The document describes the synthesis and characterization of 9-anilinoacridine derivatives substituted with oxazine moieties to evaluate their antioxidant and anticancer activities. A series of 9-anilinoacridines were synthesized by reacting 1-[4-(acridin-9-ylamino)phenyl]ethanone with various aldehydes using Claisen-Schmidt condensation. The structures of the synthesized compounds were confirmed using spectroscopic techniques like IR, NMR, mass spectrometry. The compounds were evaluated for their antioxidant activity by hydrogen peroxide and superoxide radical scavenging assays. Their anticancer potential was studied against Dalton's lymphoma ascites cell line using trypan blue dye exclusion and

1. Presented by,
Anbu Dinesh Jayakumar,
M.Pharmacy II Year (SEM-III),
Dept of Pharmaceutical Chemistry,
College of Pharmacy ,SRIPMS.
Source : 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
EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY 56 (2012) 217-224

2. 2

3. 3
ACRIDINE derivatives are widely used as antimicrobial, antioxidant,
anticancer , antimalarial, anti-inflammatory, & other biological activities .
Heterocycles containing the OXAZINE NUCLEUS were found to possess
biological properties like analgesic, anti-inflammatory, anti-leukemic,
antipyretic, anticonvulsant and antimicrobial activities.
A series of 9-anilinoacridines substituted with oxazine derivatives were
synthesized to evaluate their ANTIOXIDANT STUDIES AND ANTICANCER
ACTIVITY STUDIES against DALTONS LYMPHOMA ASCITES (DLA) CELL
GROWTH by in-vitro method.
INTRODUCTION

4.  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 synthesised compounds were characterised by various SPECTROPHOTOMETRIC methods.
 The docking studies of the synthesized compounds were performed towards the key Topoisomerase II (1QZR)
by using Schrodinger Maestro 9.2 version.
 The results revealed that the newly synthesized derivatives exhibited significant in vitro cytotoxicity.
4

5.  Antioxidants are compounds that inhibit oxidation .
 Oxidation is a chemical reaction that can produce free radicals leading to chain
reactions that may damage the cells of organisms.
 Antioxidants such as thiols or ascorbic acid are used to terminate these chain
reactions.
 They are also called as "free-radical scavengers.“
 In this research anti-oxidant activity was evaluated by two methods.
i) Scavenging of hydrogen peroxide radicals,
ii) Scavenging of superoxide radical by alkaline DMSO method.
5

6. 6

7. METHODS OF SCREENING ANTI-CANCER ACTIVITY
IN-VITRO
1. Tryphan blue dye exclusion assay
2. LDH (Lactic dehydrogenase) Assay
3. MTT assay
4. XTT assay
5. Sulporhodamine B assay
IN-VIVO
1. Carcinogen induced models
2. Viral infection models
3. Transplantation Models
4. Genetically Engineered Mouse Models
5. In vivo hollow fibre assay
8

8. Melting points were obtained on Veego VMP-1 apparatus .
The reactions were monitored by TLC on silica gel thin layer
plates.
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.
Mass spectra of the final compounds were recorded on a JEOL
GC mate Mass Spectrometer.
9

9. 10

10. SYNTHESIS OF 9-CHLOROACRIDINE
 4.06 g (0.03 mol) of 4-aminoacetophenone was refluxed with 5.4528 g (0.0256 mol)
of 9 chloroacridine is taken in 250ml round bottom flask to this 80 mL of 2-butanol was
added and kept for 3 h and cooled RT to this 150 mL of ice water added.
 The precipitate formed was filtered by suction, washed with water, dried and
recrystallized from ethanol.
SYNTHESIS OF 1-[4-(ACRIDIN-9-YLAMINO)PHENYL]ETHANONE
 9-Chloroacridine was synthesized by the cyclization of N-arylanthranilic acid
with phosphorus oxychloride
11

11. GENERAL PROCEDURE FOR SYNTHESIS OF
CHALCONES 4A-J
 Chalcones were synthesized by using general Claisen Schimdt condensation .
 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.
12

