mL
ref: 10g
ref: 10f
Figure 2.Structures of some coumarin derivatives exhibiting potent anti-tubercularactivity.
3

ACCEPTED MANUSCRIPT
It is known that most of the currently existing anti-tubercular drugs such as Isoniazid,
Cycloserin, Pyrazinamide, Rifampicin, Amikacin and Capreomycin IA (Figure 1) constitute side
aliphatic region wherein carbonyl group and nitrogen atom are in common; we anticipate that
these groups may contribute in enhancing the anti-tubercular activity. Hence in search of new
anti-tubercular agents containing both nitrogen and carbonyl group particularly in aliphatic
region, our attention curved in the direction of oxime moiety containing a carbonyl group and a
nitrogen atom (Butane-2,3-dione monoxime – starting material), in expectancy that it may lead
to more potent anti-TB agents. Further, oxime-based compounds have long since contributed to
various fields of chemistry, especially medicinal chemistry and are currently attractive synthetic
target molecules used in the molecular design of drugs [13]. In many cases, the oxime derived
compounds have been found to exert substantially increased biological effects compared to their
parent compounds [14]. Recent studies have shown that oxime motifs possess excellent DNA
photo-cleavage ability (examples shown in Figure 3) [15], and are stable to chemical
degradation in vivo [16]. In addition, oxime-based linkers have been exploited as a general strategy for
the rapid generation and screening of multidentate inhibitors [17]. The potential biological
significance of oximes and its important functional groups which is found mostly in the aliphatic
region of anti-tubercular drugs intrigued our interest in utilizing these scaffolds as conjugates to
the bioactive molecules for the development of effective anti-tubercular agents. Here, we
conjugated oximes to coumarin moiety, since from literature review as well as from our previous
studies [10-12] it is evident that conjugation of coumarin with various bio-active molecules has
produced novel hybrid molecules which are endowed with excellent anti-tubercular activity [10-
1
2] (Figure 2).The conjugation was done mainly at C-4 position of coumarin [10, 12a], since
this position is sterically more favored region for enhancing the activity of the compound [10c]
and has also shown to exhibit better selectivity over 3-substituted coumarins as they don’t inhibit
nuclear receptors [9c]. Hence we predict that the additive effect at C-4 position of coumarin with
oxime might produce synergetic effect in enhancing the bioactivity of the compound and could
be a good starting point for the development of new lead anti-tubercular drugs.
4

ACCEPTED MANUSCRIPT
X
NO2
N
O
N
K
O
Ar
N
O
N
O
N
O
N
O
O
O
R
O
H
O
N
N
CH3
Z
X
Z
X
Y
N
N
O
O
R = H or NH2
X = N, Z = CH
X = CH, Z = N
X= CH, Z=C NO2
O
X = N, Y = Z = CH
X = CH, Y = N, Z = CH
X= Y = CH, Z = N,
K = C NO2
K = N
Ar = 4-FPh, ref: 23b
Ar = 4-CNPh, ref: 23c
X
ref: 23a
ref: 23a
ref: 23a
X = H, CH3, F, CN
ref: 23d
Figure 3. Examples of oxime-derived compounds possessing DNA photo-cleavage ability.
In our continuous efforts to discover novel anti-tubercular scaffolds [12], we, herein report the
synthesis and in vitro evaluation of novel coumarin-oxime ether analogs as possible anti-
tubercular agent’s with their DNA cleavage ability, cytotoxicity and interactions with drug-
carrier protein BSA are also presented.
2
. RESULTS AND DISCUSSION
2
.1. Synthesis of coumarin-oxime ether conjugates
In order to tether the oxime moiety to the coumarin core, a simple protocol using SN2
substitution was undertaken. For this, the requisite 4-bromomethyl coumarins (a–j) [18] were
synthesised by the Pechman cyclization of phenols (iii) with 4-bromoethylacetoacetate (ii)
(
(
(
Table 1). Condensation of 4-bromomethyl coumarins (a–j) with butane-2,3-dione monoxime
iv) in the presence of anhydrous K CO in absolute ethanol afforded oxime-derived coumarins
2
3
1a-1j) in 63-84% yields (Table 1). All the newly synthesised compounds were characterized
1
13
using ATR-IR, H NMR, C NMR Mass spectral and elemental analysis. Further, the structures
of compounds (1d) and (1h) were also elucidated by X-ray crystallography. Spectral data of the
newly synthesised compounds (1a–1j) are provided in the experimental section, and are in
accordance with the assigned structures of the compounds.
5

ACCEPTED MANUSCRIPT
a
Table 1.Synthetic route to coumarin-oximeconjugates1a-1j.
1
5
iv
1
O
2
1
4
iii
OH
Br HO
O
O
N
11
N
R
16
O
O
O
ii
O
5
13
Br2
Et O
4
K CO
2
6
7
10
17
3
3
Br
O
i
O
O
R
R
H SO ,
°C to rt
8 h
ethanol,
rt, 12 h
2
2
4
9
O
2
1
O
0
O
O
8
0
-5 °C
4
a-j
2
4 h
1a-j
O
O
O
O
O
O
O
O
N
O
N
N
N
O
N
Me
Cl
MeO
Me
O
O
O
O
O
O
O
O
O
Me 1e, 76%
1a, 73%
1b, 67%
1c, 64%
1d, 78%
O
O
O
O
O
O
O
O
1e
O
N
O
N
N
Me
N
N
Br
Cl
O
O
Me
O
O
Me
O
O
O
O
O
O
1f, 63%
1g, 81%
1h, 84%
1i, 73%
1j, 74%
a
Yields of the purified products.
2
.2. X-ray diffraction analysis of compounds (1d) and (1h)
Three-dimensional X-ray data were collected on a Bruker Kappa Apex CCD diffractometer at
low temperature by the scan method. Reflections were measured from a hemisphere of data
collected from frames, each of them covering 0.3º in .A total of 37225 for (1d) and 9116 for
1h), reflections measured were corrected for Lorentz and polarization effects and for absorption
by multi-scan methods based on symmetry-equivalent and repeated reflections. Of the total, 3298
for (1d) and 2973 for (1h), independent reflections exceeded the significance level ( F /  F )
4.0. After data collection, in each case an multi-scan absorption correction (SADABS) [19]
φ-ω
ω
(
σ
>
was applied, and the structure was solved by direct methods and refined by full matrix least-
2
squares on F data using SHELX suite of programs [20]. Hydrogen atoms were included in
calculation position and refined in the riding mode in the two structures. Refinements were done
with allowance for thermal anisotropy of all non-hydrogen atoms. A final difference Fourier map
-3
-3
showed no residual density outside: 0.408 and -0.259 e.Å for (1d) and 0.376 and -0.221 e.Å
2
2
o
2
for (1h).A weighting scheme w = 1/[σ (F ) + (0.062000 P) + 0.483400 P] for (1d) andw =
2
2
2
2
2
1
/[σ (F ) + (0.053800 P) + 0.206400 P] for (1h), where P = (|F | + 2|F | )/3, were used in the
o o c
latter stages of refinement. Further details of the crystal structures determination are given in
Table 2.
6

