Efficient and easy synthesis of new Benzo[h]chromene and Benzo[h]quinoline derivatives as a new class of cytotoxic agents

Article abstract of DOI:10.1016/j.molstruc.2019.05.081

The present study presents easy and rapid methods to synthesize new benzo[h]chromene and benzo[h] quinoline derivatives. Cytotoxic evaluations of most of the examined compounds indicated that they had significant cytotoxic activities against HepG-2 (human

Full text of DOI:10.1016/j.molstruc.2019.05.081

Accepted Manuscript
Efficient and easy synthesis of new Benzo[h]chromene and Benzo[h]quinoline
derivatives as a new class of cytotoxic agents
Mogedda E. Haiba, Ebtehal S. Al-Abdullah, Nesreen S. Ahmed, Hazem A. Ghabbour,
Hanem M. Awad
PII:
S0022-2860(19)30651-9
DOI:
https://doi.org/10.1016/j.molstruc.2019.05.081
MOLSTR 26588
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 November 2018
Revised Date: 7 May 2019
Accepted Date: 19 May 2019
Please cite this article as: M.E. Haiba, E.S. Al-Abdullah, N.S. Ahmed, H.A. Ghabbour, H.M. Awad,
Efficient and easy synthesis of new Benzo[h]chromene and Benzo[h]quinoline derivatives as a
new class of cytotoxic agents, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.05.081.
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ACCEPTED MANUSCRIPT
Efficient and Easy Synthesis of New Benzo[h]chromene and Benzo[h]quinoline
Derivatives as a New Class of Cytotoxic Agents
2,3
Mogedda E. Haiba1,*, Ebtehal S. Al-Abdullah2, Nesreen S. Ahmed1, Hazem A. Ghabbour
Hanem M. Awad 4
and
1Department of Therapeutic Chemistry, Pharmaceutical and Drug Industries Research Division, National Research
Center, El Buhouth Street, Dokki, Cair, 12622, Egypt
2Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi
Arabia
3Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
4 Tanning Materials and Leather Technology Department, National research Centre, 12622, Dokki, Cairo, Egypt
*
Author to whom correspondence should be addressed; E-Mail: Mogedda.haiba@yahoo.com
Abstract:
The present study presents easy and rapid methods to synthesize new benzo[h]chromene and
benzo[h] quinoline derivatives. Cytotoxic evaluations of most of the examined compounds
indicated that they had significant cytotoxic activities against HepG-2 (human cancer cells) and
MCF-7 (breast cancer cells). Compounds 4 and 11 had stronger cytotoxic activity against HepG-
2 human cancer cells than the reference drug Doxorubicin. All the examined compounds were
significantly active against MCF-7 human cancer cells and were more potent than Doxorubicin.
The structures of the new compounds were established from their spectral and elemental data. In
addition the structures of benzo[h] chromene 3 and benzo[h]quinoline 7 were recognized by x-
ray crystallography. Herein detailed syntheses, spectroscopic information and biological actions
of the tested compounds are reported.
Key words: Benzo[h]chromene, Benzo[h]quinoline, Cytotoxicity, X-ray crystallography.
INTRODUCTION
Cancer is considered to be one of the most common causes of death worldwide. One of the most
primary bases for cancer treatment is chemotherapy. The undesirable side effects of
chemotherapy treatment, promote researchers for developing new chemotherapeutic agents with
(1)
more potent anticancer activity. Our recent literature report
revealed significant cytotoxic
activity of benzo[h] chromene 2a and benzo[h]quinoline 6a against human glioblastoma cells.
These results endorsed us to design a new series of benzo[h]chromene and benzo [h] quinoline
1

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derivatives, which were easily, synthesized describing efficient, clean reaction profiles and short
reaction times. Most reactions were carried out under microwave irradiation or through solvent-
free conditions. Literature reports demonstrated the importance of chromene derivatives as
(9,10)
anticancer (1-4), antimicrobial (5,6), antioxidant (7,8), anti-inflammatory
and in treatment of
Alzheimer’s disease(11). On the other hand, literature survey reported that numerous quinoline
derivatives were recognized as potent antimicrobial (12-14), antimalarial (15), anticancer (16,17), HIV-
integrase inhibitory (18), antifungal (19), antitubercular (20) and anti-inflammatory (21). The main
objective of our research was to synthesize new benzo[h] chromene and benzo[h]quinoline
derivatives, to examine their biological activity, hoping to develop therapeutic drugs for
treatment of different types of cancer, using 6-methoxytetralone as the starting material.
RESULTS AND DISCUSSION
The synthesized compounds were constructed via the routes illustrated in schemes 1-3.
Condensation of 6-methoxy-1-tetralone 1 with different substituted arylidenemalononitriles,
namely, 2-(benzylidenemalononitrile), 2-((furan-2-yl) methylene) malononitrile, 2-(4-
chlorobenzylidene) malononitrile, 2-(3,4,5-trimethoxybenzylidene) malononitrile and 2-(p-
methoxybenzylidene) malononitrile, in the presence of catalytic quantities of piperidine under
microwave irradiation, gave the corresponding chromene carbonitriles (2a-d), within 15 minutes
in 90-65% yields. Condensation of the chromene carbonitrile 2a with dimethylformamide-
dimethylacetal (DMF-DMA), under solvent-free conditions produced chromene derivative 3
within 1h., in 90% yield, which was reacted with formamide to give the pyrimidine derivative 4
within 1 h., in 90% yield. Reaction of 6-methoxy-1-tetralone 1 with the same substituted
arylidenemalononitriles, under similar conditions as formerly described, but in the presence of
ammonium acetate, gave the corresponding quinoline carbonitriles (6a-d), within 15 minutes in
90-65% yields. Reaction of the derivative 6a with DMF-DMA under solvent-free conditions
produced the enamine derivative 7 within 1 h., in 85% yield, which was reacted with formamide
to form the pyrimido quinoline derivative 8 in 85% yield. Stirring compounds 2a or 6a with
o
concentrated sulfuric acid at 20-22 C yielded the targeting carboxamide derivatives 5 and 9
within 30 minutes in 85-90% yields respectively (Scheme 1). Reaction of formamidine
derivative 3 with heterocyclic amines namely; morpholine, 4-aminopyridine, 2-amino-5-
chloropyridine or 2-amino-4-chlorobenzothiazole, in the presence of glacial acetic acid generated
2

