Development of acridine derivatives as selective Mycobacterium tuberculosis DNA gyrase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.01.011

In this study we have designed p-phenylene diamine linked acridine derivative from our earlier reported quinoline-aminopiperidine hybrid MTB DNA gyrase inhibitors with aiming more potency and less cardiotoxicity. We synthesized thirty six compounds using four step synthesis from 2-chloro benzoic acid. Among them compound 4-chloro-N-(4-((2-methylacridin-9-yl)amino)phenyl)benzenesulphonamide (6) was found to be more potent with MTB DNA gyrase super coiling IC₅₀of 5.21 ± 0.51 μM; MTB MIC of 6.59 μM and no zHERG cardiotoxicity at 30 μM and 11.78% inhibition at 50 μM against mouse macrophage cell line RAW 264.7.

Full text of DOI:10.1016/j.bmc.2016.01.011

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of acridine derivatives as selective Mycobacterium
tuberculosis DNA gyrase inhibitors
Brahmam Medapi a, Nikhila Meda a, Pushkar Kulkarni b, Perumal Yogeeswari a, Dharmarajan Sriram a,
⇑
a Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Jawahar Nagar, Shameerpet, Hyderabad 500078, India
b Dr Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study we have designed p-phenylene diamine linked acridine derivative from our earlier reported
quinoline–aminopiperidine hybrid MTB DNA gyrase inhibitors with aiming more potency and less car-
diotoxicity. We synthesized thirty six compounds using four step synthesis from 2-chloro benzoic acid.
Among them compound 4-chloro-N-(4-((2-methylacridin-9-yl)amino)phenyl)benzenesulphonamide (6)
Received 4 November 2015
Revised 29 December 2015
Accepted 7 January 2016
Available online xxxx
was found to be more potent with MTB DNA gyrase super coiling IC50 of 5.21 0.51
6.59 M and no zHERG cardiotoxicity at 30 M and 11.78% inhibition at 50 M against mouse macro-
phage cell line RAW 264.7.
lM; MTB MIC of
l
l
l
Keywords:
Tuberculosis
DNA gyrase
Ó 2016 Elsevier Ltd. All rights reserved.
Acridine derivatives
1. Introduction
Presently, we focused on to develop new MTB DNA gyrase
inhibitors.4–8 DNA gyrase is a type II topoisomerase that intro-
duces negative supercoiling of native bacterial DNA, maintains
its topology, plays a major role in DNA replication for its survival
and its absence in eukaryotic cell makes interesting target for
developing potential new anti-TB drugs.9 DNA gyrase not only
involves in supercoiling of DNA but also helps in relaxation,
cleavage and catenation activities. DNA gyrase is a tetrameric
holo enzyme with two A and B subunits together forming a het-
erodimer structure (A2B2 complex). Whereas Gyr A involves in
nicking and ligation of DNA, on the other hand, Gyr B portion
plays an important role in ATPase activity acts as power house
in DNA replication process. Fluoroquinolones are the well-known
DNA gyrase inhibitors; they were still in clinical practise that
target Gyr A portion specifically. Novobiocin was licenced during
1960’s is the one which acts as competitive inhibitor of ATPase
activity of Gyr B protein portion of DNA gyrase enzyme.10 The
potency of Novobiocin is considerably higher than the fluoro-
quinolones that also acts on DNA gyrase but it was withdrawn
because of its adverse side effects and poor pharmacological
properties. Aminopiperidine based novel bacterial topoisomerase
inhibitors (NBTIs) inhibitors targets MTB DNA gyrase enzyme
and they also possess cardiac toxicity.5,11 In this work we modify
our earlier reported quinoline–aminopiperidine linker hybrid
MTB DNA gyrase molecules5 with considerable cardiotoxicity
into acridine-p-phenylenediamine linker and evaluated its
biological properties.
Tuberculosis (TB) is an air-borne contagious disease that infects
majorly lungs and caused by Mycobacterium tuberculosis (MTB)
bacillus. Resistance to powerful first-line antibiotic drugs is making
worst situation for the treatment of TB resulting in prolonged ill-
ness caused to severe death rate. Further emergence of more resis-
tance strains like multi-drug resistance TB (MDR-TB), extensively
drug resistance TB (XDR-TB) and totally drug resistance TB (TDR-
TB) are major concerns to public health and scientific communities
worldwide, signifies the importance of development of new, safer
and more efficacious inhibitors/drugs to prevent TB. According to
2015 WHO Global TB reports1 revealed that approximately 9 mil-
lion people get infected with this MTB bacillus, 1.5 million died
from the TB also revealed that the existence of MDR-TB in almost
all countries. From past 40 years the only drug has been approved
by US-FDA was bedaquiline (SIRTURO/TMC-207) for treatment of
drug sensitive as well as MDR-TB2 which acts on an ATP synthase
enzyme even its showing adverse effects like QT prolongation,
increased mortality.3 Still there are no available drugs in the mar-
ket that treat effectively and shorten the current treatment regi-
men. All these factors necessitate the design of new
antimycobacterial agents with fewer side effects, more effective
and synthetically feasible ones for the development of efficient
anti-TB drugs.
⇑
Corresponding author. Tel.: +91 40663030506; fax: +91 4066303998.
E-mail address: dsriram@hyderabad.bits-pilani.ac.in (D. Sriram).
http://dx.doi.org/10.1016/j.bmc.2016.01.011
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Medapi, B.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.011

2
B. Medapi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
zoicacid (1), it was reacted with different para-substituted anilines
2. Results and discussion
using stille coupling conditions, that is, Copper, DMF (N,N
Dimethylformamide) heating the reactants at 140 °C for 6 h result-
ing in formation of 2-(p-tolyl/4-chlorophenyl/phenylamino)ben-
zoic acid derivatives (2a–c). Further these 2a–c intermediates
were cyclized intra molecularly in refluxing phosphorousoxy chlo-
ride (POCl3) for 6 h to get corresponding 9-chloro-2-substituted
acridine derivatives (3a–c). The formed acridine intermediates
(3a–c) were reacted with p-phenylenediamine using microwave
conditions at 130 °C, PTSA (para toluenesulphonic acid) as catalyst
in methanol for 1 h resulting in formation of N1-(2-methyl/2-
chloro/simple acridin-9-yl)benzene-1,4-diamine derivatives (4a–
c). These amine intermediates were further reacted with different
substituted aryl sulfonylchorides and aryl isothio/iso cyanates to
get desired final sulphonamide (5–16) and thio/urea (17–28, 29–
40) derivatives. All the reactions were monitored by thin layer
chromatography. Purity of the synthesized compounds was
checked using LC–MS, structure characterized by 1H NMR, 13C
NMR and elemental analysis. In the spectra (1H NMR and 13C
NMR), the signals of the respective protons of the synthesized
derivatives were verified on the basis of their chemical shifts, mul-
tiplicities, and coupling constants. The elemental analysis results
were within 0.4% of the theoretical values.
2.1. Designing of the molecules
The chemical structures of NBTIs comprise a bicyclic hetero aro-
matic left-hand side (LHS) ring, a mono- or bicyclic hydrophobic
right-hand side (RHS) ring and a middle aminopiperidine linker
part (Fig. 1). As per literature, it is reported that aminopiperidines
having a bicyclic aromatic moiety, generally show potent broad-
spectrum antibacterial activity, but usually suffer from potent
hERG inhibition leading to hERG or cardiotoxicity.12,13 In order to
overcome this toxicity issues, we have made an attempt to synthe-
size tricyclic hetero aromatic acridine as LHS with p-phenylenedi-
amine based linker replacing the aminopiperidine as shown in
Figure 1 and check its efficacy and cardiotoxicity profile.
2.2. Chemistry
The titled compounds were synthesized by a series of reactions
that has been shown in Figure 2. Starting with simple 2-chloroben-
r
e
k
n
i
L
H
N
NH
2.3. Biological evaluation
NO2
RHS
RHS
X
HN
N
X
In the preliminary screening all the thirty six synthesized com-
pounds were subjected to screening for their biological inhibition
studies using MTB DNA gyrase supercoiling assay kit (Inspiralis,
Norwich, UK). Novobiocin and Moxifloxacin was used as positive
control in these assays, as it had been shown to be a potent inhibi-
tor of DNA supercoiling of mycobacterial DNA gyrase (Table 1).
Among the compounds tested, all compounds showed activity at
X = S, O
R
R
N
H
HN
L
ink
er
H
N
O
N
S
O
R
Increase of
Hydrophobicity
LHS
HN
N
N
<50
lM concentration, twenty six compounds showed IC50 value
LHS
of <25
l
M, ten compounds showed IC50 value of 610 l
M. Com-
Reported
pound 4-chloro-N-(4-((2-methylacridin-9-yl)amino)phenyl)ben-
quinoline-aminopiperidine based
DNA gyrase inhibitors
Designed
zenesulphonamide (6) emerged as more potent with MTB DNA
gyrase super coiling IC50 of 5.21 0.51
times more potent than standard antitubercular DNA gyrase
acridine-p-phenylene diamine
l
M (Fig. 3); and was ꢀ2
based DNA gyrase inhibitors
Figure 1. Design of compounds.
inhibitor Moxifloxacin [IC50 of 11.2 0.36
l
M].
