Dinaphthonaphthyridines - A class of novel molecules with potent antioxidant and anticancer activity

Article abstract of DOI:10.1039/c2md20270k

A series of novel dinaphtho[1,2-b:1′,2′-h][1,6]naphthyridine derivatives bearing aliphatic, aromatic and heteroaromatic substituents at the C₇ position were designed and synthesized conveniently from 4-chloro-2-methylbenzo[h]quinoline. All compounds were screened for in vitro antioxidant activities against DPPH, ABTS⁺, superoxide and hydroxyl radicals. Among the compounds screened, 13, 15, 16, 19 and 20 showed excellent antioxidant activity against OH. In addition, the cytotoxicity of the dinaphthonaphthyridine derivatives was evaluated by MTT assay and compared with Adriamycin. From screening, we identified that compounds 6, 16 and 20 showed IC₅₀s ranging from 1.20 to 5.61 μM for growth inhibition of HeLa, HCT116 and AGS cancer cell lines. The results documented that varying of the chemical entity at C₇ can fine-tune the enhancement of cell growth inhibition strategy. The Royal Society of Chemistry.

Full text of DOI:10.1039/c2md20270k

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Experimental protocols
General
Melting points (m.p.) were determined on Mettler FP 51 apparatus (Mettler Instruments,
Switzerland) and are uncorrected. They are expressed in degree centigrade (°C). A Nicolet
1
Avatar Model FT-IR spectrophotometer was used to record the IR spectra (4000–400 cm-1). H
13
NMR and C NMR spectra were recorded on Bruker AV 500 (500 MHz (1H) and 125 MHz
(13C)) spectrometer using tetramethylsilane (TMS) as an internal reference. The chemical shifts
are expressed in parts per million (ppm). Mass spectra (MS) were recorded on Auto Spec EI+
Shimadzu QP 2010 PLUS GC-MS mass spectrometer. Microanalyses were performed on a Vario
EL III model CHNS analyzer (Vario, Germany) at the Department of Chemistry, Bharathiar
University, Coimbatore - 46, India. The solvent and reagents used for the preparations were of
reagent grade and were purified by standard methods, petroleum ether used was of boiling range
60-80C. Anhydrous sodium sulphate was used to dry the solution of organic extracts. Thin layer
chromatography (TLC) was performed using glass plates coated with silica gel-G containing
13% calcium sulphate as binder. Ethyl acetate and petroleum ether were used as developing
solvents. A chamber containing iodine vapour was used to locate the spots. Separation and
purification of the crude products was carried out using chromatographic columns packed with
activated silica gel (60-120 mesh). In the case of mixture of solvents used for elution, the ratio of
the mixture is given in brackets.
Synthesis
Preparation of 4-chloro-2-methylbenzo[h]quinoline (5)
1-Naphthylamine (0.25 mol) and ethylacetoacetate (0.25 mol) were mixed together and to it 1:1
HCl (5 drops) was added and the mixture was shaken well. It was left aside and within a few
minutes the mixture became turbid indicating the liberation of water due to the condensation
reaction. At this stage the mixture was kept inside in a vacuum dessicator over conc.H2SO4 for 2
days to get the corresponding anilide. The anilide was then cyclised using boiling diphenyl ether
under 250 °C for half an hour. The reaction mixture was cooled to get white solid. It was washed
several times with petroleum ether to get 2-methyl-4-hydroxybenzo[h]quinoline. This was
treated with POCl3 to get 4-chloro-2-methylbenzo[h]quinoline (5).
1
White solid; m.p. 62-64 °C; IR νmax (cm-1): 1581 (C=N), 1537, 1172, 1073; H NMR (δ, ppm,
CDCl3): 2.80 (s, 3H, C2-CH3), 7.48 (s, 1H, C3-H), 7.68-7.74 (m, 2H, C8, C9-H), 7.84 (d, 1H, C6-

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H, J = 9.20 Hz), 7.92 (dd, 1H, C7-H, Jo = 8.80 Hz, Jm = 2.40 Hz), 8.09 (d, 1H, C5-H, J = 9.20
Hz), 9.32 (dd, 1H, C10-H, Jo = 7.60 Hz, Jm = 2.00 Hz); Anal. Calcd. for C14H10ClN: C, 73.85; H,
4.43; N, 6.15; Found: C, 73.81; H, 4.45; N, 6.17 %.
General procedure Preparation of 2-methyl-N-phenylbenzo[h]quinolin-4-amine (6)
Method A (Solvent condition)
An appropriate mixture of 4-Chloro-2-methylbenzo[h]quinoline (5, 0.004 mol) was reacted with
1-Naphthylamine, (0.004 mol) using CuI and heated in DMSO at 120 °C for an hour. After the
reaction completion was indication by the TLC, the reaction mixture was washed with water,
dried, adsorbed and purified using silica gel column chromatography and eluted with
ethylacetate:methanol (99:1) mixture to get 6 which was then recrystallised using methanol
Method B (Thermal condition)
An appropriate mixture 4-Chloro-2-methylbenzo[h]quinoline (5, 0.004 mol) was reacted with 1-
Naphthylamine, (0.004 mol) was heated under neat condition (without any solvent) at 190 °C for
an hour. After the reaction completion was indication by the TLC, the reaction mixture was
washed with water, dried, adsorbed and purified using silica gel column chromatography and
eluted with ethylacetate:methanol (95:5) mixture to get 6 which was then recrystallised using
methanol.