ACCEPTED MANUSCRIPT
Table 2. Crystal data and structure refinement for the compounds (1d) and (1h).
Compound
Formula
1d
1h
C H N O
C H N O4
16 17
18
15
4
Formula weight
T, K
309.31
287.31
100(2)
100(2)
Wavelength, Å
Crystal system
Space group
a/Å
0.71073
Monoclinic
0.71073
Triclinic
P 1
P2 /c
1
7.1714(2)
19.3308(6)
10.6997(3)
90
7.5936(6)
7.6065(6)
12.8938(9)
102.148(5)
98.695(5)
98.493(5)
707.19(9)
2
b/Å
c/Å
α
/º
β/º
γ/º
102.4205(10)
90
3
V/Å
Z
1448.57(7)
4
F000
648
304
-3
Dcalc/g cm
1.418
1.349
-1
µ/mm
0.101
0.097
θ
/ (º)
2.11 to 28.39
0.0272
3.54 to 28.34
0.0202
Rint
3
Crystal size/ mm
0.49 x 0.31 x 0.28
1.050
0.34 x 0.29 x 0.28
1.075
2
Goodness-of-fit on F
a
R [I>2σ(I)]
1
0.0386
0.0423
b
wR (all data)
0.1101
0.1150
2
Largest differences peak and
0.408 and -0.259
0.376 and -0.221
-3
hole (eÅ )
a
b
2
2
2
2 2 1/2
R = Σ F  - F  /Σ F  . wR = {Σ[w( F  -F   ) ] /Σ[w(F ) ]}
1
o
c
o
2
o
c
o
The single crystals for compound (1d) and (1h) were developed by slow evaporation of DMF
and ethanol mixture (80:20) at room temperature. Compound (1d) was crystallized in monoclinic
7

ACCEPTED MANUSCRIPT
with space group P2(1)/c. Compound (1h) was crystallized intriclinic system with space group
P 1.ORTEP diagrams of (1d)and (1h)are shown in Figures 4 and 5, respectively. π-π stacking
interactions are present in the two structures in the crystal packing. Compound (1d) present weak
π-π interactions and the distance between centroids, dc1-c2 = 4.001(10)Å, [c1, C(1A)-C(2A)-
C(3A)-C(4A)-C(13A)-O1A, c2, C(5G)-C(6G)-C(7G)-C(8G)-C(9G)-C(10G)], (see Figure S1 in
Supporting Information only labeling for A molecule are drawing for clarity). Nevertheless,
compound (1h) present strong π-π interactions and the distance between centroids, dc3-c4
=
3
.484(12) Å, [c3, C(4A)-C(5A)-C(6A)-C(7A)-C(8A)-C(9A); c4, C(1M)-C(2M)-C(3M)-C(4M)-
C(9M)-O(1M)] (see Figure S3 in Supporting Information).Coumarin and oxime groups are
planes as expected [mean deviation from planarity for C(1)-C(2)-C(3)-C(4)-C(5)-C(10)-C(11)-
C(12)-C(13)-O(1), 0.0616(13)Å and for N(1)-C(15)-C(16)-C(17)-C(18)-O(3), 0.0537 Å in 1d,
C(1)-C(2)-C(3)-C(4)-C(5)-C(6)-C(7)-C(8)-C(9)-O(1), 0.0171(11) Å and for N(1)-C(13)-C(14)-
C(15)-C(16)-O(3), 0.0241(10) Å, in (1h), respectively] and the angles between planes are
8
9.40(3)º in (1d) and 88.58(3)º in (1h). Atropoisomers by rotation around O-N bond of oxime
group are present in the structure. Figures S2 and S4 (see Supporting Information) show the two
atropoisomers observed in each compound. Hydrogen bonds are not present in the structures.
Selected Bond lengths [Å] and angles [°] for the compounds (1d) and (1h) are given in Table 3.
Table 3. Selected Bond lengths [Å] and angles [°] for the compounds 1d and 1h.
Bond lengths
O(1)-C(13)
1d
Bond lengths
O(1)-C(1)
1h
1.3687(13)
1.3710(13)
1.2781(13)
1.4038(11)
1.2066(14)
1.4277(12)
1.2167(13)
1d
1.3613(14)
1.3774(13)
1.2855(16)
1.3962(13)
1.2116(14)
1.4329(14)
1.2144(16)
1h
O(1)-C(1)
O(1)-C(9)
N(1)-C(15)
N(1)-C(13)
N(1)-O(3)
N(1)-O(3)
C(1)-O(2)
C(1)-O(2)
O(3)-C(14)
O(3)-C(12)
O(4)-C(14)
Angles
O(4)-C(17)
Angles
C(13)-O(1)-C(1)
C(15)-N(1)-O(3)
O(2)-C(1)-O(1)
O(2)-C(1)-C(2)
122.05(8)
110.66(8)
117.47(10)
126.76(11)
C(1)-O(1)-C(9)
C(13)-N(1)-O(3)
O(2)-C(1)-O(1)
O(2)-C(1)-C(2)
122.34(9)
111.24(10)
117.43(10)
125.97(11)
8

ACCEPTED MANUSCRIPT
O(1)-C(1)-C(2)
N(1)-O(3)-C(14)
O(3)-C(14)-C(3)
115.76(9)
109.28(7)
112.07(8)
O(1)-C(1)-C(2)
116.60(10)
108.73(8)
112.11(9)
N(1)-O(3)-C(12)
O(3)-C(12)-C(3)
Figure 4.ORTEP of the compound (1d). All the non-hydrogen atoms are presented by their 50%
probability ellipsoids. Hydrogen atoms are omitted for clarity.
Figure 5.ORTEP of the compound (1h). All the non-hydrogen atoms are presented by their 50%
probability.Hydrogen atoms are omitted for clarity.
9