R2
O
S O
HN
NH
NH2
R
d
N
NH
N
Cl
N
COOH
NH
COOH
5-16
R
R
R 1
a
c
b
Cl
X
HN
NH
e
R
3a-c
R = CH3, Cl, HX = S, O
4a-c
2a-c
1
NH
N
R1 = H, 4-Cl, 4-NO2, 4-CH3
R
R2 = Ph, 4-Cl Ph, 4-NO2 Ph,
Tolyl
17 - 28, X = S
29 - 40, X = O
Figure 2. Synthetic protocol for the entitled substituted N-[4-(substituted-9-acridinyl)amino]phenyl analogues. Reagents and conditions: (a) (sub) aniline, Cu powder, DMF,
140 °C, 6 h; (b) POCl3 reflux 6 h; (c) 1,4-phenylenediamine, p-toluenesulfonic acid, MeOH, MW 13 °C, 1 h; (d) (sub) alkyl/aryl sulfonyl chlorides, Et3N, DMF, 0 to rt, 3 h; (e)
(sub) aryl isothiocyanate/isocyanate, Et3N, 80 °C, 8 h.
Please cite this article in press as: Medapi, B.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.011

B. Medapi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Table 1
In vitro biological evaluation of the acridine derivatives
H
N
O
S
O
R2
X
R2
NH
NH
HN
HN
N
R1
R1
N
5-16
X = S, 17-28, X = O, 29-40
Compd
R1
R2
MTB supercoiling assay (IC50 in
lM)
MTB MIC (lM)
Cytotoxicity at 50 lM (% Inhib.)
5
6
7
8
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
H
H
H
H
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
H
H
H
H
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
H
Phenyl
5.86 0.35
5.21 0.51
8.34 0.59
9.8 0.33
9.2 0.28
12.4 0.66
8.1 0.61
22.8 0.42
15.1 0.59
23.8 1.24
22.4 0.29
9.3 0.65
6.3 0.58
11.2 0.83
24.8 0.29
26.9 0.48
29.3 0.34
31.5 0.81
28.8 1.94
33.9 1.44
8.2 0.29
8.4 0.44
13.1 0.59
31.1 0.54
22.1 0.82
11.4 0.64
7.4 0.29
29.6 0.55
23.6 1.82
11.8 0.34
7.3 0.43
12.5 0.59
32.6 0.61
10.4 0.46
26.8 1.59
13.1 0.88
46 10 nM
>50
28.44
6.59
39.57
11.78
26.98
27.81
14.46
14.19
30.39
38.54
42.06
34.21
34.12
28.44
10.81
26.00
28.83
34.99
11.69
6.70
30.37
25.51
29.24
12.54
20.80
27.41
24.78
25.86
33.94
43.11
27.67
17.12
45.88
29.20
30.45
26.25
19.25
18.37
NT
4-Chlorophenyl
4-Nitrophenyl
4-Toluyl
12.90
27.56
27.17
25.08
24.75
>52.75
14.69
27.18
24.64
28.44
7.20
26.70
>52.13
55.73
27.47
>51.08
>50.00
>53.30
14.86
13.74
13.43
>57.53
29.87
27.60
13.48
57.80
28.48
13.20
6.46
9
Phenyl
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
4-Chlorophenyl
4-Nitrophenyl
4-Toluyl
Phenyl
4-Chlorophenyl
4-Nitrophenyl
4-Toluyl
Phenyl
4-Chlorophenyl
4-Nitrophenyl
4-Toluyl
Phenyl
4-Chlorophenyl
4-Nitrophenyl
4-Toluyl
Phenyl
4-Chlorophenyl
4-Nitrophenyl
4-Toluyl
Phenyl
4-Chlorophenyl
4-Nitrophenyl
4-Toluyl
Phenyl
4-Chlorophenyl
4-Nitrophenyl
4-Toluyl
13.80
>61.81
28.48
27.81
14.93
>50
Phenyl
H
H
H
4-Chlorophenyl
4-Nitrophenyl
4-Toluyl
Novobiocin
Isoniazid
0.66
NT
Rifampicin
Moxifloxacin
>50
11.2 0.36
0.23
1.26
NT
1.26
NT indicates not tested.
Further we tested these compounds for their in vitro activity
using Mycobacterium smegmatis (MS) GyrB ATPase assay as ATPase
activity of GyrB protein from MTB was found to be very low when
compared to MS. None of the compounds were shown any appre-
ciable inhibition in GyrB assay indicates that these molecules
specifically interact with GyrA subunit of DNA gyrase like other
reported NBTIs. These molecules were more also selective towards
MTB DNA gyrase only and did not inhibit DNA supercoiling activity
of Staphylococcus aureus, Escherichia coli and Pseudomonas aerugi-
nosa DNA gyrases. These molecules are structurally similar to m-
Amsacrine (Fig. 4), an acridine derivative, act as a eukaryotic topoi-
somerase II poison14 was also reported as MTB topoisomerase I
pounds showed any inhibition at 50 lM. This result indicates the
importance of methoxyl group present in the phenyl ring linker
of m-Amsacrine.
With respect to structure activity relationship we have not
found good correlation in SAR. To describe SAR in general; we have
prepared twelve sulphonamides, urea and thiourea and in general
order of activity sulphonamides (5–16) > thiourea (17–28) > urea
(29–40). Among sulphonamides derivatives, we prepare three
derivatives with H, Cl, and CH3 groups at 2nd position of acridine
moiety (LHS). Among them methyl derivatives (5–8) showed better
activity (IC50 ranges from 5.21 0.51 to 9.88 0.33
by chloro derivatives (9–12) with IC50 ranges from 8.1 0.61 to
22.8 0.42 M and then unsubstituted acridine derivatives (13–
16) with IC50 ranges from 9.3 0.65 to 23.8.8 1.24 M (except
compound 16). Among compounds 5–16 we also prepared H, Cl,
lM) followed
inhibitor with IC50 of 10
l
M.15 As synthesized molecules were
l
structurally related to m-Amsacrine we also tested our compounds
l
MTB topoisomerase 1 relaxation assay16 and none of the com-
Please cite this article in press as: Medapi, B.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.011

4
B. Medapi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3.125
12.5
25
6.25
B
C
S
Figure 3. Picture depicting the supercoiling assay of compound 5. B—relaxed DNA;
C—relaxed DNA + enzyme; S—relaxed DNA + enzyme + Novobiocin; 25, 12.5, 6.25,
3.125—relaxed DNA + enzyme + compd 5 at different concentrations.
O
S
O
O
NH
HN
Figure 5. Mean ( S.E.M.) of the heart rates of atria and ventricles of compound 6
treatment groups (*p <0.05, **p <0.01 and ***p <0.001). Statistical significance was
analysed comparing control group versus treated groups.
N
Figure 4. Structure of m-Amsacrine.
and CH3 groups in the phenyl ring of RHS; among them the order of
activity H > NO2 > CH3 and Cl groups at the 4th position of RHS
phenyl ring.
The compounds were further screened for their in vitro antimy-
cobacterial activity against M. tuberculosis H37Rv by microplate
alamar blue assay method. Isoniazid, Rifampicin and Moxifloxacin
were used as a positive control and for comparison. The minimum
inhibitory concentration (MIC) was determined for each compound
which was measured as the minimum concentration of compound
required to completely inhibit the bacterial growth. Compounds 6,
7, 13–15, 17, 21, 27, 29, 33–36, and 39–40 showed good correlation
between the MTB DNA gyrase super coiling assay IC50 and in vitro
MTB MIC (Table 1); whereas other compounds showed more than
two times higher than DNA gyrase super coiling IC50; this might be
because of less penetration of these molecules towards the MTB
cell wall or efflux pump present in the cytoplasmic membrane of
MTB may pump out these compounds efficiently. Among thirty
six compounds tested twelve compounds showed MIC < 20 lM
and three compounds (6, 17, and 35) showed MTB MIC of <10 lM.
The safety profile of the synthesized compounds were also
accessed by testing their in vitro cytotoxicity against RAW 264.7
Figure 6. Mean ( S.E.M.) score of atrio ventricular ratio of compound 6 treatment
groups (*p <0.05, **p <0.01 and ***p <0.001). Statistical significance was analysed
comparing control group versus all groups.
cells at 50 lM concentration using (4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay. Selection of mouse
macrophage cell line RAW 264.6 is based to check the toxicity in
macrophages; as MTB resides in its. Percentage inhibitions of cells
are reported in Table 1. Most of the tested compounds demon-
strated good safety profile with very low inhibitory potential
(<50%).
The most potent compound 6 was further examined for hERG
channel inhibition by elucidating arrhythmogenic potential on
Zebrafish ether-a-go-go-related gene (zERG) which was orthologus
to the human ether-a-go-go-related gene (hERG).17 This method
has significant advantages over current conventional animal mod-
els include ethical issues, low compound requirement and cost of
described in more detail in the Section 4. The compound was found
to be safe when compared to positive control (20
lM terfenadine),
not showing any significant cardiotoxicity until 30
l
M (Figs. 5 and
6). Significant changes in the heart rate as well as AV ratio were
also not observed, compared to control group making them rela-
tively safe. In our earlier study with aminopiperidine based MTB
DNA gyrase molecules5 at 30
lM was toxic to fish and died. To con-
clude replacement of aminopiperidine linker with p-phenylenedi-
amine linker reduces cardiac toxicity.