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products 10-13 (Scheme 2). Moreover, quinoline derivative 7 was reacted with 4-aminopyridine
or morpholine to produce formamidine derivatives 14 and 15 within 1h., in 85-80% yields
respectively (Scheme 3).
N
N
N
N
CN
O
O
NH2
iii
H3CO
H3CO
4
3
ii
2a
NH2
O
NH2
CN
Ar
O
NH2
O
iv
i
H3CO
H3CO
2a-d
5
a, Ar = phenyl
b, Ar = 2-furyl
O
c, Ar = p-chlorophenyl
d, Ar = 3,4,5-trimethoxyphenyl
e, Ar = p-methoxyphenyl
H3CO
1
NH2
O
NH2
CN
viii
Ar
N
NH2
N
v
H3CO
H3CO
6a-d
9
6a
vi
N
CN
N
N
N
N
N
NH2
vii
H3CO
H3CO
Scheme 1: Synthetic routes of compounds 2a-e-9
7
8
Reagents and conditions: (i) microwave, arylidenemalononitriles, ethanol, 15 min; (ii) and (vi) DMF-DMA, reflux,
1h; (iii) and (vii) formamide, reflux, 1h; (iv) and (viii) H2SO4, r.t., 1h; (v) microwave, arylidene malononitriles,
ethanol, ammoniumacetate, 15 min.
3

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N
O
N
CN
i
O
H3CO
10
N
HN
O
N
CN
ii
H3CO
N
O
11
N
CN
Cl
N
N
HN
O
H3CO
3
CN
iii
H3CO
12
S
N
HN
O
Cl
N
iv
CN
H3CO
13
Scheme 2: Synthetic routes of compounds 10-13
Reagents and conditions: (i), (ii), (iii), (iv) CH3COOH, reflux, 2-3 h; morpholine, 4-aminopyridine,
2-amino-5-chloropyridine, 2-amino-5-chlorobenzoyhiazole respectively
4

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N
N
N
H
i
CN
N
N
N
H3CO
N
14
CN
H3CO
7
N
O
N
ii
CN
N
H3CO
15
Scheme 3: Synthetic routes of compounds 14 and 15
Reagents and conditions: (i), (ii) reflux, 15-20 min; morpholine and 4-aminopyridine respectively
X-ray crystallographic analysis
Products 3 and 7 were obtained as single crystals by slow evaporation of ethanol from the pure
compounds at room temperature. Data were collected on a Bruker APEX-II D8 Venture area
diffractometer fitted with graphite monochromatic Mo Kα radiation at 293 (2) and 100 (2) K,
respectively Cell improvement and data reduction were carried out with Bruker SAINT.
SHELXS-97 (22, 23) was used to resolve the structures. Refinements were made by the full-matrix
least-squares techniques using anisotropic thermal data for non-hydrogen atoms on ̀. CCDC
1404393 and CCDC 1543562 contain the supplementary crystallographic data for these compounds
and are available free of charge from the Cambridge Crystallographic Data Centre at
http://www.ccdc.cam.ac.uk/data_request/cif.
5

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The crystallographic records and refinement information for C24H23N3O2 compound 3 and
C24H22N4O compound 7 are summarized in (Table 1). The asymmetric units of compounds 3 and
7 consist of one independent molecule as shown in Figure 1. All bond lengths and angles are
within normal ranges (24). Selected bond lengths and angles are listed in (Table 2). Figure 2a
shows that the molecules of compound 3 are packed together with no hydrogen interactions. In
Figure 2b, the molecules of compound 7 are linked by single intermolecular non-classical
hydrogen interactions (Table 3).
3
7
Fig. 1 ORTEP diagrams of the products 3 and 7. Displacement ellipsoids are plotted
at the 40% probability level for non-H atoms.
6

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Table 1: X-Ray crystallography specifics for products 3 and 7
Crystal data
3
7
Chemical formula
Mr
C24H23N3O2
385.45
C24H22N4O
382.46
Crystal system, space group
Temperature (K)
a, b, c (Å)
Triclinic, P-1
293
8.3125 (5), 10.0564 (6), 13.5434
Monoclinic, P21/n
100
10.8672 (5), 14.3735
(6), 12.9222 (5)
(9)
α, β, γ (°)
69.197 (2), 78.218 (3),
76.214 (2)
1018.90 (11)
2
90, 99.561 (2), 90
V (Å3)
Z
1990.41 (15)
4
Radiation type
Mo Kα
0.08
Mo Kα
0.08
µ (mm−1)
Crystal size (mm)
Data collection
Diffractometer
0.32 × 0.25 × 0.21
0.51 × 0.40 × 0.15
Bruker APEX-II D8
venture
Bruker APEX-II D8
venture
diffractometer
diffractometer
Absorption correction
Multi-scan
Multi-scan
SADABS Bruker 2014
0.975, 0.983
SADABS Bruker 2014
0.960, 0.988
Tmin, Tmax
No. of measured, independent
and observed [I > 2σ(I)]
reflections
41937, 4227, 2182
0.187
24576, 4558, 3773
0.081
Rint
Refinement
R[F2 > 2σ( F2)], wR( F2), S
No. of reflections
No. of parameters
No. of restraints
ꢀρmax, ꢀρmin (e Å−3)
CCDC No.
0.074, 0.228, 1.04
4227
266
0
0.058, 0.171, 1.03
4558
265
0
0.28, −0.32
0.40, −0.33
1543562
1404393
7

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Fig. 2a (3)
Fig. 2b (7)
Fig. 2 Molecular packing of titled compounds. Hydrogen interactions are drawn as dashed lines.
Table 2: Selected geometric parameters (Å, °)
Compound 7
Bond Lengths
O1—C5
1.3676 (18)
1.4307 (18)
1.3408 (18)
1.3383 (18)
1.149 (2)
N3—C13
N3—C21
N4—C21
N4—C22
N4—C23
1.3913 (18)
1.3017 (19)
1.3362 (19)
1.450 (2)
O1—C24
N1—C1
N1—C13
N2—C20
1.457 (2)
Bond angles
C5—O1—C24
C1—N1—C13
C13—N3—C21
C21—N4—C22
C21—N4—C23
C22—N4—C23
N1—C1—C2
N1—C1—C10
117.13 (12)
119.00 (12)
116.97 (12)
121.53 (12)
120.85 (13)
116.95 (12)
116.89 (12)
123.62 (12)
O1—C5—C4
O1—C5—C6
115.79 (13)
124.25 (13)
121.93 (13)
121.17 (12)
116.86 (12)
178.59 (17)
122.02 (13)
N1—C13—N3
N1—C13—C12
N3—C13—C12
N2—C20—C12
N3—C21—N4
Compound 3
Bond Lengths
O1—C1
O1—C13
O2—C5
1.389 (4)
1.375 (4)
1.375 (5)
1.415 (6)
N2—C13
N2—C22
N3—C22
N3—C23
1.359 (4)
1.292 (4)
1.319 (4)
1.434 (5)
O2—C14
8