ACCEPTED MANUSCRIPT
3
. BIOLOGICAL STUDIES
The synthesised compounds were screened for their in vitro anti-tubercular activity using the
Microplate Alamar Blue Assay (MABA) [21] and CT-DNA cleavage study by gel
electrophoresis method [22]. The active compounds were also tested for their cytotoxicity
against Vero cells by MTT assay [23]. The interactions of the most active compound (1h) were
studied with the model serum protein BSA through spectroscopic techniques. pKa studies of all
the compounds were assessed using potentiometric titration method. UV-Visible spectral studies
were also carried out at pH conditions varying from 4.2–8.6 to check the suitability and stability
of the compounds under physiological conditions
3
.1. Anti-tubercularactivity
All the compounds (1a-1j) were initially screened at a single concentration of 6.25µg/mL
against MTBH37Rv (ATCC-27294) strain in BACTEC 12 B medium, using the Microplate
Alamar Blue Assay (MABA). In preliminary screening, compounds (1b, 1f, 1i & 1j) were found
to be inactive againstMTBH Rv strain; hence these compounds were not evaluated further. It
3
7
was observed in the initial screening (6.25 µg/mL concentration), the –Br substituted coumarin
derivative (1j) restrained the MTB by 81%, but in case of –Cl substituted coumarin (1b) and (1f)
the MTB strains were restricted by only 26 and 21% respectively at same concentration. This
could be due to the reason that the –Br group are comparatively more toxic than –Cl and thereby
the inhibition rate of MTB strain were increased when substituted with–Br group. Compounds
exhibiting ≥ 90% inhibition (1a, 1c, 1d, 1e, 1g and 1h) in the initial screening were tested in a
-1
dose range starting from 6.25µg mL concentration with serial dilution of 2-fold to determine
the actual minimum inhibitory concentration (MIC) value. From, Table 4 its clear that most of
the tested compounds exhibited significant anti-tubercular activity with MICs in the range 0.04–
-1
3
.12µg mL . In the secondary level of screening, the methyl substituted analogs, 1a (6-Me), and
g (7-Me) exhibited similar anti-tubercular activity with MIC value of 0.39 µg/mL. A lower MIC
1
value (0.19µg/mL) was observed in case of 6-OMe analog (1c), whereas the 5,6-Benzocoumarin
analog (1d) exhibited a high MIC value of 3.12 µg/mL. Interestingly, the dimethyl substituted
coumarins 1e (7,8-diMe) and 1h (5,7-diMe) proved beneficial for activity, exhibited the most
pronounced anti-tubercular activities with MIC 0.09 and 0.04µg/mL, respectively. These values
were very close to the MIC for isoniazid (0.02 µg/mL), a standard anti-tubercular drug.
10

ACCEPTED MANUSCRIPT
a
Table 4. In vitro anti-tubercular activity, cytotoxicity, lipophilicity and pK of compounds 1a–1j.
%
Inhibition at
% Survival of Vero
pK
a
a
MIC
c
Compound
R
concentration
cells at concentration Log P
(
µg/mL)
b
6
.25 µg/mL
(10 x MIC)
1
a
6-CH
3
92
26
94
90
95
21
91
97
87
81
0.39
N.D
0.19
3.12
0.09
N.D
0.39
0.04
N.D
N.D
72%
N.D
78%
67%
84%
N.D.
59%
89%
N.D
N.D
2.15
2.38
1.76
2.89
2.53
2.38
2.15
2.53
2.89
2.51
5.7
5.4
5.8
5.6
5.5
5.4
5.5
5.5
5.6
5.4
1
b
6-Cl
1
c
6-OCH
3
1
d
5,6-benzo
1
e
7,8- diCH
7-Cl
3
1
f
1
g
7-CH
5,7-diCH
3
3
1
h
1
i
7,8-benzo
6-Br
1
j
1
.8,
d
Isoniazid
-
100
0.02
98%
-0.97
3
.5, 9.5
a
b
Minimum inhibitory concentration against H37Rv strain of MTB (µg/mL).
Compound is considered toxic if it causes over 50% inhibition of Vero cells at concentration10
fold higher than its MIC [24].
c
Calculated by http://www.molinspiration.com; N.D. - Not determined.
d
Isoniazid pK values [25].
a
3
.1.1. Cytotoxicity study
Having identified a good number of active antimycobacterial compounds the next step was to
examine their toxicity against the Vero cell, at a concentration 10 times their actual MIC value
[24]. From the results (Table 4), it is clear that the tested compounds (1a, 1c, 1d, 1e, 1g and 1h)
exhibited moderate to low levels of cytotoxicity with a percentage survival of the Vero cells in
the range of 59 to 89%. A compound is considered to be safe for in vivo assay, only if the
percentage survival of Vero cells is above 50%, at concentration of compound 10times its actual
MIC value (10 x MIC) [24]. From the cytotoxicity results it is clear that tested compounds (1a,
1
c, 1d, 1e, 1g and 1h) exhibited good safety profile, suggesting great potential for their in vivo
11

ACCEPTED MANUSCRIPT
use as antitubercular agents. Among the tested compounds, the highest cytotoxicity against Vero
cells were observed by 7-CH (1g) and 5,6-benzo(1d)substituted coumarins, with percentage
3
survival of 59 and 67% respectively.
3 3
The second line of cytotoxicity was observed for –CH (1a) and –OCH (1c) substituted analogs
at C-6 position, with a percentage survival of 72 and 78% respectively. The di-CH substituted
3
compounds, (1e) and (1h) exhibited the highest safety profile with percentage survival of 84 and
8
9% respectively, suggesting the potential of these compounds as leads for the development of
new antitubercular drugs. Figure 6 gives the comparison between % survivals of Vero cells at
concentrations of the compounds which is 10 fold than its actual MIC values (µg/mL).
Figure 6. Comparison between percent survival of Vero cells at concentration of the compound
1
0 times than their actual MIC value (µg/mL).
Further, to assess the drug like properties of the compounds, their physicochemical analyses
were performed using free online software (http://www.molinspiration.com), for calculation of
important molecular properties (logP, polar surface area, number of hydrogen bond donors and
acceptors and others) [12a,b]. All compounds were found to follow Lipinski rule or the rule of
five (RO5) [26] i.e. molecular weights were below 500 daltons, lipophilicity expressed as a log P
(http://www.molinspiration.com), was less than 5 (Table 4); the number of hydrogen bond
donors as well as acceptors were also less than 5. A significantly low MIC value (0.04 µg/mL)
together with its physiochemical properties offers compound (1h) as a potential anti-tubercular
drug candidate.
12

ACCEPTED MANUSCRIPT
3
.2. Electrophoresis analysis
The design and development of small or medium-size molecules targeting nucleic acid cleavage
can lead to novel therapeutic agents for infectious diseases. Interactions of small molecules with
specific sites of a DNA strand as reactive models for protein-nucleic acid bindings provide paths
toward rational drug design as well as means to develop sensitive chemical probes for DNA. In
particular, designing of the compound based on their ability to cleave DNA is of great
importance not only from the primary biological point of view but also in terms of photodynamic
therapeutic approach to develop potent drugs [30]. Hence to understand the mode of action of the
synthesised compounds (1a-1j) and to identify new molecules that induce apoptosis we carried
out CT-DNA cleavage assay by agarose gel electrophoresis method (Figure7). The cleavage
potentials of the test compounds were assessed by comparing the bands appeared in test
compounds and control at 5µM concentration. DNA without the test compounds was used as a
control which does not show any cleavage of DNA even after a long time exposure.
It was observed that compounds (1a, 1b, 1c, 1e, 1h and 1j) cleaved DNA more efficiently
as indicated by the formation of smear in the gel without any sharp bands as shown in the
control. Whereas DNA treated with compound (1d) developed smear and one light band with
low molecular weight indicating the partial shearing of DNA. DNA treated with compound (1f)
showed similar sharp band as that of control but one additional band with low base also appeared
indicating the trimming activity of the compound. DNA treated with compounds (1g) and (1i)
showed three and four bands, respectively, indicating the partial cleavage activity of the
compounds. From Figure 7, it is clear that compounds (1a, 1b, 1c, 1e, 1h and 1j) cleave DNA
more efficiently, as no traces of DNA were found. Cleavage of DNA by these compounds may
be attributed to the denaturation of double-stranded DNA by cleavage of hydrogen bonds
between nitrogen bases and phosphodiester bond between pentose sugars at various sites. In
addition, the cleavage of DNA might have occurred frequently leading to low molecular weight
fragments that migrated away from the gel during electrophoresis. The remaining compounds
(1f), (1g) and (1i) were found to be inactive to cleave DNA efficiently, whereas little tailing in
band was observed in DNA treated with compound (1d) indicating the shearing of DNA. From
the structural point of view, it is observed that the substituent’s at C-6 position of the coumarin
moiety (viz.1a, 1b, 1c and 1j) as well as di-CH substituents at 5,7- and 7,8-positions (viz.1e and
3
13

