Synthesis of novel chromene derivatives of expected antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2012.11.011

Inhibition of tubulin polymerization is one of the important tactics in cancer therapy. Since 4-aryl-4H-chromene derivatives are found to be microtubule-binding agents via interfering with tubulin polymerization so we decide to concentrate our exploration efforts on the combination of this nucleus with 5-, 6-, and/or 7-memebered heterocyclic moieties in a novel series of compounds to explore the effect that might result from this combination. Ten novel compounds were selected for anticancer screening assay against MCF-7 breast cancer cell line in comparison to colchicine as positive control and most of them showed excellent activity.

Full text of DOI:10.1016/j.ejmech.2012.11.011

European Journal of Medicinal Chemistry 59 (2013) 183e193
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel chromene derivatives of expected antitumor activity
,
b
b
Manal M. Kandeel a, Aliaa M. Kamal a , Eman K.A. Abdelall , Heba A.H. Elshemy
*
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11561 Egypt
b Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2012
Received in revised form
6 November 2012
Accepted 11 November 2012
Available online 17 November 2012
Inhibition of tubulin polymerization is one of the important tactics in cancer therapy. Since 4-aryl-4H-
chromene derivatives are found to be microtubule-binding agents via interfering with tubulin poly-
merization so we decide to concentrate our exploration efforts on the combination of this nucleus with
5-, 6-, and/or 7-memebered heterocyclic moieties in a novel series of compounds to explore the effect
that might result from this combination. Ten novel compounds were selected for anticancer screening
assay against MCF-7 breast cancer cell line in comparison to colchicine as positive control and most of
them showed excellent activity.
Keywords:
Ó 2012 Elsevier Masson SAS. All rights reserved.
Microtubule-binding agents
4-Aryl-4H-chromenes
Heterocyclic moieties
Breast cancer
Cytotoxic activity
1. Introduction
destabilizers, binding at or nearly at the binding site of colchicine.
They were also active in drug-resistant cancer cell lines and highly
Microtubule-binding agents (MBAs) are widely used in cancer
chemotherapy [1]. There are two classes of MBAs; those that
stabilize microtubules and promote polymerization, and those that
destabilize the microtubules and promote depolymerization. Both
types interfere with the mitotic spindle assembly during cell divi-
sion, resulting in cell death. Colchicine (I), podophyllotoxin (II), and
combretastatin A-4 (III) (Fig. 1) are representatives of compounds
that inhibit microtubule assembly [2,3] and share a common
structural feature; two aromatic rings directly bonded or separated
by 1e4 carbon atoms, in such a way that they are close in space but
not coplanar [4].
active as single agents or in combination with other anticancer
agents in several tumor models, so they could be developed into
new therapeutic anticancer regimes [15e19]. On the other hand,
numerous triazolo, triazino and triazepino pyrimidine derivatives
have been reported as cell cycle arrest agents that act via interfering
with the polymerization [20,21] or depolymerization [22] of
microtubules assembly leading to cell death. Prompted by these
findings, we intended to synthesize novel series of compounds
containing both 4-aryl-4H-chromenes and triazolo, triazino and
triazepino pyrimidine moieties to explore the synergistic effect that
might result from this combination (Schemes 1e5).
A major turning point in the development of MBAs as antitumor
agents is the identification of the natural compound namely com-
bretastatin A-4 (III) [4]. Compound III strongly inhibits the poly-
merization of tubulin by binding to the colchicine binding site [2,3].
Besides, it can inhibit angiogenesis, a process essential for tumor
growth [5e7]. The synthesis of different 4-arylcoumarin analogs of
combretastatin A-4 (III) lead to the discovery of promising series of
potent apoptosis inducing agents possessing vascular targeting
activity [8e14]. These compounds were found to be tubulin
2. Results and discussion
2.1. Chemistry
2-Amino-4-(4-chlorophenyl)-7-hydroxy-4H-chromene-3-
carbonitrile (1) was synthesized according to the reported method
[23,24] and alkylated using methyl or ethyl iodide to afford
compounds 2a and 2b. The IR spectra of 2a and 2b assured the
presence of both amino functionality and cyano group at 3432 (2a),
3332 (2b) cmꢀ1 and 2196 (2a), 2185 (2b) cmꢀ1 respectively.
