Docking studies, synthesis, characterization of some novel oxazine substituted 9-anilinoacridine derivatives and evaluation for their antioxidant and anticancer activities as topoisomerase II inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.08.025

A series of 9-anilinoacridines substituted with oxazine derivatives were synthesized to evaluate their antioxidant and anticancer activity against Daltons Lymphoma Ascites (DLA) cell growth by in vitro method. It was revealed that these conjugates exhibited significant antioxidant and anticancer activity (inhibition of DLA cell proliferation). Among these agents, compounds 5a, 5h, 5i, 5j were the most cytotoxic with CTC₅₀ value of 140-250 μg/mL. The docking studies of the synthesized compounds were performed towards the key Topoisomerase II (1QZR) by using Schrodinger Maestro 9.2 version. The oxazine substituted 9-anilinoacridine derivatives 5a, 5h, 5i, 5j have significant anticancer activity as topoisomerase II inhibitors.

Full text of DOI:10.1016/j.ejmech.2012.08.025

European Journal of Medicinal Chemistry 56 (2012) 217e224
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Docking studies, synthesis, characterization of some novel oxazine substituted
9-anilinoacridine derivatives and evaluation for their antioxidant and
anticancer activities as topoisomerase II inhibitors
*
R. Kalirajan , Vivek Kulshrestha, S. Sankar, S. Jubie
Department of Pharmaceutical Chemistry, JSS College of Pharmacy, (Off Campus, JSS University, Mysore), Udhagamandalam 643001, Tamilnadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 9-anilinoacridines substituted with oxazine derivatives were synthesized to evaluate their
antioxidant and anticancer activity against Daltons Lymphoma Ascites (DLA) cell growth by in vitro
method. It was revealed that these conjugates exhibited significant antioxidant and anticancer activity
(inhibition of DLA cell proliferation). Among these agents, compounds 5a, 5h, 5i, 5j were the most
Received 8 June 2012
Received in revised form
15 August 2012
Accepted 17 August 2012
Available online 31 August 2012
cytotoxic with CTC50 value of 140e250 mg/mL. The docking studies of the synthesized compounds were
performed towards the key Topoisomerase II (1QZR) by using Schrodinger Maestro 9.2 version. The
oxazine substituted 9-anilinoacridine derivatives 5a, 5h, 5i, 5j have significant anticancer activity as
topoisomerase II inhibitors.
Keywords:
Acridine
Chalcone
Oxazine
Ó 2012 Elsevier Masson SAS. All rights reserved.
Antioxidant
Anticancer
1. Introduction
DNA base pairs. The chemical modification of acridines such as the
introduction of different substitutions or hetero cyclic rings were
Acridine derivatives are one of the most studied chemothera-
peutic compounds, widely used as antimicrobial [1], antioxidant
[2], anticancer [3e7], antimalarial [8,9], anti-inflammatory [10],
analgesic [11], antileishmanial [12], antinociceptive [13], acetyl
cholinesterase inhibitors [14] and antiherpes [15] etc. Antitumour
cytotoxic agents with DNA-intercalative properties have been
studied. Amsacrine is the best-known compound of
9-anilinoacridines series. It was one of the first DNA-intercalating
agents to be considered as a topoisomerase II inhibitor. The inter-
calation process is the strongest type of reversible binding to the
double helical DNA in compounds with sufficiently large coplanar
aromatic chromophore. Moreover, the cytotoxicity of most of the
clinically useful DNA-intercalating agents involves the inhibition of
the enzyme DNA-topoisomerase I or II. Several detailed SAR studies
of acridine-based DNA-intercalating agents suggest that the mode
of binding is important and the chromophore intercalate with the
allowed expansion of research on the structure activity relationship
to afford new insight into molecular interactions at the receptor
level [16]. In fact, it is well established that slight structural
modification on 9-anilinoacridines may bring various pharmaco-
logical effects. Similarly oxazine derivatives also have various bio-
logical activities [17e20] like antimicrobial, anticancer etc. In vitro
cytotoxicity against Daltons Lymphoma Ascites (DLA) cell lines are
described. As a part of our ongoing research on searching new
potent cytotoxic agents, we have synthesized 9-anilinoacridine
analogues bearing the oxazine residue on anilino rings for anti-
cancer evaluation. The results revealed that the newly synthesized
derivatives exhibited significant in vitro cytotoxicity.
2. Chemistry
Our synthetic pathway (Scheme 1) was based on the preparation
of 9-chloroacridine 2, which was refluxed with p-amino-
acetophenone to yield 1-(4-(acridine-9-ylamino) phenyl)ethanone 3
[10]. The various chalcone substituted 9-anilinoacridines 4aej [21]
were prepared by the reaction of 3 with various aldehydes and
* Corresponding author. Tel.: þ91 423 2443393x223; fax: þ91 423 2442937.