ACCEPTED MANUSCRIPT
1
h) play a critical role in DNA cleavage. However, the nature of reactive intermediates involved
in the DNA cleavage by the compounds is not yet clear.
Figure 7. CT DNA Cleavage study by Agarose gel electrophoreses.lane C: control
DNA(untreated sample), lane 1a: DNA+1a,lane 1b: DNA+1b,lane 1c: DNA+1c, lane 1d:
DNA+1d,lane 1e: DNA+1e,lane 1f: DNA+1f,lane 1g: DNA+1g,lane 1h: DNA+1h,lane 1i:
DNA+1i,lane 1j: DNA+1j.
3
.3. Interaction study with BSA
BSA protein acts as a major reservoir of drug molecules which affects the overall bioavailability
of the drug. Therefore, studying the binding behavior between BSA and bioactive drug alike
compounds through spectroscopic techniques is of high significance. Compound (1h) which
exhibited excellentanti-tubercular activity with low MIC value and a minimal cytotoxicity was
further studied to gain insight into the mode of interaction with a model serum protein, bovine
serum albumin (BSA).
3
.3.1. UV-Visible spectroscopy studies
UV–Visible absorption spectroscopy is used as an informative tool to assess the binding between
adrug moleculeand bio-macromolecules such as proteins [31]. The absorbance shown by BSA
protein around 280nm due to the presence of tryptophanresidues [32] is an important factor in
observing the complex formation between the protein and drug molecule. The absorption spectra
of titration of BSA (5µM) with different concentrations of compound1h (0.83 µM to 8.26 µM) in
°
phosphate buffer of pH 7.4 at 27 Cis shown in Figure8a. The absorbance of BSA increased with
increasing concentrations of (1h) which suggested a static interaction [33] between (1h) and
BSA. In order to find out the extent of binding, the binding constant or association constant Ka
was calculated from the double reciprocal plot (A /(A-A ) vs 1/c) obtained from the UV-
0
0
absorbance data as shown in Figure 8b. The corresponding Gibb’s free energy change ∆G at 27
°
C was calculated using the Gibbs-Helmholtz equation (Eq. 1)
14

ACCEPTED MANUSCRIPT
∆
ꢀ = −ꢁꢂ ln ꢃꢄ
(1)
-1
-1
Where R=8.314JK mol is the universal gas constant, T=Temperature (298K) and K =
a
2
association constant/binding constant. The coefficient of determination (R ) upon linear fitting
a
was found to be 0.999. The calculated association constant (K ) and molar free energy (∆G)
4
-1
-1
values were 1.24×10 Lmol and –23.51 kJmol , respectively. The negative value of free energy
of binding indicated spontaneous interaction [33] between BSA and (1h).
(
a)
(b)
Figure 8. (a) UV-Visible spectra of BSA (5µM) titrated with 10 successive concentrations of
1h) (0.83 µM to 8.26 µM), (b) double reciprocal plot for binding between BSA and (1h).
(
3
.3.2. Fluorescence spectroscopy studies
Fluorescence spectroscopy provides information about quenching of fluorescence of the protein
upon binding with a bioactive molecule. This yields the number of binding sites available and
therefore provides a measure of extent of binding between protein and the molecule [34].
Fluorescence emission spectra of BSA (5µM) in the presence of different concentrations of the
°
compound1h (0.83 µM to 8.26 µM) in phosphate buffer of pH 7.4 at 27 C was recorded. As
shown in Figure 9, BSA emission maximum was observed at 347nm. Addition of compound
(1h) gradually decreased the fluorescence intensity. The extent of fluorescence quenching may
be due to different molecular interactions such as collisions between molecules or the ground-
state complex formation between the fluorophore and the quencher. To evaluate this, Stern-
Volmer plot [35] was drawn as shown in Figure 10a and the quenching constant was calculated
using the Stern-Volmer equation (Eq. 2),
ꢅꢆ
=
1 + ꢇ ꢉ [ꢋ] = 1 + ꢃ [ꢋ]
(2)
ꢈ ꢊ
ꢌꢍ
ꢅ
15

ACCEPTED MANUSCRIPT
where F
0
and F are the fluorescence intensities of BSA in the absence and presence of (1h)
at different concentrations. K refers to the Stern-Volmer quenching constant, k is the quenching
sv
q
rate constant of the biomolecular reaction, [Q] is the concentration of the compound and τ is the
0
-9
0
average lifetime of the pure BSA molecules (≈ 10 s). The plot of F /F vs [Q] gave a linear graph
2
with R =0.999 which provides an additional evidence of static quenching of the fluorophore
BSA by the ground state complex formation [36]. The values of Ksv and k
q
were calculated to be
4
-1
13
-1 -1
6
.19x10 mol and 1.07x10 mol s . The high values of the Stern-Volmer quenching constant
1
0
KSV and the biomolecular quenching constant k (˃10 ) suggest quenching of BSA by ground
q
a
state complex formation [35] with 1h. The binding constant K and the number of binding sites
(
n) available were also calculated by plotting log (F -F)/F vs log [Q] as shown in Figure 10b.
0
ꢒꢊ − ꢒ
ꢒ
ꢎꢏꢐ ꢑ
ꢓ = ꢎꢏꢐꢃ + ꢔꢎꢏꢐ[ꢋ]
ꢄ
Where F and F are the fluorescence intensities of pure BSA and BSA after titrations with
0
2
varying concentrations of (1h). Linear fitting gave R value as 0.954. The value of n was
calculated to be nearly 1, suggesting a 1:1 binding between the protein and (1h) which means
that there is an average of one binding site in BSA protein for compound (1h). The value of
4
-1
a
binding constant K calculated (1.37x10 Lmol ) was nearly same as that obtained from the UV-
Visible studies.
Figure 9. Intrinsic fluorescence spectra of BSA (5µM) titrated with 10 successive concentrations
of the compound 1h (0.83 µM to 8.26 µM).
16