Meanwhile, 1H NMR spectra of compounds 2a and 2b revealed the
* Corresponding author. Tel.: þ20 1280007280 (mobile); fax: þ20 2 23628426.
presence of one exchangeable singlet signal at
d 6.98 and 6.54 ppm
E-mail
addresses:
K_aliaa2511@hotmail.com,
K_aliaa2@hotmail.com
(A.M. Kamal).
respectively corresponding to NH2 protons. The 13C NMR for
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.011

184
M.M. Kandeel et al. / European Journal of Medicinal Chemistry 59 (2013) 183e193
OH
O
CH3
O
O
O
O
H3C
H3C
O
O
O
NHCOCH3
O
H3C
CH3
CH3
O
O
O
CH3
OH
CH3
O
CH3
CH3
O
O
CH3
O
(I)
(II)
Fig. 1. Colchicine I, podophyllotoxin II, and combretastatin A-4 III.
(III)
compound 2a disclosed three signals at
114.2 ppm corresponding to the stereogenic carbon of the chro-
mene nucleus, CH3 and CN, sequentially.
Compounds 2a and 2b were reacted with triethyl orthoformate
to give 3a and 3b. IR spectra of compounds 3a and 3b revealed the
disappearance of NH2 group while, their 1H NMR spectra showed
d
39.9 ppm,
d
55.5 ppm and
4b showed appearance of D2O exchangeable broad signal at
d 4.74 ppm assigned to two protons of NH2 group. It is worth to
d
mention that, upon hydrazinolysis of 3a and 3b using excess
hydrazine hydrate 99% instead of equimolar amounts, compounds
2a and 2b were recovered. This may be attributed to, after the
reaction of 1 mol of hydrazine with the iminoether 3a and 3b
a preferential elimination of ethyl formate hydrazone rather than
cyclization to give the pyrimidine ring took place (Fig. 2).
a triplet at
d
1.39, 1.36 ppm (OCH2CH3), a quartet at
d 4.43, 3.49 ppm
(OCH2CH3) and a singlet at
d
8.30, 8.38 ppm indicated the presence
of imino group (N]CH). Interaction of 3a and 3b with equimolar
amount of either methylamine or hydrazine hydrate in absolute
ethanol at ambient temperature gave the key intermediates 4ae
d (Scheme 1).
The structures of compounds 4aed were established on the
basis of elemental analyses and spectral data. IR spectra of
compounds 4aed showed no absorption band for CN group
meanwhile revealed characteristic absorption bands at 3336e
3156 cmꢀ1 indicating the presence of NH, NH2 functionality.
Furthermore, 1H NMR spectra of compounds 4aed revealed the
Schemes 2e5 summarizes the synthesis of the tri-
azolochromenopyrimidines with different substitution at C2 using
different one-carbon donor cyclizing agents to react with the key
intermediates 4b and 4d. Cyclization of 4b and 4d with either
triethyl orthoformate, acetic anhydride or diethyl oxalate gave the
corresponding 1,2, 4-triazolo[1,5-c]pyrimidines 5a and 5b; 5c and
5d and 5e and 5f respectively. Neither IR nor 1H NMR spectra
supported the presence of the NH or NH2 protons that existed in the
precursor 4b and 4d. IR spectra of compounds 5e and 5f showed
a band at 1741, 1745 cmꢀ1 confirming the presence of (C]O) of the
ester moiety. Moreover, the 1H NMR spectra of 5aef showed two
appearance of singlet signals at
d 7.79e8.17 ppm corresponding to
C2H of the pyrimidine ring. 1H NMR spectrum of compound 4a
singlet signals at
d 5.63, 5.81 ppm and d 9.00, 9.75 ppm indicating
revealed two singlets at
d
3.40 and 3.77 ppm assigned to NeCH3
C12H and C5H, respectively. The increased chemical shift for these
signals-compared to their values in tricyclic system 4b and 4d
and OeCH3 protons, respectively, 1H NMR spectrum of compound
Cl
Cl
Cl
CN
CN
CN
N
RI
CH(OC2H5)3
HO
O
NH2
RO
O
NH2
CH3
RO
O
O
1
and
Cl
and
3a
3b
2a
2b
R = CH
R = C H
2a
R1NH2
3
2b
2
5
NH
N
R1
N
O
OR
4a-d
Scheme 1. Synthesis of compounds 2a and 2b, 3a and 3b and 4aed.