E-mail addresses: rkalirajan@ymail.com, rkalirajan@jsscpooty.org (R. Kalirajan).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.025

218
R. Kalirajan et al. / European Journal of Medicinal Chemistry 56 (2012) 217e224
OH
O
Anhyd K2CO3 + CuO
H2N
NH
HO
N-phenyl anthranilic acid(1)
O
reflux for 2 hr at oil bath/
Cl
aniline
O
2-chlorobenzoic acid
reflux for 3 hr
at oil bath/ 455 Watt
(65%intensity) for 3 min
POCl3
2-BuOH +
Cl
HN
4-Amino acetophenone
reflux for 3 hr at oil bath
N
N
1-(4-(acridin-9-ylamino)phenyl)ethanone (3)
9-Chloroacridine (2)
EtOH +
Stirr at room
10% NaOH
temperature for 8 hr
R-CHO
O
R
HN
cyclization with urea
stirred for 2-3 hours
R
N
Chalcone derivatives (4a-j)
Oxazine substituted 9-anilino acridine derivatives (5a-j)
Cl
Cl
R =
CH3-
Cl
OCH3
NO2
OCH3
OCH3
4j,5j
NO2
4f,5f
Cl
OH
4i,5i
Cl
4h,5h
4g,5g
4b,5b
4c,5c
4e,5e
4d,5d
4a,5a
Scheme 1.
these chalcone derivatives were allowed to cyclized with urea afford
the corresponding oxazine substituted 9-anilinoacridines 5aej [22].
Synthesis, characterization and evaluation of biological activities of
novel chalcone and oxazine substituted 9-anilinoacridines are
described in this paper. The synthesized compounds were purified by
column chromatography. The final yield of the derivatives was in the
range of 50e75%. The compounds obtained were stable in thesolid as
well as in the solution state. The new compounds were completely
characterized by IR, 1H NMR, 13C NMR, mass spectral data and
elemental analysis. The IR spectra of compounds 5aej showed
intense bands in the region 1200e1300 cmꢀ1 due to carbonyl
stretching and broad bands in theregion3200e3400 cmꢀ1 due to NH
stretching. The 1H NMR spectra also support the structure of the
compounds5aej. The NHprotonappeared at7.9e8.1 andNH2 proton
at 5.9e6.3. The mass spectra of all compounds 5aej showed molec-
ular ion peaks confirming their molecular weight.
in vitro anticancer activity against Daltons Lymphoma Ascites (DLA)
cells. Many of the synthesized final compounds 5aej have signifi-
cant activities.
3.1. Antioxidant activity
3.1.1. Scavenging of hydrogen peroxide radicals [26]
Hydrogen peroxide was generated in vivo by several oxidase
enzymes and by activated phagocytes and it is known to play an
important role in the killing of several bacterial and fungal
strains. There are increasing evidences that, H2O2, either directly
or indirectly, OHþ can act as a messenger molecule in the
synthesis and activation of several inflammatory mediators.
When a scavenger is incubated with H2O2 using a peroxides
assay system, the loss of H2O2 can be measured. H2O2 itself is
a rather weak oxidant and most organic compounds (except for
some sulphur containing molecules) were virtually inert to
attack by it at ordinary environmental or cellular concentration
and temperature.
3. Pharmacology
Acridine derivatives possess a diverse range of pharmacological
activities [23e25]. The objectives of this study were to determine
whether the oxazine substituted 9-anilinoacridine derivatives
posses the antioxidant and cytotoxic activity. Aiming at this goal,
we have proved the impact of the acridines on the cytotoxicity of
cells by intercalating DNA. Hence all the chalcone and oxazine
substituted 9-anilinoacridine derivatives 4aej, 5aej were screened
for antioxidant activity and 5aej were screened for short term
3.1.2. Scavenging of superoxide radical by alkaline DMSO method
[27]
Superoxide was generated in vitro and the reduction of NBT by
superoxide was determined in the presence and absence of sample
and standard shows scavenging ability of different samples and
standard against superoxide radicals. The results are shown in
Table 1.

R. Kalirajan et al. / European Journal of Medicinal Chemistry 56 (2012) 217e224
219
Table 1
were scaled by Van der Waals radii of 1.0 Å with the partial
atomic charge less than 0.25 defaults. The active site was defined
as an enclosing box at the centroid of the workspace ligand as
selected in the receptor folder. The ligands similar in size to the
workspace ligand were allowed to dock into the active site. No
constraints either positional, H-bonding or hydrophobic were
defined.
Antioxidant activity of synthesized compounds 4aej and 5aej by H2O2 method and
alkaline DMSO method
S. No.
Compound
IC50 values (
for H2O2 method
m
g/mL)
IC50 values (mg/mL)
for alkaline DMSO method
1
2
3
4a
4b
4c
>1000
>1000
>1000
>500
>500
>500
Ligand docking was performed using OPLS force field. The
receptor grid defined in the receptor grid generation folder was
selected for the docking of ligands prepared by using Ligprep.
Flexible docking was performed using the Extra Precision (XP)
feature of Glide module. The structure output format was set to
pose viewer file so as to view the output of the resulting docking
studies from pose-viewer. The XP-Glide predicted pose of active
compounds in the scheme are shown in Fig. 1.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
4d
4e
4f
4g
4h
4i
4j
5a
5b
5c
5d
5e
5f
5g
5h
5i
>1000
>1000
>1000
>1000
>1000
>1000
>1000
20.03 ꢃ 0.2583
17.23 ꢃ 1.148
30.01 ꢃ 0.028
36.01 ꢃ 0.1028
25.01 ꢃ 1.123
24.03 ꢃ 0.1402
12.00 ꢃ 0.5660
14.20 ꢃ 0.3564
14.50 ꢃ 0.2764
18.06 ꢃ 0.6568
18.60 ꢃ 1.039
>500
>500
>500
>500
>500
>500
>500
28.02 ꢃ 0.6583
22.27 ꢃ 0.5308
68.13 ꢃ 0.4144
37.43 ꢃ 0.3296
32.05 ꢃ 0.4825
47.50 ꢃ 0.5518
29.03 ꢃ 1.265
26.30 ꢃ 0.1219
24.02 ꢃ 0.3567
28.01 ꢃ 0.504
24.77 ꢃ 0.2404
5. Results and discussion
Results are summarized in Tables 1e3 and schematised in
Scheme 1. They showed that the pharmacological properties of the
compounds greatly depended on the number and the chemical
nature of the substituents.
