Facile synthesis and antiproliferative activity of 7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amines

Article abstract of DOI:10.1016/j.ejmech.2016.12.037

A series of 7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amines 6a-t were synthesized as new potential antiproliferative agents. The in?vitro antiproliferative activity evaluation of title compounds using MTT assay revealed that most compounds showed significa

Full text of DOI:10.1016/j.ejmech.2016.12.037

Accepted Manuscript
Facile synthesis and antiproliferative activity of 7H-
benzo[7,8]chromeno[2,3-d]pyrimidin-8-amines
Hamidreza Akrami, Maliheh Safavi, Bibi Fatemeh Mirjalili, Mahmood Dehghani
Ashkezari, Farhang Dadfar, Najme Mohaghegh, Saeed Emami, Fahimeh Salehi,
Hamid Nadri, Sussan K. Ardestani, Loghman Firoozpour, Mehdi Khoobi, Alireza
Foroumadi
PII:
S0223-5234(16)31043-1
10.1016/j.ejmech.2016.12.037
EJMECH 9128
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 November 2016
Revised Date: 2 December 2016
Accepted Date: 18 December 2016
Please cite this article as: H. Akrami, M. Safavi, B.F. Mirjalili, M.D. Ashkezari, F. Dadfar, N. Mohaghegh,
S. Emami, F. Salehi, H. Nadri, S.K. Ardestani, L. Firoozpour, M. Khoobi, A. Foroumadi, Facile synthesis
and antiproliferative activity of 7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amines, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.037.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
Facile synthesis and antiproliferative activity of 7H-benzo[7,8]chromeno[2,3-
d]pyrimidin-8-amines
Hamidreza Akrami, Bibi Fatemeh Mirjalili, Maliheh Safavi, Mahmood Dehghani Ashkezari,
Farhang Dadfar, Najme Mohaghegh, Saeed Emami, Fahimeh Salehi, Hamid Nadri, Sussan K.
Ardestani, Mehdi Khoobi*, Alireza Foroumadi*
OMe
MeO
HN
F
N
O
N
6l
Compound 6l
A series of 7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amines 6a-t were synthesized as
potential antitumor agents. The 2-fluoro-aniline derivative 6l showed the best profile of
activity against A549, MOLT-4 and HeLa cells (IC s= 5.2-6.9 µM).
50

ACCEPTED MANUSCRIPT
Facile synthesis and antiproliferative activity of 7H-benzo[7,8]chromeno[2,3-
d]pyrimidin-8-amines
This work is dedicated to the memory of Prof. Abbas Shafiee (1937-2016)
a
b
a
Hamidreza Akrami , Maliheh Safavi , Bibi Fatemeh Mirjalili , Mahmood Dehghani
c
c
a
d
e
Ashkezari , Farhang Dadfar , Najme Mohaghegh , Saeed Emami , Fahimeh Salehi , Hamid
f
e
g
h,i
g,h
Nadri , Sussan K. Ardestani , Loghman Firoozpour , Mehdi Khoobi *, Alireza Foroumadi *
a
Department of Chemistry, College of Science, Yazd University, Yazd, PO Box 8915813149, Iran
b
Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST),
Tehran, Iran
c
Department of Biology, Ashkezar Branch, Islamic Azad University, Ashkezar, Iran
d
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
e
Institute of Biochemistry and Biophysics, Department of Biochemistry, University of Tehran, Tehran,
Iran
f
Faculty of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
g
Drug Design and Development Research Center, Tehran University of Medical Sciences, Tehran,
Iran
h
Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical
Sciences, Tehran, Iran
i
Department of Pharmaceutical Biomaterials and Medical Biomaterials Research Center, Faculty of
Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Abstract: A series of 7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amines 6a-t were synthesized as
new potential antiproliferative agents. The in vitro antiproliferative activity evaluation of title
compounds using MTT assay revealed that most compounds showed significant activity against tested
cancer cell lines (A549, MOLT-4, and HeLa). The 2-fluoro-aniline derivatives 6e and 6l were the most
active compounds against A549 and MOLT-4 cells, respectively. The benzylamine analog 6h showed
superior activity against HeLa cells. However, compound 6l with IC values of 5.2-6.9 µM had the
5
0
best profile of activity against all tested cell lines. The morphological and flow cytometric analyses
showed that compound 6l can induce apoptosis in the MOLT-4 cells.
Keywords: Anticancer; cytotoxic agents; benzochromene; polycyclic compounds
*
Corresponding authors:
E-mail addresses: aforoumadi@yahoo.com (A. Foroumadi); khoobi@tums.ac.ir (M. Khoobi)
1

ACCEPTED MANUSCRIPT
Tel: +98-21-66954708; Fax: +98 21 66461178.
1
. Introduction
Cancer is defined as a group of complicated diseases characterized by unregulated
proliferation of abnormal cells with capability of metastasizing to healthy tissues [1]. It is a
fatal disease responsible for more than 20% of all deaths and stands after the cardiovascular
diseases in terms of morbidity and mortality [2]. More than 14 million new cases of cancer
have been reported annually around the world. The World Health Organization (WHO)
estimates that the worldwide burden of cancer will reach to 22 million per year within the next
two decades [3,4]. Anticancer drugs are important tools in the treatment of cancers. However,
severe side effects resulting from toxicity of anticancer agents on normal cells remains a
substantial drawback in their clinical usefulness [5]. Furthermore, majority of the conventional
chemotherapy fails due to drug resistance. Indeed, most of cancer-related deaths are
considered to be a result of chemotherapy failure [6]. Accordingly, there is a huge academic
and industrial interest to find new chemotherapeutic agents with high potency and optimal
selectivity and safety.
In the course of identifying various chemical entities which may serve as new leads for
designing new anticancer agents [7-12], our interest was focused on the chromene and
pyrimidine heterocyclic compounds. The chromene-based heterocyclic compounds are
attractive derivatives due to their structural and biological diversity, particularly as natural
products [13-16]. Cai and coworkers reported different types of 4-aryl-4H-chromenes I
(Figure 1) as antiproliferative agents with ability to induce apoptosis [17]. On the other hand,
4
-aminopyrimidine derivatives have also gained fame as an important scaffold in different
approved chemotherapeutic agents like gefitinib, erlotinib and lapatinib. Particularly, Cai et al.
also reported novel 4-aminopyrimidine derivatives II (Figure 1) as potent and efficacious
apoptosis inducer anticancer agents [18].
In the light of the above mentioned findings, we designed a novel series of
chromenopyrimidine derivatives 6a-t (Figure 1) as hybrids of 4-aryl-4H-chromenes I and 4-
2

ACCEPTED MANUSCRIPT
aminopyrimidines II. Thus we describe here, the synthesis and biological activity of 7H-
benzo[7,8]chromeno[2,3-d]pyrimidin-8-amines 6a-t as new potential anticancer agents.
2
. Chemistry
The synthesis of target compounds 6a–t was outlined in Scheme 1. Several procedures were
screened for the synthesis of 2-amino-4-phenyl-4H-benzo[h]chromene-3-carbonitrile
derivatives 4a–c via three-component condensation of α-naphthol (1), malononitrile (2) and
appropriate benzaldehyde 3a-c (Supplementary material). The best result was obtained in the
presence of piperidine in refluxing ethanol. Compounds 4a–c were converted to imines 5a–c
by reacting with N,N-dimethylformamide dimethyl acetal (DMF-DMA). Finally, the
cyclization of intermediates 5a–c with different amines was also screened under several
conditions to obtain target compounds 6a–t (Supplementary material). The best result was
obtained in the presence of acetic acid under microwave irradiation.
3
. Results and discussion
The inhibitory activities (IC values, µM) of compounds 6a–t against proliferation of cancer
5
0
cell lines A549, MOLT-4, and HeLa were listed in Table 1.
All compounds showed significant activity against cancer cells proliferation at micromolar
range. Among them, the 2-fluoro-aniline derivatives 6e (5.4 ± 0. 2 µM) and 6l 5.2 ± 0.02 µM)
(
were the most active compounds against A549 and MOLT-4 cells, respectively. It should be
noted that the IC values of all 2-fluoro-aniline congeners towards all cell lines were less than
5
0
1
3 µM, indicating substantial cytotoxic profile of fluoro-aniline derivatives. Moreover their 4-
fluoro- regioisomer (compound 6m) had good activity against all cell lines (IC s = 6.8-10.5
5
0
µM).
A survey on the IC s of test compounds against HeLa cells revealed that the benzylamine
5
0
derivative 6h had superior activity (IC50
= 5.9 ± 1.4 µM). In addition, the unsubstituted aniline
derivative 6a with IC50 value of 6.4 ± 0.3 µM was equipotent as 6a towards HeLa cells.
According to the activities of the other compounds having benzylamine or aniline substitution,
it can be concluded that the introduction of substituent on aniline or benzylamine moiety
diminished the activity against HeLa cells. Moreover, increasing the distance between
aromatic phenyl group and nitrogen of amine dramatically decrease the activity as seen in
3