M.M. Kandeel et al. / European Journal of Medicinal Chemistry 59 (2013) 183e193
185
Cl
2
1
N
11
8
CH(OC2H5)3
3
12
or
N
10
N
and
4b
4d
4
(C2H5O) CH = C (CN) COOC2H5
5
9
N
6
O
7
OR
R = CH3, C2H5
R1 = NH2
and
5a
5b
(C2H5O)CH=C(CN)COOC2H5
N
N
N
COOEt
NH2
O
N
RO
Cl
Compounds
A
Scheme 2. Synthesis of compounds 5a and 5b.
d
4.88e5.22(C5H)and
deshielding effect of the diamagnetic current of the aryl
The 1H NMR spectra of compounds 5a and 5b revealed a singlet
signal at
8.28 ppm which was assigned to the C2H. While, 1H NMR
spectra of 5c and 5d, the methyl protons (C2eCH3) were found as
a singlet signal at
2.55, 2.57 ppm. Also, the 1H NMR spectra of
d
8.09e8.11(C2H)ppm-canbeattributedtothe
A suggested mechanism for the formation of 5g and 5h is shown
(Fig. 5).
p-electrons.
Preparation of compounds D was intended to be via the reaction
of the key intermediates 4b and 4d with different chloro acid
chlorides but interestingly, treating compounds 4b and 4d with
equimolar amount of chloro acid chloride in dioxane containing
catalytic amount of triethylamine did afford neither the starting
materials nor compounds D but compounds 6aed were the prod-
ucts and this might be ascribed to the stability and aromaticity of
the triazole ring system over the triazepine ring system (Scheme 5).
The structure of 6aed was suggested by different spectroscopic
data. IR spectra displayed the absence of absorption bands corre-
sponding to (C]O and NH) groups existing in the compounds D. 1H
NMR spectra of compounds 6a and 6c showed a singlet signal at
d
d
5e and 5f showed triplet and quartet signals corresponding to
ester protons.
In the course of this study, attempts of synthesis of chromeno-
pyrimidinotriazepines A via reacting compounds 4b and 4d with
ethyl ethoxymethylene cyanoacetate were not successful. Instead,
this reaction gave a product which was identical in all respects
(m.p., mixed m.p., IR and 1H NMR) with compounds 5a and 5b
(Scheme 2).
The suggested mechanism for the reaction of 4b and 4d with
ethyl ethoxymethylene cyanoacetate might be illustrated in Fig. 3.
Reacting compounds 4b and 4d with acetyl acetone for
obtaining compounds B was unsuccessful and compounds 5c and
5d were resulted instead (Scheme 3).
d
4.73 assigned to two protons of (CH2Cl). Inspection of the 1H NMR
spectra of compounds 6b and 6d revealed the presence of pentet
and two triplet signals at 2.22e2.38, 3.06e3.07 and 3.66 ppm due
d
to (CH2eCH2eCH2) protons respectively (Scheme 5). Compounds
7aef were prepared from the interaction of the key intermediates
4b and 4d with different arylidene malononitriles in the presence
of piperidine under reflux in absolute ethanol. The structure of the
given compounds was consistent with the IR, 1H NMR and mass
spectra. The absence of NH group as well as NH2 group from the IR
and 1H NMR spectra of 7aef showed the increase in aromatic
The suggested mechanism for the reaction of 4b and 4d with
acetyl acetone might be illustrated in Fig. 4.
An attempt to prepare hydroxypyrimidotriazines C via heating
compounds 4b and 4d with ethyl cyanoacetate in refluxing ethanol
took place but unfortunately different products were obtained.
These compounds were suggested to be the triazolopyrimidines 5g
and 5h according to all available spectroscopic data and elemental
analysis.
protons at
C12H and C5H of compounds 7aef showed increase in chemical
shift at 5.72e5.76 (C12H) and 9.05e9.09 (C5H) ppm e compared
to their values in tricyclic system 4b and 4d 4.88e5.22 (C5H) and
8.09e8.11 (C2H) ppm e and this can be attributed to the
d 6.71e8.28 ppm confirmed their structures. Once more
d
d
The IR spectra showed the presence of cyano group at 2251,
d
2259 cmꢀ1. Furthermore, 1H NMR spectra revealed a singlet signal
d
at
d
4.00 corresponding to two protons (CH2CN) and no signals
deshielding effect of the diamagnetic current of the aryl
electrons.
p-
corresponding to OH either in IR or 1H NMR spectra.