For in vitro antioxidant activity, all the synthesized chalcone and
oxazine substituted 9-anilinoacridines were screened by H2O2
method and alkaline DMSO method. Almost all the oxazine
substituted derivatives exerted an interesting antioxidant activity.
Among these compounds 5a, 5b, 5g, 5h, 5i and 5j were more potent
antioxidant activity when compared to standard ascorbic acid. The
IC50 values, which are the concentration of the sample required to
increase 50% of reducing capacity of the compounds are summa-
rized in Table 1.
The docking studies of the ligands to protein active sites were
performed by an advanced molecular docking program Schro-
dinger Maestro 9.2 version for determining the binding affinities
of the compounds. The designed analogues are docked towards
the Topoisomerase II (1QZR) in order to ascertain their anti-
cancer activity. The analogues show best fit Root Mean Square
Difference (rmsd) value of 0.000. The results obtained in the
molecular docking studies showed a good correlation between
their short term in vitro anticancer activity and Glide score. The
compounds 5h, 5i, 5j showed good affinity to the receptor due to
more lipophilic character and hydrogen bonding when
compared with standard. The results are summarized in the
Table 2.
5j
Standard
ascorbic acid
3.2. Short term study for antitumour activity [28]
Short term antitumour activity of the compounds were assayed
by determining the percentage viability of DLA cells using trypan
blue dye exclusion technique. DLA cells were cultured in the
peritoneal cavity of healthy albino mice weighing 25e30 g by
injecting a suspension of DLA cells (1 ꢁ 106 cells/mL) intraperito-
neally. The cells were aspirated aseptically from the peritoneal
cavity of the mice on day 15. The cells were washed with Hank’s
balanced salt solution (HBSS) and centrifuged for 10e15 min at
1500 rpm in the cooling centrifuge. The pellet was re-suspended
with HBSS and the process was repeated three times. Finally the
cells were suspended in a known quantity of HBSS and the cell
count was adjusted to 2 ꢁ 106 cells/mL. 0.1 mL of the diluted cell
suspension was distributed in to Eppendorf tubes and exposed
0.1 mL each of the different concentration of the drug in phosphate
buffer saline and incubated at 37 ꢂC, 5% CO2 for 3 h. After 3 h,
trypan blue dye exclusion test was performed to determine
percentage viability.
The synthesized final compounds 5aej were subjected to short
term study for in vitro antitumour activity against Daltons
Lymphoma Ascites (DLA) cells. The compounds 5a, 5h, 5i, 5j exer-
ted significant anticancer activity against DLA cells at the concen-
For testing viability using dye exclusion method, the pooled cells
from wells of each concentration were mixed with 0.4% trypan blue
in a ratio of 1:1 and the number of stained, non-stained and total
number of cells were counted using haemocytometer. The
percentage inhibition and CTC50 values were calculated.
tration of 140e250
mg/mL (0.352e0.512 mM). The results are
summarized in the Table 3. The aliphatic methyl at 4th position
of oxazine increases the cytotoxic activity than aromatic substitu-
tion. 4th position of oxazine has di substituted aromatic ring which
increases the cytotoxic activity than mono substituted aromatic
ring.
The compounds 5h, 5i and 5j exhibited the highest Glide
score ꢀ5.37, ꢀ6 and ꢀ6.22 respectively are having good in vitro
anticancer activity. The best affinity modes of the docked
compounds (5a, 5h, 5i, 5j) with Topoisomerase II were shown in
Fig. 1.
The compound 5j has more hydrogen bonding with receptor
and lipophilic factors for their good affinity to the receptor site
which was shown in the Fig. 2. The compounds 5h and 5i also have
more lipophilic factors for their good affinity to the receptor site. So
this research work can be used for further study to synthesize many
useful medicinal compounds.
4. Docking studies [29]
Protein preparation was performed by using protein preparation
Wizard of Maestro 9.2 software. The protein structure 1QZR was
taken from PDB data bank. The A chain was treated to add missing
hydrogen, assign proper bond orders and delete water molecules
which were more than 5 Å from the heterogeneous groups. Finally,
the protein structure was minimized to the default Root Mean
Square Deviation (RMSD) value of 0.30.
Ligand preparation was accomplished on all the 10 compounds
using Ligprep module to clean the structure and generate tauto-
mers as described.
Receptor was defined and the co-crystallized ligand was
differentiated from the active site of receptor A chain. The atoms

220
R. Kalirajan et al. / European Journal of Medicinal Chemistry 56 (2012) 217e224
Fig. 1. Docking pictures of compounds with topoisomerase II (1QZR).