ACCEPTED MANUSCRIPT
2
compounds 6j and 6k. The data indicated that derivatives having substitution on R group and
,5-dimethoxy substituent on 7-phenyl ring are more potent against HeLa cell. While insertion
2
of additional methoxy group (3,4,5-trimethoxy derivatives) reduce the activity.
The screening data indicated that substitution of 2,5-dimethoxy on 7-phenyl ring is promising
for improved activity against MOLT-4. For example, 2,5-dimethoxy analogs 6l and 6n were
more potent than their corresponding unsubstituted congeners (6e and 6c, respectively). In
contrast, the 3,4,5-trimethoxy functionality on the 7-phenyl ring (as was found in compounds
6
q-t) could not improve the cytotoxic activity. In the 2,5-dimethoxyphenyl series, the order of
activity by considering the substituent on aniline moiety was as follow: 2-F > 4-F > 4-OMe >
-Cl > 4-Br. The di-substituted congeners (6f, 6g) were more potent than mono-substituted
4
compounds 6b-e against MOLT-4 cell line.
Generally it was revealed that 2-fluoro derivatives expressed desired activity on all cell lines.
After cytotoxic screening of designed compounds, the representative compound 6l with IC50
value of 5.2 µM against MOLT-4 cell line and compound 6e with IC50 value of 5.4 µM
against A549 cell line were selected for further studies. Firstly, compound 6e and 6l were
studied morphologically to identify their potential for induction of apoptosis in A549 and
MOLT-4 cells. The fluorescence microscopy after double staining with acridine
orange/ethidium bromide demonstrated that compound 6e and 6l can reduce cell viability and
induce apoptosis in A549 and MOLT-4 cells (Figure 2 and Figure 3).
In the next step, flow cytometry analysis with annexin V-FITC/PI double staining was carried
out to confirm apoptosis induction by the compounds 6e and 6l. The results of flow cytometric
analysis showed that exposure of A549 and MOLT-4 cells to the IC50 concentration of
compound 6e and 6l respectively, resulted in apoptosis. As depicted in Figure 4, about 47% of
cells were at the early stage of apoptosis after 24 h treatment, while 44% of them underwent
late stage of apoptosis. According to the Figure 5, it was revealed that about 35% of A549
cells were at the early stage of apoptosis after 24 h treatment while, only 3% of them were
dead.
4
. Conclusion
4

ACCEPTED MANUSCRIPT
We designed and synthesized a new series of 7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-
amines as potential anticancer agents. The inhibitory activities of compounds 6a–t against
proliferation of cancer cells A549, MOLT-4, and HeLa demonstrated the substantial potency
of compounds. Particularly, 2-fluoro-aniline derivative 6l showed better profile of activity
against tested cell lines (IC values = 5.2-6.9 µM). The focused SAR study on the 7-phenyl,
5
0
7
-(2,5-dimethoxyphenyl), and 7-(3,4,5-trimethoxyphenyl) series revealed that the introduction
of 2-fluoroanilino on the 8 position associated with improvement of cytotoxicity. The
morphological and flow cytometric analyses showed that compound 6l can induce apoptosis
in the MOLT-4 cells.
5
. Experimental
All commercially available reagents were purchased from Merck AG or Aldrich and used
without further purification. TLC was conducted on silica gel 250 micron. Melting points
were measured on the Buchi Melting point B-540. FT-IR spectra were run on a Bruker,
1
Eqinox 55 spectrometer (ATR). H NMR spectra were recorded on a Bruker 400 or 500 MHz
NMR instruments. The chemical shifts (δ) and coupling constants (J) are expressed in parts
per million and Hertz, respectively. The experiments were performed using a microwave oven
(ETHOS 1600, Milestone) with a power of 600 W specially designed for an organic synthesis
and modified with a condenser and mechanical stirrer.
5
.1. General procedure for the synthesis of 2-amino-4-aryl-4H-benzo[h]chromene-3-
carbonitrile (4a-c)
A mixture of benzaldehyde or 2,5-dimethoxybenzaldehyde or 3,4,5-trimethoxybenzaldehyde
(1 mmol), malononitrile (1 mmol), α-naphthol (1 mmol) and three drop of piperidine was
refluxed in ethanol for 2-4 h. After completion of the reaction (monitored by TLC), the
reaction mixture was cooled, filtered and washed with cold ethanol to obtain white solid.
5
.1.1. 2-Amino-4-phenyl-4H-benzo[h]chromene-3-carbonitrile (4a)
-1
White solid; yield 91%; mp 208-210 ºC; IR (KBr, cm ) νmax: 3465, 3318, 2200, 1660, 1600
1
and 1550. H NMR (400 MHz, CDCl ) δ: 4.76 (s, 2H, NH ), 4.90 (s, 1H, H-4), 7.07-7.12 (m,
3
2
6
H, H-5 and Ar-H2,3,4,5,6), 7.56-7.66 (m, 3H, H-6, H-8 and H-9), 7.94 (d, J = 8.4 Hz, 1H, H-7),
5

ACCEPTED MANUSCRIPT
8
.23 (d, J = 8.4 Hz, 1H, H-10). Anal. Calcd for C H N O (298.34): C, 80.52; H, 4.73; N,
20
14
2
9
.39. Found: C, 80.79; H, 4.96; N, 9.69.
5
.1.2. 2-Amino-4-(2,5-dimethoxyphenyl)-4H-benzo[h]chromene-3-carbonitrile (4b)
-1
White solid; yield 89%; mp 216-219 ºC; IR (KBr, cm ) νmax: 3418, 3340, 2190, 1636 and
1
1
590. H NMR (400 MHz, CDCl ) δ: 3.67 (s, 3H, -OCH ), 3.83 (s, 3H, -OCH ), 4.72 (s, 2H,
3
3
3
NH ), 5.39 (s, 1H, H-4), 6.61 (s, 1H, Ar-H ), 6.72-6.74 (m, 1H, H-5), 6.87 (d, J = 8.2 Hz, 1H,
2
6
Ar-H ), 7.14 (d, J = 8.2 Hz, 1H, Ar-H ), 7.48-7.56 (m, 3H, H-6, H-8 and H-9), 7.78 (d, J = 7.4
4
3
Hz, 1H, H-7), 8.17 (d, J = 7.4 Hz, 1H, H-10). Anal. Calcd for C H N O (358.39): C, 73.73;
2
2
18
2
3
H, 5.06; N, 7.82. Found: C, 73.50; H, 4.86; N, 7.60.
5
.1.3. 2-Amino-4-(3,4,5-trimethoxyphenyl)-4H-benzo[h]chromene-3-carbonitrile (4c)
-1
White solid; yield 88%; mp 201-202 ºC; IR (KBr, cm ) νmax: 3444, 3336, 2187, 1667 and
1
1
590. H NMR (400 MHz, CDCl ) δ: 3.77 (s, 6H, 2×CH O), 3.83 (s, 3H, -OCH ), 4.76 (s, 2H,
3
3
3
NH ), 4.83 (s, 1H, H-4), 6.43 (s, 2H, Ar-H ), 7.08 (d, J = 8.4 Hz, 1H, H-5), 7.53-7.59 (m,
2
2,6
3
H, H-6, H-8 and H-9), 7.82 (d, J = 6.8 Hz, 1H, H-7), 8.20 (d, J = 7.6 Hz, 1H, H-10). Anal.
Calcd for C H N O (388.14): C, 71.12; H, 5.19; N, 7.21. Found: C, 71.46; H, 5.39; N, 7.53.
2
3
20
2
4
5
.2. General procedure for the preparation of compounds 5a-c
A mixture of compound 4 (10 mmol) in N,N-dimethylformamide dimethylacetal (15 ml) was
heated under reflux for 3-4 h. After concentration of the reaction mixture, the residue was
triturated with diethyl ether to give compounds 5a-c.
5
.2.1. N'-(3-Cyano-4-phenyl-4H-benzo[h]chromen-2-yl)-N,N-dimethylformimidamide (5a)
-1
1
White solid; yield 91%; mp 242-244 ºC; IR (KBr, cm ) ν : 2191, 1623, 1604 and 1576. H
max
NMR (400 MHz, CDCl ) δ: 3.17 (s, 3H, -CH ), 3.22 (s, 3H, -CH ), 4.95 (s, 1H, H-4), 7.03-
3
3
3
7
.05 (m, 1H, H-5), 7.27 (br s, 5H, Ar-H2,3,4,5,6), 7.48-7.58 (m, 3H, H-6, H-8 and H-9), 7.80 (d,
J = 6.8 Hz, 1H, H-7), 8.20 (d, J = 7.2 Hz, 1H, H-10), 8.42 (s, 1H, -N=CH-). Anal. Calcd for
C H N O (353.42): C, 78.16; H, 5.42; N, 11.89. Found: C, 78.46; H, 5.08; N, 11.72.
23
19
3
5
.2.2. N'-(3-Cyano-4-(2,5-dimethoxyphenyl)-4H-benzo[h]chromen-2-yl)-N,N-
dimethylformimidamide (5b)
-1
White solid; yield 90%; mp 223-225 ºC; IR (KBr, cm ) νmax: 2924, 2195, 1625, 1607 and
1
1
588. H NMR (400 MHz, CDCl ) δ: 3.18 (s, 3H, -CH ), 3.22 (s, 3H, -CH ), 3.65 (s, 3H, -
3
3
3
6