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N1—C21
1.142 (5)
N3—C24
1.445 (5)
Bond angles
C1—O1—C13
C5—O2—C14
C13—N2—C22
C22—N3—C23
C22—N3—C24
C23—N3—C24
O1—C1—C2
O1—C1—C10
118.8 (2)
117.2 (3)
119.8 (3)
120.3 (3)
121.9 (3)
117.8 (3)
112.6 (3)
123.2 (3)
O2—C5—C4
O2—C5—C6
115.7 (4)
124.3 (4)
116.2 (3)
120.9 (3)
122.9 (3)
176.3 (4)
122.4 (3)
O1—C13—N2
O1—C13—C12
N2—C13—C12
N1—C21—C12
N2—C22—N3
Table 3: Hydrogen interactions geometry (Å, °) for compound 7
Compound 7
D—H···A
D—H
0.9500
H···A
2.6100
D···A
3.304 (2)
D—H···A
130.00
C19—H19A···N2i
Symmetry codes: (i) −x+1/2, y+1/2, −z+5/2.
Cytotoxicity screening
Anti-tumor activity
Thirteen compounds were scanned in vitro for their activities against HepG-2 and MCF-7 using
MTT assay. The percentages of intact cells were calculated and compared to those of the control
(Fig. 3). Activities of these products against the two carcinoma cell lines were compared to the
activity of doxorubicin. All compounds suppressed both tumor cells in a dose-dependent manner.
Figure 3 shows that, compounds 4 and 11 had significant cytotoxic activity against HepG-2 at
100 µM and were more potent than doxorubicin. Compounds 2d, 10, 12 and 15 also presented
with significant cytotoxic activity. All other compounds were moderately active against HepG-2.
All thirteen investigated compounds were significantly active against MCF-7 and were more
potent than doxorubicin. The IC50 of all investigated compounds are shown in Table 4.
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120
100
80
60
40
20
0
HepG-2
MCF-7
Figure 3: Cytotoxic actions of the thirteen products against two cancer cell lines
according to the MTT assay at 100 µM.
Table 4: IC50 of the thirteen products against the two cancer cell lines
according to the MTT assay
HepG-2
MCF-7
Compound
IC50 (µM) ± SD
2a
70.5 ± 3.1
72.4 ± 5.3
2d
2e
3
4
5
6a
6d
7
9
11
12
15
62.3 ± 5.2
66.7 ± 7.4
65.5 ± 3.8
54.7 ± 4.1
64.8 ± 2.5
75.3 ± 2.9
70.4 ± 1.8
64.6 ± 3.2
73.8 ± 2.7
55.8 ± 4.5
59.8 ± 3.6
59.6 ± 3.5
57.7 ± 2.1
70.1 ± 2.9
74.5 ± 5.1
68.1 ± 4.7
64.1 ± 2.7
66.8 ± 4.1
71.2 ± 3.2
70.6 ± 3.5
67.1 ± 5.1
70.1 ± 3.9
65.3 ± 2.5
66.7 ± 5.5
65.5 ± 4.7
74.3 ± 4.2
Doxorubicin
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Experimental
Chemistry
Melting points were uncorrected and recorded on a Barnstead 9001 Electrothermal melting point
apparatus (Cole-Parmer, Vernon Hills, IL, USA). IR spectra were recorded with KBr discs on a
PerkinElmer FT-IR Spectrum BX Spectrometer (PerkinElmer, Inc., Waltham, MA, USA) at a
1
cm-1 scale. The H-NMR or 13C-NMR spectra were plotted on a JEOL 500 MHz spectrometer
(JEOL, Ltd., Akishima, Tokyo Japan). Chemical shifts were expressed in δ (ppm) relative to a
tetramethylsilane internal standard. Coupling constants are given in Hz. The mass spectra were
recorded on a GCMC-QP 1000 EX Shimadzu gas chromatograph-mass spectrometer (GC-MS;
Shimadzu Corp. Kyoto, Japan) at electron ionization (EI) of 70 eV. Elemental analyses (C, H,
and N) were conducted at the Microanalytical Center of the Faculty of Science of Cairo
University, Cairo, Egypt. They aligned with the proposed structures within ±0.1-0.2% of the
theoretical values. All reagents were commercial grade and used without further purification.
Reaction progress was monitored by thin layer chromatography (TLC) on precoated (0.75 mm)
silica gel GF254 plates (Merck Group, Darmstadt, Germany). Products were visualized under
ultraviolet (UV) light. Reactions that carried out by microwave irradiation were done using
Biotage microwave reactor (Biotage® Initiator+, EXP EU, 400 W).
General procedure for the synthesis of 2-amino-5,6-dihydro-8-methoxy-4-phenyl (or
substituted phenyl)-4H-benzo [h] chromene-3-carbonitrile 2a-e
A mixture of 2-substituted arylidenemalononitriles (0.001mol), namely; 2-Benzylidene
malononitrile, 2-((furan-2-yl) methylene) malononitrile, 2-(4-chlorobenzylidene) malononitrile,
2-(3,4,5-trimethoxybenzylidene) malononitrile, or 2-(4-methoxybenzylidene) malononitrile, in
ethanol (3 mL), were added to a mixture of 6-methoxy-1-tetralone (1) (0.001 mol), and few
drops of piperidine. The reaction mixture was subjected to microwave irradiation at 130 oC, (400
W) for ~ 15 minutes. Completion of the reaction (was monitored by TLC). After cooling the
product was filtered, and recrystallized from dilute ethanol to give compounds 2a-e.
2-amino-5,6-dihydro-8-methoxy-4-phenyl-4H-benzo[h]chromene-3-carbonitrile 2a (1)
Compound 2a was prepared and characterized as described in our literature report (1)
11