12. S.NO COMPOUND
CODE
CHALCONES
APPEARANCE
(POWDER)
MELTING
POINT(°C)
YIELD (%) S.NO COMPOUN
D CODE
ANI.ACRID
INE
APPEARANCE(
POWDER)
MELTING
POINT(°C)
YIELD (%)
1 4a YELLOW 195-197 79 11 5a YELLOW 170-172 56
2 4b YELLOW 179-181 78 12 5b BROWNISH
YELLOW
215-217 78
3 4c YELLOW 188-190 65 13 5c YELLOW 175-177 67
4 4d YELLOW 196-198 65 14 5d YELLOWISH
GREEN
185-186 75
5 4e CHERRY RED 190-192 53 15 5e GREEN 132-133 66
6 4f YELLOW 208-210 65 16 5f YELLOW 208-209 70
7 4g YELLOW 188-190 65 17 5g YELLOW 210-211 61
8 4h YELLOW 210-212 73 18 5h YELLOW 168-170 57
9 4i ORANGE 188-190 61 19 5i YELLOW 175-177 51
10 4j YELLOW 228-230 54 20 5j DARK YELLOW 160-161 71
13

13. IR VALUES OF CHALCONES IR VALUES OF ANILOACRIDINE
1 4a 3269 (N-H), 3063-3000 (Ar C-H), 1649 (α,β-unsat.
C=O), 1607 & 1512 (ArC-C), 1231 (C-N), 761 (Ar C-H)
11 5a 3340(N-H), 2993 (Ar st C-H), 1600 & 1473 (Ar C=C), 1157 (Ar C=N),
1226 (C-O), 742 (Ar C-H)
2 4b 3269 (N-H),3057-3000 (Ar C-H), 1647 (α,β-unsat. C=O),
1604 & 1516 (Ar C=C), 1226 (C-N), 759 (Ar C-H)
12 5b 3340(N-H), 2991 (Ar st C-H), 1600 & 1473 (Ar C=C), 1157 (Ar C=N),
1226 (CO), 742 (Ar C-H)
3 4c 3302(N-H), 3100-3000 (Ar C-H), 1651 (α,β-unsaturated
C=O), 1606 &1518 (Ar C=C), 1267 (C-N), 748 (Ar C-H)
13 5c 3218 (N-H), 3008 (Ar st C-H),1573 & 1545 (Ar C=C),1157
(Ar C=N), 1227 (C-O), 756 (Ar C-H), 815 (C-Cl)
4 4d 3302(N-H), 3100-3000 (Ar C-H), 1624 (α,β-unsat. C=O),
1606 & 1518 (Ar C=C),1267 (C-N), 748 (Ar C-H)
14 5d 3218 (N-H), 3055 (Ar st C-H), 1606 & 1593 (Ar C=C), 1707 (Ar
C=N), 1274 (C-O), 744 (Ar C-H), 831 (C-Cl)
5 4e 3279 (N-H), 3109-2999 (Ar C-H),1647 (α,β-unsat.
C=O),1591 & 1496 (Ar C=C),1273 (C-N), 746 (Ar C-H)
15 5e 3351 (N-H),3064 (Ar st C-H), 1593 (Ar C=C), 1707 (Ar C=N), 1274 (C-O),
744 (Ar C-H), 831 (C-Cl
6 4f 3034(N-H), 3100-3000 (Ar C-H), 1622 (α,β-unsat. C=O),
1577 & 1498(Ar C=C), 1529 & 1348 (NO2), 1280 (C-N),
748 (ArC-H)
16 5f 3463(N-H), 3080 (Ar st C-H), 1599 & 1583 (Ar C=C), 1635 (Ar C=N),
1232 (C-O), 746 (Ar C-H)
7 4g 3034(N-H), 3100-3000 (Ar C-H), 1626 (α,β-unsat. C=O),
1577 & 1498(Ar C=C), 1529 & 1348 (NO2), 1280 (C-N),
748 (ArC-H)
17 5g 3336 (N-H), 3064 (Ar st C-H), 1589 & 1564 (Ar C=C), 1157 (Ar C=N),
1232(C-O), 746 (Ar C-H)
8 4h 3304 (N-H), 3100-3000 (Ar st C-H),1626 (α,β-unsat.
C=O),1587 & 1568 (ArC=C), 3327 (Ar-OH), 748 (Ar C-
H);
18 5h 3332(N-H), 2993 (Ar st C-H), 1589 & 1563 (Ar C=C), 3227 (Ar-OH),
1653 (Ar C=N), 1178 (C-O), 750 (Ar C-H);
9 4i 3347 (N-H), 3100-3000 (Ar st C-H), 1622 (α,β-unsat.
C=O), 1604 & 1473 (ArC=C)
19 5i 3343 (N-H), 3067 (Ar st C-H), 1589 & 1563 (Ar C=C), 1159 (Ar C=N),
1178(C-O), 742 (Ar C-H
10 4j 3044(N-H), 3100-3000 (Ar st C-H), 1626 (α,β- 20 5j 3352 (N-H), 3005 (Ar st C-H), 1600 & 1583 (Ar C=C), 1642 (Ar C=N),