2-Methyl-N-(naphthalen-1-yl)benzo[h]quinolin-4-amine (6)
1
Brown solid; m.p. >300 °C; Yield: (75 %); IR νmax (cm-1): 3134 (NH), 1629 (C=N), 1267; H
NMR (δ, ppm, DMSO-d6): 2.67 (s, 3H, C2-CH3), 6.34 (s, 1H, C3-H), 7.57-7.97 (m, 7H, C2'-C8'-
H), 8.13 (d, 2H, C6, C7 -H J = 8.00 Hz), 8.23-8.26 (m, 2H, C8, C9 -H J = 9.00 Hz ), 8.82 (d, 1H,
C5-H J = 8.00 Hz,), 9.45 (d, 1H, C10-H J = 9.00 Hz), 11.12 (s, 1H, C4-NH amino form), 13.70
13
(bs, 1H, C1-NH imino form, ratio of amino form : imino form is 1 : 1); C NMR (δ, ppm,
DMSO-d6): 20.4 (C2-CH3), 103.9 (C3), 113.9 (C6'), 119.5 (C4a), 122.6 (C4'), 123.0 (C8'), 124.4
(C8a'),125.9 (C5), 126.3 (C7'), 127.4 (C6'), 127.7 (C3'), 127.9 (C10), 128.5 (C6), 128.6 (C9), 129.1
(C8), 129.2 (C7), 129.3 (C5'), 130.7 (C4a'), 133.6 (C10a), 134.8 (C6a) , 134.9 (C1'), 137.2 (C10b),
154.2 (C4), 156.4 (C2); MS: m/z (%) 334 (M+, 100); Anal. Calcd. for C24H18N2: C, 86.20; H,
5.43; N, 8.38; Found: C, 86.17; H, 5.44; N, 8.40 %.
General procedure for the synthesis of compound (7)

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2-methyl-N-(naphthalen-1-yl)benzo[h]quinolin-4-amine (6, 0.002 mol) and acetic acid (0.0025
mol) were added to polyphosphoric acid (6 g of P2O5 in 3 mL of H3PO4) and heated at 160 °C
for 3 hours. The reaction was monitored by using TLC. After the completion of the reaction, it
was poured into ice water, neutralized with saturated sodium bicarbonate solution to remove
excess of benzoic acid, extracted with ethyl acetate, purified by column chromatography using
silica gel and product eluted with petroleum ether: ethyl acetate (99:1) mixture to get 7 which
was recrystallised using methanol.
6, 7-Dimethyldinaphtho[1,2-b:1',2'-h][1,6]naphthyridine (7)
Pale yellow solid; m.p. 230-232 °C; Yield: (79 %); IR νmax (cm-1) : 1635, 1598 (C=N), 1571,
1469; 1H NMR (δ, ppm, CDCl3): 2.30 (s, 3H, C6-CH3), 3.20 (s, 3H, C7-CH3) 7.60 - 7.76 (m, 5H,
C8, C9, C10, C11, C12-H), 7.82 (d, 1H, C16-H J = 8.00 Hz), 7.92-7.98 (m, 3H, C1, C2, C3-H), 9.30
(d, 1H, C4-H, J = 8.00 Hz), 9.38 (d, 1H, C15-H, J = 9.00 Hz), 9.62 (d, 1H, C13-H, Jo = 9.50 Hz);
13C NMR (δ, ppm, CDCl3): 19.2 (C7-CH3), 31.5 (C6-CH3), 120.7 (C6a), 121.8 (2C, C8 &C14b),
122.0 (C13), 124.6 (C4), 125.1 (C7a), 126.0 (C16), 126.4 (C12), 126.9 (C3) 127.3 (C10), 127.4 (C2),
127.6 (C15), 127.7 (C9), 127.8 (C1), 129.4 (C11), 131.0 (C4a), 131.7 (C13a), 133.9 (C9a), 134.4
(C16a), 141.3 (C4b), 143.9 (C7) 147.0 (C14a), 147.5 (C13b), 157.9 (C6); MS: m/z (%) 358 (M+, 100);
Anal. Calcd. for C26H18N2: C, 87.12; H, 5.06; N, 7.82; Found: C, 87.16; H 5.04; N, 7.80 %.
General procedure for the synthesis of compound (8-20)
2-methyl-N-(naphthalen-1-yl)benzo[h]quinolin-4-amine (6, 0.002 mol) and various aromatic
carboxylic acids and hetero carboxylic acids (0.0025 mol) were added to polyphosphoric acid (6
g of P2O5 in 3 mL of H3PO4). The reaction time, temperature maintained and various acid used
for the synthesis of respective product are mention in the Table 2. The reaction was monitored
by using TLC. After the completion of the reaction, it was poured into ice water, neutralized with
saturated sodium bicarbonate solution to remove excess of carboxylic acids, extracted with ethyl
acetate, purified by column chromatography using silica gel and product eluted with petroleum
ether:ethyl acetate (97:3) mixture to get 8-20 which was recrystallised using methanol.