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2-amino-5,6-dihydro-8-methoxy-4-furyl-4H-benzo [h] chromene-3-carbonitrile 2b
o
1
Yield: 90%; m.p.: >300 C; IR (υmax / cm-1): 3440, 3332 (NH2), 2192 ( CN); H-NMR (DMSO-
d6): δ, 2.05 (m, 2H, CH2-5), 2.4 (m, 2H, CH2-6), 3.8 (s, 3H, OCH3), 3.9 (s, 1H, CH, CH-4), 5.1
(s, 2H, NH2), 6.6 (s, 1H, CH-7), 6.7-6.8 (m, 4H, Ar-H), 7.9 (d, J=7.5 Hz, 1H, CH-10); 13C-NMR
(DMSO-d6): δ, 24.1 (CH2, C-5), 27.08 (CH2, C-6), 39.8 (CH, C-4) 55.1 (OCH3), 56.4 (C-3)
108.8, 111.2, 113.4, 120.5, 121.1, 121.9, 126.9, 127.7, 128.5, 137.1, 139.7, 144.00, 159.1, 159.8
(Ar-C); MS: m/z (%): 320 (M+1, 1.09) consistent with the molecular formula C19H16N2O3.
2-amino-5,6-dihydro-8-methoxy-4-p-chlorophenyl-4H-benzo[h]chromene-3-carbonitrile 2c
Yield: 65%; m.p.: >300 oC; IR (υmax / cm-1): 3354, 3310 (NH2), 2194 (CN); 1H-NMR (CDCl3): δ,
2.55 (t, J =7.2 Hz, 2H, CH2-5), 2.75 (t, J =7.2 Hz, 2H, CH2-6), 3.7 (s, 1H, CH-4), 3.8 (s, 3H,
OCH3), 6.7 (s, 1H, CH-7), 6.8 (d, J =7 Hz, 1H, CH-9), 7.3 (d,d, J =7.5 Hz, 2H, CH-2`, 6`) 7.4
(d,d, J =7.5 Hz, 2H, CH-3`, 5`), 8.01 (d, J =7 Hz,1H, CH-10); 13C-NMR (CDCl3): δ, 25.9 (CH2-
5), 28.3 (CH2-6), 37.9 (CH-4), 50.1 (C-3), 55.3 (OCH3), 93.3, 112.6, 112.8, 119.4, 127.01, 127.9,
128.6, 128.9, 130.06, 131.05, 134.8, 141.03, 153.4, 161.3 (Ar-C and CN); MS: m/z (%): 364,
366 (M+, 2.5) consistent with the molecular formula C21H17ClN2O2.
2-amino-5,6-dihydro-8-methoxy-4-(trimethoxyphenyl)-4H-benzo[h]chromene-3-arbonitrile
2d
o
1
Yield: 90%; m.p.: 185-187 C; IR (υmax / cm-1): 3454, 3331 (NH2), 2196 (CN); H-NMR
(DMSO-d6): δ, 2.1 (m, 2H, CH2-5), 2.5 (m, 2H, CH2-6 ), 3.80 (s, 3H, OCH3), 3.86 (s, 1H, CH-4),
3.90 (s, 3H, OCH3), 3.97 (s, 6H, 2OCH3), 6.5 (s, 1H, CH-7), 6.6 (d, J=7.5 Hz, 1H, CH-9), 7.1 (s,
13
2H, CH-2`, 6`), 7.6 (s, 2H, NH2), 8.2 (d, J=7.5 Hz, 1H, CH-10); C-NMR (DMSO-d6): δ, 24.3
(CH2-5), 28.5 (CH2-6), 42.0 (CH, C-4), 50.9 (C-3), 55.1 (OCH3 ), 56.2 (3OCH3), 102.1, 102.5,
106.2, 108.2, 112.7, 113.1, 113.9, 125.9, 127.9, 132.8, 143.9, 153.2, 153.5, 159.3, (Ar-C and
CN); MS: m/z (%): 420 (M+, 1.8) consistent with the molecular formula C24H22N4O3.
2-amino-5,6-dihydro-8-methoxy-4-(p-methoxyphenyl)-4H-benzo[h]chromene-3-carbonitrile
2e
Yield: 88%; m.p.: >300 oC; IR (υmax / cm-1): 3448, 3354 (NH2), 2189 (CN); 1H-NMR (CDCl3): δ,
2.2 (m, 2H, CH2-5), 2.6 (m, 2H, CH2-6), 3.7 (s, 1H, CH, C-4), 3.8 (s, 3H, OCH3), 3.89 (s, 3H,
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OCH3), 6.7 (d, J=7 Hz, 1H, CH-9), 6.85 (m, 3H, Ar-H), 6.9 (d, J=7 Hz, 2H, CH-3`,5`), 7.7 (d,
13
J=7 Hz, 1H, CH-10), 8.1 (s, 2H, NH2); C-NMR (CDCl3): δ, 25.1 (CH2-5), 27.8 (CH2-6), 41.3
(CH, C-4), 50.4 (C-3) 55.2 (OCH3 ), 55.3 (OCH3), 93.3, 112.6, 112.8, 119.4, 127.01, 127.9,
128.6, 128.9, 130.06, 131.05, 134.8, 141.03, 153.4, 161.3MS: m/z (%): 361 (M+1, 1.09)
consistent with the molecular formula C22H20N2O3.
General procedure for the synthesis of (1E)-N`-(3-cyano-5,6-dihydro-8-methoxy-4-phenyl
benzo[h]chromene or quinoline-2-yl) N,N-dimethylformamidine 3, 7
A mixture of 2a or 6a (0.001 mol) and DMF-DMA (2 mL) was refluxed for ~ 1 h., after cooling
the solid product was filtered, washed with petroleum ether, dried, and recrystallized from
ethanol to give compounds 3 and 7, respectively.
N`-(3-cyano-5,6-dihydro-8-methoxy-4-phenylbenzo[h]chromene-2-yl) N,N-dimethyl form-
amidine 3
Yield: 90%; m.p.: 202-204 oC; IR (υmax / cm-1): 2191 (CN); 1H-NMR (DMSO-d6): δ, 1.7-2.1 (m,
2H, CH2-5), 2.5-2.7 (m, 2H, CH2-6), 2.9, 3.1 (2s, 6H, -N(CH3)2), 3.7 (s, 3H, OCH3), 4.1 (s, 1H,
CH-4), 6.7 (s, 1H, CH-7), 6.7 (d, J=8.4 Hz, 1H, CH-9), 7.2-7.3 (m, 5H, Ar-H), 7.5 (d, J=8.4 Hz,
1H, CH-10), 8.3 (s, 1H, N=CH); 13C-NMR (DMSO-d6): δ, 24.8 (CH2-5), 27.6 (CH2-6), 34.6
(N(CH3)2), 44.1 (CH, C-4), 55.5 (OCH3), 73.0 (C-3), 108.8, 111.8, 113.7, 120.7, 121.9, 123.0,
127.6, 128.3, 129.0, 137.7, 140.7, 143.8, 154.7, 159.5, 159.7(Ar-C and CN); MS: m/z (%): 385
(M+, 100) consistent with the molecular formula C24H23N3O2.
General procedure for the synthesis of 6,7-dihydro-4-amino-9-methoxy-5-phenyl-5H-benzo
[h]chromeno[2,3-d]pyrimidine 4
A mixture of compound 3 (0.001 mol) and formamide (3 mL) was refluxed for 1 h., after
cooling, the solid product was filtered, washed with dilute ethanol, dried, and recrystallized from
ethanol to give compound 4.
o
1
Yield: 90%; m.p.: 260-262 C; IR (υmax / cm-1): 3400, 3298 (NH2); H-NMR (DMSO-d6): δ, 2.4
(m, 2H, CH2-5), 2.7 (m, 2H, CH2-6), 3.8 (s, 3H, OCH3), 4.5 (s, 1H, CH-5), 6.85 (s, 1H, CH-8),
6.9 (d, J=7.5 Hz, 1H, CH-10), 7.4-7.7 (m, 5H, Ar-H), 8.3 (d, J=7.5 Hz, 1H, CH-11), 8.4 (s, 1H,
pyrimidine), 13C-NMR (DMSO-d6): δ, 25.5 (CH2-6), 27.6 (CH2-7), 40.4 (CH-5), 55.7 (OCH3),
13