186
M.M. Kandeel et al. / European Journal of Medicinal Chemistry 59 (2013) 183e193
Cl
H3C
N
or
Ac2O
N
and
4 b
4d
N
CH3COCH2COCH3
R = CH3, C2H5
R1 = NH2
N
O
OR
and
5c
5d
CH3COCH2COCH3
CH3
CH3
N
N
N
O
N
Cl
RO
Compounds
B
Scheme 3. Synthesis of compounds 5c and 5d.
2.2. Antitumor activity
chromenes and different triazolopyrimidinyl moieties and investi-
gate the anticancer activity of these new compounds. It is worth to
mention, many of unexpected compounds were resulted during the
reaction of the key intermediates 4b and 4d with different organic
reagents and this may be attributed to the electronic nature of this
aminocyanochromene ring system where the amino group is a very
weak basic center and carbon number 2 is carrying a highly positive
charge. The preliminary biological evaluations revealed that the
newly synthesized chromene derivatives 4a, 4b, 5f, 7c and 7f
showed superior in vitro antitumor activity when compared to
colchicine as a reference drug. We might conclude that, this
superior in vitro anticancer activity and good potential of causing
cancer cells death might be attributed to their structure similarity
to colchicine (I) and podophyllotoxin (II) (Fig. 1).
In this study, ten of the newly synthesized compounds were
subjected to cytotoxic evaluation against human breast tumor cell
line (MCF-7) using Colchicine as the reference drug. The response
parameter calculated was IC50 value (Table 1), which corresponds
to the compound concentration causing 50% mortality in net cells.
Compounds 4a, 4b, 5f, 7c and 7f turned to be particularly
promising for the development of new pharmacological inhibitors
for breast tumor cells. The most promising results were obtained
with compounds 4a and 5f (IC50 ¼ 0.007
m
M) that showed almost
double the activity of colchicine (IC50 ¼ 0.013
mM) (Fig. 6).
Three of the tested compounds 4b, 7c and 7f (IC50 ¼ 0.008
mM)
(Fig. 7) showed nearly one and half the activity of colchicine.
Compounds 5a and 5h (IC50 ¼ 0.011
equal inhibitory effect on breast tumor cells as than the positive
control (IC50 ¼ 0.013 M).
Moreover, compound 6d (IC50 ¼ 0.029
nearly half the activity of the positive control. Finally compounds 5c
mM) (Fig. 8) showed almost
4. Experimental protocols
m
m
M) (Fig. 9) revealed
4.1. Chemistry
and 5g (IC50 ¼ 0.033
mM, 0.039 mM) (Fig. 10) are the least reactive
Progress of the reactions was monitored using thin layer chro-
matography (TLC) sheets that precoated with UV fluorescent silica
gel MERCK 60 F 254 that was visualized by UV lamp and I2 vapor.
Solvent system was chloroform: methanol (in different ratios). 1H
NMR and 13C-NMR spectra were carried out on Varian Gemini
300 MHz Spectrometer, at the Microanalytical Center, Cairo
University, Egypt. Using TMS as internal standard and chemical
ones.
3. Conclusion
In summary, the objective of the present study was to design,
synthesize library of novel hybrid compounds comprising
a

M.M. Kandeel et al. / European Journal of Medicinal Chemistry 59 (2013) 183e193
187
Cl
EtOOC
N
N
(COOC2H5)2
N
and
4b
4d
R = CH3, C2H5
R1= NH2
N
O
OR
and
5e
5f
CNCH2COOC2H5
Cl
N
CNCH2COOC2H5
N
OH
H
N
N
N
N
N
O
N
N
O
OR
Cl
and
5g
5h
RO
Compounds
C
Scheme 4. Synthesis of compounds 5eeh.