6-Methyl-7-phenyldinaphtho[1,2-b:1',2'-h][1,6] naphthyridine (8)
Yellow prisms; m.p. 270-272 °C; Yield: (83 %); IR νmax (cm-1) : 1566, 1536 (C=N), 1494,
1438; 1H NMR (δ, ppm, CDCl3): 2.52 (s, 3H, C6-CH3), 7.41 (d, 1H, C9-H J = 9.00 Hz), 7.48-7.69

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(m, 6H, C2', C3', C4', C5', C6' & C8-H), 7.72-7.85 (m, 3H, C10, C11, C12)7.89-7.93 (m, 2H, C2, C3-
H), 8.07 (d, 1H, C16-H, J = 8.00 Hz), 8.16 (d, 1H, C1-H, J = 9.00 Hz), 9.42 (d, 1H, C4-H, J = 8.80
13
Hz), 9.65 (d, 1H, C15-H, J = 9.00 Hz), 9.85 (d, 1H, C13-H, J = 8.50 Hz); C NMR (δ, ppm,
CDCl3): 29.5 (C6-CH3), 120.6 (C6a), 121.0 (C8), 121.5 (C14b), 122.2 (C13), 124.7 (C4), 125.3
(C7a), 126.1 (C16), 126.5 (C12), 126.8 (C3), 127.1 (C10), 127.5 (C2), 127.7 (C15), 127.8 (C9), 127.9
(C1), 128.5 (C2' & C6'), 128.6 (C3', C4' & C5'), 129.3 (C11), 130.0 (C1') 131.1 (C4a), 131.4 (C13a),
133.8 (C9a), 134.1 (C16a), 141.1 (C4b), 143.8 (C7) 147.1 (C14a), 147.9 (C13b), 159.0 (C6); MS: m/z
(%) 420 (M+, 100); Anal. Calcd. for C31H20N2: C, 88.55; H, 4.79; N, 6.66; Found: C, 88.52; H
4.80; N, 6.68 %.
6-Methyl-7-tolyldinaphtho[1,2-b:1',2'-h][1,6]naphthyridine (9)
Pale yellow solid; m.p. 249-251 °C; Yield: (86 %); IR νmax (cm-1): 1650, 1602 (C=N), 1441;
1H NMR (δ, ppm, CDCl3): 2.43 (s, 3H, C6-CH3), 2.57 (s, 3H, C6'-CH3), 7.42 (d, 1H, C9-H J=
9.00 Hz), 7.44-7.82 (m, 8H, C8, C10, C11, C12, C2', C3', C5' & C6'-H), 7.85-7.90 (m, 2H, C2, C3-H),
8.07 (d, 1H, C16-H, J = 7.20 Hz), 8.12 (d, 1H, C1-H, J = 8.80 Hz), 9.39 (d, 1H, C4-H, J = 8.00
13
Hz), 9.61 (d, 1H, C15-H, J = 8.80 Hz), 9.81 (d, 1H, C13-H, J = 8.00 Hz); C NMR (δ, ppm,
CDCl3): 21.5 (C4'-CH3), 29.6 (C6-CH3), 120.5 (C6a), 121.3 (C8), 121.7 (C14b), 122.1 (C13), 124.8
(C4), 125.4 (C7a), 126.2 (C16), 126.7 (C12), 126.9 (C3), 127.2 (C10), 127.5 (C2), 127.7 (C15), 127.8
(C9), 127.9 (C1), 128.4 (C2' & C6'), 128.8 (C3' & C5'), 129.1 (C11), 130.3 (C1') 131.4 (C4a), 131.6
(C13a), 133.7 (C9a), 134.2 (C16a), 135.6 (C4'), 141.0 (C4b), 143.6 (C7), 147.3 (C14a), 148.0 (C13b),
159.5 (C6); MS: m/z (%) 434 (M+, 100); Anal. Calcd. for C32H22N2: C, 88.45; H, 5.10; N, 6.45;
Found: C, 88.43; H, 5.09; N, 6.48 %.
6-Methyl-7-(2'-chlorophenyl)dinaphtho[1,2-b:1',2'-h][1,6] naphthyridine (10)
Yellow solid; m.p. 221-223 °C; Yield: (77 %); IR νmax (cm-1): 1663, 1567 (C=N), 1535, 1434;
1H NMR (δ, ppm, CDCl3): 2.49 (s, 3H, C6-CH3), 7.33 7.84 (m, 11H, C2, C3, C8, C9, C10, C11,
C12, C2', C3', C5' & C6'-H), 7.98 (d, 1H, C16-H, J = 7.20 Hz), 8.21 (d, 1H, C1-H, J = 8.80 Hz),
9.33 (d, 1H, C4-H, J = 8.00 Hz), 9.56 (d, 1H, C15-H, J = 8.80 Hz), 9.75 (d, 1H, C13-H, J = 8.00
13
Hz); C NMR (δ, ppm, CDCl3): 29.9 (C6-CH3), 120.8 (C6a), 121.1 (C8), 121.4 (C14b), 122.3
(C13), 124.9 (C4), 125.2 (C7a), 126.3 (C16), 126.6 (C12), 126.8 (C3), 127.3 (C10), 127.5 (C2), 127.7
(C15), 127.8 (C9), 127.9 (C1), 128.1 (C6'), 128.7 (C3', C4' & C5'), 129.0 (C11), 130.1 (C1') 131.3
(C4a), 131.5 (C13a), 133.6 (C9a), 134.4 (C16a), 135.7 (C2') 141.2 (C4b), 143.7 (C7), 147.2 (C14a),

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147.7 (C13b), 159.6 (C6); MS: m/z (%) 454 (M+, 100), 456 (M+2, 100) ; Anal. Calcd. for
C31H19ClN2: C, 81.84; H, 4.21; N, 6.16; Found: C, 81.78; H, 4.26; N, 6.10 %.