3. Conclusion
experiment. Compound was treated starting from 1 lM to 30 lM
concentrations with 0.1% DMSO as a vehicle. The compound was
analysed for the heart rate variations and AV ratio using a protocol
In conclusion, we identified a new class of acridine derivatives
that selectively inhibit MTB DNA gyrase enzyme with promising
Please cite this article in press as: Medapi, B.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.011

B. Medapi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
attributes of synthetic accessibility, and anti-TB activity. With new
4.1.2.1. 9-Chloro-2-methylacridine (3a).
Pale brown solid;
anti-TB agents desperately needed, we believe that the class of
MTB DNA gyrase inhibitors reported in this work, would be inter-
esting as potential leads for further development.
yield 71%; ESI-MS was found at 228.64 (M+H)+.
4.1.2.2. 2,9-Dichloroacridine (3b).
Pale brown solid; yield
74%; ESI-MS was found at 249.34 (M+H)+.
4. Experimental section
4.1. Chemistry
4.1.2.3. 9-Chloroacridine (3c).
Pale orange solid; yield 69%;
ESI-MS was found at 228.47 (M+H)+.
4.1.3. Preparation of N-(2-methoxy-4-nitrophenyl)acridin/2-
methylacridine-9-amine intermediates (4a–b)
All commercially available chemicals and solvents were used
without further purification. Melting points of the synthesized
compounds were determined by Buchi B-540 open capillary
instrument and were uncorrected. The homogeneity of the com-
pounds was monitored by TLC (Thin layer chromatography) on sil-
ica gel 40 F254 coated on aluminium plates, visualized by UV or
iodine chamber and KMnO4 treatment. All 1H and 13C NMR spectra
were recorded on a Bruker AM-300 (300.12 MHz, 75.12 MHz) NMR
spectrometer and BrukerBioSpin Corp, Germany, respectively.
Molecular weights of the synthesized compounds were checked
by SHIMADZU LCMS-2020 series in ESI mode. Chemical shifts are
reported in ppm (d) with reference to the internal standard TMS.
The signals were designated as follows: s, singlet; d, doublet; dd,
doublet of doublets; t, triplet; m, multiplet. Elemental analyses
were carried out on ElementarVario MICRO CUBE CHN Analyser.
The 9-chloroacridine derivatives (3a–c) (10 mmol) (3a–b) were
further reacted with p-phenylenediamine (1.8 g, 10.7 mmol) under
microwave conditions in methanol using PTSA (para-toluene sul-
phonic acid) as a catalyst (5–10 mg) at 130 °C for 1 h, reaction
was monitored by TLC. After completion of reaction, methanol
was evaporated under vacuum the crude was dissolved in ethyl
acetate, washed with saturated bicarbonate solution. The organic
layer was dried over anhydrous sodium sulphate and evaporated
under reduced pressure obtained crude was further purified by
flash column chromatography in 60–120 silica gel using 10–35%
ethyl acetate: hexane as eluent to get an orange to yellow solid
in moderate yields.
4.1.3.1. N1-(2-Methylacridin-9-yl)benzene-1,4-diamine (4a).
Brown solid; yield 69%; ESI-MS was found at 300.38 (M+H)+.
4.1.1. General procedure for the synthesis of 2-(p-tolylamino)/2-
((4-chlorophenyl)amino)/2-(phenylamino)benzoic acid
intermediates (2a–c)
4.1.3.2. N1-(2-Chloroacridin-9-yl)benzene-1,4-diamine (4b).
Pale brown solid; yield 68%; ESI-MS was found at 318.89
(MÀH)À.
To a stirred solution of 2-chlorobenzoic acid (5 g, 32 mmol) in
20 mL of DMF (N,N-dimethylformamide) potassium carbonate
(7.51 g, 54.4 mmol) corresponding aniline (38.4 mmol) and copper
powder (0.81 g, 12.8 mmol) were added and heated up to 140 °C
for 6 h. Progress of reaction was monitored by thin layer chro-
matography after completion of the reaction, reaction mass was fil-
tered through celite, celite bed was washed with ethyl acetate. The
combined organic layer was evaporated under reduced pressure,
obtained crude was purified by flash column chromatography
using 5–20% ethyl acetate: hexane as eluent in 100–200 mesh sil-
ica gel to get the corresponding phenylamino benzoic acid deriva-
tives in good yield. The intermediates (2a–c) were confirmed by
mass analysis (ESI mode) and proceed to next step.
4.1.3.3. N1-(2-Chloroacridin-9-yl)benzene-1,4-diamine (4c).
Pale brown solid; yield 70%; ESI-MS was found at 286.42 (M+H)+.
4.1.4. Preparation of N-(3-methoxy-4-((acridin/2-
methylacridin-9-yl)amino)phenyl)methane/substituted
benzene sulphonamide derivatives (5–16)
For the preparation of desired sulphonamide derivatives (5–16)
corresponding acridine amine derivatives (4a–c) (0.6 mmol) were
taken in (3 mL) DMF to this triethylamine (1.38 mmol) and corre-
sponding substituted alkyl/aryl sulphonyl chlorides (0.6 mmol)
were added at 0 °C and allowed to stir at room temperature for
3 h. After completion of reaction crushed ice was added to the
crude reaction mass the solid precipitated was filtered, dried and
further purified by flash column chromatography in 100–200 mesh
silica gel using 5–10% methanol: dichloromethane as eluent to get
desired sulphonamide derivatives (5–16) in good yields.
4.1.1.1. 2-(p-Tolylamino) benzoic acid (2a). Pale brown solid;
yield 77%; ESI-MS was found at 228.47 (M+H)+.
4.1.1.2. 2-((4-Chlorophenyl) amino) benzoic acid (2b).
Off-
white solid; yield 78%; ESI-MS was found at 248.78 (M+H)+.
4.1.4.1.
sulphonamide (5).
N-(4-((2-Methylacridin-9-yl)amino)phenyl)benzene-
Yellow solid; yield 68%; mp 236–238 °C;
4.1.1.3. 2-(Phenylamino) benzoic acid (2c). Off-white solid; yield
75%; ESI-MS was found at 212.32 (MÀH)À.
1H NMR (DMSO-d6): dH. 2.35 (s, 3H, CH3), 6.41 (m, 4H, Ar-H),
7.52–7.83 (m, 10H, Ar-H), 8.01–8.19 (m, 2H, Ar-H), 10.69 (s, 1H,
Ar-NH-Ar), 10.96 (s, 1H, Ar-NH-S). 13C NMR (DMSO-d6): dC. 149.9,
142.7, 140.4(2C), 136.6(2C), 132.7(3C), 130.5, 129.6(3C), 128.5,
128.0(3C), 122.2, 121.4, 119.8, 118.9(2C), 117.7(2C), 108.5, 22.4.
EI-MS m/z (Calcd for C26H21N3O2S: 439.14); found: 440.19 (M
+H)+. Anal. Calcd for C26H21N3O2S: C, 71.25; H, 4.82; N, 9.56. Found:
C, 71.19; H, 4.79; N, 9.54.
4.1.2. Preparation of 9-chloroacridine derivatives from
substituted phenylamino benzoic acid intermediates (3a–c)
The phenyl amino benzoic acid derivatives (47 mmol) (2a–c)
were cyclized intra molecularly to 9-chloroacridine derivatives
(3a–c) by refluxing them in 60 mL of POCl3 for 4 h and the reaction
was monitored by TLC. After completion, the excess POCl3 was
removed by rota evaporator under reduced pressure to the crude
reaction mass crushed ice was added, pH was adjusted ꢀ7 by add-
ing saturated bicarbonate solution. The solid separated was fil-
tered, dried and purified by flash column chromatography using
5–10% ethyl acetate: hexane as eluent in 60–120 mesh silica gel
to get the corresponding 9-chloroacridine derivatives in good yield.
These intermediates were confirmed by mass analysis (ESI mode)
and proceed to next step.
4.1.4.2.
benzenesulphonamide (6).
4-Chloro-N-(4-((2-methylacridin-9-yl)amino)phenyl)
Pale yellow solid; yield 71%; mp
245–247 °C; 1H NMR (DMSO-d6): dH. 2.32 (s, 3H, CH3), 6.36 (m,
4H, Ar-H), 7.53–7.87 (m, 9H, Ar-H), 8.02–8.22 (m, 2H, Ar-H),
10.68 (s, 1H, Ar-NH-Ar), 10.94 (s, 1H, Ar-NH-S). 13C NMR (DMSO-
d6): dC. 150.0, 142.8, 140.2, 138.5(2C), 136.7(2C), 132.2(2C),
Please cite this article in press as: Medapi, B.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.011

6
B. Medapi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
130.6, 130.0(3C), 129.4(2C), 128.5(2C), 122.1, 121.4, 119.8, 118.9
(2C), 118.7(2C), 108.3, 22.3. EI-MS m/z (calcd for C26H20ClN3O2S:
473.10); found: 472.14 (MÀH)À. Anal. Calcd for C26H20ClN3O2S:
C, 65.89; H, 4.25; N, 8.87. Found: C, 66.05; H, 4.27; N, 8.85.