6. Conclusion
exerted potent anticancer activities. It was revealed that these
agents exhibited significant cytotoxicity against DLA cell growth
in vitro as Topoisomerase II inhibitor. Results observed in the
present study clearly demonstrated that some derivatives of the
oxazine substituted 9-anilinoacridine family could exert interesting
anticancer and antioxidant activities. The molecular docking
studies show a good correlation between their biological activities
screened and Glide score. The compounds 5a, 5h, 5i, 5j have
Acridine family includes a wide range of tricyclic molecules with
various biological properties. Considered as potential anticancer
agents since the 1980s, numerous acridine derivatives have been
synthesised and successfully assessed for their anticancer activity.
On this basis, authors recently demonstrated that diverse
compounds of the oxazine substituted 9-anilinoacridine series

R. Kalirajan et al. / European Journal of Medicinal Chemistry 56 (2012) 217e224
221
Table 2
Docking studies of synthesized compounds with Topoisomerase II (1QZR).
7.2. General procedure for synthesis of chalcones 4aej
The chalcones were synthesized by using general Claisene
Schimdt condensation [21]. In a 100 mL flat bottomed flask
25 mL of the 10% sodium hydroxide and 25 mL of ethanol were
taken with a magnetic stirring bar and it was stirred on the
magnetic stirrer. To this 0.01 mol of corresponding aldehyde was
added, then 2.99 g (0.0096 mol) of 1-[4-(acridin-9-ylamino)phenyl]
ethanone was added at the last. The solution was allowed to stir for
8 h at room temperature. After completion of the reaction 100 mL of
water was added, formed precipitate was filtered washed three
times with 50 mL of water each time to remove sodium hydroxide,
dried and crystallized from ethanol.
S. No.
Compound no
G-score
Lipophilic Evdw
H-bond
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
5f
5g
5h
ꢀ3.45
ꢀ5.26
ꢀ5.44
ꢀ5.41
ꢀ5.05
ꢀ4.50
ꢀ3.3
ꢀ2.13
ꢀ3.48
ꢀ3.86
ꢀ2.2
ꢀ0.03
ꢀ0.69
ꢀ0.42
ꢀ1.26
ꢀ0.83
0
ꢀ3.45
ꢀ3.39
ꢀ3.68
ꢀ4.68
ꢀ4.53
ꢀ3.91
ꢀ3.95
0
ꢀ5.37
ꢀ6
ꢀ0.52
ꢀ0.91
ꢀ1.35
ꢀ0.53
9
10
11
5i
5j
ꢀ6.22
ꢀ5.13
Ledacrine (std)
7.2.1. (E)-1-(4-(Acridin-9-ylamino)phenyl)-3-(2,4-dichloro phenyl)
prop-2-en-1-one (4a)
significant anticancer activity and are likely to be useful as drugs
after further refinement. These derivatives will encourage helping
to design future anticancer agents with therapeutic potentials.
This compound was obtained as a yellow powder; m.p.: 195e
197 ꢂC; Yield: 79%; Anal. Calc. for C28H18Cl2N2O: C, 71.68; H, 3.73;
N, 5.90; Found: C, 71.73; H, 3.77; N, 5.92; IR (KBr,
H), 3063e3000 (Ar CeH), 1649 ( -unsat. C]O), 1607 & 1512 (Ar
C]C), 1231 (CeN), 761 (Ar CeH); MS: m/z 468.16 (Mþ); 1H NMR (in
y
, cmꢀ1): 3269 (Ne
a,b
7. Experimental protocol
ppm): 6.65e8.02 (m, ArH), 7.90 and 7.56 (s, a,b-unsaturated), 11.21
Melting points were obtained on Veego VMP-1 apparatus in
open capillary tubes and are uncorrected. The reactions were
monitored by TLC on silica gel thin layer plates. Compounds were
analysed for C, H, N and analytical results obtained for these
elements were within ꢃ0.5% of the calculated values for the
formula shown. All reagents were of commercial quality or were
purified before use. Organic solvents were of analytical grade or
were purified by standard procedures. IR spectra were obtained
using a Shimadzu 8400 FT-IR spectrometer. 1H NMR and 13C NMR
were recorded on Bruker A VIII 500 MHz Spectrometer. Chemical
shifts are in parts per million (ppm). Mass spectra of the final
compounds were recorded on a JEOL GC mate Mass Spectrometer.
(s, NH); 13C NMR (in ppm) :184.4, 153.5, 150.8, 143.2, 141.2, 136.3,
136.1, 132.4, 131.7, 130.8, 129.6, 130.5, 128.6, 127.2, 127.1, 127.2, 121.6,
119.5, 116.3.
7.2.2. (E)-1-(4-(Acridin-9-ylamino)phenyl)-3-phenyl prop-2-en-1-
one (4b)
This compound was obtained as a yellow powder; m.p.: 179e
181 ꢂC; Yield: 78%; Anal. Calc. for C28H20N2O: C, 83.98; H, 5.03; N,
7.07; Found: C, 83.84; H, 5.12; N, 7.13; IR (KBr,
3057e3000 (Ar CeH), 1647 ( -unsat. C]O), 1604 & 1516 (Ar C]
C), 1226 (CeN), 759 (Ar CeH); MS: m/z 400.16 (Mþ); 1H NMR (in
y
, cmꢀ1): 3269 (NeH),
a,b
ppm): 6.65e8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s, a,b-unsatu-
rated), 11.21 (1H, s, NH); 13C NMR (in ppm) :183.4, 153.8, 149.8,
142.8,140.5,136.1,135.4,131.7,131.3,130.4,129.3,129.8,128.9,127.8,
126.7, 126.7, 121.9, 119.3, 115.7.