6-Methyl-7-(4'- methoxyphenyl)dinaphtho[1,2-b:1',2'-h][1,6]naphthyridine (11)
Pale yellow solid; m.p. 262-264 °C; Yield: (71 %); IR νmax (cm-1): 1597, 1521 (C=N), 1445,
1402; 1H NMR (δ, ppm, CDCl3): 2.44 (s, 3H, C6-CH3), 4.00 (s, 3H, C4'-OCH3), 7.32-7.82 (m,
11H, C2, C3, C8, C9, C10, C11, C12, C2', C3', C5' & C6'-H), 8.06 (d, 1H, C16-H, J = 8.00 Hz), 8.22
(d, 1H, C1-H, J = 8.80 Hz), 9.38 (d, 1H, C4-H, J = 8.00 Hz), 9.62 (d, 1H, C15-H, J = 8.80 Hz),
9.84 (d, 1H, C13-H, J = 8.00 Hz); 13C NMR (δ, ppm, CDCl3): 29.8 (C6-CH3), 55.6 (OCH3), 120.9
(C6a), 121.4 (C8), 121.9 (C14b), 122.2 (C13), 124.6 (C4), 125.1 (C7a), 126.0 (C16), 126.7 (C12),
126.9 (C3), 127.4 (C10), 127.5 (C2), 127.7 (C15), 127.8 (C9), 127.9 (C1), 128.2 (C2' & C6'), 128.9
(C3' & C5'), 129.4 (C11), 130.5 (C1'), 131.2 (C4a), 131.7 (C13a), 133.9 (C9a), 134.5 (C16a), 135.9
(C4'), 141.5 (C4b), 143.5 (C7), 147.4 (C14a), 148.1 (C13b), 159.7 (C6); MS: m/z (%) 450 (M+,
100);Anal. Calcd. for C32H22N2O: C, 85.31; H, 4.92; N, 6.22, Found: C, 85.35; H, 4.90; N, 6.27
%.
6-Methyl-7-(3'-nitrophenyl)dinaphtho[1,2-b:1',2'-h][1,6] naphthyridine (12)
Dark yellow solid; m.p. 282-284 °C; Yield: (68 %); IR νmax (cm-1): 1591, 1590 (C=N), 1513
(asym NO2), 1472, 1341 (sym NO2); 1H NMR (δ, ppm, CDCl3): 2.41 (s, 3H, C6-CH3), 7.39-7.90
(m, 11H, C2, C3, C8, C9, C10, C11, C12, C2', C4', C5' & C6'-H), 7.90 (d, 1H, C16-H, J = 7.20 Hz),
8.30 (d, 1H, C1-H, J = 8.00 Hz), 9.31(d, 1H, C4-H, J = 8.00 Hz), 9.63 (d, 1H, C15-H, J = 8.80
13
Hz), 9.86 (d, 1H, C13-H, J = 8.00 Hz); C NMR (δ, ppm, CDCl3): 29.8 (C6-CH3), 120.4 (C6a),
121.1 (C8), 121.5 (C14b), 122.4 (C13), 124.7 (C4), 125.3 (C7a), 126.1 (C16), 126.7 (C12), 126.8
(C3), 127.1 (C10), 127.5 (C2), 127.7 (C15), 127.8 (C9), 127.9 (C1), 128.0 (C5'), 128.4 (C4'), 128.5
(C4'), 129.1 (C11), 129.9 (C2'), 130.3 (C1'), 131.4 (C4a), 131.8 (C13a), 133.7 (C9a), 134.3 (C16a),
136.0 (C3'), 141.6 (C4b), 144.0 (C7), 147.5 (C14a), 147.8 (C13b), 159.3 (C6); MS: m/z (%) 465 (M+,
100); Anal. Calcd. for C31H19N3O2: C, 79.99; H, 4.11; N, 9.03; Found: C, 80.05; H, 4.07; N, 9.09
%.