(2C), 130.4, 129.9(2C), 128.7(2C), 128.0(2C), 122.8, 121.4, 119.3
(3C), 117.6(2C), 109.7, 22.0. EI-MS m/z (Calcd for C26H20ClN3O2S:
473.10); found: 472.05 (MÀH)À. Anal. Calcd for C26H20ClN3O2S:
C, 65.89; H, 4.25; N, 8.87. Found: C, 65.61; H, 4.27; N, 8.85.
4.1.4.3. N-(4-((2-Methylacridin-9-yl)amino)phenyl)-4-nitroben-
zenesulphonamide (7).
4.1.4.9. N-(4-(Acridin-9-ylamino)phenyl)benzenesulphonamide
(13).
Brown gammy; yield 69%; 1H NMR
Orange solid; yield 72%; mp 187–189 °C; 1H NMR
(DMSO-d6): dH. 2.36 (s, 3H, CH3), 6.42 (m, 4H, Ar-H), 7.53–7.97
(m, 6H, Ar-H), 8.14–8.41 (m, 5H, Ar-H), 10.67 (s, 1H, Ar-NH-Ar),
10.88 (s, 1H, Ar-NH-S). 13C NMR (DMSO-d6): dC. 151.9, 149.7,
146.3, 142.6, 140.1, 139.5(2C), 132.0(2C), 130.3(2C), 128.8(2C),
128.0(2C), 124.9(2C), 122.2, 121.5, 120.0, 119.2(2C), 117.9(2C),
108.4, 22.6. EI-MS m/z (Calcd for C26H20N4O4S: 484.12); found:
485.31 (M+H)+. Anal. Calcd for C26H20N4O4S: C, 64.45; H, 4.16; N,
11.56. Found: C, 64.54; H, 4.18; N, 11.54.
(DMSO-d6): dH. 6.36 (m, 4H, Ar-H), 7.56–7.94 (m, 11H, Ar-H),
8.19 (m, 2H, Ar-H), 10.56 (s, 1H, Ar-NH-Ar), 10.89 (s, 1H, Ar-NH-
S). 13C NMR (DMSO-d6): dC. 150.4, 142.9(2C), 140.3, 136.5, 132.2,
130.6(2C), 129.8(2C), 128.5(3C), 127.8(2C), 126.9(2C), 121.5(2C),
118.8(2C), 117.6(2C), 115.4(2C). EI-MS m/z (Calcd for C25H19N3O2
S: 425.12); found: 426.29 (M+H)+. Anal. Calcd for C25H19N3O2S: C,
70.57; H, 4.50; N, 9.88. Found: C, 70.76; H, 4.48; N, 9.85.
4.1.4.10.
sulphonamide (14).
N-(4-(Acridin-9-ylamino)phenyl)-4-chlorobenzene-
Pale yellow solid; yield 77%; mp 220–
4.1.4.4.
benzenesulphonamide (8).
4-Methyl-N-(4-((2-methylacridin-9-yl)amino)phenyl)
Yellow solid; yield 66%; mp
222 °C; 1H NMR (DMSO-d6): dH. 6.42 (m, 4H, Ar-H), 7.61–7.96 (m,
10H, Ar-H), 8.15 (m, 2H, Ar-H), 10.53 (s, 1H, Ar-NH-Ar), 10.91 (s,
1H, Ar-NH-S). 13C NMR (DMSO-d6): dC. 150.2, 142.8(2C), 138.4
(2C), 136.3, 130.5(2C), 129.7(2C), 129.4(2C), 128.6(3C), 126.9(2C),
121.4(2C), 119.0(2C), 117.8(2C), 115.5(2C). EI-MS m/z (Calcd for
252–254 °C; 1H NMR (DMSO-d6): dH. 2.32 (s, 6H, CH3), 6.37 (m,
4H, Ar-H), 7.39–7.83 (m, 9H, Ar-H), 8.01–8.23 (m, 2H, Ar-H),
10.67 (s, 1H, Ar-NH-Ar), 10.93 (s, 1H, Ar-NH-S). 13C NMR (DMSO-
d6): dC. 149.9, 142.7, 140.1, 138.3, 137.2, 136.4(2C), 132.6(2C),
130.7(2C), 130.1(2C), 129.0(2C), 128.5(2C), 121.9(2C), 119.8,
118.9(2C), 117.7(2C), 108.3, 22.6(2C). EI-MS m/z (Calcd for C27H23
N3O2S: 453.15); found: 454.32 (M+H)+. Anal. Calcd for C27H23N3O2
S: C, 71.50; H, 5.11; N, 9.26. Found: C, 71.72; H, 5.09; N, 9.23.
C
25H18ClN3O2S: 459.08); found: 460.15 (M+H)+. Anal. Calcd for C25
H18ClN3O2S: C, 65.28; H, 3.94; N, 9.14. Found: C, 65.46; H, 3.96; N,
9.11.
4.1.4.11.
sulphonamide (15).
N-(4-(Acridin-9-ylamino)phenyl)-4-nitrobenzene-
Brown solid; yield 68%; mp 187–
4.1.4.5.
sulphonamide (9).
N-(4-((2-Chloroacridin-9-yl)amino)phenyl)benzene-
Orange solid; yield 63%; mp 181–183 °C;
189 °C; 1H NMR (DMSO-d6): dH. 6.39 (m, 4H, Ar-H), 7.57–7.93 (m,
6H, Ar-H), 8.10–8.41 (m, 6H, Ar-H), 10.49 (s, 1H, Ar-NH-Ar),
10.87 (s, 1H, Ar-NH-S). 13C NMR (DMSO-d6): dC. 152.7, 150.5,
146.2, 142.9(2C), 136.6, 130.4(2C), 128.8(2C), 128.2(3C), 127.0
(2C), 124.7(2C), 121.5(2C), 119.1(2C), 117.9(2C), 115.3(2C). EI-MS
m/z (Calcd for C25H18N4O4S: 470.10); found: 469.08 (MÀH)À. Anal.
Calcd for C25H18N4O4S: C, 63.82; H, 3.86; N, 11.91. Found: C, 63.65;
H, 3.88; N, 11.94.
1H NMR (DMSO-d6): dH. 6.41 (m, 4H, Ar-H), 7.61–7.79 (m, 9H, Ar-
H), 7.99–8.17 (m, 3H, Ar-H), 10.71 (s, 1H, Ar-NH-Ar), 10.90 (s,
1H, Ar-NH-S). 13C NMR (DMSO-d6): dC. 149.3, 143.8, 140.4(2C),
136.6, 132.3, 131.9(2C), 131.3(2C), 130.7, 129.8(2C), 128.3, 127.9
(3C), 122.7, 121.4, 119.2(3C), 117.8(2C), 109.9. EI-MS m/z (Calcd
for C25H18ClN3O2S: 459.08); found: 458.11 (MÀH)À. Anal. Calcd
for C25H18ClN3O2S: C, 65.28; H, 3.94; N, 9.14. Found: C, 65.45; H,
3.92; N, 9.16.
4.1.4.12.
sulphonamide (16).
N-(4-(Acridin-9-ylamino)phenyl)-4-methylbenzene-
Brown solid; yield 75%; mp 206–
4.1.4.6.
4-Chloro-N-(4-((2-chloroacridin-9-yl)amino)phenyl)
Yellow solid; yield 71%; mp
benzenesulphonamide (10).
208 °C; 1H NMR (DMSO-d6): dH. 2.36 (s, 3H, CH3), 6.41 (m, 4H,
Ar-H), 7.43–7.92 (m, 10H, Ar-H), 8.21 (m, 2H, Ar-H), 10.53 (s, 1H,
Ar-NH-Ar), 10.97 (s, 1H, Ar-NH-S). 13C NMR (DMSO-d6): dC. 150.4,
142.8(2C), 138.3, 137.5, 136.8, 130.5(2C), 129.9(2C), 129.0(2C),
128.6(3C), 127.0(2C), 121.4(2C), 119.1(2C), 117.8(2C), 115.4(2C),
22.0. EI-MS m/z (Calcd for C26H21N3O2S: 439.14); found: 440.31
(M+H)+. Anal. Calcd for C26H21N3O2S: C, 71.05; H, 4.82; N, 9.56.
Found: C, 71.24; H, 4.84; N, 9.52.
189–191 °C; 1H NMR (DMSO-d6): dH. 6.39 (m, 4H, Ar-H), 7.59–
7.81 (m, 8H, Ar-H), 8.01–8.22 (m, 3H, Ar-H), 10.67 (s, 1H, Ar-NH-
Ar), 10.89 (s, 1H, Ar-NH-S). 13C NMR (DMSO-d6): dC. 149.2, 143.9,
140.2, 138.4(2C), 136.7, 132.0(2C), 131.7(2C), 130.4, 129.8(2C),
129.4(2C), 128.2(2C), 122.9, 121.6, 119.3(3C), 117.9(2C), 109.7.