ACCEPTED MANUSCRIPT
(
a)
(b)
Figure 10.(a) Stern-Volmerplot to calculateKSV and k , (b) graphical plot to calculate binding
q
sites (n) and Ka.
3
.4. pK and Stability studies
a
The physicochemical properties of drugs such as lipophilicity and biological membrane
permeability are greatly affected by their pK value. The pK values of all the compounds (1a-1j)
a
a
were assessed using potentiometric titration method [27]. UV-Visible spectral studies were also
carried out at pH conditions varying from 4.2–8.6 to check the stability of the most active
compounds (1a, 1c. 1e and 1h) under physiological conditions [28]. All the compounds (1a-1j)
showed a pH between 6.4 to 7.3 and this pH was slowly brought down to about pH 2 by
gradually titrating with HCl (25mM). Henceforth; the pH variation trend was carefully studied
with the slow addition of KOH solution from pH 2 to 12. A graph of pH vs volume of KOH
a
added was plotted to find the equivalence point and thereby pK . (Figure S5 to S14 in
Supporting Information). The pK value was found to be in between 5.4 to 5.8 for all the
a
compounds (Table 4). The pK
cross the lipid membrane barrier, get absorbed and also may enter to the target cells for action
29].
a
values suggest that the potential drugs moieties have ability to
[
17

ACCEPTED MANUSCRIPT
Figure 11.
Titration of 1h with HCl. The spectra were recorded after successive addition of 1
–
4
drop portion of HCl to 10 mL of ca. 10 solution of 1h.
The stability study of the most active compounds (1h) was established by electronic
absorption spectroscopy in the pH range of 4.4 – 8.2. Addition of HCl (25mM) to the compound
1
h results in the increase intensity of the absorption band at 290 nm while shoulder band at 315
nm slowly decrease in intensity (pH = 7.3 to 4.4, given in Figure 11). The reaction can be
reversed by treatment with KOH (25mM) (pH = 4.4 to 8.2, given in Figure 12). The reversibility
of the reaction suggests protonation of the imine nitrogen occurs during titration with HCl.
Absorbance bands maintained their positions throughout the experiments, no additional bands
were detected and the original absorbance bands stayed intact which illustrates that the
compounds 1h is stable in the pH range of 4.4 – 8.2.
18

ACCEPTED MANUSCRIPT
Figure 12.
Titration of 1h with KOH. The spectra were recorded after successive addition of
–
4
1
drop portion of KOH to 10 mL of ca. 10 solution of 1h.
The stability studies of the other active compounds (1a, 1c, 1e) were also established by
electronic absorption spectroscopy in the pH range of 4.2 – 8.6. Addition of HCl leads to slight
change in global absorbance that reverts back by addition of KOH suggests the protonation of
the imine nitrogen occurs during titration with HCl (Figure S15 to S20 in Supporting
Information). Absorbance bands maintained their positions which evident, the other active
compounds (1a, 1c, 1e) are stable in the pH range of 4.2 – 8.6.
4
. EXPERIMENTAL SECTION
4
.1. Materials and Methods
All solvents and chemicals were used as purchased without further purification. The
progress of all reactions was monitored on precoated silica gel plates (Merck, with fluorescence
indicator UV254) using Hexane: Ethyl acetate (70: 30) system as mobile phase. Spots were
visualized by irradiation with ultraviolet light (254 nm). Melting points were determined with an
1
13
open capillary method on a Buchi apparatus and are uncorrected. Proton ( H) and carbon ( C)
19

ACCEPTED MANUSCRIPT
1
NMR spectra were recorded on a BrukerAvance400 (400.13 MHz for H and 125.76 MHz for
1
3
C) using CDCl and for some compounds DMSO-d as solvent. Chemical shifts are given in
3
6
1
13
parts per million (ppm) (δ relative to residual solvent peak for H and C). IR spectra were
recorded on a Bruker, ALPHA, ATR-IR Spectrometer. Mass spectra were recorded using a
Shimadzu GCMSQP2010S. The elemental analyses were carried out using a Hereaus CHN rapid
analyzer. UV-Vis spectrawere recorded on a SHIMADZU, UV-1800 instrument. Fluorescence
spectra were recorded on a Spectroflouro photometer SHIMADZU, RF-5301pc instrument.
Three-dimensional X-ray data for (1d)and(1h) were collected on a Bruker SMART Apex CCD
diffractometer. Complex scattering factors were taken from the program package SHELXTL.
The pKa of the compounds was determined by means of potentiometric titration experiments
using a pH meter (HI5000 Series, Hanna) calibrated with standard buffers of pH 4 and 7. The
absorbance spectra of the compounds with varied pH conditions were studied using UV
Spectrophotometer (UV-1800, SHIMADZU).
4
4
.2. Chemistry
.2.1. General Procedure for Synthesis of Compounds (1a−1j)
Take 50 mL round bottom (R.B) flask fitted with drying tube. Add 2,3-butane dionemonoxime
0.01mol) and10mL of absolute ethanol (99% purity) to R.B flask and stir for 5min. Then add
powdered anhydrous K CO (0.03mol) and stirr for 40 min at room temperature. After 40min
(
2
3
add 4-bromomethylcoumarin (0.01mol) and stir the reaction mixture for 12 h at room
temperature. The progress of the reactions was examined using thin layer chromatography
(TLC), by using Hexane: Ethyl acetate system in the ratio of 70: 30 as mobile phase. After
completion of the reaction, the reaction mixture was quenched in crushed ice; the solid product
was filtered and given water wash. Finally, the product was recrystallized from aqueous ethanol
and some in DMF and ethanol mixture (80:20). The schematic representation to prepare
coumarin-oxime analogs and their respective yields are given in Table1.
6
-Methyl-4-({[(E)-(3-oxobutan-2-ylidene)amino]oxy}methyl)-2H-1-benzopyran-2-one
1a).2,3-butanedionemonoxime: 1.01 g (0.01 mol); 4-(bromomethyl)-6-methyl-2H-chromen-2-
one (a): 2.53g (0.01 mol); K CO : 4.14 g (0.03mol); absolute ethanol: 10 mL. Yield: 1.84 g
(
2
3
o
(
73%), colourless crystals; compound recrystallized in aqueous ethanol; mp 167–169 C; ATR-
20