6-Methyl-7-(4'-hydroxyphenyl)dinaphtho[1,2-b:1',2'-h][1,6] naphthyridine (13)
Pale Brown solid; m.p. 272-274 °C; Yield: (32 %); IR νmax (cm-1): 3431 (OH), 1631, 1591
1
(C=N); H NMR (δ, ppm, CDCl3): 2.45 (s, 3H, C6-CH3), 5.36 (bs, 1H, C4'- OH), 7.35-7.88 (m,

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11H, C2, C3, C8, C9, C10, C11, C12, C2', C3', C5' & C6'-H), 8.00 (d, 1H, C16-H, J = 8.50 Hz), 8.15
(d, 1H, C1-H, J = 8.00 Hz), 9.35 (d, 1H, C4-H, J = 8.00 Hz), 9.66 (d, 1H, C15-H, J = 8.50 Hz),
13
9.83 (d, 1H, C13-H, J = 8.00 Hz); C NMR (δ, ppm, CDCl3): 28.8 (C6-CH3), 120.3 (C6a), 121.2
(C8), 121.9 (C14b), 122.0 (C13), 124.9 (C4), 125.6 (C7a), 126.3 (C16), 126.7 (C12), 126.8 (C3),
127.1 (C10), 127.4 (C2), 127.6 (C15), 127.8 (C9), 127.9 (C1), 128.0 (C2' & C6'), 128.7 (C3' & C5'),
129.2 (C11), 130.4 (C1'), 131.3 (C4a), 131.5 (C13a), 133.6 (C9a), 134.2 (C16a), 135.3 (C4'), 141.4
(C4b), 144.0 (C7), 147.4 (C14a), 148.0 (C13b), 159.9 (C6); MS: m/z (%) 436 (M+, 100); Anal.
Calcd. for C31H20N2O: C, 85.30; H, 4.62; N, 6.42; Found: C 85.27; H 4.67; N 6.46 %.
2-(6-methyldinaphtho[1,2-b:1',2'-h][1,6]naphthyridin-7-yl)aniline (14)
Yellow spongy solid; m.p. 263-265 °C; Yield: (61 %); IR νmax (cm-1): 3326, 3273 (NH2),
1630, 1600 (C=N); 1H NMR (δ, ppm, CDCl3): 2.50 (s, 3H, C6-CH3), 5.65 (s, 2H, C2'-NH2), 7.43
(d, 1H, C9-H J= 9.00 Hz), 7.50-7.70 (m, 5H, C3', C4', C5', C6' & C8-H), 7.74-7.86 (m, 3H, C10,
C11, C12), 7.90-7.96 (m, 2H, C2, C3-H), 8.03 (d, 1H, C16-H, J = 8.00 Hz), 8.20 (d, 1H, C1-H, J =
9.00 Hz), 9.40 (d, 1H, C4-H, J = 8.50 Hz), 9.59 (d, 1H, C15-H, J = 9.00 Hz), 9.77 (d, 1H, C13-H, J
= 8.50 Hz);13C NMR (δ, ppm, CDCl3): 28.9 (C6-CH3), 120.1 (C6a), 121.0 (C8), 121.6 (C14b),
122.3 (C13), 124.6 (C4), 125.7 (C7a), 126.0 (C16), 126.6 (C12), 126.9 (C3), 127.1 (C10), 127.4 (C2),
127.6 (C15), 127.8 (C9), 127.9 (C1), 128.2 (C6'), 128.8 (C3', C4' & C5'), 129.0 (C11), 130.1 (C1'),
131.2 (C4a), 131.2 (C13a), 133.7 (C9a), 134.4 (C16a), 138.6 (C2'), 141.0 (C4b), 144.0 (C7), 147.6
(C14a), 147.5 (C13b), 158.7 (C6); MS: m/z (%) 435 (M+, 100); Anal. Calcd. for C31H21N3: C,
85.49; H, 4.86; N, 9.65; Found: C, 88.52; H, 4.81; N, 6.67 %.
2-(6-methyldinaphtho[1,2-b:1',2'-h][1,6]naphthyridin-7-yl)-4-nitroaniline (15)
Yellow solid; m.p. 285-287 °C; Yield: (68 %); IR νmax (cm-1): 1632, 1602 (C=N), 1520 (asym
1
NO2), 1349 (sym NO2); H NMR (δ, ppm, CDCl3): 2.43 (s, 3H, C6-CH3), 5.17 (s, 2H, NH2),
7.42-7.89 (m, 10H, C2, C3, C8, C9, C10, C11, C12, C3', C4' & C6' -H), 8.01 (d, 1H, C16-H, J = 8.50
Hz), 8.10 (d, 1H, C1-H, J = 8.00 Hz), 9.35 (d, 1H, C4-H, J = 8.00 Hz), 9.57 (d, 1H, C15-H, J =
8.50 Hz), 9.70 (d, 1H, C13-H, J = 8.00 Hz); 13C NMR (δ, ppm, CDCl3): 28.5 (C6-CH3), 119.5
(C3'), 120.3 (C6a), 121.1 (C8), 121.5 (C14b), 122.4 (C13), 124.7 (C4), 125.5 (C7a), 126.1 (C16),
126.7 (C12), 126.8 (C3), 127.3 (C10), 127.5 (C2), 127.7 (C15), 127.8 (C9), 127.9 (C1), 128.0 (C6'),
128.3 (C4'), 129.1 (C11), 130.3 (C1'), 131.4 (C4a), 131.6 (C13a), 133.9 (C9a), 134.5 (C16a), 137.6
(C5'), 138.3 (C2'), 141.3 (C4b), 143.6 (C7), 147.7 (C14a), 148.1 (C13b), 158.5 (C6); MS: m/z (%)

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480 (M+, 100); Anal. Calcd. for C31H20N4O2: 77.49; H, 4.20; N, 11.66;; Found: C, 77.46; H,
4.23; N, 11.61 %.