EI-MS m/z (Calcd for C25H17Cl2N3O2S: 493.04); found: 494.25 (M
+H)+. Anal. Calcd for C25H17Cl2N3O2S: C, 60.73; H, 3.47; N, 8.50.
Found: C, 60.92; H, 3.46; N, 8.53.
4.1.5. Preparation of 1-(3-methoxy-4-((acridin/2-methylacridin-
9-yl)amino)phenyl)-3-phenylthiourea/ urea derivatives (17–40)
For the preparation of desired thiourea (17–28) and urea (29–
40) derivatives corresponding acridin amine derivative (4a–c)
(0.6 mmol), triethylamine (1.38 mmol) (Et3N) and substituted phe-
nyl isothiocyanate/isocyanate (0.6 mmol) were dissolved in etha-
nol and heated up to 80 °C for 8 h reaction was monitored by
TLC, after completion of the reaction the solid precipitated was fil-
tered, dried and recrystallized from ethanol to get the desired cor-
responding thiourea (17–28) and urea (29–40) derivatives.
4.1.4.7. N-(4-((2-Chloroacridin-9-yl)amino)phenyl)-4-nitroben-
zenesulphonamide (11).
Brown gammy; yield 67%; 1H NMR
(DMSO-d6): dH. 6.43 (m, 4H, Ar-H), 7.63–7.97 (m, 5H, Ar-H),
8.07–8.41 (m, 6H, Ar-H), 10.57 (s, 1H, Ar-NH-Ar), 10.92 (s, 1H,
Ar-NH-S). 13C NMR (DMSO-d6): dC. 152.8, 149.3, 146.5, 143.9,
140.3, 136.7, 132.2(2C), 131.5(2C), 130.5, 129.0(2C), 128.4(2C),
124.8(2C), 122.9, 121.5, 119.1(3C), 117.7(2C), 109.9. EI-MS m/z
(Calcd for C25H17ClN4O4S: 504.07); found: 505.31 (M+H)+. Anal.
Calcd for C25H17ClN4O4S: C, 59.47; H, 3.39; N, 11.10. Found: C,
59.62; H, 3.40; N, 11.12.
4.1.5.1. 1-(4-((2-Methylacridin-9-yl)amino)phenyl)-3-phenylth-
4.1.4.8. N-(4-((2-Chloroacridin-9-yl)amino)phenyl)-4-methyl-
iourea (17).
Orange solid; yield 67%; mp 120–122 °C; 1H NMR
benzenesulphonamide (12).
Yellow solid; yield 69%; mp
(DMSO-d6): dH. 2.36 (s, 3H, CH3), 6.24–6.79 (m, 5H, Ar-H), 7.21–
7.81 (m, 9H, Ar-H), 8.01–8.19 (m, 2H, Ar-H), 8.67 (s, 2H, Ar-NH),
10.54 (s, 1H, Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 180.3, 149.7,
142.4(2C), 139.9, 138.8, 135.6, 131.7(2C), 130.1(2C), 129.5(2C),
128.9(2C), 127.7(3C), 126.8(2C), 122.0, 121.2, 119.7, 118.3(2C),
214–216 °C; 1H NMR (DMSO-d6): dH. 2.35 (s, 3H, CH3), 6.41 (m,
4H, Ar-H), 7.39–7.78 (m, 8H, Ar-H), 7.99–8.19 (m, 3H, Ar-H),
10.49 (s, 1H, Ar-NH-Ar), 10.91 (s, 1H, Ar-NH-S). 13C NMR (DMSO-
d6): dC. 149.5, 143.8, 140.4, 128.3, 137.5, 136.8, 132.3(2C), 131.7
Please cite this article in press as: Medapi, B.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.011

B. Medapi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
108.5, 22.2. EI-MS m/z (Calcd For C27H22N4S: 434.16); found:
435.09 (M+H)+. Anal. Calcd for C27H22N4S: C, 74.63; H, 5.10; N,
12.89. Found: C, 74.81; H, 5.08; N, 12.85.
118.4(2C), 108.7. EI-MS m/z (Calcd for C26H18ClN5O2S: 499.09);
found: 500.21 (M+H)+. Anal. Calcd for C26H18ClN5O2S: C, 62.46;
H, 3.63; N, 14.01. Found: C, 62.64; H, 3.61; N, 14.05.
4.1.5.2. 1-(4-Chlorophenyl)-3-(4-((2-methylacridin-9-yl)amino)
4.1.5.8. 1-(4-((2-Chloroacridin-9-yl)amino)phenyl)-3-(p-tolyl)
phenyl)thiourea (18).
Yellow solid; yield 68%; mp 138–
thiourea (24).
Pale orange solid; yield 70%; mp 149–153 °C;
140 °C; 1H NMR (DMSO-d6): dH. 2.32(s, 3H, CH3), 6.19–6.56 (m,
6H, Ar-H), 7.25–7.84 (m, 7H, Ar-H), 8.0–8.17 (m, 2H, Ar-H), 8.71
(s, 2H, Ar-NH), 10.61 (s, Ar-NH-Ar). 13C NMR (DMSO-d6): dC.
180.1, 149.8, 142.5(2C), 140.0, 137.2, 135.7, 134.1, 132.0(2C),
131.5(2C), 130.1(2C), 129.8(2C), 128.8, 127.9(3C), 121.9, 121.0,
120.2, 118.4(2C), 108.3, 22.1. EI-MS m/z (Calcd for C27H21ClN4S:
468.12); found: 469.01 (M+H)+. Anal. Calcd for C27H21ClN4S: C,
69.14; H, 4.51; N, 11.95. Found: C, 69.28; H, 4.55; N, 11.97.
1H NMR (DMSO-d6): dH. 2.36 (s, 3H, CH3), 6.23–6.95 (m, 8H, Ar-
H), 7.65–8.14 (m, 7H, Ar-H), 8.63 (s, 2H, Ar-NH), 10.71 (s, 1H, Ar-
NH-Ar). 13C NMR (DMSO-d6): dC. 180.5, 149.2, 143.9, 142.5,
140.3, 237.8, 136.2, 132.0(2C), 131.5(2C), 130.5, 129.9(2C), 129.2,
127.8(2C), 127.4, 126.9(2C), 122.7, 121.4, 118.9, 118.3(2C), 109.7,
21.8. EI-MS m/z (Calcd for C27H21ClN4S: 468.12); found: 467.05
(MÀH)À. Anal. Calcd for C27H21ClN4S: C, 69.14; H, 4.51; N, 11.95.
Found: C, 69.33; H, 4.49; N, 11.92.
4.1.5.3. 1-(4-((2-Methylacridin-9-yl)amino)phenyl)-3-(4-nitro-
4.1.5.9.
(25).
1-(4-(Acridin-9-ylamino)phenyl)-3-phenylthiourea
phenyl)thiourea (19).
Pale orange solid; yield 65%; mp
Brown solid; yield 67%; mp 156–158 °C; 1H NMR
131–133 °C; 1H NMR (DMSO-d6): dH. 2.35 (s, 3H, CH3), 6.23–6.67
(m, 6H, Ar-H), 7.58–7.89 (m, 6H, Ar-H), 8.03–8.18 (m, 3H, Ar-H),
8.57 (s, 2H, Ar-NH), 10.61 (s, 1H, Ar-NH-Ar). 13C NMR (DMSO-d6):
dC. 180.3, 150.1, 145.0, 144.4(2C), 142.1(2C), 136.7, 131.9, 130.2
(2C), 128.9, 127.8(2C), 127.0, 126.3, 125.6(2C), 124.9(2C), 124.5,
122.0, 121.7, 118.4(2C), 1.8.8, 22.3. EI-MS m/z (Calcd for C27H21N5
O2S: 479.14; found: 478.25 (MÀH)À. Anal. Calcd for C27H21N5O2S:
C, 67.62; H, 4.41; N, 14.60. Found: C, 67.43; H, 4.39; N, 14.56.
(DMSO-d6): dH. 6.25–6.78 (m, 5H, Ar-H), 7.23–7.71 (m, 8H, Ar-H),
7.97–8.15 (m, 4H, Ar-H), 8.69 (s, 2H, Ar-NH), 10.71 (s, 1H, Ar-
NH-Ar). 13C NMR (DMSO-d6): dC. 180.2, 150.4, 143.0(2C), 142.5,
139.1, 130.3(2C), 129.7(2C) 129.0(2C), 128.4(2C), 127.9(2C), 127.0
(2C), 126.8(2C), 121.4(2C), 118.6(2C), 115.3(2C). EI-MS m/z (Calcd
for C26H20N4S: 420.14); found: 421.28 (M+H)+. Anal. Calcd for
C26H20N4S: C, 74.26; H, 4.79; N, 13.32. Found: C, 74.04; H, 4.83;
N, 13.34.
4.1.5.4. 1-(4-((2-Methylacridin-9-yl)amino)phenyl)-3-(p-tolyl)
thiourea (20).
4.1.5.10. 1-(4-(Acridin-9-ylamino)phenyl)-3-(4-chlorophenyl)
thiourea (26).