shifts were recorded in ppm on
d
scales. IR spectra were recorded
1H, ArH);6.66(d, J¼ 8.7 Hz,1H, ArH);6.90(d, J¼ 8.7Hz,1H, ArH);6.98
(s, 2H, NH2, D2O exchangeable); 7.18 (d, J ¼ 8.1 Hz, 2H, ArH); 7.35 (d,
on a Shimadzu 435 Spectrometer, using KBr discs and values were
represented in cmꢀ1. GC Mass spectra were run on Shimadzu QP-
2010 spectrometer and Mass spectra were run on Hewlett Pack-
ard 5988 spectrometer at the Microanalytical Center, Cairo
University, Egypt and National Research Center, Giza, Egypt.
Melting points were determined on a Griffin instrument and are
uncorrected. All products reported showed 1H NMR spectra in
agreement with the assigned structures. Elemental Analyses were
performed by the Micro-analytical Laboratory, Cairo University,
Egypt. Compound 1 was prepared according to reported method
[23,24].
J ¼ 8.1 Hz, 2H, ArH) ppm; 13C NMR (DMSO-d6):
d 39.9, 55.5, 76.5,101.4,
111.7, 114.2, 119.5, 128.9, 129.2, 130.1, 133.0, 143.2, 149.1, 159.0, 159.5;
EIMS: m/z (%) ¼ 314 (M þ 2
, 2.8), 312 (M
, 24.3); Anal. Calcd. for C17H13ClN2O2
, 8.4), 201 (Me
C6H4Cl , 100), 75 (C6H3
(312.76): C 65.29, H 4.19, N 8.96; Found: C 65.50, H 4.31, N 9.16%.
4.1.1.2. (RS)-2-Amino-4-(4-chlorophenyl)-7-ethoxy-4H-chromene-3-
carbonitrile (2b). Yield 85%. mp 181e182 ꢁC. IR (KBr): 3424, 3332
(forked, NH2), 3070 (CH arom.), 2976 (CH aliph.), 2196(CN) cmꢀ1
;
1H NMR (DMSO-d6):
d 1.29 (t, 3H, CH3); 3.98 (q, 2H, CH2); 4.72 (s,
1H, C4H); 6.54 (s, 1H, ArH); 6.65 (d, 1H, ArH þ 2H, NH2, D2O
exchangeable); 6.90 (d, 1H, ArH); 7.18 (d, J ¼ 8.4 Hz, 2H, ArH); 7.36
(d, J ¼ 8.4 Hz, 2H, ArH) ppm; Anal. Calcd. for C18H15ClN2O2 (326.79):
C 66.16, H 4.63, N 8.57; Found: C 66.42, H 4.42, N 8.43%.
4.1.1. General procedure for the preparation of compounds 2a and
2b
A mixture of compound 1 (2.98 g, 0.01 mol) and potassium
hydroxide (0.56 g, 0.01 mol) in ethanol (20 mL) was stirred at room
temperature for 1 h, then the appropriate methyl or ethyl iodide
(0.015 mol) was added and the mixture was heated under reflux for
24 h. The reaction mixture was cooled, filtered and the formed
precipitate was washed with ethanol and crystallized from absolute
ethanol to yield 2a and 2b.
4.1.2. General procedure for the preparation of compounds (3a and
3b)
A mixture of compound 2a and 2b (0.01 mol) and triethyl
orthoformate (20 mL) was heated under reflux for 4 h. The excess of
orthoformate was removed under reduced pressure, and then the
residue was washed with ethanol and crystallized from the
appropriate solvent to give 3a and 3b.
4.1.1.1. (RS) 2-Amino-4-(4-chlorophenyl)-7-methoxy-4H-chromene-
3-carbonitrile (2a). Yield 84%. mp 167e168 ꢁC. IR (KBr): 3432, 3347
(forked, NH2), 3055 (CH arom.), 2960 (CH aliph.), 2185 (CN) cmꢀ1
;
4.1.2.1. Ethyl (RS) (ZE) N-4-(4-chlorophenyl)-3-cyano-7-methoxy-
4H-chromen-2-ylformimidate (3a). Crystallized from absolute
1H NMR (DMSO-d6):
d
3.72 (s, 3H, OCH3); 4.72 (s, 1H, C4H); 6.55 (s,

188
M.M. Kandeel et al. / European Journal of Medicinal Chemistry 59 (2013) 183e193
4.1.3.2. (ZE) (RS)-5-(4-Chlorophenyl)-4,5-dihydro-4-imino -8-
methoxy-3H-chromeno[2,3-d]pyrimidin-3-amine (4b). Yield 93%.