ACCEPTED MANUSCRIPT
OCH ), 3.84 (s, 3H, -OCH ), 5.51 (s, 1H, H-4), 6.67-6.71 (m, 2H, H-5 and Ar-H ), 6.86 (d, J
3
3
6
=
8.4 Hz, 1H, Ar-H ), 7.14-7.17 (m, 1H, Ar-H ), 7.45-7.55 (m, 3H, H-6, H-8 and H-9), 7.82
4 3
(d, J = 6.8 Hz, 1H, H-7), 8.17 (d, J = 6.8 Hz, 1H, H-10), 8.41 (s, 1H, -N=CH-). Anal. Calcd
for C H N O (413.47): C, 72.62; H, 5.61; N, 10.16. Found: C, 72.90; H, 5.36; N, 10.47.
2
5
23
3
3
5
.2.3. N'-(3-Cyano-4-(3,4,5-trimethoxyphenyl)-4H-benzo[h]chromen-2-yl)-N,N-
dimethylformimidamide (5c)
-1
1
White solid; yield 89%; mp 252-254 ºC; IR (KBr, cm ) ν : 2191, 1632, 1604 and 1588. H
max
NMR (400 MHz, CDCl ) δ: 3.18 (s, 3H, -CH ), 3.23 (s, 3H, -CH ), 3.79 (s, 6H, 2×CH O),
3
3
3
3
3
.81 (s, 3H, -OCH ), 4.90 (s, 1H, H-4), 6.45 (s, 2H, Ar-H ), 7.07 (d, J = 7.6 Hz, 1H, H-5),
3 2,6
7
.51-7.59 (m, 3H, H-6, H-8 and H-9), 7.81 (d, J = 7.6 Hz, 1H, H-7), 8.18 (d, J = 7.6 Hz, 1H,
H-10), 8.43 (s, 1H, -N=CH-). Anal. Calcd for C H N O (443.49): C, 70.41; H, 5.68; N,
2
6
25
3
4
9
.47. Found: C, 70.18; H, 5.37; N, 9.84.
5
.3. General procedure for the preparation of compounds 6a-t
A mixture of compound 5 (0.5 mmol) and appropriate amine (1 mmol) in acetic acid (2 ml)
was irradiated with microwaves at 700 W for 5-15 min. Then, water was added to the reaction
mixture and the crude product was extracted with dichloromethane. After evaporation of
organic solvent under reduced pressure, the residue was crystallized from ethanol to give pure
compound 6.
5
.3.1. N,7-Diphenyl-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amine (6a)
-1
1
Yellow solid; yield 89%; mp 242-244 ºC; IR (KBr, cm ) ν : 3406, 1655, 1633, 1592; H
max
NMR (500 MHz, CDCl ) δ: 5.25 (s, 1H, H-7), 7.11-7.13 (m, 2H, Ar-H ), 7.22-7.23 (m, 3H,
3
2,6
Ar-H and N-Ar-H ), 7.29-7.32 (m, 2H, N-Ar-H ), 7.35-7.37 (m, 1H, Ar-H ), 7.42-7.43 (m,
3
,5
4
3,5
4
4
H, H-6, N-Ar-H and NH), 7.50-7.58 (m, 2H, H-3 and H-5), 7.60-7.64 (m, 1H, H-2), 7.77
2,6
1
3
(
d, J = 8.0 Hz, 1H, H-4), 8.51-8.54 (m, 2H, H-1 and H-10). C NMR (CDCl , 125 MHz) δ:
3
4
1
8
0.9, 121.0, 121.2, 121.6, 121.9, 124.1, 124.2, 125.7, 126.0, 126.7, 126.8, 126.9, 127.4, 128.0,
28.2, 128.4, 128.9, 129.3, 143.3, 157.1, 159.6. Anal. Calcd for C H N O (401.46): C,
2
7
19
3
0.78; H, 4.77; N, 10.47. Found: C, 80.45; H, 4.98; N, 10.16.
5
.3.2. N-(4-Ethylphenyl)-7-phenyl-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amine (6b)
7

ACCEPTED MANUSCRIPT
-1
Yellow solid; yield 86%; mp 233-235 ºC; IR (KBr, cm ) νmax: 3422, 1645, 1591, 1562 and
1
1
511; H NMR (500 MHz, CDCl ) δ: 1.21 (t, J = 7.6 Hz, 3H, -CH ), 2.61 (q, J = 7.6 Hz, 2H, -
3
3
CH ), 5.38(s, 1H, H-7), 7.08 (d, J = 8.5 Hz, 1H, H-6), 7.13 (s, 4H, N-Ar-H and Ar-H2,6),
2
3,5
7
.31-7.33 (m, 2H, Ar-H and NH), 7.37-7.39 (m, 4H, Ar-H and N-Ar-H ), 7.49 (d, J = 8.5
4 3,5 2,6
Hz, H-5), 7.51-7.53 (m, 1H, H-3), 7.56-7.59 (m, 1H , H-2), 7.74 (d, J = 8.0 Hz, 1H, H-4), 8.41
1
3
(
s, 1H, H-10), 8.49 (d, J = 8.2 Hz, 1H, H-1). C NMR (CDCl , 125 MHz) δ: 15.6, 28.3, 40.7,
3
9
6.9, 116.8, 121.7, 121.9, 122.2, 123.9, 124.5, 125.7, 126.7, 127.0, 127.4, 128.2, 128.3, 128.4,
29.8, 133.3, 135.5, 140.7, 143.1, 156.5, 159.7. Anal. Calcd for C H N O (429.5): C, 81.09;
1
2
9
23
3
H, 5.40; N, 9.78. Found: C, 81.38; H, 5.06; N, 9.52.
5
.3.3. N-(4-Chlorophenyl)-7-phenyl-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amine (6c)
-1
Yellow solid; yield 92%; mp 206-208 ºC; IR (KBr, cm ) νmax: 3418, 1649, 1631, 1606 and
1
1
587; H NMR (500 MHz, CDCl ) δ: 5.28 (s, 1H, H-7), 6.60 (s br, 1H, NH), 7.13 (d, J = 8.6
3
Hz, 1H, H-6), 7.18-7.26 (m, 5H, N-Ar-H2,6 and Ar-H2,4,6), 7.33-7.46 (m, 4H, N-Ar-H3,5 and
Ar-H ), 7.51 (d, J = 8.6 Hz, 1H, H-5), 7.54 (t, J = 7.6, 1H, H-3), 7.60 (t, J = 7.6 Hz, 1H, H-
3
,5
1
3
2
), 7.77 (d, J = 8.0 Hz, 1H, H-4), 8.50-8.53 (m, 2H, H-1 and H-10). C NMR (CDCl , 125
3
MHz) δ: 40.6, 121.8, 122.4, 124.4, 125.6, 126.1, 126.3, 126.8, 127.1, 127.4, 128.2, 128.5,
28.9, 129.2, 129.9, 133.3, 143.0, 143.3, 156.4, 159.3, 161.1, 161.6. Anal. Calcd for
C H ClN O (435.90): C, 74.39; H, 4.16; N, 9.64. Found: C, 74.69; H, 4.44; N, 9.95.
1
27
18
3
5
.3.4. N-(4-Bromophenyl)-7-phenyl-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amine (6d)
-1
Yellow solid; yield 87%; mp 237-239ºC; IR (KBr, cm ) νmax: 3407, 1645, 1608, 1586 and
1
1
7
1
570; H NMR (500 MHz, CDCl ) δ: 5.27 (s, 1H, H-7), 7.13-7.16 (m, 2H, N-Ar-H ), 7.35-
3
2,6
.40 (m, 4H, N-Ar-H and Ar-H ), 7.42-7.44 (m, 4H, Ar-H and NH), 7.50 (d, J = 8.0,
2,4,6
3
,5
3,5
H, H-6), 7.54-7.56 (m, 2H, H-2 and H-3), 7.58-7.60 (m, 1H, H-5), 7.77 (d, J = 8.0 Hz, 1H,
1
3
H-4), 8.51-8.53 (m, 2H, H-1 and H-10). C NMR (CDCl , 125 MHz) δ: 29.6, 96.1, 121.9,
3
1
1
6
22.7, 122.9, 124.5, 125.6, 126.0, 126.9, 127.0, 127.4, 127.5, 127.8, 128.0, 128.2, 128.6,
28.9, 130.0, 131.8, 132.3, 143.3, 143.4, 159.3. Anal. Calcd for C H BrN O (480.36): C,
2
7
18
3
7.51; H, 3.78; N, 8.75. Found: C, 67.82; H, 3.46; N, 8.39.
5
.3.5. N-(2-Fluorophenyl)-7-phenyl-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amine (6e)
-1
1
Yellow solid; yield 89%; mp 225-227 ºC; IR (KBr, cm ) ν : 3421, 1648, 1625, 1594; H
max
NMR (500 MHz, CDCl ) δ: 5.23 (s, 1H, H-7), 6.81-6.82 (m, 1H, NH), 6.97 -7.07 (m, 2H, N-
3
Ar-H ), 7.11-7.15 (m, 1H, N-Ar-H ), 7.27-7.32 (m, 1H, Ar-H ), 7.36-7.39 (m, 2H, Ar-H ),
4
,6
3,5
4
3,5
8