Orange solid; yield 63%; mp 138–140 °C; 1H
Orange solid; yield 65%; mp 147–149 °C; 1H
NMR (DMSO-d6): dH. 2.36 (s, 6H, CH3), 6.26–6.94 (m, 8H, Ar-H),
7.51–7.95 (m, 6H, Ar-H), 8.16 (m, 1H, Ar-H), 8.59 (s, 2H, Ar-NH),
10.63 (s, 1H, Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 180.0, 149.8,
142.3(2C), 140.1, 137.7, 135.9(2C), 132.2(2C), 130.5(2C), 129.8
(2C), 129.0, 127.9(2C), 126.8(2C), 121.7, 121.3(2C), 119.6, 118.2
(2C), 108.4, 22.5(2C). EI-MS m/z (Calcd for C28H24N4S: 448.17);
found: 449.19 (M+H)+. Anal. Calcd for C28H24N4S: C, 74.97; H,
5.39; N, 12.49. Found: C, 75.18; H, 5.37; N, 12.45.
NMR (DMSO-d6): dH. 6.19–6.56 (m, 6H, Ar-H), 7.27–7.774 (m, 6H,
Ar-H), 7.99–8.18 (m, 4H, Ar-H), 8.82 (s, 2H, Ar-NH), 10.61 (s, 1H,
Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 180.4, 150.5, 142.9(2C),
142.5, 137.3, 134.2, 131.8(2C), 130.4(2C), 129.7(2C), 129.1, 128.5
(2C), 127.9(2C), 126.7(2C), 121.4(2C), 118.3(2C), 115.5(2C). EI-MS
m/z (Calcd for C26H19ClN4S: 454.10); found: 455.03 (M+H)+. Anal.
Calcd for C26H19ClN4S: C, 68.64; H, 4.21; N, 12.31. Found: C,
68.83; H, 4.23; N, 12.28.
4.1.5.5. 1-(4-((2-Chloroacridin-9-yl)amino)phenyl)-3-phenylth-
iourea (21).
4.1.5.11.
thiourea (27).
1-(4-(Acridin-9-ylamino)phenyl)-3-(4-nitrophenyl)
Orange solid; yield 67%; mp 159–161 °C; 1H
Orange solid; yield 66%; mp 108–110 °C; 1H NMR
(DMSO-d6): dH. 6.22–6.35 (m, 4H, Ar-H), 6.85–7.18 (m, 3H, Ar-H),
7.62–7.97 (m, 7H, Ar-H), 8.07–8.21 (m, 2H, Ar-H), 8.55 (s, 2H, Ar-
NH), 10.48 (s, 1H, Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 180.1,
149.3, 143.7, 142.4, 140.2, 139.0, 132.3(2C), 131.9(2C), 130.5,
129.7(2C), 129.2(2C), 128.0(2C), 127.7, 127.1(2C), 122.8, 121.4,
NMR (DMSO-d6): dH. 6.22–6.65 (m, 6H, Ar-H), 7.61–7.92 (m, 6H,
Ar-H), 8.04–8.19 (m, 4H, Ar-H), 8.78 (s, 2H, Ar-NH), 10.73 (s, 1H,
Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 180.2, 150.4, 145.0, 144.5,
142.9(2C), 142.6, 130.4(2C), 129.1, 128.3(2C), 127.9(2C), 127.0
(2C), 125.3(2C), 124.9(2C), 121.5(2C), 118.2(2C), 115.6(2C). EI-MS
m/z (Calcd for C26H19N5O2S: 465.13); found: 466.21 (M+H)+. Anal.
Calcd for C26H19N5O2S: C, 67.08; H, 4.11; N, 15.04. Found: C,
67.26; H, 4.13; N, 15.08.
119.2, 118.5(2C), 109.7. EI-MS m/z (Calcd for
C26H19ClN4S:
454.10); found: 453.01 (MÀH)À. Anal. Calcd for C26H19ClN4S: C,
68.64; H, 4.21; N, 12.31. Found: C, 68.82; H, 4.19; N, 12.34.
4.1.5.6. 1-(4-((2-Chloroacridin-9-yl)amino)phenyl)-3-(4-chloro-
4.1.5.12.
(28).
1-(4-(Acridin-9-ylamino)phenyl)-3-(p-tolyl)thiourea
Brown solid; yield 64%; mp 190–192 °C; 1H NMR
phenyl)thiourea (22).
Brown solid; yield 66%; mp 135–
137 °C; 1H NMR (DMSO-d6): dH. 6.19–6.55 (m, 6H, Ar-H), 7.21–
7.75 (m, 6H, Ar-H), 8.01–8.19 (m, 3H, Ar-H), 8.76 (s, 2H, Ar-NH),
10.57 (s, 1H, Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 180.5, 149.1,
143.9, 142.3, 140.0, 137.2, 134.4, 132.0(2C), 131.8(2C), 131.4(2C),
130.1, 129.6(2C), 128.9, 127.7(2C), 127.3, 122.8, 121.2, 118.9,
(DMSO-d6): dH. 2.36 (s, 3H, CH3), 6.23–6.95 (m, 8H, Ar-H), 7.62–
7.91 (m, 6H, Ar-H), 8.21 (m, 2H, Ar-H), 8.79 (s, 2H, Ar-NH), 10.66
(s, 1H, Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 180.3, 150.4, 142.6
(3C), 137.7, 136.2, 130.6(2C), 129.8(2C), 128.9, 128.2(2C), 127.6
(2C), 126.9(2C), 126.7(2C), 121.5(2C), 118.3(2C), 115.5(2C), 21.7.
EI-MS m/z (Calcd for C27H22N4S: 434.16); found: 435.19 (M+H)+.
Anal. Calcd for C27H22N4S: C, 74.63; H, 5.10; N, 12.89. Found: C,
74.85; H, 5.08; N, 12.92.
118.4(2C), 109.9. EI-MS m/z (Calcd for
C26H18Cl2N4S:488.06);
found: 489.12 (M+H)+. Anal. Calcd for C26H18Cl2N4S: C, 63.81; H,
3.71; N, 11.45. Found: C, 63.62; H, 3.73; N, 11.49.
4.1.5.7. 1-(4-((2-Chloroacridin-9-yl)amino)phenyl)-3-(4-nitro-
4.1.5.13. 1-(4-((2-Methylacridin-9-yl)amino)phenyl)-3-pheny-
lurea (29).
phenyl)thiourea (23).
Orange solid; yield 68%; mp 142–
Brown gummy; yield 69%; 1H NMR (DMSO-d6):
144 °C; 1H NMR (DMSO-d6): dH. 6.22–6.67 (m, 6H, Ar-H), 7.61–
7.89 (m, 5H, Ar-H), 8.02–8.18 (m, 4H, Ar-H), 8.56 (s, 2H, Ar-NH),
10.64 (s, 1H, Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 180.3, 150.1,
145.3, 144.5(2C), 142.3(2C), 132.9, 132.0, 130.8, 130.1, 129.2,
127.9(2C), 126.4(2C), 125.2(2C), 124.8(2C), 124.5, 122.0(2C),
dH. 2.31 (s, 3H, CH3), 6.65 (m, 2H, Ar-H), 7.22–7.95 (m, 13H, Ar-
H), 8.19 (d, J = 8.1 Hz, 1H, Ar-H), 8.79 (s, 2H, Ar-NH), 10.58 (s, 1H,
Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 153.5, 149.7, 142.3(2C),
140.1(2C), 135.6, 132.0(2C), 130.4, 129.9(2C), 129.3(2C), 128.6,
127.4, 122.9(2C), 122.2(2C), 122.0, 121.3, 119.9, 118.4(2C), 108.6,
Please cite this article in press as: Medapi, B.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.011

8
B. Medapi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
22.3. EI-MS m/z (Calcd For C27H22N4O: 418.18); found: 417.06
(MÀH)À. Anal. Calcd For C27H22N4O: C, 77.49; H, 5.30; N, 13.39.
Found: C, 77.25; H, 5.32; N, 13.35.
for C26H18ClN5O3: 483.11); found: 482.05 (MÀH)À. Anal. Calcd for
C
26H18ClN5O3: C, 64.53; H, 3.75; N, 14.47. Found: C, 64.71; H,
3.73; N, 14.44.
4.1.5.14.
amino)phenyl)urea (30).
1-(4-Chlorophenyl)-3-(4-((2-methylacridin-9-yl)
Pale yellow solid; yield 66%; mp
4.1.5.20. 1-(4-((2-Chloroacridin-9-yl)amino)phenyl)-3-(p-tolyl)
urea (36).
Yellow solid; yield 62%; mp 253–255 °C; 1H NMR
163–165 °C; 1H NMR (DMSO-d6): dH. 2.35 (s, 3H, CH3), 6.59 (m,
2H, Ar-H), 7.41–7.96 (m, 12H, Ar-H), 8.21 (d, J = 7.8 Hz, 1H, Ar-H),
8.84 (s, 2H, Ar-NH), 10.58 (s, 1H, Ar-NH-Ar). 13C NMR (DMSO-d6):
dC. 153.6, 149.7, 142.3(2C), 140.1, 138.3, 135.8, 133.9, 132.1(2C),
130.5, 130.0(2C), 129.7(2C), 127.5, 122.9(2C), 122.1, 121.4(2C),
121.3, 119.8, 118.6(2C), 108.3, 22.5. EI-MS m/z (Calcd for C27H21
ClN4O: 452.14); found: 453.23 (M+H)+. Anal. Calcd for C27H21ClN4
O: C, 71.60; H, 4.67; N, 12.37. Found: C, 71.79; H, 4.65; N, 12.41.