ACCEPTED MANUSCRIPT
-1
1
IR ( ν max/cm ): 1701 (C=O of lactone), 1570 (C=N of imine); H NMR (400 MHz , CDCl
3
, δ
ppm) 2.00 (s, 3H, -CH ), 2.32 (s, 3H, -CH ), 2.41 (s, 3H, -CH ), 5.43 (s, 2H, -CH ), 6.39 (s, 1H,
3
3
3
2
1
3
=
CH), 7.24-7.36 (m, 3H, Ar-H); C NMR (100 MHz,CDCl , δ ppm): 9.0, 21.9, 25.0, 72.5,
3
+
1
13.7, 117.1, 117.1, 123.4, 133.0, 134.1, 150.1, 151.9, 157.2, 160.6, 195.7;GC-MS: 273 [M] ;
anal. calcd for C H NO : C, 65.92; H, 5.53; N, 5.13. Found: C, 65.88; H, 5.49; N, 5.17.
1
5
15
4
6
-Chloro-4-({[(E)-(3-oxobutan-2-ylidene)amino]oxy}methyl)-2H-1-benzopyran-2-one (1b).
2
,3-butanedionemonoxime: 1.01 g (0.01 mol); 4-(bromomethyl)-6-chloro-2H-chromen-2-one
(
b): 2.73g (0.01 mol); K CO : 4.14 g (0.03mol); absolute ethanol: 10 mL. Yield: 1.82 g (67%) ,
2 3
o
pale yellow solid; compound recrystallized in aqueous ethanol; mp 191–193 C; ATR-IR (ν
-1
1
max/cm ): 1713 (C=O of lactone), 1602 (C=N of imine); H NMR (400 MHz , CDCl , δ ppm)
3
2
.00 (s, 3H, -CH ), 2.33 (s, 3H, -CH ), 5.40 (s, 2H, -CH ), 6.45 (s, 1H, =CH), 7.49-7.57 (m, 2H,
3 3 2
1
3
Ar-H), 7.67 (s, 1H, Ar-H); C NMR (100 MHz,CDCl
18.3, 123.5, 132.3, 134.1, 149.2, 152.4, 157.5, 159.7, 195.75;GC-MS: 293 [M] ; anal. calcd for
: C, 57.25; H, 4.12; N, 4.77. Found: C, 57.22; H, 4.16; N, 4.73.
3
, δ ppm): 9.0, 25.1, 72.3, 115.2, 117.2,
+
1
14 4
C H12ClNO
6
-Methoxy-4-({[(E)-(3-oxobutan-2-ylidene)amino]oxy}methyl)-2H-1-benzopyran-2-one(1c).
2
,3-butanedionemonoxime: 1.01 g (0.01 mol); 4-(bromomethyl)-6-methoxy-2H-chromen-2-one
(
c): 2.69 g (0.01 mol); K CO : 4.14 g (0.03 mol); absolute ethanol: 10 mL.Yield: 1.72 g (64%),
2 3
o
-1
white solid; compound recrystallized in aqueous ethanol; mp 173–175 C; ATR-IR (ν max/cm ):
1
1
3
7
1
715 (C=O of lactone), 1606 (C=N of imine); H NMR (400 MHz , DMSO-d , δ ppm) 2.13 (s,
6
3 3 3 2
H, -CH ), 2.22 (s, 3H, -CH ), 3.55 (s, 3H, -OCH ), 5.41 (s, 2H, -CH ), 6.18 (s, 1H, =CH), 7.29-
1
3
.41 (m, 3H, Ar-H); C NMR (100 MHz, DMSO-d , δ ppm): 9.2, 25.2, 55.4, 72.7, 111.6, 115.0,
6
+
21.8, 124.4, 126.4, 142.5, 151.4, 152.4, 157.1, 161.2, 196.8; GC-MS: 289 [M] ; anal. calcd for
15 5
C H15NO : C, 62.28; H, 5.23; N, 4.84. Found: C, 62.31; H, 5.19; N, 4.80.
1
-({[(E)-(3-Oxobutan-2-ylidene)amino]oxy}methyl)-3H-naphtho[2,1-b]pyran-3-one(1d).2,3-
butane dionemonoxime(1): 1.01 g (0.01 mol); 1-(bromomethyl)-3H-benzo[f]chromen-3-one (d):
2
.89 g (0.01 mol); K
2
CO
3
: 4.14 g (0.03mol); Absolute ethanol: 10 mL. Yield: 2.25 g (78%),
o
brown crystals; compound recrystallized in DMF and ethanol mixture (80:20); mp 203–205 C;
-1
1
ATR-IR (ν max/cm ): 1700 (C=O of lactone), 1643 (C=N of imine); H NMR (400 MHz ,
CDCl ,δ ppm) 2.03 (s, 3H, -CH ), 2.35 (s, 3H, -CH ), 5.86 (s, 2H, -CH ), 6.62 (s, 1H, =CH),
3
3
3
2
21

ACCEPTED MANUSCRIPT
13
7
.47-7.70 (m, 3H, Ar-H), 7.92-8.01 (m, 2H, Ar-H ), 8.19 (d, 1H, J =8.8 Hz); C NMR
(
100MHz,CDCl , δ ppm): 9.2, 25.1, 75.7, 112.9, 114.1, 117.9, 124.9, 125.7, 128.4, 129.3, 129.9,
3
+
1
31.3, 134.0, 152.3, 155.0, 157.5, 160.2, 195.8;GC-MS: 309 [M] ; anal. calcd for C H NO :
1
8
15
4
C, 69.89; H, 4.89; N, 4.53. Found: C, 69.91; H, 4.87; N, 4.49.
7
,8-dimethyl-4-({[(E)-(3-oxobutan-2-ylidene)amino]oxy}methyl)-2H-1-benzopyran-2-one
(1e).2,3-butanedionemonoxime: 1.01 g (0.01 mol); 4-(bromomethyl)-7,8-dimethyl-2H-chromen-
2
-one (e): 2.67g (0.01 mol); K
2
CO
3
: 4.14 g (0.03mol); Absolute ethanol: 10 mL. Yield: 2.02 g
o
(
76%), white solid; compound recrystallized in aqueous ethanol; mp 163–165 C; ATR-IR (ν
-1
1
max/cm ): 1696 (C=O of lactone), 1604 (C=N of imine); H NMR (400 MHz , CDCl
3
, δ ppm)
2
6
1
.00 (s, 3H, -CH ), 2.31 (s, 3H, -CH ), 2.36 (s, 3H, -CH ), 2.64 (s, 3H, -CH ), 5.54 (s, 2H, -CH ),
3
3
3
3
2
1
3
.34 (s, 1H, =CH), 7.72-7.80 (m, 2H, Ar-H) ; C NMR (100 MHz,CDCl , δ ppm): 4.4, 16.3,
3
8.9, 20.2, 70.3, 108.4, 109.9, 111.5, 124.9, 130.5, 137.5, 147.3, 150.5, 152.4, 155.6, 191.0 ;GC-
+
MS: 287 [M] ; anal. calcd for C H NO : C, 66.89; H, 5.96; N, 4.88. Found: C, 66.92; H, 5.92;
1
6
17
4
N, 4.85.
7
-Chloro-4-({[(E)-(3-oxobutan-2-ylidene)amino]oxy}methyl)-2H-1-benzopyran-2-one
(1f).2,3-butanedionemonoxime: 1.01 g (0.01 mol); 4-(bromomethyl)-7-chloro-2H-chromen-2-
one (f): 2.73g (0.01 mol); K
2
CO
3
: 4.14 g (0.03mol); absolute ethanol: 10mL. Yield: 1.71 g
o
(
63%), pale yellow solid; compound recrystallized in aqueous ethanol; mp 197–198 C; ATR-IR
-1
1
(
ν max/cm ): 1709 (C=O of lactone), 1579 (C=N of imine); H NMR (400 MHz , DMSO-d , δ
6
ppm) 2.17 (s, 3H, -CH
3
), 2.28 (s, 3H, -CH
3
), 4.78 (s, 2H, -CH
2
), 6.51 (s, 1H, =CH), 7.30-7.38
1
3
(
m, 2H, Ar-H), 7.69 (s, 1H, Ar-H ); C NMR (100 MHz, DMSO-d , δ ppm): 9.2, 25.6, 72.3,
6
+
1
15.5, 116.2, 117.1, 125.4, 126.9, 137.3, 144.2, 150.9, 153.8, 160.4, 195.3; GC-MS: 293 [M] ;
anal. calcd for C H ClNO : C, 57.25; H, 4.12; N, 4.77. Found: C, 57.23; H, 4.15; N, 4.75.
1
4
12
4
7
-Methyl-4-({[(E)-(3-oxobutan-2-ylidene)amino]oxy}methyl)-2H-1-benzopyran-2-one
1g).2,3-butanedionemonoxime: 1.01 g (0.01 mol); 4-(bromomethyl)-7-methyl-2H-chromen-2-
one (g): 2.53g (0.01 mol); K CO : 4.14 g (0.03mol); absolute ethanol: 10mL. Yield: 2.04 g
(
2
3
o
(
81%), white solid; compound recrystallized in aqueous ethanol; mp 171–172 C; ATR-IR (ν
-1
1
max/cm ): 1702 (C=O of lactone), 1605 (C=N of imine); H NMR (400 MHz , CDCl
3
, δ ppm)
2
.10 (s, 3H, -CH ), 2.34 (s, 3H, -CH ), 2.42 (s, 3H, -CH ), 5.44 (s, 2H, -CH ), 6.37 (s, 1H,
3
3
3
2
1
3
=
3
CH), 7.25-7.26 (m, 1H, Ar-H), 7.34-7.37(m, 2H, Ar-H); C NMR (100 MHz,CDCl , δ ppm):
22