14. S.NO COMPOUND CODE
CHALCONES
MS (M+) S.NO COMPOUND CODE
ANILO ACRIDINE
MS (M+)
1 4a 468.16 11 5a 511.15
2 4b 400.16 12 5b 442.57
3 4c 434.12 13 5c 476.56
4 4d 434.52 14 5d 476.78
5 4e 434.62 15 5e 476.92
6 4f 445.34 16 5f 487.47
7 4g 445.37 17 5g 487.47
8 4h 450.34 18 5h 488.47
9 4i 338.18 19 5i 380.47
10 4j 460.58 20 5j 502.52
15

15. S.NO CODE H-NMR C-13 NMR
1 4a 6.65-8.02 (m, ArH), 7.90 and 7.56 (s, α,β-unsaturated),
11.21(s, NH)
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.
2 4b 6.65-8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s, α,β-
unsaturated),11.21 (1H, s, NH)
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.
3 4c 6.65-8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s, α,β
unsaturatedcarbonyl), 11.21 (1H, s, NH);
187(C=O), 114.8-149.7 (aromatic carbons).
4 4d 6.65-8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s, α,β
unsaturatedcarbonyl), 11.21 (1H, s, NH);
189(C=O), 114.8-149.7 (aromatic carbons).
5 4e 6.65-8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s, α,β-
unsaturatedcarbonyl), 11.21 (1H, s, NH)
189 (C=O), 114.8-149.7 (aromatic carbons).
6 4f 6.65-8.02 (16H, m, ArH), 7.90 and
7.56 (2H, s, α,β-unsaturated carbonyl),11.21 (1H, s, NH)
189 (C=O), 114.8-149.7 (aromatic carbons
7 4g 6.65-8.02 (16H, m, ArH), 7.90 and
7.56 (2H, s, α,β-unsaturated carbonyl),11.21 (1H, s, NH);
189 (C=O), 114.8-149.7 (aromatic carbons).
8 4h 6.65-8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s, α,β
unsaturatedcarbonyl), 11.21 (1H, s, NH);
176(C=O), 42.03 (OCH3), 110.2-169.3 (aromatic
carbons).
9 4i 6.65-8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s, α,β-
unsaturated carbonyl), 11.21 (1H, s,NH)
189 (C=O), 3.72 (CH3), 114.8-149.7(aromatic carbons)
10 4j 6.65-8.02 (16H, m, ArH), 7.90 and 7.56 (2H, s, α,β
unsaturatedcarbonyl), 11.21 (1H, s, NH).
181(C=O), 53.49 (OCH3), 54.31 (OCH3), 104.8-158.6
(aromatic carbons).
16

16. S.NO COMPOUND CODE H-NMR C-13 NMR
1 5a 6.85-7.94 (16H, m, ArH), 7.95 (1H, s, NH), 6. 19
(2H, s, NH2
114.3-178.4 (aromatic carbons
2 5b 6.83-8.92 (16H, m, ArH), 7.95 (1H, s, NH), 6. 19
(2H, s, NH2
112.3-166.1 (aromatic carbons).
3 5c 7.36-7.70 (16H, m, ArH), 7.77-8.05 (2H, m,
CH), 7.91 (1H, s, NH), 6. 19 (2H, s, NH2);
113.4-175.1 (aromatic carbons)
4 5d 6.85-7.97 (18H, m, ArH), 8.02 (1H, s,
NH), 6. 21 (2H, s, NH2)
108.3-157.8 (aromatic carbons).
5 5e 6.24-7.98 (18H, m, ArH), 8.22 (1H, s, NH), 6. 24
(2H, s, NH2
108.8-171.4 (aromatic carbons).
6 5f 6.61-7.21 (18H, m, ArH), 7.94 (1H, s, NH), 6. 02
(2H, s, NH2);
109.7-168.7 (aromatic carbons
7 5g 7.67-7.9 (18H, m, ArH), 8.28 (1H, s, NH), 6. 27
(2H, s, NH2);
107.8-176.5 (aromatic carbons).
8 5h 7.27-7.71 (18H, m, ArH), 8.28 (1H, s, NH), 6.
27(2H, s, NH2)
40.03 (OCH3), 112.4-163.9(aromatic carbons).
9 5i 7.23-7.85 (18H, m, ArH), 8.38 (1H, s, NH), 6. 18
(2H, s, NH2
2.50 (CH3), 112.8-175.2 (aromatic carbons).
10 5j 6.85-7.97 (16H, s, ArH), 7.48 (1H, s, NH), 6.12
(2H, s, NH2), 3.80, 3.86
(6H, d, OCH3)
55.69 (OCH3), 55.53 (OCH3),
Oth-r aromatic carbons ar- 110.49, 111.54, 112.7,
119.97, 123.21,125.55, 127.97, 130.98, 141.87,17