7.1. Synthesis and characterization of the compounds
7.1.1. Synthesis of 9-chloroacridine 2
9-Chloroacridine was synthesized by the cyclization of N-ary-
lanthranilic acid with phosphorus oxychloride as reported [21].
7.2.3. (E)-1-(4-(Acridin-9-ylamino)phenyl)-3-(4-chlorophenyl)
prop-2-en-1-one (4c)
This compound was obtained as a yellow powder; m.p.: 188e
190 ꢂC; Yield: 65%; Anal. Calc. for C28H19ClN2O: C, 77.33; H, 4.40;
7.1.2. Synthesis of 1-[4-(acridin-9-ylamino)phenyl]ethanone 3
In a 250 mL round bottomed flask 4.06 g (0.03 mol) of
4-aminoacetophenone was refluxed with 5.4528 g (0.0256 mol) of
9-chloroacridine in 80 mL of 2-butanol for 3 h [10]. After comple-
tion of reaction the reaction mixture was allowed to cool to room
temperature then it was poured into 150 mL of ice water. A
precipitate formed was filtered by suction, washed with water,
dried and recrystallized from ethanol.
N, 6.44; Found: C, 77.25; H, 4.29; N, 6.62; IR (KBr,
(NeH), 3100e3000 (Ar CeH), 1651 ( -unsaturated C]O), 1606 &
1518 (Ar C]C), 1267 (CeN), 748 (Ar CeH); MS: m/z 434.12 (Mþ); 1H
y
, cmꢀ1): 3302
a,b
NMR (in ppm): 6.65e8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s, a,b-
unsaturated carbonyl), 11.21 (1H, s, NH); 13C NMR (in ppm) :187
(C]O), 114.8e149.7 (aromatic carbons).
7.2.4. (E)-1-(4-(Acridin-9-ylamino)phenyl)-3-(3-chlorophenyl)
prop-2-en-1-one (4d)
This compound was obtained as a yellow powder; m.p.: 196e
Table 3
198 ꢂC; Yield: 65%; Anal. Calc. for C28H19ClN2O: C, 77.32; H, 4.42;
Short term in vitro anticancer activity of synthesized compounds against Daltons
Lymphoma Ascites (DLA) cells.
N, 6.45; Found: C, 77.25; H, 4.29; N, 6.62; IR (KBr,
(NeH), 3100e3000 (Ar CeH), 1624 ( -unsat. C]O), 1606 & 1518
(Ar C]C),1267 (CeN), 748 (Ar CeH); MS: m/z 434.52 (Mþ); 1H NMR
y
, cmꢀ1): 3302
a,b
S. No.
Compound name
CTC50 (mg/mL)
1
2
3
4
5
6
7
8
9
10
5a
5b
5c
5d
5e
5f
5g
5h
5i
180
425
640
820
590
725
600
140
250
190
(in ppm): 6.65e8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s,
a,b-
unsaturated carbonyl), 11.21 (1H, s, NH); 13C NMR (in ppm) :189
(C]O), 114.8e149.7 (aromatic carbons).
7.2.5. (E)-1-(4-(Acridin-9-ylamino)phenyl)-3-(2-chlorophenyl)
prop-2-en-1-one (4e)
This compound was obtained as a cherry red powder; m.p.: 190e
192 ꢂC; Yield: 53%; Anal. Calc. for C28H19ClN2O: C, 77.33; H, 4.40; N,
5j
CTC50 e concentration required to reduce viability by 50%.
6.43; Found: C, 77.59; H, 4.20; N, 6.25; IR (KBr, y
, cmꢀ1): 3279 (NeH),

222
R. Kalirajan et al. / European Journal of Medicinal Chemistry 56 (2012) 217e224
Fig. 2. Lipophilic evidence and hydrogen bonding for compound 5j.
3109e2999 (Ar CeH),1647 (
a
,
b
-unsat. C]O),1591 & 1496 (Ar C]C),
(Ar C]C), 1529 & 1348 (NO2), 1280 (CeN), 748 (Ar CeH); MS: m/z
445.34 (Mþ); 1H NMR (in ppm): 6.65e8.02 (16H, m, ArH), 7.90 and
7.56 (2H, s,
-unsaturated carbonyl),11.21 (1H, s, NH); 13C NMR (in
1273 (CeN), 746 (Ar CeH); MS: m/z 434.62 (Mþ); 1H NMR (in ppm):
6.65e8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s,
a
,b
-unsaturated
a,b
carbonyl), 11.21 (1H, s, NH); 13C NMR (in ppm) :189 (C]O), 114.8e
ppm) :189 (C]O), 114.8e149.7 (aromatic carbons).
149.7 (aromatic carbons).