ACCEPTED MANUSCRIPT
9
.0, 21.0, 25.0, 72.5, 113.7, 117.0, 117.1, 123.4, 133.0, 134.1, 150.1, 151.8, 157.2, 160.7, 195.8;
+
GC-MS: 273 [M] ; anal. calcd for C H NO : C, 65.92; H, 5.53; N, 5.13. Found: C, 65.95; H,
1
5
15
4
5
.49; N, 5.11.
5
,7-Dimethyl-4-({[(E)-(3-oxobutan-2-ylidene)amino]oxy}methyl)-2H-1-benzopyran-2-one
(1h). 2,3-butanedionemonoxime: 1.01 g (0.01 mol); 4-(bromomethyl)-5,7-dimethyl-2H-
chromen-2-one (h): 2.67g (0.01 mol); K CO : 4.14 g (0.03mol); absolute ethanol: 10mL. Yield:
2
3
2
.24 g (84%),colourless crystal; compound recrystallized in DMF and ethanol mixture (80:20);
o
-1
1
mp 160–161 C; ATR-IR (ν max/cm ): 1698 (C=O of lactone), 1604 (C=N of imine); H NMR
400 MHz , CDCl ,δ ppm) 1.99 (s, 3H, -CH ), 2.32 (s, 3H, -CH ), 2.35 (s, 3H, -CH ), 2.63 (s,
H, -CH ) 5.53 (s, 2H, -CH ), 6.33 (s, 1H, =CH), 6.89 (s, 1H, Ar-H), 7.00 (s, 1H, Ar-H ); C
(
3
3
3
3
1
3
3
3
2
NMR (100 MHz,CDCl , δ ppm): 4.4, 16.4, 18.9, 20.3, 70.3, 108.4, 110.0, 111.5, 124.9, 130.6,
3
+
1
6
37.6, 147.4, 150.6, 152.5, 155.7, 191.2; GC-MS: 287 [M] ; anal. calcd for C16
6.89; H, 5.96; N, 4.88. Found: C, 66.91; H, 5.94; N, 4.91.
4
H17NO : C,
4
-({[(E)-(3-Oxobutan-2-ylidene)amino]oxy}methyl)-2H-naphtho[1,2-b]pyran-2-one (1i).2,3-
butanedionemonoxime: 1.01 g (0.01 mol); 4-(bromomethyl)-2H-benzo[h]chromen-2-one (i):
.89g (0.01 mol); K CO : 4.14 g (0.03mol); absolute ethanol: 10mL. Yield: 2.10 g (73%), light
2
2
3
o
-
brown solid; compound recrystallized in aqueous ethanol; mp 211–213 C; ATR-IR (ν max/cm
1
1
)
: 1697 (C=O of lactone), 1615 (C=N of imine); H NMR (400 MHz , CDCl ,δ ppm) 2.02 (s,
3
3
7
2
1
H, -CH ), 2.35 (s, 3H, -CH ), 5.84 (s, 2H, -CH ), 6.61(s, 1H, =CH), 7.42-7.69 (m, 3H, Ar-H),
3
3
2
1
3
3
.89-7.99 (m, 2H, Ar-H ), 8.17(d, 1H, J = 8.8 Hz); C NMR (100 MHz,CDCl , δ ppm): 9.2,
5.1, 75.7, 112.5, 114.1, 117.80, 124.79, 125.5, 128.5, 128.6, 129.9, 131.7, 134.0, 152.3, 154.9,
+
57.5, 160.2, 195.7;GC-MS: 309 [M] ; anal. calcd for C18
H
15NO
4
: C, 69.89; H, 4.89; N, 4.53.
Found: C, 69.92; H, 4.85; N, 4.49.
6
2
3
-Bromo-4-({[(E)-(3-oxobutan-2-ylidene)amino]oxy}methyl)-2H-1-benzopyran-2-one(1j)
,3-butanedionemonoxime: 1.01 g (0.01 mol); 6-bromo-4-(bromomethyl)-2H-chromen-2-one (j):
.17g (0.01 mol); K CO : 4.14 g (0.03mol); absolute ethanol: 10mL. Yield: 2.34 g (74%), pale
2
3
o
-1
yellow; compound recrystallized in aqueous ethanol; mp 207–208 C; ATR-IR (ν max/cm ):
1
1
714 (C=O of lactone), 1602 (C=N of imine); H NMR (400 MHz , CDCl
3
,δ ppm) 2.03 (s, 3H, -
CH ), 2.35 (s, 3H, -CH ), 5.85 (s, 2H, -CH ), 6.62 (s, 1H, =CH), 7.46-7.54 (m, 2H, Ar-H),
3
3
2
1
3
7
3
.71(s, 1H, Ar-H ); C NMR (100 MHz,CDCl , δ ppm): 9.2, 25.1, 75.7, 114.1, 117.9, 128.4,
23