(DMSO-d6): dH. 2.25 (s, 3H, CH3), 6.62 (m, 2H, Ar-H), 7.23–7.59
(m, 8H, Ar-H), 7.81–8.15 (m, 5H, Ar-H), 8.79 (s, 2H, Ar-NH), 10.68
(s, 1H, Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 153.6, 149.2, 144.1,
142.3, 140.5, 137.4(2C), 132.2(3C), 131.1, 130.3(2C), 129.8(2C),
127.7, 123.0(3C), 122.2(2C), 121.3, 119.0, 118.5(2C), 109.9, 21.7.
EI-MS m/z (Calcd for C27H21ClN4O: 452.14); found: 453.45 (M
+H)+. Anal. Calcd for C27H21ClN4O: C, 71.60; H, 4.67; N, 12.37.
Found: C, 71.58; H, 4.65; N, 12.41.
4.1.5.15. 1-(4-((2-Methylacridin-9-yl)amino)phenyl)-3-(4-nitro-
phenyl)urea (31).
4.1.5.21.
(37).
1-(4-(Acridin-9-ylamino)phenyl)-3-phenylurea
Brown gummy; yield 61%; 1H NMR
Orange solid; yield 65%; mp 162–164 °C; 1H NMR
(DMSO-d6): dH. 2.32 (s, 3H, CH3), 6.62 (m, 2H, Ar-H), 7.42–7.89
(m, 10H, Ar-H), 8.19–8.27 (m, 3H, Ar-H), 8.79 (s, 2H, Ar-NH),
10.61 (s, 1H, Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 153.4, 149.8,
146.1, 144.3(2C), 142.0(2C), 136.9, 131.8, 130.5(3C), 127.3, 126.1,
124.7(3C), 122.9(2C), 122.2, 121.6, 120.3(2C), 118.5(2C), 108.7,
22.2. EI-MS m/z (Calcd for C27H21N5O3: 463.16); found: 462.14
(MÀH)À. Anal. Calcd for C27H21N5O3: C, 69.97; H, 4.57; N, 15.11.
Found: C, 70.16; H, 4.55; N, 15.08.
(DMSO-d6): dH. 6.65 (m, 2H, Ar-H), 7.19–7.59 (m, 9H, Ar-H),
7.81–8.15 (m, 6H, Ar-H), 8.79 (s, 2H, Ar-NH), 10.51 (s, 1H, Ar-
NH-Ar). 13C NMR (DMSO-d6): dC. 153.4, 150.2, 142.9(2C), 142.0,
139.8, 130.3(3C), 120.5(3C), 128.3(2C), 127.0(2C), 123.2(2C),
122.1(2C), 121.5(2C), 118.1(2C), 115.4(2C). EI-MS m/z (Calcd for
C
C
26H20N4O: 404.16); found: 405.29 (M+H)+. Anal. Calcd for
26H20N4O: C, 77.21; H, 4.98; N, 13.85. Found: C, 77.45; H, 5.01;
N, 13.82.
4.1.5.16. 1-(4-((2-Methylacridin-9-yl)amino)phenyl)-3-(p-tolyl)
urea (32).
4.1.5.22. 1-(4-(Acridin-9-ylamino)phenyl)-3-(4-chlorophenyl)
urea (38).
Pale orange gummy; yield 67%; 1H NMR (DMSO-
Brown solid; yield 68%; mp 241–243 °C; 1H NMR
d6): dH. 2.32 (s, 6H, CH3), 6.64 (m, 2H, Ar-H), 7.23–7.61 (m, 9H,
Ar-H), 7.80–8.21 (m, 4H, Ar-H), 8.78 (s, 2H, Ar-NH), 10.68 (s, 1H,
Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 153.6, 149.7, 142.3(2C),
140.1, 137.3(2C), 135.8, 132.0(2C), 130.7, 130.1(2C), 129.6(2C),
127.8, 123.0(2C), 122.1(3C), 121.3, 119.8, 118.4(2C), 108.3 22.5
(2C). EI-MS m/z (Calcd for C28H24N4O: 432.20); found: 433.12 (M
+H)+. Anal. Calcd for C28H24N4O: C, 77.75; H, 5.59; N, 12.95. Found:
C, 77.51; H, 5.61; N, 12.99.
(DMSO-d6): dH. 6.59 (m, 2H, Ar-H), 7.41–7.92 (m, 12H, Ar-H),
8.19 (m, 2H, Ar-H). 13C NMR (DMSO-d6): dC. 153.6, 150.4, 143.1
(2C), 141.9, 138.2, 133.8, 130.5(3C), 129.5(2C), 128.2(2C), 127.0
(2C), 122.9(2C), 121.4(2C), 121.0(2C), 118.3(2C), 115.5(2C). EI-MS
m/z (Calcd for C26H19ClN4O: 438.12); found: 437.01 (MÀH)À. Anal.
Calcd for C26H19ClN4O: C, 71.15; H, 4.36; N, 12.77. Found: C, 70.95;
H, 4.38; N, 12.74.
4.1.5.23.
urea (39).
1-(4-(Acridin-9-ylamino)phenyl)-3-(4-nitrophenyl)
4.1.5.17. 1-(4-((2-Chloroacridin-9-yl)amino)phenyl)-3-pheny-
Brown solid; yield 62%; mp 252–254 °C; 1H NMR
lurea (33).
Brown solid; yield 68%; mp 168–170 °C; 1H NMR
(DMSO-d6): dH. 6.62 (m, 2H, Ar-H), 7.43–7.94 (m, 10H, Ar-H),
8.15–8.27 (m, 4H, Ar-H), 8.73 (s, 2H, Ar-NH), 10.77 (s, 1H, Ar-
NH-Ar). 13C NMR (DMSO-d6): dC. 153.5, 150.3, 146.1, 144.3, 142.9
(2C), 141.7, 130.5(3C), 128.3(2C), 127.0(2C), 124.8(2C), 123.1(2C),
121.6(2C), 120.4(2C), 118.5(2C), 115.3(2C). EI-MS m/z (Calcd for
(DMSO-d6): dH. 6.62 (m, 2H, Ar-H), 7.23–7.62 (m, 9H, Ar-H),
7.81–8.22 (m, 5H, Ar-H), 8.68 (s, 2H, Ar-NH), 10.71 (s, 1H, Ar-
NH-Ar). 13C NMR (DMSO-d6): dC. 153.4, 149.3, 143.9, 142.1, 140.4
(2C), 132.2(2C), 131.8(2C), 130.5, 129.9, 129.3(3C), 127.7, 122.9
(3C), 122.3(2C), 121.4, 119.0, 118.5(2C), 109.8. EI-MS m/z (Calcd
for C26H19ClN4O: 438.12); found: 439.25 (M+H)+. Anal. Calcd for
C
C
26H19N5O3: 449.15); found: 450.45 (M+H)+. Anal. Calcd for
26H19N5O3: C, 69.48; H, 4.26; N, 15.58. Found: C, 69.68; H, 4.27;
C26H19ClN4O: C, 71.15; H, 4.36; N, 12.77. Found: C, 70.98; H,
N, 15.53.
4.34; N, 12.81.
4.1.5.24.
(40).
1-(4-(Acridin-9-ylamino)phenyl)-3-(p-tolyl)urea
4.1.5.18. 1-(4-((2-Chloroacridin-9-yl)amino)phenyl)-3-(4-chlor-
Brown gummy; yield 64%; 1H NMR (DMSO-d6): dH.
ophenyl)urea (34).
Brown solid; yield 69%; mp 234–236 °C;
2.31 (s, 3H, CH3), 6.64 (m, 2H, Ar-H), 7.19–7.74 (m, 10H, Ar-H),
7.98–8.15 (m, 4H, Ar-H), 8.67 (s, 2H, Ar-NH), 10.59 (s, 1H, Ar-
NH-Ar). 13C NMR (DMSO-d6): dC. 153.4, 150.2, 142.9(2C), 142.1,
137.3(2C), 130.5(2C), 129.9(3C), 128.3(2C), 127.0(2C), 122.8(2C),
122.1(2C), 121.6(2C), 118.3(2C), 115.6(2C), 21.9. EI-MS m/z (Calcd
for C27H22N4O: 418.18); found: 419.27 (M+H)+. Anal. Calcd for
1H NMR (DMSO-d6): dH. 6.59 (m, 2H, Ar-H), 7.41–7.79 (m, 10H,
Ar-H), 9.01–8.23 (m, 3H, Ar-H), 8.65 (s, 2H, Ar-NH), 10.59 (s, 1H,
Ar-NH-Ar). 13C NMR (DMSO-d6): dC. 153.5, 149.1, 143.8, 142.0,
140.1, 138.3, 133.9, 132.3(2C), 131.7(2C), 130.3(2C), 129.4(2C),
127.5, 122.9(3C), 121.6(3C), 119.1, 118.4(2C), 109.8. EI-MS m/z
(Calcd for C26H18Cl2N4O: 472.09); found: 473.17 (M+H)+. Anal.