ACCEPTED MANUSCRIPT
7
.42-7.43 (m, 2H, Ar-H ), 7.49-7.54 (m, 2H, H-5 and H-6), 7.59-7.62 (m, 1H, H-3), 7.76 (d,
2
,6
J = 8.2 Hz, 1H, H-4), 8.34-8.39 (m, 1H, H-2), 8.54 (d, J = 8.4 Hz, 1H, H-1), 8.57 (s, 1H, H-
1
3
1
1
1
4
0). C NMR (CDCl , 125 MHz) δ: 40.8, 98.4, 114.7, 114.9, 117.0, 121.8, 122.2, 122.3,
3
23.7, 123.8, 124.3, 124.4, 125.7, 126.7, 127.0, 127.4, 128.1, 128.2, 129.7, 133.3, 142.8,
43.4, 152.0, 154.0, 156.6, 159.2, 162.2. Anal. Calcd for C H FN O (419.45): C, 77.31; H,
2
7
18
3
.33; N, 10.02. Found: C, 77.70; H, 4.64; N, 10.37.
5
.3.6.
N-(2,4-Dimethylphenyl)-7-phenyl-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amine
(6f)
-1
Yellow solid; yield 89%; mp 187-188 ºC; IR (KBr, cm ) νmax: 3410, 1645, 1589, 1562 and
1
1
7
7
509; H NMR (500 MHz, CDCl ) δ: 1.64 (s, 3H, -CH ), 2.29 (s, 3H, -CH ), 5.25 (s, 1H, H-
3
3
3
), 6.93 (s, 1H, N-Ar-H ), 7.01 (d, J = 8.0 Hz, 1H, N-Ar-H ), 7.16 (d, J = 8.5 Hz, 1H, H-6),
3
6
.19 (d, J = 8.0 Hz, 1H, N-Ar-H ), 7.31-7.39 (m, 6H, NH and Ar-H2,3,4,5,6), 7.51 (d, J = 8.5
5
Hz, 1H, H-5), 7.54-7.55 (m, 1H, H-2), 7.61 (t, J = 8.0 Hz, 1H, H-3), 7.77 (d, J = 8.0 Hz, 1H,
1
3
H-4), 8.44 (s, 1H, H-10), 8.54 (d, J = 8.5 Hz, 1H, H-1). C NMR (CDCl , 125 MHz) δ: 17.6,
3
2
0.9, 38.5, 97.5, 119.5, 121.2, 123.7, 124.6, 126.7, 126.9, 127.1, 127.3, 127.5, 128.1, 129.1,
31.2, 133.2, 134.2, 134.9, 135.3, 144.4, 144.5, 156.9, 160.5, 162.7. Anal. Calcd for
1
C H N O (429.51): C, 81.09; H, 5.40; N, 9.78. Found: C, 81.38; H, 5.73; N, 9.47.
29
23
3
5
.3.7. N-(2,4-Dimethoxyphenyl)-7-phenyl-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amine
(6g)
-1
1
Yellow solid; yield 83%; mp 206-207 ºC; IR (KBr, cm ) ν : 3399, 1604, 1564, 1528; H
max
NMR (500 MHz, CDCl ) δ: 3.71 (s, 3H, -OCH ), 3.78 (s, 3H, -OCH ), 5.22(s, 1H, H-7), 6.43
3
3
3
(
d, J = 2.6 Hz, 1H, N-Ar-H ), 6.48-6.50 (dd, J = 9.0 Hz, J = 2.6 Hz, 1H, N-Ar-H ), 6.96 (s,
3
5
1
H, NH), 7.17-7.19 (d, J = 9.0 Hz, 1H, N-Ar-H ), 7.27-7.28 (m, 1H, Ar-H ), 7.35 (t, J = 7.6
6 4
Hz, 2H, Ar-H ), 7.43-7.44 (m, 2H, Ar-H ), 7.46-7.53 (m, 2H, H-3 and H-6), 7.59 (t, J = 7.6
3
,5
2,6
Hz, 1H, H-2), 7.76 (d, J = 8.2 Hz, 1H, H-5), 8.23 (d, J = 8.8 Hz, 1H, H-4), 8.52 (s, 1H, H-10),
1
3
8
.54 (d, J = 8.2 Hz, 1H, H-1). C NMR (CDCl , 125 MHz) δ: 40.7, 55.5, 57.1, 97.6, 98.7,
3
1
03.7, 117.3, 121.7, 121.9, 123.9, 124.1, 125.8, 126.6, 126.8, 127.3, 127.8, 128.3, 129.3,
33.2, 143.2, 143.5, 149.9, 156.1, 156.8, 159.3, 162.1. Anal. Calcd for C H N O (461.51):
1
2
9
23
3
3
C, 75.47; H, 5.02; N, 9.10. Found: C, 75.74; H, 5.36; N, 9.37.
5
.3.8. N-Benzyl-7-phenyl-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amine (6h)
9

ACCEPTED MANUSCRIPT
-1
1
Yellow solid; yield 82%; mp 210-211 ºC; IR (KBr, cm ) ν : 3426, 1645, 1589, 1569; H
max
NMR (500 MHz, CDCl ) δ: 4.48 (dd, J = 14.9 Hz, J = 4.7 Hz, 1H, -CH ), 4.72 (dd, J = 14.9
3
2
Hz, J = 4.7 Hz, 1H, -CH ), 5.11 (s, 2H, NH and H-7), 6.85-6.86 (m, 2H, N-Ar-H ), 7.09 (d, J
2
2,6
=
8.5, 1H, H-6), 7.20-7.24 (m, 3H, N-Ar-H3,4,5), 7.26-7.32 (m, 5H, Ar-H2,3,4,5,6), 7.47 (d, J =
8
4
4
1
.5 Hz, 1H, H-5), 7.51-7.54 (m, 1H, H-3), 7.58-7.61 (m, 1H, H-2), 7.75 (d, J = 8.1 Hz, 1H, H-
1
3
), 8.44 (s, 1H, H-10), 8.52 (d, J = 8.5 Hz, 1H, H-1). C NMR (CDCl , 125 MHz) δ: 40.5,
3
5.3, 95.9, 117.0, 121.9, 123.9, 124.0, 125.8, 126.6, 126.8, 127.0, 127.35, 127.39, 127.9,
28.0, 128.6, 129.6, 133.2, 137.9, 143.5, 143.6, 157.0, 161.2, 161.7. Anal. Calcd for
C H N O (415.49): C, 80.94; H, 5.09; N, 10.11. Found: C, 80.63; H, 5.38; N, 9.86.
28
21
3
5
.3.9. N-(2-Chlorobenzyl)-7-phenyl-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amine (6i)
-1
1
Yellow solid; yield 83%; mp 196-198 ºC; IR (KBr, cm ) ν : 3418, 1650, 1608, 1586; H
max
NMR (500 MHz, CDCl ) δ: 4.63 (dd, J = 15.5 Hz, J = 5.5 Hz, 1H, -CH ), 4.75 (dd, J = 15.5
3
2
Hz, J = 5.5 Hz, 1H, -CH ), 5.11 (s, 1H, H-7), 5.26 (br s, 1H, NH), 5.26 (br s, 1H, NH), 6.84
2
(
d, J = 6.7 Hz, 1H, N-Ar-H ), 7.07 (t, 1H, J = 7.7 Hz, Ar-H ), 7.10 (d, J = 8.5 Hz, 1H, H-6),
6
4
7
.15-7.18 (m, 1H, N-Ar-H ), 7.24-7.26 (m, 1H, N-Ar-H ), 7.30-7.35 (m, 5H, Ar-H
and N-
5
3
2,3,5,6
Ar-H ), 7.46 (d, J = 8.5 Hz, 1H, H-5), 7.50-7.52 (m, 1H, H-3), 7.57-7.60 (m, 1H, H-2), 7.73
4
1
3
(
d, J = 8.1 Hz, 1H, H-4), 8.40 (s, 1H, H-10), 8.51 (d, J = 8.5 Hz, 1H, H-1). C NMR (CDCl ,
3
1
1
1
4
25 MHz) δ: 40.3, 43.1, 96.1, 116.9, 121.9, 123.8, 124.2, 125.7, 126.7, 126.8, 126.9, 127.3,
28.0, 128.7, 128.9, 129.1, 129.4, 129.6, 133.2, 133.3, 135.3, 143.2, 143.3, 143.5, 156.1,
61.0. Anal. Calcd for C H ClN O (449.93): C, 74.74; H, 4.48; N, 9.34. Found: C, 74.99; H,
2
8
20
3
.80; N, 9.63.
5
.3.10. N-Phenethyl-7-phenyl-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-amine (6j)
-1
Yellow solid; yield 74%; mp 194-195 ºC; IR (KBr, cm ) νmax: 3426, 1593, 1610, 1570 and
1
1
496; H NMR (500 MHz, CDCl ) δ: 2.73-2.78 (m, 2H, -NCH CH ), 3.70-3.72 (m, 1H, -
3
2
2
NCH ), 3.80-3.83 (m, 1H, -NCH ), 4.87 (s, 1H, H-7), 7.03 (d, J = 8.5 Hz, 1H, H-6), 7.07 (d, J
2
2
=
6.6 Hz, 2H, N-Ar-H ), 7.10-7.12 (m, 3H, N-Ar-H ), 7.23-7.26 (m, 3H, Ar-H and NH),
2,6 3,4,5 3,5
7
.27-7.32 (m, 3H, Ar-H2,4,6), 7.46 (d, J = 8.5 Hz, 1H, H-5), 7.51-7.53 ( m, 1H, H-3), 7.57-7.60
(m, 1H, H-2 ), 7.74 (d, J = 8.1 Hz, 1H, H-4), 8.45 (s, 1H, H-10), 8.50 ( d, J = 8.5 Hz, 1H, H-
1
3
1
1
). C NMR (CDCl , 125 MHz) δ: 111.3, 111.5, 117.2, 121.9, 124.0, 125.7, 126.5, 126.8,
3
27.3, 127.7, 128.7, 129.4, 130.7, 133.2, 138.4, 143.2, 147.8, 149.2, 156.7, 161.2, 161.6.
Anal. Calcd for C H N O (429.51): C, 81.09; H, 5.40; N, 9.78. Found: C, 81.46; H, 5.69; N,
2
9
23
3
9
.99.
1
0