ACCEPTED MANUSCRIPT
+
1
29.2, 131.3, 134.0, 152.3, 155.0, 157.5, 160.2, 195.8;GC-MS: 338 [M] ; anal. calcd for
C H BrNO : C, 49.73; H, 3.58; N, 4.14. Found: C, 49.69; H, 3.61; N, 4.12.
14
12
4
4
.3. Biological Assay
4
.3.1. Anti-tubercularAssay
All the compounds were first screened at a minimum inhibition concentration of 6.25µg/mL
against M. tuberculosis H Rv (ATCC-27294) in BACTEC 12 B medium using the
3
7
MicroplateAlamar Blue Assay (MABA). Compounds exhibiting < 90% inhibition in the primary
screening were not evaluated further, while compounds exhibiting> 90% inhibition were re-
tested against MTBH37Rv at lower concentrations in order to determine the actual minimum
inhibitory concentration (MIC) in the MABA. The experiment was carried out in triplicate using
a 96-welled plate, to each well100µL of Middlebrook 7H9 broth was added and a serial dilution
of compounds was made directly on the plate. Firstly, 200µL of the sterile deionized water was
added to all outer perimeter wells of sterile 96-well plate to minimize evaporation of medium in
the test wells during incubation. Isoniazid was included as a positive drug control. The test
compounds were serially diluted by two-fold serial dilution method. The plates were covered and
sealed with para film and incubated at 37º C for 5 days. After this, 25 µL of freshly prepared1:1
mixture of Almar Blue reagent and 10% tween 80 were added to the plate and incubated for 24
h. The blue color in the wells indicated the inhibition of bacterial growth while the pink color
was scored as growth. Further, the minimum concentration of the compound required to inhibit
the bacterial growth was determined.
4
.3.2. Assay for in vitroCytotoxicity Against Verocells
The cytotoxicity of the compounds (1a, 1c, 1d, 1e, 1g and 1h) with a MIC ≤ 6.25µg/mLwas
evaluated using Vero cells. The Vero cells were cultured in Dulbecco Modified Eagle Medium
(
DMEM) containing 2 mMNa CO supplemented with 10% (v/v) fetal bovine serum (FBS).The
2 3
o
cells were incubated at 37 C under 5% CO and 95% air in a humidified atmosphere until
2
6
confluent and then diluted with phosphate-buffered saline to obtain 10 cells per mL. Stock
solutions were prepared in dimethylsulfoxide (DMSO) and further dilutions were made with
fresh culture medium. The medium was removed and replaced by 180 mL of fresh medium
containing the test compound at a concentration 10 fold its actual MIC value. After incubation at
o
3
7 C for 72 h, the medium was removed and the monolayer was washed twice with 100 µL of
24

ACCEPTED MANUSCRIPT
warm Hanks’ balanced salt solution (HBSS). One hundred micro liters of warm medium and 20
µL of freshly made MTS-PMS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium and phenylmethasulfazone](100 : 20) (Promega) were added to
each well, the plates were incubated for 3 h, and the absorbance was determined at 560 nm using
a microplate reader. The percentage of cell survival was calculated after considering the control
wells (cells incubated in DMSO-containing medium).
4
.3.3. AgaroseGel Electrophoresis
1
% agarose gel buffered by tris–acetate-EDTA was run to indicate the integrity of the
DNA. The gel was prepared by adding 1g of agarose in 100 mL of TAE buffer. The mixture was
heated to dissolve the agarose completely. 5 µL of ethidium bromide was added to a slightly
cooledmoltanagarose. Then the moltanagarose was poured into the casting tray and allowed to
solidify. After solidification the comb was removed and the tray was placed in the tank wherein
the tank buffer (TAE) was poured. Then the wells were loaded with 10 µl of CT-DNA mixed
with 5 µg of test compounds solution and 5 µL of the tracking dye. Electrophoresis was done at
1
00 volts for 45mins till the loaded gel solution reaches end of the gel. The gel was visualized on
gel documentation system under UV light for analysis of the DNA fragmentation.
4
.3.4. Interaction Studyof compound 1h with BSA
Standard solutionof compound 1h (1mM) was prepared in DMSO. BSA standard solution (5µM)
was prepared by gentle dissolution in sodium phosphate buffer of pH 7.4 followed by incubation
for 24 h. The final concentration of the BSA solution was calculated using UV
Spectrophotometer by measuring the absorbance of BSA solution at 280nm (λmax of BSA) and
-1
-1
using the molar extinction coefficient of BSA (ε=43824M cm ) Absorption spectra was
recorded on a UV-1800 SHIMADZU UV-VIS Spectrophotometer using quartz cuvettes of 1.0cm
pathlength. The fluorescence spectra were measured using a RF-5301pc SHIMADZU
Spectrofluorophotometer equipped with a 150W xenon lamp. The excitation was set at 280nm
and the emission spectrum was measured between 290nm to 500nm with the slit width at 5nm.
4
.4. pH titrimetric assay
Stock solutions of the compounds 1a-1j (0.5 mM), HCl and KOH (25 mM each) were prepared
in acetonitrile and water mixture (9:1) [37]. The pK of the compounds was determined by
a
25

ACCEPTED MANUSCRIPT
means of potentiometric titration experiments using a pH meter (HI5000 Series, Hanna)
calibrated with standard buffers of pH 4 and 7. The absorbance spectra of the compounds with
varied pH conditions were studied using UV Spectrophotometer (UV-1800, SHIMADZU).
5
. CONCLUSION
The present study describes the synthesis and evaluation of new coumarin-oxime analogs(1a–
1
j) as potent anti-tubercular agents. Most of the compounds exhibited significant activity with
-1
MIC in the range0.04–3.12µg mL along with good safety profile against Vero cells. Single X-
ray crystals of compounds (1d) and (1h) were developed and their crystal parameters were
evaluated, which can contribute to the understanding of the reactivity, affinity and binding
properties of the molecules. Electrophoresis analysis revealed an excellent nuclease activity for
six analogs (1a, 1b, 1c, 1e, 1h and 1j) leading to a complete DNA cleavage. Among the
screened, compound 5,7-Dimethyl-4-({[(E)-(3-oxobutan-2-ylidene)amino]oxy}methyl)-2H-1-
-
1
benzopyran-2-one(1h) was identified as a hit candidate exhibiting MIC of 0.04µg mL along
with good safety profile against Vero cells suggesting great potential for their in vivo use as
antitubercular agents. The interaction studies of the most active analog (1h) with model serum
protein, BSA displayed an efficient binding to the protein without disrupting its secondary
structure and therefore, maintaining its function. The obtained results suggest that the potent
compound (1h) can act as promising lead for the development of new anti-tubercular agents.
Moreover, compounds (1h) and (1e) which were found to be the most active in vitro against the
MTBH Rv strain with good safety profile were also found to cleave DNA completely, indicating
3
7
that compounds (1h) and (1e) are biologically more potent in vitro agents and can be further
evaluated for other biological activities. The pKa studies, suggest that the compounds have
ability to cross the lipid membrane barrier, get absorbed and may also enter to the target cells for
action. The obtained results suggest that the potent coumarin-oxime analogs may serve as lead
chemical entities in search for new classes of potential anti-tubercular drugs. Further, this study
may help in exploring the oxime based linkages in the field of tubercular treatment and could be
a good starting point for the development of effective anti-tubercular drugs.
26

ACCEPTED MANUSCRIPT
Acknowledgements
Amit Kumar and Rakesh Kumar are grateful to SERB and Department of Science and
Technology for research grants (Ref. CS/097/2013 and letter no. SB/FT/CS-100/2013) and
(YSS/2015/001893), and Centre for Nano and Material Sciences, Jain University,
Bangalore,India for providing financial support and the necessary infrastructure.
Supplementary data
Crystallographic data has been deposited with the Cambridge Crystallographic Data Center with
CCDC No. 1556035 and 1556036 for compounds (1d) and (1h),respectively. These data can be
obtained free of charge via the URL http:// www.ccdc.cam.ac.uk/conts/retrieving.html (or from
the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336033; e-mail:
deposit@ccdc.cam.ac.uk).
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