Calcd for C26H18Cl2N4O: C, 65.97; H, 3.83; N, 11.84. Found: C,
66.19; H, 3.81; N, 11.86.
C27H22N4O: C, 77.49; H, 5.30; N, 13.39. Found: C, 77.25; H, 5.32;
N, 13.43.
4.2. Biological activity
4.1.5.19. 1-(4-((2-Chloroacridin-9-yl)amino)phenyl)-3-(4-nitro-
phenyl)urea (35).
Brown gummy; yield 68%; 1H NMR
4.2.1. MTB DNA super coiling assay
(DMSO-d6): dH. 6.64 (m, 2H, Ar-H), 7.43–7.89 (m, 9H, Ar-H),
8.13–8.26 (m, 4H, Ar-H), 8.76 (s, 2H, Ar-NH), 10.67 (s, 1H, Ar-
NH-Ar). 13C NMR (DMSO-d6): dC. 153.7, 150.5, 146.1, 144.0(2C),
142.3(2C), 132.8, 132.0, 140.9, 130.3(2C), 126.7(2C), 124.9(3C),
123.0(2C), 12.3(2C), 120.5(2C), 118.3(2C), 108.8. EI-MS m/z (Calcd
Supercoiling assay was performed using MTB DNA supercoiling
assay kits (Inspiralis Limited, Norwich). Briefly, the assay was done
in 30 lL reaction volume for 30 min at 37 °C in an assay buffer
containing 50 mM HEPES. KOH (pH 7.9), 6 mM magnesium acetate,
4 mM DTT, 1 mM ATP, 100 mM potassium glutamate, 2 mM sper-
Please cite this article in press as: Medapi, B.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.011

B. Medapi et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
midine and 0.05 mg/mL of albumin. During the assay 2 U of DNA
gyrase was incubated with 0.5 g of relaxed pBR322 in the assay
each well of 24 well plate along with 250
well was then added with 250 ll of working concentration of the
l
l of 0.1% DMSO. Each
l
buffer for 30 min, then the reaction was quenched by the addition
of equal volume of 30 mL of chloroform/isoamylalcohol (24:1) and
STEB buffer, with a brief vortex and followed by centrifugation. The
products were analysed by electrophoresis on 1% agarose gels after
staining with ethidium bromide. Using Image lab software (Bio-
rad), intensity of bands were measured and analysed to determine
the enzyme inhibition by relative band intensity.
drug. Embryos were allowed to incubate at the optimum tempera-
ture for 4 h. After 4 h of incubation, embryos were treated with tri-
caine and immediately used for reading the heart rate. 30 heart
beats were counted for atrium and ventricle per embryo and the
corresponding time was noted, from which number of heart beats
per minute was calculated by the formula: 1800/X = beats/minute
(where X = time in seconds).
4.2.2. In vitro MTB screening
Acknowledgements
Two-fold serial dilutions of each test compound/drug were pre-
pared and incorporated into Middle-brook 7H11 agar medium
with oleic acid, albumin, dextrose, and catalase (OADC) growth
supplement to get final concentrations of 50, 25, 12.5, 6.25, 3.13,
DS acknowledges University Grants Commission, New Delhi,
India for UGC Research Award. Authors also thankful to J.P.S. Devi
for her assistance in MTB screening.
1.56, and 0.78 lg/mL. Inoculum of M. tuberculosis H37Rv ATCC
27294 was prepared from fresh Middlebrook 7H11 agar slants with
OADC (Difco) growth supplement adjusted to 1 mg/mL (wet
weight) in Tween 80 (0.05%) saline diluted to 10À2 to give a con-
centration of ꢀ107 cfu/mL. Five microlitres of this bacterial suspen-
sion was spotted onto 7H11 agar tubes containing different
concentrations of the drug as discussed above. The tubes were
References and notes
1. Global Tuberculosis Report 2015. <http://www.who.int/tb/publications/global_
report/en/> (last accessed 3rd November 2015).
2. Mahajan, R. Int. J. Appl. Basic Med. Res. 2013, 3, 1.
3. Field, S. K. Ther. Adv. Chronic Dis. 2015, 6, 170.
4. Medapi, B.; Suryadevara, P.; Renuka, J.; Sridevi, J. P.; Yogeeswari, P.; Sriram, D.
Eur. J. Med. Chem. 2015, 103, 1.
incubated at 37 °C, and final readings (as MIC in
lg/mL) were
5. Medapi, B.; Renuka, J.; Saxena, S.; Sridevi, J. P.; Medishetti, R.; Kulkarni, P.;
Yogeeswari, P.; Sriram, D. Bioorg. Med. Chem. 2015, 23, 2062.
6. Saxena, S.; Samala, G.; Renuka, J.; Sridevi, J. P.; Yogeeswari, P.; Sriram, D. Bioorg.
Med. Chem. 2015, 23, 1402.
7. Jeankumar, V. U.; Reshma, R. S.; Janupally, R.; Saxena, S.; Sridevi, J. P.; Medapi,
B.; Kulkarni, P.; Yogeeswari, P.; Sriram, D. Org. Biomol. Chem. 2015, 13, 2423.
8. Jeankumar, V. U.; Kotagiri, S.; Janupally, R.; Suryadevara, P.; Sridevi, J. P.;
Medishetti, R.; Kulkarni, P.; Yogeeswari, P.; Sriram, D. Bioorg. Med. Chem. 2015,
23, 588.
9. Mdluli, K.; Ma, Z. Infect. Disord. Drug Targets 2007, 7, 159.
10. Chopra, S.; Matsuyama, K.; Tran, T.; Malerich, J. P.; Wan, B.; Franzblau, S. G.;
Lun, S.; Guo, H.; Maiga, M. C.; Bishai, W. R.; Madrid, P. B. J. Antimicrob.
Chemother. 2012, 67, 415.
11. Hameed, P. S.; Manjrekar, P.; Raichurkar, A.; Shinde, V.; Puttur, J.; Shanbhag, G.;
Chinnapattu, M.; Patil, V.; Rudrapatana, S.; Sharma, S.; Kumar, C. N. N.;
Nandishaiah, R.; Madhavapeddi, P.; Sriram, D.; Solapure, S.; Sambandamurthy,
V. K. ACS Med. Chem. Lett. 2015, 6, 746.
determined after 28 days. The MIC is defined as the minimum con-
centration of compound required to give complete inhibition of
bacterial growth. This method is similar to that recommended by
the National Committee for Clinical Laboratory Standards for the
determination of MIC in triplicate.
4.2.3. In vitro cytotoxicity screening
Some compounds were further examined for toxicity in a RAW
264.7 cell line at the concentration of 50 lM. After 72 h of expo-
sure, viability was assessed on the basis of cellular conversion of
MTT into a formazan product using the Promega Cell Titer 96
non-radioactive cell proliferation assay.
12. Hameed, P. S.; Manjrekar, P.; Raichurkar, A.; Shinde, V.; Puttur, J.; Shanbhag, G.;
Chinnapattu, M.; Patil, V.; Rudrapatana, S.; Sharma, S.; Kumar, C. N. N.;
Nandishaiah, R.; Madhavapeddi, P.; Sriram, D.; Solapure, S.; Sambandamurthy,
V. K. ACS Med. Chem. Lett. 2014, 6, 741.
13. Reck, F.; Alm, R.; Brassil, P.; Newman, J.; Dejonge, B.; Eyermann, C. J.; Breault,
G.; Breen, J.; Comita-Prevoir, J.; Cronin, M.; Davis, H.; Ehmann, D.; Galullo, V.;
Geng, B.; Grebe, T.; Morningstar, M.; Walker, P.; Hayter, B.; Fisher, S. J. Med.
Chem. 2011, 54, 7834.
14. Ketron, A. C.; Denny, W. A.; Graves, D. E.; Osheroff, N. Biochemistry 2012, 51,
1730.
15. Godbole, A. A.; Ahmed, W.; Bhat, R. S.; Bradley, E. K.; Ekins, S.; Nagaraja, V.
Biochem. Biophys. Res. Commun. 2014, 446, 916.
4.2.4. zERG channel inhibition screening
All experiments were performed as per the guidelines published
by the National Institutes of Health for care and use of zebrafish. In
brief, male and female fishes were maintained separately in the re-
circulatory system with 14 h light and 10 h dark cycle, with 28 °C
as optimum temperature. Breeding was carried out using 2
females: 3 males in a separate breeding cage. Embryos were col-
lected into petridishes and allowed to grow in the incubator in
E3 medium at 28 °C temperature. On the 3rd day embryos were
removed and washed. Stock solution of the drug was prepared in
100% DMSO and the working concentrations were prepared
accordingly by serial dilutions. 6 embryos were transferred into
16. Sridevi, J. P.; Suryadevara, P.; Janupally, R.; Sridhar, J.; Soni, V.; Anantaraju, H.
S.; Yogeeswari, P.; Sriram, D. Eur. J. Pharm. Sci. 2015, 72, 81.
17. Chaudhari, G. H.; Chennubhotla, K. S.; Chatti, K.; Kulkarni, P. J. Pharmacol.
Toxicol. Methods 2013, 67, 115.
Please cite this article in press as: Medapi, B.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.011