ACCEPTED MANUSCRIPT
5
.3.11. N-(2,4-Dimethoxyphenethyl)-7-phenyl-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-
amine (6k)
-1
Yellow solid; yield 82%; mp 154-156 ºC; IR (KBr, cm ) νmax: 3407, 1643, 1605,1586 and
1
1
570; H NMR (500 MHz, CDCl ) δ: 2.72 (t, J = 6.5 Hz, 2H, N-CH -CH ), 2.82-2.84 (m, 1H,
3
2
2
N-CH ), 3.61-3.63 (m, 1H, N-CH ), 3.84 (s, 3H, -OCH ), 3.93 (s, 3H, -OCH ), 4.75 (br s, 1H,
2
2
3
3
NH), 4.85 (s, 1H, H-7), 6.60-6.62 (m, 2H, N-Ar-H ), 6.82 (d, J = 8.5 Hz, 1H, H-6), 7.03 (d, J
3
,5
=
8.6 Hz, 1H, N-Ar-H ), 7.07-7.09 (m, 2H, Ar-H ), 7.20-7.22 (m, 3H, Ar-H2,4,6), 7.45 (d, J =
6 3,5
8
1
3
1
1
5
.5 Hz, 1H, H-5), 7.50-7.53 (m, 1H, H-3), 7.58 (t, J = 8.0 Hz, 1H, H-2), 7.74 (d, J = 8.0 Hz,
1
3
H, H-4), 8.46 (s, 1H, H-10), 8.50 (d, J = 8.5 Hz, 1H, H-1). C NMR (CDCl , 125 MHz) δ:
3
4.7, 40.5, 42.1, 55.8, 55.9, 96.0, 111.3, 111.5, 117.2, 120.6, 121.9, 123.9, 124.1, 125.7,
26.6, 126.8, 127.3, 127.7, 129.3, 130.7, 133.2, 143.1, 143.6, 147.8, 149.2, 156.6, 161.1,
61.6. Anal. Calcd for C H N O (489.56): C, 76.05; H, 5.56; N, 8.58. Found: C, 76.36; H,
3
1
27
3
3
.45; N, 8.28.
5
.3.12. 7-(2,5-Dimethoxyphenyl)-N-(2-fluorophenyl)-7H-benzo[7,8]chromeno[2,3-
d]pyrimidin-8-amine (6l)
-1
Yellow solid; yield 86%; mp 228-230 ºC; IR (KBr, cm ) νmax: 3396, 1648, 1620, 1608 and
1
1
7
6
580; H NMR (500 MHz, CDCl ) δ: 3.55 (s,3H, -OCH ), 4.12 (s,3H, -OCH ), 5.78 (s, 1H, H-
3
3
3
), 6.56 (d, J = 2.7 Hz, 1H, Ar-H ), 6.71-6.73 (m, 1H, Ar-H ), 6.74-6.78 (m, 1H, N-Ar-H ),
6
4
5
.98 (d, J = 8.9 Hz, 1H, Ar-H ), 7.05 (d, J = 8.4 Hz, 1H, H-6), 7.18 (d, J = 8.0 Hz, 1H, N-Ar-
3
H ), 7.24-7.27 (m, 3H, H-5 and N-Ar-H ), 7.50-7.56 (m, 1H, H-1, H-3), 7.62 (t, J = 7.5 Hz,
6
2,4
1
H, H-2), 7.75 -7.79 (m, 2H, H-4 and NH), 8.49 (s, 1H, H-10), 8.55 (d, J = 8.4 Hz, 1H, H-1).
1
3
C NMR (CDCl , 125 MHz) δ: 31.5, 55.5, 57.2, 98.8, 108.1, 108.3, 110.1, 110.3, 112.4,
3
1
13.5, 115.9, 116.9, 117.7, 121.8, 123.9, 124.5, 125.9, 126.6, 126.9, 127.4, 129.8, 129.9,
33.1, 133.3, 144.7, 148.4, 155.2, 156.1, 159.0, 161.9. Anal. Calcd for C H FN O (479.50):
1
2
9
22
3
3
C, 72.64; H, 4.62; N, 8.76. Found: C, 72.95; H, 4.34; N, 8.47.
5
.3.13. 7-(2,5-Dimethoxyphenyl)-N-(4-fluorophenyl)-7H-benzo[7,8]chromeno[2,3-
d]pyrimidin-8-amine (6m)
-1
1
Yellow solid; yield 91%; mp 195-196 ºC; IR (KBr, cm ) ν : 3384, 1632, 1601, 1516; H
max
NMR (500 MHz, CDCl ) δ: 3.55 (s, 3H, -OCH ), 4.08 (s, 3H, -OCH ), 5.78 (s, 1H, H-7),
3
3
3
6
.54-6.57 (d, J = 3 Hz, 1H, Ar-H ), 6.72-6.75 (dd, J =9.0 Hz, J = 3.0 Hz, 1H, Ar-H ), 6.97 (d,
6 4
J = 9.0 Hz, 1H, Ar-H ), 7.01-7.06 (m, 2H, N-Ar-H ), 7.41-7.44 (m, 2H, N-Ar-H ), 7.52 (d,
3
3,5
2,6
1
1