7.2.7. (E)-1-(4-(Acridin-9-ylamino)phenyl)-3-(3-nitro phenyl)
prop-2-en-1-one (4g)
7.2.6. (E)-1-(4-(Acridin-9-ylamino)phenyl)-3-(4-nitro phenyl)
prop-2-en-1-one (4f)
This compound was obtained as a yellow powder; m.p.: 188e
This compound was obtained as a yellow powder, m.p.: 208e
190 ꢂC; Yield: 65%; Anal. Calc. for C28H19N3O3: C, 75.47; H, 4.30;
210 ꢂC; Yield: 65%; Anal. Calc. for C28H19N3O3: C, 75.49; H, 4.32;
N, 9.45; Found: C, 75.26; H, 4.52; N, 9.51; IR (KBr,
(NeH), 3100e3000 (Ar CeH), 1626 ( -unsat. C]O), 1577 & 1498
(Ar C]C), 1529 & 1348 (NO2), 1280 (CeN), 748 (Ar CeH); MS: m/z
y
, cmꢀ1): 3034
N, 9.45; Found: C, 75.26; H, 4.52; N, 9.51; IR (KBr,
y
, cmꢀ1): 3034
a,b
(NeH), 3100e3000 (Ar CeH), 1622 ( -unsat. C]O), 1577 & 1498
a,b

R. Kalirajan et al. / European Journal of Medicinal Chemistry 56 (2012) 217e224
223
445.37 (Mþ); 1H NMR (in ppm): 6.65e8.02 (16H, m, ArH), 7.90 and
7.56 (2H, s,
-unsaturated carbonyl),11.21 (1H, s, NH); 13C NMR (in
6.83e8.92 (16H, m, ArH), 7.95 (1H, s, NH), 6. 19 (2H, s, NH2); 13C
NMR (in ppm): 112.3e166.1 (aromatic carbons).
a,b
ppm) :189 (C]O), 114.8e149.7 (aromatic carbons).
7.3.3. N-(4-(2-Amino-4-(4-chloro phenyl)-2H-1,3-oxazin-6yl)
phenyl)acridin-9-amine (5c)
7.2.8. (E)-1-(4-(Acridin-9-ylamino)phenyl)-3-(4-hydroxy-3-
methoxy phenyl)prop-2-en-1-one (4h)
This compound is obtained as a yellow powder; m.p.: 175e
177 ꢂC; Yield: 67%; Anal. calc. for C29H21ClN4O: C, 73.18; H, 4.45;
Cl, 7.52; N, 11.68; Found: C, 73.12; H, 4.49; Cl, 7.56; N, 11.73; IR (KBr,
This compound was obtained as a yellow powder; m.p.: 210e
212 ꢂC; Yield: 73%; Anal. Calc. for C29H26N2O3: C, 77.42; H, 5.85;
N, 6.33; Found: C, 77.46; H, 5.92; N, 6.37; IR (KBr,
H), 3100e3000 (Ar st CeH), 1626 ( -unsat. C]O), 1587 & 1568 (Ar
C]C), 3327 (AreOH), 748 (Ar CeH); MS: m/z 450.34 (Mþ); 1H NMR
y
, cmꢀ1): 3304 (Ne
y
, cmꢀ1): 3218 (NeH), 3008 (Ar st CeH), 1573 & 1545 (Ar C]C),1157
a,
b
(Ar C]N), 1227 (CeO), 756 (Ar CeH), 815 (CeCl); MS: m/z 476.56
(Mþ); 1H NMR (in ppm): 7.36e7.70 (16H, m, ArH), 7.77e8.05 (2H, m,
CH), 7.91 (1H, s, NH), 6. 19 (2H, s, NH2); 13C NMR (in ppm): 113.4e
175.1 (aromatic carbons).
(in ppm): 6.65e8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s,
a,b-
unsaturated carbonyl), 11.21 (1H, s, NH); 13C NMR (in ppm) :176
(C]O), 42.03 (OCH3), 110.2e169.3 (aromatic carbons).
7.3.4. N-(4-(2-Amino-4-(3-chloro phenyl)-2H-1,3-oxazin-6yl)
phenyl)acridin-9-amine (5d)
7.2.9. (E)-1-(4-(Acridin-9-ylamino)phenyl)but-2-en-1-one (4i)
This compound was obtained as an orange powder; m.p.: 188e
190 ꢂC; Yield: 61%; Anal. Calc. for C23H18N2O: C, 81.49; H, 5.33; N,
This compound is obtained as a yellowish green powder; m.p.:
185e186 ꢂC; Yield: 75%; Anal. calc. for C29H21ClN4O: C, 73.18; H,
8.23; Found: C, 81.53; H, 5.37; N, 8.13; IR (KBr,
3100e3000 (Ar st CeH), 1622 ( -unsat. C]O), 1604 & 1473 (Ar
C]C); MS: m/z 338.18 (Mþ); 1H NMR (in ppm): 6.65e8.02 (16H, m,
y
, cmꢀ1): 3347 (NeH),
4.45; N, 11.78; Found: C, 73.23; H, 4.48; N, 11.68; IR (KBr, y
, cmꢀ1):
a
,b
3218 (NeH), 3055 (Ar st CeH), 1606 & 1593 (Ar C]C), 1707 (Ar
C]N), 1274 (CeO), 744 (Ar CeH), 831 (CeCl); MS: m/z 476.78
(Mþ); 1H NMR (in ppm): 6.85e7.97 (18H, m, ArH), 8.02 (1H, s,
NH), 6. 21 (2H, s, NH2); 13C NMR (in ppm): 108.3e157.8 (aromatic
carbons).