ACCEPTED MANUSCRIPT
106.1, 113.03, 113.8, 126.7, 128.4, 128.6, 129.6, 130.1, 137.7, 142.1, 145.9, 157.3, 157.9, 158.4,
161.8, 162.5 (Ar-C and CN); MS: m/z (%): 357(M+, 2.9) consistent with the molecular formula
C22H19N3O2.
General procedure for the synthesis of 2-amino-5,6-dihydro-8-methoxy-4-phenyl benzo[h]
chromene-3-carboxamide 5
Compound 2a (0.001mol) in concentrated sulfuric acid (5 mL) was stirred for ~ 30 min., at room
o
temperature ~ 22 C. After completion of the reaction, the mixture was poured onto cold water.
The solid product was filtered and recrystallized from ethanol to give compound 5.
Yield: 85%; m.p.: 195-197 oC; IR (υmax / cm-1): 3311 (br, NH2), 1670 (C=O); 1H-NMR (DMSO-
d6): δ, 2.4 (m, 2H, CH-5), 2.7 (m, 2H, CH-6), 3.8 (s, 3H, OCH3), 3.9 (br. s, 1H, CH-4), 6.8 (s,
1H, CH-7), 6.9 (d, J=7 Hz, 1H, CH-9), 7.3-7.5 (m, 6H, Ar-H), 8.3, 8.5 (2s, 4H, 2 NH2), 8.5 (s,
2H, NH2); 13C-NMR (DMSO-d6): δ, 24.3 (CH2-5), 28.01 (CH2-6), 55.9 (OCH3), 56.2 (CH-4),
88.4 (C-3), 111.3, 117.2, 117.7, 124.2, 127.1, 128.5, 128.8, 129.2, 135.06, 136.5, 142.7, 153.6,
158.5, 163.7 (Ar-C, CO and CN); MS: m/z (%): 348(M+, 2.9) consistent with the molecular
formula C21H20N2O3.
General procedure for the synthesis of 2-amino-5,6-dihydro-8-methoxy-4-phenyl (or
substituted phenyl) benzo [h] quinoline-3-carbonitrile 6a-e
A mixture of suitable 2-substituted arylidenemalononitriles (0.001mol), namely; 2-Benzylidene
malononitrile, 2-((furan-2-yl) methylene) malononitrile, 2-(4-chlorobenzylidene) malononitrile,
2-(3,4,5-trimethoxybenzylidene) malononitrile, and/or 2-(4-methoxybenzylidene) malononitrile,
in ethanol (3 mL), were added to a mixture of 6-methoxy-1-tetralone (1) (0.001 mol),
ammonium acetate (0.002 mol), and few drops of piperidine. The reaction mixture was subjected
o
to microwave irradiation at 130 C, (400 W) for ~ 15 minutes. Completion of the reaction (was
monitored by TLC), after cooling the product was filtered, and recrystallized from dilute ethanol
to give compounds 6a-e.
2-amino-5,6-dihydro-8-methoxy-4-phenyl benzo[h]quinoline-3-carbonitrile 6a (1)
Compound 6a was prepared and characterized as described in our literature report (1)
14