17. 3D STRUCTURE OF LIGAND
USING MARWIN SKETCH
AND CONVERTED TO PDB
LIGAND PRPN USING
SCHRODINGER
(PDBQT)
Docking was performed with Schrodinger Maestro and glide scores are
obtained
PARAMETER AND GRID FILES
ARE DONE USING GLIDE
CHARGES ARE ADDED AND
OUTPUT IS SAVED IN PDBQT
FORMAT
USING Schrodinger Maestro
SAVED AS PDB
PDB ID : 1QZR
DOWNLOADED FROM RCSB
RECEPTOR
+
LIGAND
LIGAND
RECEPTOR
18

18. 19

19. 20

20. 21
 A solution of hydrogen peroxide (40 mM) was prepared in phosphate buffer (pH 7.4).
 Different concentrations (250, 500, and 1000 μg/mL) of the synthesized compounds
(ascorbic acid as the control) were added to a hydrogen peroxide solution (0.6 mL, 40 mM).
 The absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a
blank solution .
 The hydrogen peroxide percentage scavenging activity was then calculated using the
following equation:
 Ao is the absorbance of the control reaction and
 A is the absorbance in the presence of the samples or standards.
A) SCAVENGING OF HYDROGEN PEROXIDE RADICALS

21. 22
B) SCAVENGING OF SUPEROXIDE RADICAL BY ALKALINE DMSO METHOD
 Superoxide is generated according to the alkaline DMSO method.
 The reduction of Nitro Blue Tetrazolium (NBT) by superoxide was determined in the
presence and absence of the compound.
 To the reaction mixture containing 1 mL of alkaline DMSO, 0.3 mL of the drug samples
and standard was added in DMSO at various concentrations followed by 0.1 mL of
NBT (0.1 mg) to give a final volume of 1.4 mL.
 IC50 values are calculated.

22. 23
ANTITUMOUR ACTIVITY of the compounds were assayed by using trypan
blue dye exclusion technique.
DLA cells were cultured in the peritoneal cavity of mice by injecting a
suspension of DLA cells (1x106 cells/mL) intraperitoneally for 15 days
Cells were washed with Hank’s balanced salt solution (HBSS) and
centrifuged for 10-15 min at 1500 rpm in the cooling centrifuge.
Finally the cells were suspended in a known quantity of HBSS and the cell
count was adjusted to 2 x 106 cells/mL.

23. 24
• 0.1 mL each of the different concentration of the drug in phosphate buffer saline
and incubated at 37 ºC, 5% Carbon dioxide for 3 h.
• After 3 h, trypan blue dye exclusion test was performed to determine percentage
viability.
• 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.

24. 25

25. RESULTS ARE SUMMARIZED IN TABLES 1-3

26. 27
GOOD
ANTI-CANCER
ACTIVITY

27.  Oxazine substituted derivatives 5a, 5b, 5g, 5h, 5i and 5j shows antioxidant
activity when compared to standard ascorbic acid.
The docking studies and binding energies was calculated using Schrodinger
Maestro 9.2 version and evaluated for anticancer activity.
The synthesized compounds 5a-j were subjected to short term study for in vitro
antitumour activity against Daltons Lymphoma Ascites (DLA) cells.
The compounds 5a, 5h, 5i, 5j exerted significant anticancer activity against
DLA cells at the concentration of 140-250 mg/mL (0.352-0.512 mM).
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.
Synthesised compounds can be used for further study to many useful
medicinal compounds.
28

28. Oxazine substituted 9-anilinoacridine series have been synthesised and exerts potent
anticancer activities.
These agents exhibited significant cytotoxicity against DLA cell growth in vitro as
Topoisomerase II inhibitor.
The molecular docking studies show a good correlation between their biological activities
screened and Glide score.
The compounds 5a, 5h, 5i, 5j have 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.
29

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