ArH), 7.90 and 7.56 (2H, s, a,b-unsaturated carbonyl), 11.21 (1H, s,
NH); 13C NMR (in ppm) :189 (C]O), 3.72 (CH3), 114.8e149.7
(aromatic carbons).
7.2.10. (E)-1-(4-(Acridin-9-ylamino)phenyl)-3-(3,4-dimethoxy
phenyl)prop-2-en-1-one (4j)
7.3.5. N-(4-(2-Amino-4-(2-chloro phenyl)-2H-1,3-oxazin-6yl)
phenyl)acridin-9-amine (5e)
This compound was obtained as a yellow powder; m.p.: 228e
This compound is obtained as a green power; m.p.: 132e133 ꢂC;
Yield: 66%; Anal. calc. for C29H21ClN4O: C, 73.18; H, 4.38; N, 11.68;
230 ꢂC; Yield: 54%; Anal. Calc. for C30H24N2O3: C, 78.29; H, 5.33;
N, 6.13; Found: C, 78.25; H, 5.36; N, 6.12; IR (KBr,
(NeH), 3100e3000 (Ar st CeH), 1626 ( -unsaturated C]O),
1577 & 1498 (Ar C]C), 748 (Ar CeH); MS: m/z 460.58 (Mþ); 1H
y
, cmꢀ1): 3044
Found: C, 73.23; H, 4.28; N, 11.64; IR (KBr, y
, cmꢀ1): 3351 (NeH),
a
,
b
3064 (Ar st CeH), 1593 (Ar C]C), 1707 (Ar C]N), 1274 (CeO),
744 (Ar CeH), 831 (CeCl); MS: m/z 476.92 (Mþ); 1H NMR (in
ppm): 6.24e7.98 (18H, m, ArH), 8.22 (1H, s, NH), 6. 24 (2H, s, NH2);
13C NMR (in ppm): 108.8e171.4 (aromatic carbons).
NMR (in ppm): 6.65e8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s, a,b-
unsaturated carbonyl), 11.21 (1H, s, NH). 13C NMR (in ppm):181
(C]O), 53.49 (OCH3), 54.31 (OCH3), 104.8e158.6 (aromatic
carbons).
7.3.6. N-(4-(2-Amino-4-(4-nitro phenyl)-2H-1,3-oxazin-6yl)
phenyl)acridin-9-amine (5f)
7.3. General procedure for synthesis of oxazine substituted 9-
anilinoacridines 5aej
This compound is obtained as a yellow powder; m.p.: 208e
209 ꢂC; Yield: 70%; Anal. calc. for C29H21N5O3: C, 71.37; H, 4.28;
N, 14.29; Found: C, 71.41; H, 4.23; N, 14.35; IR (KBr, y
, cmꢀ1): 3463
A mixture of chalcone 4aej (0.02 mol), urea (0.02) were dis-
solved in ethanolic sodium hydroxide (10 mL) was stirred about for
2e3 h with a magnetic stirrer. This was then poured into 400 mL of
cold water with continuous stirring for an hour. This was kept in
refrigerator for 24 h. The precipitate obtained was filtered, washed
and recrystallized using petroleum ether:benzene (5:5). The reac-
tion was monitored by TLC using methanol:water (5:3).
(NeH), 3080 (Ar st CeH), 1599 & 1583 (Ar C]C), 1635 (Ar C]N),
1232 (CeO), 746 (Ar CeH). MS: m/z 487.47 (Mþ); 1H NMR (in ppm):
6.61e7.21 (18H, m, ArH), 7.94 (1H, s, NH), 6. 02 (2H, s, NH2); 13C
NMR (in ppm): 109.7e168.7 (aromatic carbons).
7.3.7. N-(4-(2-Amino-4-(3-nitro phenyl)-2H-1,3-oxazin-6yl)
phenyl)acridin-9-amine (5g)
This compound is obtained as a yellow powder; m.p.: 210e
7.3.1. N-(4-(2-Amino-4-(2,4-dichloro phenyl)-2H-1,3-oxazin-6yl)
phenyl)acridin-9-amine (5a)
211 ꢂC; Yield: 61%; Anal. calc. for C29H21N5O3: C, 71.31; H, 4.29; N,
14.48; Found: C, 71.25; H, 4.42; N, 14.51; IR (KBr, y
, cmꢀ1): 3336 (Ne
This compound is obtained as a yellow powder; m.p.: 170e
H), 3064 (Ar st CeH), 1589 & 1564 (Ar C]C), 1157 (Ar C]N), 1232
(CeO), 746 (Ar CeH); MS: m/z 487.47 (Mþ); 1H NMR (in ppm):
7.67e7.9 (18H, m, ArH), 8.28 (1H, s, NH), 6. 27 (2H, s, NH2); 13C NMR
(in ppm): 107.8e176.5 (aromatic carbons).