ACCEPTED MANUSCRIPT
J = 8.5 Hz, 1H, H-6), 7.53-7.56 (m, 1H, H-3), 7.61-7.64 (m, 1H, H-2), 7.68 (br s, 1H, NH),
7
8
1
1
.79 (d, J = 8.1 Hz, 1H, H-5), 7.96 (d, J = 8.1 Hz, 1H, H-4), 8.43 (s, 1H, H-10), 8.56 (d, J =
13
.4 Hz, 1H, H-1). C NMR (CDCl , 125 MHz) δ: 31.6, 55.5, 57.1, 112.3, 113.5, 115.4, 115.6,
3
16.2, 117.0, 123.0, 121.8, 123.1, 123.9, 124.4, 124.8, 126.0, 126.6, 126.8, 127.4, 127.7,
33.4, 133.6, 134.9, 144.8, 148.6, 155.1, 156.4, 159.3, 162.4. Anal. Calcd for C H FN O
2
9
22
3
3
(479.50): C, 72.64; H, 4.62; N, 8.76. Found: C, 72.35; H, 4.34; N, 8.97.
5
.3.14. N-(4-Chlorophenyl)-7-(2,5-dimethoxyphenyl)-7H-benzo[7,8]chromeno[2,3-
d]pyrimidin-8-amine (6n)
-1
1
Yellow solid; yield 90%; mp 215-217 ºC; IR (KBr, cm ) ν : 3358, 1618, 1591, 1498; H
max
NMR (500 MHz, CDCl ) δ: 3.54 (s, 3H, -OCH ), 4.09 (s, 3H, -OCH ), 5.77 (s, 1H, H-7), 6.56
3
3
3
(
d, J = 3.0 Hz, 1H, Ar-H ), 6.71-6.74 (dd, J = 9.0 Hz, J = 3.0 Hz, 1H, Ar-H ), 6.97 (d, J = 9.0
6
4
Hz, 1H, Ar-H ), 7.05 (d, J = 8.5 Hz, 1H, H-6), 7.26-7.30 (m, 2H, N-Ar-H ), 7.47 (d, J = 8.8
3
2,6
Hz, 2H, N-Ar-H ), 7.51 (d, J = 8.5 Hz, 1H, H-5), 7.53-7.56 (m, 1H , H-3), 7.62 (t, J = 8.0
3
,5
Hz, 1H , H-2), 7.70 (s, 1H, NH), 7.79 (d, J = 7.8 Hz, 1H , H-4), 8.45 (s, 1H, H-10), 8.55 (d, J
1
3
=
8.2 Hz, 1H, H-1). C NMR (CDCl , 125 MHz) δ: 31.6, 55.5, 57.2, 98.6, 112.4, 113.5,
3
1
1
17.0, 117.7, 121.8, 122.3, 123.9, 124.5, 125.9, 126.6, 126.9, 127.4, 128.7, 128.9, 133.1,
33.3, 137.5, 144.7, 148.5, 155.1, 156.2, 159.1, 162.3. Anal. Calcd for C H ClN O
3
2
9
22
3
(495.96): C, 70.23; H, 4.47; Cl, 7.15; N, 8.47; Found: C, 70.57; H, 4.62; N, 8.24.
5
.3.15. N-(4-Bromophenyl)-7-(2,5-dimethoxyphenyl)-7H-benzo[7,8]chromeno[2,3-
d]pyrimidin-8-amine (6o)
-1
1
Yellow solid; yield 87%; mp 214-215 ºC; IR (KBr, cm ) ν : 3367, 1645, 1607, 1561; H
max
NMR (500 MHz, CDCl ) δ: 3.55 (s, 3H, -OCH ), 4.09 (s,3H, -OCH ), 5.77 (s, 1H, H-7), 6.56
3
3
3
(
d, J = 3.0 Hz, 1H, Ar-H ), 6.71-6.74 (dd, J = 9.0 Hz, J = 3.0 Hz, 1H, Ar-H ), 6.97 (d, J = 9.0
6
4
Hz, 1H, Ar-H ), 7.05 (d, J = 8.5 Hz, 1H, H-6), 7.40-7.45 (m, 4H, N-Ar-H2,3,5,6), 7.51 (d, J =
3
8
.5 Hz, 1H, H-5), 7.52-7.54 (m, 1H , H-3), 7.56-7.62 (m, 1H , H-2), 7.71 (s, 1H, NH), 7.79 (d,
1
3
J = 8.1 Hz, 1H , H-4), 8.45 (s, 1H, H-10), 8.55 (d, J = 8.4 Hz, 1H, H-1). C NMR (CDCl ,
3
1
25 MHz) δ: 31.5, 55.5, 57.2, 112.4, 113.5, 116.3, 117.0, 117.6, 121.8, 122.6, 123.8, 124.5,
25.9, 126.6, 126.9, 127.4, 131.8, 133.1, 133.3, 138.0, 148.4, 155.1, 156.1, 159.0, 159.5,
62.2. Anal. Calcd for C H BrN O (540.41): C, 64.45; H, 4.10; N, 7.78; Found: C, 64.80;
1
1
2
9
22
3
3
H, 4.38; N, 7.45.
1
2

ACCEPTED MANUSCRIPT
5
.3.16. 7-(2,5-Dimethoxyphenyl)-N-(4-methoxyphenyl)-7H-benzo[7,8]chromeno[2,3-
d]pyrimidin-8-amine (6p)
-1
1
Yellow solid; yield 89%; mp 247-249 ºC; IR (KBr, cm ) ν : 3324, 1629, 1610, 1580; H
max
NMR (500 MHz, CDCl ) δ: 3.55 (s, 3H, -OCH ), 3.80 (s, 3H, -OCH ), 4.06 (s, 3H, -OCH ),
3
3
3
3
5
.77 (s, 2H, H-7 and NH), 6.58 (d, J = 2.9 Hz, 1H, Ar-H ), 6.72-6.74 (dd, J = 8.9 Hz, J = 2.9
6
Hz, 1H, Ar-H ), 6.88 (d, J = 8.9 Hz, 1H, Ar-H ), 6.96 (d, J = 8.8 Hz, 1H, N-Ar-H ), 7.07 (d,
4
3
2,6
J = 8.4 Hz, 1H, N-Ar-H ), 7.34 (d, J = 8.7 Hz, 1H, H-6), 7.49-7.63 (m, 3H , H-2, H-3 and H-
3
,5
1
3
5
), 7.79 (d, J = 8.1 Hz, 1H , H-4), 8.40 (s, 1H, H-10), 8.56 (d, J = 8.4 Hz, 1H, H-1). C NMR
CDCl , 125 MHz) δ: 30.9, 55.5, 56.9, 112.0, 113.4, 114.1, 114.4, 117.0, 117.9, 120.8, 121.9,
(
3
1
23.5, 124.0, 124.3, 124.4, 124.7, 126.0, 126.1, 126.5, 126.7, 127.4, 127.6, 131.8, 133.3,
48.7, 156.6, 159.6. Anal. Calcd for C H N O (491.54): C, 73.30; H, 5.13; N, 8.55; Found:
1
30
25
3
4
C, 73.64; H, 4.78; N, 8.31.
5
.3.17. N-Phenyl-7-(3,4,5-trimethoxyphenyl)-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-
amine (6q)
-1
1
Cream solid; yield 89%; mp 214-216 ºC; IR (KBr, cm ) ν : 3425, 1645, 1608, 1592; H
max
NMR (500 MHz, CDCl ) δ: 3.82-3.83 (m, 9H, 3×CH O), 5.17 (s, 1H, H-7), 6.63 (s, 2H, Ar-
3
3
H ), 6.70 (s, 1H, NH), 7.08 (t, J = 7.2 Hz, 1H, N-Ar-H ), 7.18 (d, J = 8.7 Hz, 1H, H-6), 7.26-
2
,6
4
7
.32 (m, 4H, N-Ar-H2,3,5,6), 7.52-7.56 (m, 2H, H-3 and H-5), 7.61 (t, J = 7.5 Hz, 1H, H-2),
1
3
7
.79 (d, J = 8.0 Hz, 1H, H-4), 8.52-8.54 (m, 2H, H-1 and H-10). C NMR (CDCl , 125 MHz)
3
δ: 41.2, 56.3, 60.9, 97.0, 105.2, 116.5, 121.0, 121.9, 123.9, 124.1, 124.2, 125.5, 126.7, 127.0,
27.4, 129.0, 133.4, 138.0, 138.3, 138.9, 154.4, 157.1, 159.8, 162.1. Anal. Calcd for
C H N O (491.54): C, 73.30; H, 5.13; N, 8.55. Found: C, 73.14; H, 5.38; N, 8.20.
1
30
25
3
4
5
.3.18. N-(4-Methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)-7H-benzo[7,8]chromeno[2,3-
d]pyrimidin-8-amine (6r)
-1
1
White solid; yield 85%; mp 229-231 ºC; IR (KBr, cm ) ν : 3384, 1631, 1611, 1580; H
max
NMR (500 MHz, CDCl ) δ: 3.71-3.84 (m, 12H, 4×CH O), 5.61 (s, 1H, H-7), 6.69 (s, 2H, Ar-
3
3
H ), 6.89-6.91 (m, 2H, N-Ar-H ), 6.99 (br s, 1H, NH), 7.27-7.30 (m, 2H, N-Ar-H ), 7.44-
2
,6
3,5
2,6
7
1
1
1
5
.54 (m, 3H, H-3, H-5 and H-6), 7.83-7.86 (m, 2H, H-2 and H-4), 8.15 (d, J = 8.4 Hz, 1H, H-
1
3
), 8.41 (s, 1H, H-10). C NMR (CDCl , 125 MHz) δ: 40.1, 54.5, 56.6, 60.5, 96.5, 111.3,
3
11.5, 120.1, 122.1, 123.9, 124.0, 124.1, 125.5, 126.5, 127.1, 127.2, 129.1, 133.3, 137.5,
37.8, 137.9, 154.1, 156.0, 159.7, 162.1. Anal. Calcd for C H N O (521.56): C, 71.39; H,
3
1
27
3
5
.22; N, 8.06. Found: C, 71.10; H, 5.57; N, 8.34.
1
3