ACCEPTED MANUSCRIPT
2-amino-5,6-dihydro-8-methoxy-4-p-furyl benzo [h] quinoline-3-carbo nitrile 6b
Yield: 65%; m.p.: >300 oC; IR (υmax / cm-1): 3425, 395 (NH2), 2212 (CN); 1H-NMR (DMSO-d6):
δ, 2.6 (m, 2H, CH2-5), 2.77 (m, 2H, CH2-6), 3.8 (s, 3H, OCH3), 6.5 (m, 4H, Ar-H), 7.5-7.7 (m,
13
4H, 2Ar-H and NH2); C-NMR (DMSO-d6): δ, 24.1 (CH2-5), 28.2 (CH2-6), 55.5 (OCH3), 86.1,
106.8, 107.9, 111.2, 112.4, 1140.6, 119.5. 120.5, 123.1, 126.9, 144.00, 150.6, 155.8, 159.1,
159.8, 160.2 (Ar-C); MS: m/z (%): 317 (M+1, 3) consistent with the molecular formula C19H15
N3O2.
2-amino-5,6-dihydro-8-methoxy-4-p-chlorophenyl benzo [h] quinoline-3-carbonitrile 6c
o
Yield: 65%; m.p.: >300 C; IR (υmax / cm-1): 3410, 3320 (NH2), 2218 (CN) ; 1H-NMR (CDCl3):
δ, 2.5 (t, J=7.2 Hz, 2H, CH2-5), 2.6 (t, J=7.2 Hz, 2H, CH2-6), 3.8 (s, 3H, OCH3), 6.7 (s, 1H, CH-
7), 6.8 (d, J=7.5 Hz, 1H, CH-9), 7.3 (m, 4H, Ar-H) 7.8 (d, J=7.5 Hz, 1H, CH-10), 8.7 (s, 2H,
13
NH2); C-NMR (CDCl3): δ, 26.2 (CH2-5), 28 (CH2-6), 55.2 (OCH3), 83.3, 111.4, 113.7, 116.9,
127.01, 122.5, 124.1, 127.9, 129.0, 133.2, 134.5, 136.7, 149.2, 156.8, 158.1, 160.3 (Ar-C and
CN); MS: m/z (%): 360, 361(M+, 100, 23) consistent with the molecular formula C21H16ClN3O.
2-amino-5,6-dihydro-8-methoxy-4-3,4,5-trimethoxyphenylbenzo[h]quinoline-3-carbonitrile
6d
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1
Yield: 90%; m.p.: 175-177 C; IR (υmax / cm-1): 3481, 3361 (NH2), 2202 (CN) ; H-NMR
(CDCl3): δ, 2.6 (m, 2H, CH2-5), 2.75 (m, 2H,CH2-6), 3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3),
3.9 (s, 3H, OCH3), 3.97 (s, 3H, OCH3) 5.5 (s, 2H, NH2), 6.5 (s, 1H, CH-7), 7.1 (s, 2H, CH-2`,
6`), 7.2 (d, J=7.5 Hz, 1H, CH-9), 7.6 (d, J=7.5 Hz, 1H, CH-10); 13C-NMR (CDCl3): δ, 24.3
(CH2-5), 28.5 (CH2-6), 55.3 (OCH3), 56.2, 56.3 (3O-CH3), 80.5, 102.1, 102.5, 105.7, 108.2,
112.7, 113.1, 113.9, 125.9, 126.3, 127.9, 143.9, 153.2, 153.3, 154.1, 159.3 (Ar-C and CN); MS:
m/z (%): 417 (M+, 2) consistent with the molecular formula C24H23N3O4.
2-amino-5,6-dihydro-8-methoxy-4-p-methoxyphenyl benzo [h] quinoline-3-carbonitrile 6e
Yield: 75%; m.p.: >300oC; IR (υmax / cm-1): 3456, 3373 (NH2), 2223 (CN) ; 1H-NMR (CDCl3): δ,
2.6 (t, J=6.6 Hz, 2H, CH2-5), 2.7 (t, J=6.6 Hz, 2H, CH2-6), 3.8, 3.9 (2s, 6H, 2-OCH3), 5.07 (s,
2H, NH2), 6.7 (s, 1H, CH-7), 6.8 (d, J=7 Hz, 1H, CH-9), 7.0 (d, d, J=7.5 Hz, 2H, CH3`, 5`), 7.6
13
(s, 2H, NH2), 7.9 (d, J=9 Hz, 2H, CH-2`,6`), 8.1 (d, J=7 Hz, 1H, CH-10); C-NMR (CDCl3): δ,
15

ACCEPTED MANUSCRIPT
24.5 (CH2-5), 28.5 (CH2-6), 53.8, 55.7 (2-OCH3), 99, 112.7, 114.02, 114.4, 115.1, 124.01, 126.3,
127.9, 129.8, 129.9, 133.4, 130.7, 133.4, 158.8, 160.8, 161.1 (Ar-C and CN); MS: m/z (%): 357
(M+, 4.1) consistent with the molecular formula C22H19N3O2.
N`-(3-cyano-5,6-dihydro-8-methoxy-4-phenylbenzo[h]quinoline-2-yl) N,N-dimethyl form-
amidine 7
o
1
Yield: 85%; m.p.: 173-175 C; IR (υmax / cm-1): 2212 (CN); H-NMR (DMSO-d6): δ, 2.6 (t,
J=6.5 Hz, 2H, CH2-5), 2.7 (t, J=6.5 Hz,, 2H, CH2-6), 3.09, 3.20 (2s, 6H, N(CH3)2), 3.8 (s, 3H,
OCH3), 6.83 (s, 1H, CH-7), 6.93 (d,d, J=6.5 Hz, 1H, CH-4`), 7.3 (d, J=7.5 Hz, 2H, CH3`, 5`),
7.4-7.5 (m, 3H, Ar-H), 8.2 (d, 1H, J=8 Hz, CH-9), 8.8 (s, 1H, N=CH); 13C-NMR (DMSO-d6): δ,
24.7 (CH2-5), 27.9 (CH2-6), 34.8 (N(CH3)2), 55.7 (OCH3), 99.2 (C-3) 112.9, 113.4, 117.9, 122.0,
127.0, 128.3, 128.9, 129.02, 129.09, 136.5, 141.4, 153.3, 153.7, 156.5, 161.3, 161.8 (Ar-C and
CN); MS: m/z (%): 382 (M+, 5.77) consistent with the molecular formula C24H22N4O.
Synthesis of 6,7-dihydro-4-amino-9-methoxy-5-phenylpyrimido [4,5-b]benzo[h]quinoline 8
A mixture of compound 7 (0.001 mol) and formamide (2 mL) was refluxed for ~ 1 h., after
cooling, the solid product was filtered, washed with dilute ethanol, dried, and recrystallized from
ethanol to give compound 8.
Yield: 85%; m.p.: >300 oC; IR (υmax / cm-1): 3400, 3298 (NH2); 1H-NMR (DMSO-d6): δ, 2.6 (m,
2H, CH2-5), 2.7 (m, 2H, CH2-6), 3.8 (s, 3H, OCH3), 6.8 (s, 1H, CH-8), 6.9 (d, J=7 Hz, CH-10),
13
7.4-7.7 (m, 6H, Ar-H), 8.2 (d, J=7 Hz, 1H, CH-11), 8.5 (s, 1H, pyrimidine), C-NMR (DMSO-
d6): δ, 26.2 (CH2-5), 28.1 (CH2-6), 55.7 (OCH3), 116.1, 117.03, 117.8, 126.5, 128.3, 128.6,
129.6, 136.1, 137.8, 142.4, 145.5, 158.3, 158.9, 159.4, 161.8, 162.5 (Ar-C and CN); MS: m/z
(%): 357 (M+, 2.9) consistent with the molecular formula C22H19N3O2.
Synthesis of amino-5,6-dihydro-8-methoxy-4-phenyl benzo [h] quinoline-3-carboxamide 9
A mixture of 6a (0.001mol) and concentrated sulfuric acid (5 mL) was stirred for ~ 30 min., at
room temperature at ~ 22 oC . After completion of the reaction, the mixture was poured onto cold
water the solid product was filtered and recrystallized from ethanol to give compound 9.
16