2-(6-methyldinaphtho[1,2-b:1',2'-h][1,6]naphthyridin-7-yl) benzenethiol (16)
Yellow prisms; m.p. 267-269 °C; Yield: (66 %); IR νmax (cm-1): 1628, 1596 (C=N), 2547 (S-
H); 1H NMR (δ, ppm, CDCl3): 2.47 (s, 3H, C6-CH3), 3.61 (s, 1H, C2'- SH), 7.42 (d, 1H, C9-H(J=
9.00 Hz)), 7.50-7.94 (m, 7H, C2, C3, C8, C3', C4', C5' & C6' -H), 8.00 (d, 1H, C16-H, J = 8.00 Hz),
8.19 (d, 1H, C1-H, J = 9.00 Hz), 9.42 (d, 1H, C4-H, J = 8.80 Hz), 9.65 (d, 1H, C15-H, J = 9.00
Hz), 9.87 (d, 1H, C13-H, J = 8.50 Hz); 13C NMR (δ, ppm, CDCl3): 29.9 (C6-CH3), 120.6 (C6a),
121.4 (C8), 121.8 (C14b), 122.3 (C13), 124.9 (C4), 125.5 (C7a), 126.1 (C16), 126.9 (C12), 126.9
(C3), 127.3 (C10), 127.5 (C2), 127.7 (C15), 127.8 (C9), 127.9 (C1), 128.0 (C6'), 128.7 (C3', C4' &
C5'), 129.0 (C11), 130.2 (C1'), 131.3 (C4a), 131.5 (C13a), 133.5 (C9a), 134.6 (C16a), 136.9 (C2'),
141.5 (C4b), 143.9 (C7), 147.4 (C14a), 147.6 (C13b), 158.2 (C6); MS: m/z (%) 452 (M+, 100); Anal.
Calcd. for C31H20N2S: C, 82.27; H, 4.45; N, 6.19; S, 7.09; Found: C, 82.22; H, 4.49; N, 6.24; S,
7.05 %.
6-methyl-7-(naphthalen-1-yl)dinaphtho[1,2-b:1',2'-h][1,6] naphthyridine (17)
1
Pale orange solid; m.p. 267-269 °C; Yield: (73 %); IR νmax (cm-1): 1632, 1598 (C=N); H
NMR (δ, ppm, CDCl3): 2.42 (s, 3H, C6-CH3), 7.31-7.92 (m, 14H, C2, C3, C8, C9, C10, C11, C12,
C2', C3', C4', C5', C6', C7' & C8'-H), 8.00 (d, 1H, C16-H, J = 8.50 Hz), 8.16 (d, 1H, C1-H, J = 8.00
Hz), 9.36 (d, 1H, C4-H, J = 8.00 Hz), 9.58 (d, 1H, C15-H, J = 8.50 Hz), 9.76 (d, 1H, C13-H, J =
13
8.00 Hz); C NMR (δ, ppm, CDCl3): 29.0 (C6-CH3), 119.1 (C2'), 120.1 (C6a), 121.3 (C8), 121.6
(C14b), 122.2 (C13), 124.7 (C4), 125.3 (C7a), 126.3 (C16), 126.8 (C12), 126.7 (C3), 127.4 (C10),
127.3 (C2), 127.6 (C15), 127.8 (C9), 127.9 (C1), 128.1 (C8'), 128.2 (C3') 128.6 (C4' & C6'), 128.7
(C6' & C7'), 129.2 (C11), 130.1 (C1'), 131.4 (C4a), 131.0 (C13a), 132.6 (C8a'), 133.6 (C9a), 134.4
(C16a), 135.0 (C4a'), 141.0 (C4b), 143.7 (C7), 147.3 (C14a), 148.3 (C13b), 159.1 (C6); MS: m/z (%)
470 (M+, 100); Anal. Calcd. for C35H22N2: C, 89.34; H, 4.71; N, 5.95; Found: C, 89.39; H, 4.70;
N, 5.91 %.