ACCEPTED MANUSCRIPT
5
.3.19. N-Benzyl-7-(3,4,5-trimethoxyphenyl)-7H-benzo[7,8]chromeno[2,3-d]pyrimidin-8-
amine (6s)
-1
1
Yellow solid; yield 83%; mp 167-169 ºC; IR (KBr, cm ) ν : 3393, 1648, 1589, 1572; H
max
NMR (500 MHz, CDCl ) δ: 3.64 (s, 6H, 2×CH O), 3.80 (s, 3H, -OCH ), 4.42 (dd, J = 14.3
3
3
3
Hz, J = 5.0 Hz, 1H, N-CH ), 4.65 (dd, J = 14.3 Hz, J = 5.0 Hz, 1H, N-CH ), 4.97 (s, 2H, H-7
2
2
and NH), 6.44 (s, 2H, Ar-H ), 6.97-6.99 (m, 2H, N-Ar-H ), 7.09 (d, J = 8.5 Hz, 1H, H-6),
2
,6
2,6
7
.27-7.28 (m, 3H, N-Ar-H3,4,5), 7.48 (d, J = 8.5 Hz, 1H, H-5), 7.52-7.54 (m, 1H, H-3), 7.57-
.60 (m, 1H, H-2), 7.76 (d, J = 8.0 Hz, 1H, H-4), 8.44 (s, 1H, H-10), 8.51 (d, J = 8.4 Hz, 1H,
7
1
3
H-1). C NMR (CDCl , 125 MHz) δ: 41.0, 45.8, 59.1, 60.8, 95.6, 104.9, 116.6, 121.9, 123.9,
3
1
25.68, 125.62, 126.6, 126.9, 127.4, 127.6, 128.7, 133.3, 137.5, 137.8, 139.3, 143.5, 154.1,
57.3, 161.4, 161.6. Anal. Calcd for C H N O (492.55): C, 73.15; H, 5.32; N, 8.53. Found:
1
3
0
26
3
4
C, 73.40; H, 5.06; N, 8.84.
5
.3.20. N-(2-Chlorobenzyl)-7-(3,4,5-trimethoxyphenyl)-7H-benzo[7,8]chromeno[2,3-
d]pyrimidin-8-amin (6t)
-1
1
Yellow solid; yield 80%; mp 181-182 ºC; IR (KBr, cm ) ν : 3397, 1611, 1581, 1562; H
max
NMR (500 MHz, CDCl ) δ: 3.69 (s, 6H, 2×CH O), 3.79 (s, 3H, -OCH ), 4.60-4.62 (m, 1H, N-
3
3
3
CH ), 4.79-4.68 (m, 1H, N-CH ), 5.01 (s, 1H, H-7), 5.18 (s, 1H, NH), 6.47 (s, 2H, Ar-H ),
2
2
2,6
7
.08-7.14 (m, 3H, N-Ar-H3,5,6), 7.20-7.22 (m, 1H, N-Ar-H ), 7.33 (d, J = 7.5 Hz, 1H, H-6),
4
7
.47-7.58 (m, 3H, H-2, H-3 and H-5), 7.76 (d, J = 7.0 Hz, 1H, H-4), 8.45 (s, 1H, H-10), 8.51
13
(
d, J = 7.9 Hz, 1H, H-1). C NMR (CDCl , 125 MHz) δ: 40.9, 43.4, 56.1, 60.8, 95.8, 104.9,
3
1
1
16.6, 121.9, 123.9, 124.0, 125.6, 126.6, 126.9, 127.4, 129.1, 129.5, 129.8, 133.3, 135.2,
39.0, 144.5, 148.5, 153.1, 154.2, 156.8, 156.9, 161.4. Anal. Calcd for C H ClN O
4
3
1
26
3
(540.01): C, 68.95; H, 4.85; N, 7.78. Found: C, 69.46; H, 5.09; N, 7.47.
5
.4. Cell viability assay
The cancer cell lines A549, MOLT-4, and HeLa were obtained from the National Cell Bank
of Iran. The cancer cells were maintained in RPMI 1640 medium (supplemented with 10%
FBS, and 100 units/mL penicillin G and 100 µg/mL streptomycin) and incubated at 37°C in
humidified air containing 5% CO . For preparation of stock solutions, the test compounds 6a-t
2
were dissolved in DMSO and then diluted with RPMI 1640 medium. The final concentration
of DMSO in the culture medium was less than 0.5%.
1
4

ACCEPTED MANUSCRIPT
The cell viability of cancer cell lines (A549, MOLT-4, and HeLa)
after
exposure
to
compounds 6a-t was determined by MTT assay according to the reported method [19].
Cisplatin was used as standard drug. The optical densities of samples were measured at 570
nm with background correction at 655 nm by using Bio-Rad microplate reader (Model 680).
5
.5. Acridine orange/ethidium bromide double staining test
In order to evaluate the potential of compounds 6e and 6l for induction of apoptosis, the
morphological analysis of A549 and MOLT-4 cells was used. Acridine orange/ethidium
bromide double staining test was performed according to the previously reported method [20].
5
.6. Annexin V-FITC/PI double staining and flow cytometric analysis
The A549 and MOLT-4 cells were treated with the IC concentrations of compounds 6e and
5
0
6
l, respectively, and incubated for 24 h. Then, the treated cells were collected by
centrifugation and resuspended in 500 µl of 1X Binding buffer. The cells were double stained
by using Annexin V-FITC Apoptosis Detection Kit (Bio Vision) according to the described
protocol by manufacture. Finally, Annexin V-FITC binding was analyzed by flow cytometry
(ex = 488 nm; em = 530 nm) using FITC signal detector (FL1) and PI staining by the
phycoerythrin emission signal detector (FL2) [18].
Acknowledgment
This work was supported by grants from the Iran National Science Foundation (INSF) and
Research Council of Tehran University of Medical Sciences (TUMS).
References
[
1] R.J. Boohaker, M.W. Lee, P. Vishnubhotla, J.M. Perez, A.R. Khaled, The use of
therapeutic peptides to target and to kill cancer cells. Curr. Med. Chem. 19 (2012) 3794-
3
804.
[
[
2] M. Ogris, E. Wagner, Targeting tumors with non-viral gene delivery systems. Drug
Discov. Today 7 (2002) 479-485.
3] M. Creixell, N.A. Peppas, Co-delivery of siRNA and therapeutic agents using nanocarriers
to overcome cancer resistance. Nano Today 7 (2012) 367-379.
1
5

ACCEPTED MANUSCRIPT
[
[
4] B.W. Stewart, C.P. Wild, World Cancer Report 2014, Lyon, France: International Agency
for Research on Cancer, 2014.
5] A. Coates, S. Abraham, S.B. Kaye, T. Sowerbutts, C. Frewin, R.M. Fox, M.H. Tattersall,
On the receiving end--patient perception of the side-effects of cancer chemotherapy. Eur. J.
Cancer Clin. Oncol. 19 (1983) 203-208.
[
[
6] P.D. Eckford, F.J. Sharom, ABC efflux pump-based resistance to chemotherapy drugs.
Chem. Rev. 109 (2009) 2989-3011.
7] B.H. Alizadeh, A. Foroumadi, S. Emami, M. Khoobi, F. Panah, S.K. Ardestani, A.
Shafiee, Isochaihulactone analogues: synthesis and anti-proliferative activity of novel
dibenzylbutyrolactones. Eur. J. Med. Chem. 45 (2010) 5979-5984.
[
8] M. Khoobi, A. Foroumadi, S. Emami, M. Safavi, G. Dehghan, B.H. Alizadeh, A.
Ramazani, S.K. Ardestani, A. Shafiee, Coumarin-based bioactive compounds: facile
synthesis and biological evaluation of coumarin-fused 1,4-thiazepines. Chem. Biol. Drug
Des. 78 (2011) 580-586.
[
[
[
9] L. Firoozpour, N. Edraki, M. Nakhjiri, S. Emami, M. Safavi, S.K. Ardestani, M.
Khoshneviszadeh, A. Shafiee, A. Foroumadi, Cytotoxic activity evaluation and QSAR
study of chromene-based chalcones. Arch. Pharm. Res. 35 (2012) 2117-2125.
10] M. Azizmohammadi, M. Khoobi, A. Ramazani, S. Emami, A. Zarrin, O. Firuzi, R. Miri,
A. Shafiee, 2H-chromene derivatives bearing thiazolidine-2,4-dione, rhodanine or
hydantoin moieties as potential anticancer agents. Eur. J. Med. Chem. 59 (2013) 15-22.
11] F. Molaverdi, M. Khoobi, S. Emami, M. Alipour, O. Firuzi, A. Foroumadi, G. Dehghan,
R. Miri, F. Shaki, F. Jafarpour, A. Shafiee, Polyoxygenated cinnamoylcoumarins as
conformationally constrained analogs of cytotoxic diarylpentanoids: synthesis and
biological activity. Eur. J. Med. Chem. 68 (2013) 103-110.
[
12] A. Ramazani, M. Khoobi, A. Torkaman, F.Z. Nasrabadi, H. Forootanfar, M. Shakibaie,
M. Jafari, A. Ameri, S. Emami, M.A. Faramarzi, A. Foroumadi, A. Shafiee, One-pot, four-
component synthesis of novel cytotoxic agents 1-(5-aryl-1,3,4-oxadiazol-2-yl)-1-(1H-
pyrrol-2-yl)methanamines. Eur. J. Med. Chem. 78 (2014) 151-156.
1
6