172 ꢂC; Yield: 56%; Anal. calc. for C29H20Cl2N4O: C, 68.24; H, 3.87;
N, 13.86; Found: C, 68.18; H, 3.82; N, 13.78; IR (KBr, y
, cmꢀ1): 3340
(NeH), 2993 (Ar st CeH), 1600 & 1473 (Ar C]C), 1157 (Ar C]N),
1226 (CeO), 742 (Ar CeH); MS: m/z 511.15 (Mþ); 1H NMR (in ppm):
6.85e7.94 (16H, m, ArH), 7.95 (1H, s, NH), 6. 19 (2H, s, NH2); 13C
NMR (in ppm): 114.3e178.4 (aromatic carbons).
7.3.8. 4-(6-(4-(Acridin-9-ylamino)phenyl)-2-amino-2H-1,3-
oxazin-4yl)-2methoxyphenol (5h)
This compound is obtained as a yellow powder; m.p.: 168e
7.3.2. N-(4-(2-Amino-4-(phenyl)-2H-1,3-oxazin-6yl)phenyl)
acridin-9-amine (5b)
170 ꢂC; Yield: 57%; Anal. calc. for C30H24N4O3: C, 73.68; H, 4.87;
N, 11.36; Found: C, 73.68; H, 4.87; N, 11.36; IR (KBr, y
, cmꢀ1): 3332
This compound is obtained as a Brownish yellow powder; m.p.:
(NeH), 2993 (Ar st CeH), 1589 & 1563 (Ar C]C), 3227 (AreOH),
1653 (Ar C]N), 1178 (CeO), 750 (Ar CeH); MS: m/z 488.47 (Mþ);
1H NMR (in ppm): 7.27e7.71 (18H, m, ArH), 8.28 (1H, s, NH), 6. 27
(2H, s, NH2); 13C NMR (in ppm): 40.03 (OCH3), 112.4e163.9
(aromatic carbons).
215e217 ꢂC; Yield: 78%; Anal. calc. for C29H22N4O: C, 78.74; H, 5.17;
N, 12.57; Found: C, 78.78; H, 5.23; N, 12.67; IR (KBr, y
, cmꢀ1): 3340
(NeH), 2991 (Ar st CeH), 1600 & 1473 (Ar C]C), 1157 (Ar C]N),
1226 (CO), 742 (Ar CeH); MS: m/z 442.57 (Mþ); 1H NMR (in ppm):

224
R. Kalirajan et al. / European Journal of Medicinal Chemistry 56 (2012) 217e224
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4689e4694.
7.3.9. N-(4-(2-Amino-4-(methyl)-2H-1,3-oxazin-6yl)phenyl)
acridin-9-amine (5i)
This compound is obtained as a yellow powder; m.p.: 175e
177 ꢂC; Yield: 51%; Anal. calc. for C24H20N4O: C, 75.75; H, 5.34; N,
14.68; Found: C, 75.68; H, 5.41; N,14.62; IR (KBr, y
, cmꢀ1): 3343 (Ne
H), 3067 (Ar st CeH), 1589 & 1563 (Ar C]C), 1159 (Ar C]N), 1178
(CeO), 742 (Ar CeH); MS: m/z 380.47 (Mþ); 1H NMR (in ppm):
7.23e7.85 (18H, m, ArH), 8.38 (1H, s, NH), 6. 18 (2H, s, NH2); 13C
NMR (in ppm): 2.50 (CH3), 112.8e175.2 (aromatic carbons).
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7.3.10. N-(4-(2-Amino-4-(3,4-dimethoxyphenyl)-2H-1,3-oxazin-
6yl)phenyl)acridin-9-amine (5j)
This compound is obtained as a dark yellow powder; m.p.: 160e
161 ꢂC; Yield: 71%; Anal. calc. for C31H26N4O3: C, 74.18; H, 5.16; N,
11.12; Found: C, 74.23; H, 5.12; N, 11.18; IR (KBr, y
, cmꢀ1): 3352 (Ne
H), 3005 (Ar st CeH), 1600 & 1583 (Ar C]C), 1642 (Ar C]N), 1263
(CeO), 744 (Ar CeH); MS: m/z 502.52 (Mþ); 1H NMR (in ppm):
6.85e7.97 (16H, s, ArH), 7.48 (1H, s, NH), 6.12 (2H, s, NH2), 3.80, 3.86
(6H, d, OCH3); 13C NMR (in ppm): 55.69 (OCH3), 55.53 (OCH3),
Other aromatic carbons are 110.49, 111.54, 112.7, 119.97, 123.21,
125.55, 127.97, 130.98, 141.87, 148.97, 150.71, 153.66, 185.96.
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166e172.
Acknowledgement
[20] Basappa,
Sengottuvelan
Murugan,
Chandagirikoppal,
V.
Kavitha,
We thank All India Council for Technical Education, New Delhi
for the financial support under Research Promotion Scheme. We
also thank our Vice Chancellor Dr.B. Suresh, JSS University, Mysore,
Department of Pharmaceutical analysis, Department of Pharma-
ceutical Biotechnology, Department of Pharmacology, JSS College of
pharmacy, Ooty for the technical support.
Anurag Purushothaman, G. Nevin Kottayath, Kazuyuki Sugahara, S.
Rangappa Kanchugarakoppal, Cancer Lett. 297 (2010) 231e243.
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165258, http://dx.doi.org/10.1100/2012/165258.
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K. Anandarajagopal, Int. J. Chem. Sci. 5 (1) (2007) 73e80.
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(2007) 1277e1281.
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391e395.
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