6-methyl-7-(pyridin-3-yl)dinaphtho[1,2-b:1',2'-h][1,6]naphthyridine (18)
Pale orange solid; m.p. 278-280 °C; Yield: (59 %); IR νmax (cm-1): 1608, 1598, 1590 (C=N);
1H NMR (δ, ppm, CDCl3): 2.42 (s, 3H, C6-CH3), 7.45 (t, 1H, C5'-H, J = 5.5 Hz) 7.70- 8.09 (m,

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9H, C1, C2, C3, C8, C9, C10, C11, C12, C16 - H), 8.31 (d, 1H, C6'-H, J = 5.00 Hz), 8.51 (s, 1H, C2'-
H), 8.44 (d, 1H, C4'-H, J = 5.00 Hz), 9.40 (d, 1H, C4-H, J = 8.00 Hz), 9.51 (d, 1H, C15-H, J =
13
8.50 Hz), 9.73 (d, 1H, C13-H, J = 7.50 Hz); C NMR (δ, ppm, CDCl3): 29.1 (C6-CH3), 120.0
(C6a), 121.2 (C8), 121.5 (C14b), 122.4 (C13), 124.6 (C4), 125.5 (C7a), 126.0 (C16), 126.6 (C12),
126.9 (C3), 127.3 (C10), 127.5 (C2), 127.7 (C15), 127.8 (C9), 127.9 (C1), 128.7 (C5'), 129.0 (C11),
131.3 (C4a), 131.2 (C13a), 133.1 (C9a), 133.9 (C3'), 134.5 (C16a), 135.3 (C4'), 142.0 (C4b), 144.1
(C7), 147.0 (C14a), 147.5 (C2') 148.5 (C13b), 149.0 (C6') 159.2 (C6); MS: m/z (%) 421 (M+, 100);
Anal. Calcd. for C30H19N3: C, 85.49; H, 4.54; N, 9.97; Found: C, 85.52; H, 4.52; N, 9.96 %.
4-(6-methyldinaphtho[1,2-b:1',2'-h][1,6]naphthyridin-7-yl) quinolin-2-ol (19)
Pale yellow solid; m.p. 277-279 °C; Yield: (39 %); IR νmax (cm-1): 3420 (OH), 1627, 1611,
1
1590 (C=N); H NMR (δ, ppm, CDCl3): 2.40 (s, 3H, C6-CH3),5.32 (bs, 1H, C2'-H), 6.54 (s, 1H,
C3'- H), 7.28-7.95 (m, 10H, C2, C3, C8, C9, C10, C11, C12, C6', C7' & C8'-H), 8.05 (d, 1H, C16-H, J
= 8.50 Hz), 8.22 (d, 1H, C1-H, J = 8.00 Hz), 8.67 (d, 1H, C5'-H, J = 8.50 Hz), 9.37(d, 1H, C4-H,
13
J = 8.00 Hz), 9.64 (d, 1H, C15-H, J = 8.50 Hz), 9.80 (d, 1H, C13-H, J = 8.00 Hz); C NMR (δ,
ppm, CDCl3): 29.3 (C6-CH3), 110.3 (C3'), 117.6 (C4a'), 120.7 (C6a), 121.2 (C8), 121.8 (C14b),
122.7 (C13), 124.6 (C4), 125.1 (C7a), 126.2 (C16), 126.6 (C12), 126.6 (C3), 127.2 (C10), 127.5 (C2),
127.7 (C15), 127.8 (C9), 127.9 (C1), 128.5 (C5'), 128.6 (C6'), 129.0 (C11), 129.9 (C8'), 130.1 (C1'),
131.4 (C4a), 131.6 (C13a), 133.5 (C9a), 134.4 (C16a), 136.1 (C7'), 141.9 (C4b), 143.4 (C7), 147.2
(C14a), 147.7 (C13b), 148.3 (C8a'), 151.0 (C4'), 157.1 (C2'), 159.5 (C6); MS: m/z (%) 487 (M+,
100); Anal. Calcd. for C34H21N3O: C, 83.76; H, 4.34; N, 8.62; Found: C, 83.80, H, 4.39, N, 8.57
%.
3-(6-methyldinaphtho[1,2-b:1',2'-h][1,6]naphthyridin-7-yl) pyrazin-2-amine (20)
Pale orange solid; m.p. 271-273 °C; Yield: (55 %); IR νmax (cm-1): 3420, 3332, 1635, 1509
1
(C=N); H NMR (δ, ppm, CDCl3): 2.42 (s, 3H, C6-CH3), 5.46 (s, 2H, C2'-NH2), 7.65- 8.17 (m,
9H, C1, C2, C3, C8, C9, C10, C11, C12, C16 -H), 8.40 (2d, 2H, C4' & C5'-H, J = 5.50 Hz), 9.43 (d,
1H, C4-H, J = 8.00 Hz), 9.55 (d, 1H, C15-H, J = 8.50 Hz), 9.78 (d, 1H, C13-H, J = 7.50 Hz); 13C
NMR (δ, ppm, CDCl3): 28.1 (C6-CH3), 120.1 (C6a), 121.5 (C8), 121.9 (C14b), 122.9 (C13), 124.7
(C4), 125.0 (C7a), 126.1 (C16), 126.7 (C12), 126.9 (C3), 127.3 (C10), 127.6 (C2), 127.7 (C15), 127.8
(C9), 127.9 (C1), 129.5 (C11), 131.2 (C4a), 131.5 (C13a), 132.0 (C5'), 133.9 (C9a), 134.0 (C16a),
136.0 (C3'), 141.7 (C4b), 143.2 (C7), 144.3 (C6'), 147.0 (C14a), 147.5 (C13b), 152.5 (C2'), 159.8

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(C6); MS: m/z (%) 437 (M+, 100); Anal. Calcd. for C29H19N5: C, 79.61; H, 4.38; N, 16.01;
Found: C, 85.52; H, 4.52; N, 9.96 %.