ACCEPTED MANUSCRIPT
[
13] M.N. Elinson, A. I. Ilovaisky, V.M. Merkulova, P.A. Belyakov, A.O. Chizhov, G.I.
Nikishin, Solvent-free cascade reaction: direct multicomponent assembling of 2-amino-4H-
chromene scaffold from salicylaldehyde, malononitrile or cyanoacetate and nitroalkanes.
Tetrahedron 66 (2010) 4043-4048.
[
[
14] S.A. Patil, J. Wang, X.S. Li, J. Chen, T.S. Jones, A. Hosni-Ahmed, R. Patil, W.L. Seibel,
W. Li, D.D. Miller, New substituted 4H-chromenes as anticancer agents. Bioorg. Med.
Chem. Lett. 22 (2012) 4458-4461.
15] X. Han, J. Luo, F. Wu, X.Y. Hou, G. Yan, M. Zhou, M. Zhang, C. Pu, R. Li, Synthesis
and biological evaluation of novel 2,3-dihydrochromeno[3,4-d]imidazol-4(1H)-one
derivatives as potent anticancer cell proliferation and migration agents. Eur. J. Med. Chem.
1
14 (2016) 232-243.
[
[
16] S. Emami, S. Dadashpour, Current developments of coumarin-based anti-cancer agents in
medicinal chemistry. Eur. J. Med. Chem. 102 (2015) 611-630.
17] W. Kemnitzer, J. Drewe, S. Jiang, H. Zhang, J. Zhao, C. Crogan-Grundy, L. Xu, S.
Lamothe, H. Gourdeau, R. Denis, B. Tseng, S. Kasibhatla, S.X. Cai, Discovery of 4-aryl-
4
H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high-
throughput screening assay. 3. Structure-activity relationships of fused rings at the 7,8-
positions. J. Med. Chem. 50 (2007) 2858-2864.
[
18] N. Sirisoma, A. Pervin, H. Zhang, S. Jiang, J.A. Willardsen, M.B. Anderson, G. Mather,
C.M. Pleiman, S. Kasibhatla, B. Tseng, J. Drewe, S.X. Cai, Discovery of N-(4-
methoxyphenyl)-N,2-dimethylquinazolin-4-amine, a potent apoptosis inducer and
efficacious anticancer agent with high blood brain barrier penetration. J. Med. Chem. 52
(2009) 2341-2351.
[
[
19] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J. Immunol. Methods 65 (1983) 55-63.
20] M. Banimustafa, A. Kheirollahi, M. Safavi, S. Kabudanian Ardestani, H. Aryapour, A.
Foroumadi, S. Emami Synthesis and biological evaluation of 3-(trimethoxyphenyl)-2(3H)-
thiazole thiones as combretastatin analogs. Eur. J. Med. Chem. 70 (2013) 692-702.
1
7

ACCEPTED MANUSCRIPT
Captions:
Figure 1. Design of new compounds 6a-t as anticancer agents
Figure 2. Morphological analysis of MOLT-4 cells by double staining method. a) Control
condition; b) cells treated with IC50 concentration of Cisplatin c) cells treated with IC50
concentration of compound 6l for 24 h. White arrow indicates live cells, dash arrow shows
apoptosis. The images of cells were taken with a fluorescence microscope at 400x.
Figure 3. Morphological analysis of A549 cells by double staining method. a) Control
condition; b) cells treated with IC50 concentration of Cisplatin c) cells treated with IC50
concentration of compound 6e for 24 h. White arrow indicates live cells, dash arrow shows
apoptosis.
Figure 4. Flow cytometric analysis of MOLT-4 cell line treated with compound 6l. Cells
were stained with Annexin V-FITC/PI and quantitated by flow cytometry. The cells treated
with a) DMSO 1 % (negative control); b) IC50 concentration of Cisplatin; C) IC50
concentration of compound 6l.
Figure 5. Flow cytometric analysis of A549 cell line treated with compound 6e. Cells were
stained with Annexin V-FITC/PI and quantitated by flow cytometry. The cells treated with a)
DMSO 1 % (negative control); b) IC concentration of Cisplatin; C) IC concentration of
5
0
50
compound 6e.
Scheme 1. Synthesis of compounds 6a-t.
1
8

ACCEPTED MANUSCRIPT
Table 1. Cytotoxic activities (IC values, µM, Mean ± SEM) of compounds 6a-t against cancer cell
5
0
lines.
R1
R2
HN
N
N
O
1
2
Compound
R
R
A549
MOLT-4
HeLa
6
a
H
H
H
H
H
12.5 ± 2.6
17.4 ± 2.9
13.7 ± 1.8
> 100
6.6 ± 0.2
17.6 ± 3.1
16.4 ± 1.2
29.4 ±3.2
12.8 ± 0.4
6.4 ± 0.3
6
b
14.5 ± 1.3
19.9 ± 2.6
31.6 ± 3.0
10.3 ± 1.9
CH CH
2
3
6
6
6
c
d
e
Cl
F
5.4 ± 0. 2
3
H C
6
6
f
H
H
11.2 ± 1.6
10.9 ± 0.5
8.9 ± 0.2
6.5 ± 0.1
14.6 ± 2.3
15.9 ± 2.2
CH
3
H CO
g
3
OCH
3
6
6
h
i
H
H
10.9 ± 2.2
9.6 ± 1.8
12.7 ± 1.3
11.7 ± 1.0
5.9 ± 1.4
15.4 ± 2.6
Cl
6
6
j
H
H
68.5 ± 4.4
18.9 ± 2.6
16.9 ± 1.2
14.5 ± 1.7
50.2 ± 2.8
43.5 ± 3.4
OCH
3
k
OCH3
F
6
l
2,5-(OCH3)2
6.3 ± 0.9
5.2 ± 0.02
6.9 ± 1.1
6
6
6
6
6
6
6
m
n
o
p
q
r
2,5-(OCH3)2
2,5-(OCH3)2
2,5-(OCH3)2
2,5-(OCH3)2
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
8.4 ± 1.3
14.8 ± 2.1
26.8 ± 2.4
7.2 ± 1.8
18.6 ± 4.9
46.5 ± 5.4
26.5 ± 3.7
6.8 ± 0.2
7.4 ± 1.4
20.4 ± 1.1
6.9 ± 1.5
9.8 ± 2.9
33.5 ± 4.2
10.4 ± 1.2
10.5 ± 1.7
15.9 ± 3.0
16.7 ± 2.3
10.6 ± 1.7
20.7 ± 4.5
>100
OCH3
s
44.6 ± 4.9
33.2 ± 3.2
6
t
3,4,5-(OCH3)3
-
22.2 ± 3.0
3.9 ± 0.2
14.5 ± 1.5
4.6 ± 0.7
Cl
Cisplatin
-
3.2 ± 0.4
1
9

ACCEPTED MANUSCRIPT
OMe
R1
MeO
Br
OMe
R2
N
HN
N
N
N
CN
O
O
NH2
N
R
B
6
a-t
(
I)
(II)
B= fused ring
Figure 1. Design of new compounds 6a-t as anticancer agents
2
0

ACCEPTED MANUSCRIPT
Figure 2. Morphological analysis of MOLT-4 cells by double staining method. a) Control
condition; b) cells treated with IC50 concentration of Cisplatin c) cells treated with IC50
concentration of compound 6l for 24 h. White arrow indicates live cells, dash arrow shows
apoptosis. The images of cells were taken with a fluorescence microscope at 400x.
2
1

ACCEPTED MANUSCRIPT
Figure 3. Morphological analysis of A549 cells by double staining method. a) Control
condition; b) cells treated with IC50 concentration of Cisplatin c) cells treated with IC50
concentration of compound 6e for 24 h. White arrow indicates live cells, dash arrow shows
apoptosis.
2
2

ACCEPTED MANUSCRIPT
Figure 4. Flow cytometric analysis of MOLT-4 cell line treated with compound 6l. Cells
were stained with Annexin V-FITC/PI and quantitated by flow cytometry. The cells treated
with a) DMSO 1 % (negative control); b) IC50 concentration of Cisplatin; C) IC50
concentration of compound 6l
.
2
3

ACCEPTED MANUSCRIPT
Figure 5. Flow cytometric analysis of A549 cell line treated with compound 6e. Cells were
stained with Annexin V-FITC/PI and quantitated by flow cytometry. The cells treated with a)
DMSO 1 % (negative control); b) IC concentration of Cisplatin; C) IC concentration of
5
0
50
compound 6e
.
2
4

ACCEPTED MANUSCRIPT
R1
R1
OH
5
4
3
CN
CN
Piperidine
6
DMF-DMA
reflux, 2h
+
+
Ethanol
Reflux
CN
7
8
O
2
NH2
O
H
1
1
2
10
3
a-c
9
4a-c
R1
R1
R2
HN
8
6
7
CN
N
2
5
R NH , AcOH
N9
10
2
CH3
4
MW, 5-15 min
O
N
O
N
1
1
3
CH3
1
5
a-c
2
6a-t
Scheme 1. Synthesis of compounds 6a-t
.
2
5

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
•
Some tetracyclic chromenopyrimidines were synthesized as potential anticancer agents.
The cytotoxic activity was tested against A549, MOLT-4, and HeLa cell lines.
2-Fluoroaniline derivatives 6e and 6l showed superior activity on A549 and MOLT-4.
The benzylamine analog 6h was the most potent compound against HeLa cells.
Flow cytometric analyses showed that 6l can induce apoptosis in MOLT-4 cells.