ACCEPTED MANUSCRIPT
Yield: 90%; m.p.: 290-292 oC; IR (υmax / cm-1): 3350 (br, NH2), 1685 (C=O); 1H-NMR (DMSO-
d6): δ, 2.6 (t, J=6 Hz, 2H, CH2-5), 2.8 (t, J=6 Hz, CH2-6), 3.8 (s, 3H, OCH3), 6.7 (s, 1H, CH-7),
6.8 (d, J=7 Hz, 1H, CH-9), 7.1 (br. s, 2H, NH2), 7.2-7.6 (m, 5H, Ar-H), 7.9 (d, J=7.5 Hz,, 1H,
13
CH-10), 8.3 (s, 2H, NH2); C-NMR (DMSO-d6): δ, 24.7 (CH2-5), 27.9 (CH2-6), 55.4 (OCH3),
106.5, 112.9, 113.1, 115.0, 124.7, 125.8, 128.3, 129.5, 131.7, 134.9, 141.2, 145.6, 150.6, 153.3,
155.1, 162.1 (Ar-C and CN); MS: m/z (%): 345 (M+, 1.9) consistent with the molecular formula
C21H19N3 O2.
General procedure for the synthesis of N'-(3-cyano-5,6-dihydro-8-methoxy-4-phenyl-4H-
benzo[h]chromen-2-yl)-N-(substituted) formamidine 10-13
To a solution of compound 3 (0.001 mol) in glacial acetic acid (7 mL), morpholine, 4-
aminopyridine, 2-amino-5-chloro pyridine, or 2-amino-4-chlorobenzothiazole (0.002 mol), were
added and the mixture was refluxed for 2-3 h., excess solvent was evaporated under reduced
pressure. The solid product was filtered, washed with water, dried, and recrystallized from
ethanol to give compounds 10-13.
5,6-dihydro-8-methoxy-2-(morpholinomethyleneamino)-4-phenyl-4H-benzo[h] chromene-
3-carbonitrile 10
o
1
Yield: 80%; m.p.: 86-88 C; IR (υmax / cm-1): 2222(CN); H-NMR (DMSO-d6): δ, 2.1 (m, 2H,
CH2-5), 2.4 (m, 2H, CH2-6), 2.5 (m, 4H, 2CH2-N), 3.1 (m, 4H, 2CH2-O), 3.7 (s, 3H, OCH3), 4.1
(s, 1H, CH-4), 6.7-6.9 (m, 4H, Ar-H), 7.2-7.4 (m, 3H, Ar-H), 7.5 (d, J=7 Hz, 1H, CH-10), 8.3 (s,
1H, N=CH), 13C-NMR (DMSO-d6): δ, 24.8 (CH2-5), 27.6 (CH2-6), 44.09 (CH-4), 55.5 (OCH3),
64.6 (2CH2-N), 72.9 (2CH2-O), 78.6 (C-3), 105.06, 111.8, 113.7, 122.2, 123.02, 125.8, 127.6,
128.3, 128.6, 129.07, 140.8, 147.4, 154.7, 159.5, 159.7 (Ar-C and CN); MS: m/z (%): 427 (M+1,
2) consistent with the molecular formula C26H25N3O3.
N'-(3-cyano-5,6-dihydro-8-methoxy-4-phenyl-4H-benzo[h]chromen-2-yl)-N-(pyridin-4-yl)
formamidine 11
o
Yield: 90%; m.p.: 78-80 C; IR (υmax / cm-1): 3443 (NH), 2219 (CN), 1H-NMR (CDCl3): δ, 2.07
(t, J=6.5 Hz, 2H, CH2-5), 2.8 (t, J=6.5 Hz, 2H, CH2-6), 3.8 (s, 3H, OCH3), 4.5 (s, 1H, CH-4), 6.6
(s, 1H, CH-7), 7.1-7.38 (m, 10H, Ar-H), 7.9 (s, 1H, N=CH-), 7.99 (d, J=7.5 Hz, 1H, CH-10), 8.9
17