ANTIOXIDANT ACTIVITY
Radical scavenging activity using DPPH● method
The DPPH● radical scavenging activity of synthesized compounds was measured
according to the literature method36. The concentration of compound necessary to decrease initial
concentration of DPPH by 50% (IC50) under the specified experimental condition was calculated.
Total antioxidant activity assay using radical cation (ABTS●+)
The total antioxidant activity measured by the ABTS•+ radical cation decolourization assay
involving preformed ABTS•+ radical cation. ABTS was dissolved in water to a 7 mM
concentration. ABTS radical cation (ABTS●+) was produced by reacting ABTS stock solution
with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the
dark at room temperature for 12-16 h before use. Prior to assay, the solution was diluted in
ethanol (about 1:89 v/v) and equilibrated to 30 ºC to give an absorbance at 734 nm of 0.70 ± 0.02
in a 1 cm cuvette37. The concentrations of compounds that produced between 20-80% inhibition
(50-150 µg/mL) of the blank absorbance was determined and adapted. After the addition of 1 mL
of diluted ABTS+ solution to various concentrations of compound, OD was taken at 30 ºC
exactly 30 min after the initial mixing.
Hydroxyl radical scavenging activity
The scavenging activity of synthesised compounds on hydroxyl radical was measured
according to the literature method38. Various concentrations (10 - 100 µg/mL) of compound in
test tubes were added with 1.0 mL of iron-EDTA solution (0.13% ferrous ammonium sulphate
and 0.26% EDTA), 0.5 mL of EDTA solution (0.018%), and 1.0 mL of DMSO (0.85% DMSO
(v/v) in 0.1 M phosphate buffer, pH 7.4) sequentially. The reaction was initiated by adding 0.5
mL of ascorbic acid (0.22%) and incubated at 80-90 °C for 15 min in a water bath. After
incubation, the reaction was terminated by the addition of 1.0 mL of ice-cold TCA (17.5% w/v).
Subsequently, 3.0 mL of Nash reagent was added to each tube and left at room temperature for
15 min. The reaction mixture without sample was used as control. The intensity of the colour

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formed was measured spectrophotometrically at 412 nm against reagent blank. The % hydroxyl
radical scavenging activity was calculated by the following formula:
% HRSA= [(Control OD - Sample OD)/Control OD] × 100
°-
Assay of superoxide radical (O2 ) scavenging activity
The assay was based on the capacity of the extracts to inhibit formazan formation by
scavenging the superoxide radicals generated in riboflavin-light-NBT system 39. Each 3 mL
reaction mixture contained 50 mM sodium phosphate buffer (pH 7.6), 20 µg riboflavin, 12 mM
EDTA, 0.1 mg NBT and 1 mL sample solution (50-250 µg/mL). Reaction was started by
illuminating the reaction mixture with different concentrations of compounds for 90 s.
Immediately after illumination, the absorbance was measured at 590 nm. The entire reaction
assembly was enclosed in a box lined with aluminum foil. Identical tubes with reaction mixture
kept in dark served as blanks. The percentage inhibition of superoxide anion generation was
calculated using the following formula: % of inhibition = [(A0-A1)/ A0] x 100 where A0 is the
absorbance of the control, and A1 is the absorbance of the compound.
in vitro cytotoxicity
Cell lines and cell culture
Cytotoxicity studies of the compounds along with ADR were carried out on human cervical
cancer cells (HeLa), colorectal adenocarcinoma cell line (HCT116), and Stomach
adenocarcenoma cells (AGS) which were obtained from National Centre for Cell Science, Pune,
India. Cell viability was carried out using the MTT assay method40. The HeLa, HCT8 and
HCT116 cells were grown in Eagles minimum essential medium containing 10% fetal bovine
serum (FBS). For the screening experiment, the cells were seeded into 96-well plates in 100 μL
of the respective medium containing 10% FBS, at a plating density of 10 000 cells/well, and
incubated at 37 °C, under conditions of 5% CO2, 95% air, and 100% relative humidity for 24 h
prior to the addition of compounds. The compounds were dissolved in DMSO and diluted in the
respective medium containing 1% FBS. After 24 h, the medium was replaced with the respective
medium with 1% FBS containing the compounds at various concentrations and incubated at 37
°C under conditions of 5% CO2, 95% air, and 100% relative humidity for 48 h. Triplication was
maintained, and the medium not containing the compounds served as the control. After 48 h, 10

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μL of MTT (5 mg/mL) in phosphate buffered saline (PBS) was added to each well and incubated
at 37 °C for 4 h. The medium with MTT was then flicked off, and the formed formazan crystals
were dissolved in 100 μL of DMSO. The absorbance was then measured at 570 nm using a
microplate reader. The percentage of cell inhibition was determined using the following formula,
and a graph was plotted with the percentage of cell inhibition versus concentration. From this,
the IC50 value was calculated: % inhibition = [mean OD of untreated cells (control)/mean OD of
treated cells (control)] × 100. The results were expressed as the concentration at which there was
50% inhibition (IC50).