ACCEPTED MANUSCRIPT
(s, 1H, NH); 13C-NMR (CDCl3): δ, 27.5 (CH2-5), 28.7 (CH2-6), 40.6 (CH-4), 55.4 (OCH3), 62.3
(C-3), 112.3, 113.4, 113.7, 122.8, 127.4, 127.8, 128.14, 128.19, 128.3, 128.4, 128.70, 128.78,
128.81, 128.88, 128.90, 128.97, 129.1, 129.4, 130.1, 163.8 (Ar-C and CN); MS: m/z (%): 434
(M+, 16) consistent with the molecular formula C27H22N4O2.
N-(5-chloropyridin-2-yl)-N'-(3-cyano-5,6-dihydro-8-methoxy-4-phenyl-4H-benzo[h]
chromen -2-yl)formamidine 12
Yield: 85%; m.p.: 100-102 oC; IR (υmax / cm-1): 3471 (NH), 2220 (CN); 1H-NMR (DMSO-d6): δ,
2.2 (m, 2H, CH2-5), 2.7 (m, 2H, CH2-6), 3.8 (s, 3H, OCH3), 4.4 (s, 1H, CH-4), 6.2 (d, J=7 Hz,
1H, CH-7), 7.30-7.37 (m, 9H, Ar-H), 7.6 (d, J=7 Hz, 1H, CH-10), 7.8 (s, 1H, N=CH), 10.9 (s,
1H, NH), 13C-NMR (DMSO-d6): δ, 25.02 (CH2-5), 27.9 (CH2-6), 40.8 (CH-4), 55.7 (OCH3 ),
89.5 (C-3) 107.4, 110.9, 111.3, 113.09, 113.3, 113.7, 122.8, 127.06, 127.4, 127.9, 128.3, 128.5,
128.6, 128.9, 137.6, 140.7, 141.7, 142.8, 146.9, 165.1 (Ar-C and C=N); MS: m/z (%): 467, 468,
469 (M+, 30, 8, 4) consistent with the molecular formula C27H21ClN4O2.
N-(4-chlorobenzothiazol-2-yl)-N'-(3-cyano-5,6-dihydro-8-methoxy-4-phenyl-4H-benzo [h]
chromen-2-yl)formamidine 13
Yield: 86%; m.p.: 120-122 oC; IR (υmax / cm-1): 3348 (NH), 2210 (CN); 1H-NMR (CDCl3): δ, 2.4
(t, J=6 Hz, 2H, CH2-5), 2.8 (t, J=6 Hz, 2H, CH2-6), 3.8 (s, 3H, OCH3), 4.2 (s, 1H, CH-4), 6.7 (d,
J=14 Hz, 1H, CH-9), 6.8 (s, 1H, CH-7), 7.2-7.6 (m, 9H, Ar-H), 7.8 (s, 1H, N=CH), 12.4 (s, 1H,
13
NH), C-NMR (CDCl3): δ, 24.1 (CH2-5), 29.8 (CH2-6), 55.6 (OCH3), 41.9 (CH-4), 81.6 (C-3)
108.1, 111.02, 111.3, 113.1, 113.5, 113.7, 120.2, 122.01, 122.5, 127.1, 127.8, 129.1, 137.5,
141.5, 142.2, 145.3, 145.6, 148.1, 165.1, 166.8, 165.1 (Ar-C and CN); MS: m/z (%): 524, 525,
526 (M+, 5, 4, 2) consistent with the molecular formula C29H21ClN4O2S.
General procedure for the synthesis of N'-(3-cyano-5,6-dihydro-8-methoxy-4-phenyl benzo
[h]quinolin-2-yl)-N-(substituted) formamidine 14 and 15
A mixture of compound 7 (0.001 mol) and 4-aminopyridine or morpholine (2 mL) was refluxed
for ~15-20 minutes, after cooling excess amine was evaporated and the reaction mixture was
triturated with ethanol and poured onto cold water. The solid product was dried and
recrystallized from ethanol to give compounds 14 and 15 respectively.
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ACCEPTED MANUSCRIPT
N'-(3-cyano-5,6-dihydro-8-methoxy-4-phenylbenzo[h]quinolin-2-yl)-N-(pyridin-4-yl) form
amidine 14
Yield: 85%; m.p.: 119-121 oC; IR (υmax / cm-1): 3761 (NH), 2210 (CN); 1H-NMR (CDCl3): δ, 2.7
(t, J=6.5 Hz, 2H, CH2-5), 2.8 (t, J= 6.5 Hz, 2H, CH2-6), 3.8 (s, 3H, OCH3), 6.7 (s, 1H, CH-7),
6.9 (dd, J=7.5 Hz, 2H, CH-2, 6, pyridine), 7.3 (d, J=7 Hz, 1H, CH-9), 7.5-7.3 (m, 8H, Ar-H), 8.2
(d, J=7 Hz, 1H, CH-10), 9.7 (s, 1H, NH), 13C-NMR (CDCl3): δ, 24.7 (CH2-5), 27.9 (CH2-6), 55.4
(OCH3), 98.6 (C-3), 106.5, 112.9, 113.1, 115.0, 124.7, 125.8, 128.3, 128.4, 128.9, 129.5, 131.7,
134.9, 141.2, 145.6, 150.6, 153.3, 155.1, 161.6, 162.1 (Ar-C and CN); MS: m/z (%): 431(M+, 3)
consistent with the molecular formula C27 H21 N5 O.
5,6-dihydro-8-methoxy-2-(morpholinomethyleneamino)-4-phenylbenzo[h]quinoline-3-
carbonitrile 15
o
1
Yield: 80%; m.p.: 94-96 C; IR (υmax / cm-1): 2214 (CN); H-NMR (DMSO-d6): δ, 2.5 (m, 2H,
CH2-5), 2.7 (m, 2H, CH2-6), 3.05 (m, 4H, 2CH2-N), 3.1 (m, 4H, 2CH2-O), 3.7 (s, 3H, OCH3), 6.8
(s, 1H, CH-7), 6.9 (d, J=7.5 Hz, 1H, CH-9), 7.3 (d,d, J=6, Hz 2H, CH-2`, 6`), 7.4-7.5 (m, 3H,
13
Ar-H), 8.2 (d, J=7.5 Hz, 1H, CH-10), 8.6 (s, 1H, N=CH), C-NMR (DMSO-d6): δ, 24.7 (CH2-
5), 27.9 (CH2-6), 34.8 (2CH2-N), 55.6 (OCH3), 66.4 (2CH2-O), 99.2 (C-3), 112.8, 113.4, 117.8,
122.0, 126.9, 128.3, 128.6, 128.9, 129.1, 136.4, 141.4, 153.3, 153.7, 156.5, 161.3, 161.8 (Ar-C
and CN); MS: m/z (%): 427 (M+, 1.9) consistent with the molecular formula C26H24N4O2.
Cell lines and cell culture
HepG-2 (human liver carcinoma) and MCF-7 (human breast adenocarcinoma) cell lines,
were purchased from the American Type Culture Collection (Rockville, MD, USA) and
maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-
inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin, the cells
were grown at 37 °C in a humidified atmosphere of 5% CO2.
MTT cytotoxicity assay
Cytotoxicity evaluation against HepG-2 and MCF-7 was assessed by the 3-[4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. This test is based on MTT
reduction by mitochondrial dehydrogenases in viable cells (25-27). Cells were placed in a 96 well
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ACCEPTED MANUSCRIPT
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sterile microplate (5 x 104 cells/well) and incubated at 37 C in serum-free media containing
dimethyl sulfoxide (DMSO) and either a series of various concentrations (12.5, 25, 50 and 100
µM.) of each compound or doxorubicin (positive control) for 48 h. before the MTT assay. After
incubation, the media were removed and 40 µL MTT (2.5 mg/ml) was added to each well.
Incubation was resumed for an additional 4 h. The purple formazan dye crystals were solubilized
with 200 µL DMSO. Absorbance was measured at 570 nm in a Spectra Max Paradigm Multi-
Mode microplate reader (Molecular Devices, LLC, San Jose, CA, USA) (25-27). Relative cell
viability was expressed as the mean percentage of viable cells compared to the untreated control
cells.
Statistical analysis
All experiments were conducted in triplicate and repeated on three different days. All values
were reported as mean ± SD. IC50 were determined by SPSS Inc probit analysis (IBM Corp.,
Armonk, NY, USA).
Conclusion
The present study deals with easy and rapid methods of the synthesis of new series of
benzo[h]chromene and benzo[h] quinoline derivatives. Cytotoxic evaluation of thirteen
compounds, against HepG-2 (human liver carcinoma) and MCF-7 (human breast
adenocarcinoma) cell lines was estimated. All the tested compounds, showed significant
cytotoxic activity against HepG-2 carcinoma cells, two of them were more potent than the
reference drug doxorubicin. In addition, it is obvious that all examined compounds had
significant cytotoxic activity against MCF-7, and were more potent than the effect of
doxorubicine. Benzo[h]chromene 3 and benzo[h] quinoline 7 were established by x-ray
crystallography.
Acknowledgement
This research project was supported by a grant from the “Research Center of the Center for
Female Scientific and Medical Colleges” Deanship of Scientific Research, King Saud
University.
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