mp 187e189 ꢁC. IR (KBr): 3336, 3156 (NH, NH2), 2928 (CH aliph.),
1639 (C]N) cmꢀ1; 1H NMR (CDCl3):
d 3.78 (s, 3H, OCH3); 4.74 (br s,
2H, NH2, D2O exchangeable); 4.88 (s, 1H, C5H); 6.60e7.36 (m, 7H,
ArH þ 1H, NH, D2O exchangeable); 8.09 (s, 1H, C2H) ppm Anal.
Calcd. for C18H15ClN4O2 (354.80): C 60.94, H 4.26, N 15.79; Found: C
60.76, H 4.20, N 15.49%.
4.1.3.3. (RS) (ZE) -5-(4-Chlorophenyl)-8-ethoxy-3-methyl-3H-chro-
meno[2,3-d]pyrimidin-4(5H)-imine (4c). Yield 84%. mp 169e170 ꢁC.
IR (KBr): 3324 (NH), 3028 (CH arom.), 2976 (CH aliph.), 1640 (C]N)
cmꢀ1; 1H NMR (CDCl3):
d
1.40 (t, J ¼ 7.2 Hz, 3H, CH2CH3); 3.59 (s, 3H,
NeCH3); 3.99 (q, J ¼ 7.2 Hz, 2H, CH2CH3); 5.64 (s, 1H, C5H); 6.32e
7.37 (m, 7H, ArH); 7.87 (s, 1H, NH, D2O exchangeable); 8.17 (s, 1H,
C2H) ppm; Anal. Calcd. for C20H18ClN3O2 (367.84): C 65.31, H 4.93, N
11.42; Found: C 65.54, H 4.81, N 11.12%.
4.1.3.4. (RS) (ZE) -5-(4-Chlorophenyl)-4-imino-4,5-dihydro-8-
ethoxy-3H-chromeno[2,3-d]pyrimidin-3-amine (4d). Yield 89%. mp
168e169 ꢁC. IR (KBr): 3333, 3180 (NH, NH2), 3087 (CH arom.), 2981
(CH aliph.), 1629 (C]N) cmꢀ1; 1H NMR (CDCl3):
d
1.29 (t, J ¼ 7.2 Hz,
3H, CH2CH3); 4.00 (q, J ¼ 7.2 Hz, 2H, CH2CH3); 5.22 (s, 1H, C5H);
6.65e7.36 (m, 7H, ArH þ 3H, NH, NH2, D2O exchangeable); 8.11 (s,
1H, C2H) ppm;. 13C NMR (CDCl3):
d 14.3, 25.7, 65.1, 95.0,103.2,108.9,
Scheme 5. Synthesis of compounds 6aed and 7aef.
123.1, 129.4, 129.7, 129.8, 131.8, 141.3, 157.0, 157.2, 163.0, 164.0;
EIMS: m/z (%) ¼ 370 (M þ 2 ,11.63), 368 (M , 33.67), 352 (Me
NH2 , 100); Anal. Calcd. for C19H17ClN4O2 (368.83): C 61.88, H
ethanol. Yield 88%. mp 131e132 ꢁC. IR (KBr): 3068 (CH arom.), 2965
(CH aliph.), 2200 (CN),1644 (C]N) cmꢀ1; 1H NMR (CDCl3):
1.39 (t,
4.65, N 15.19; Found: C 61.90, H 4.70, N 14.91%.
d
4.1.4. General procedure for the preparation of compounds 5a and
5b
A solution of 4b and 4d (0.001 mol) and triethyl orthoformate
(10 mL) was heated under reflux for 6 h. The precipitated solid
formed after cooling were filtered, washed with ethanol, dried and
recrystallized from absolute ethanol to afford compounds 5a and 5b.
3H, CH2CH3); 3.80 (s, 3H, OCH3); 4.43 (q, 2H, CH2); 4.80 (s,1H, C4H);
6.60 (s, 1H, ArH); 6.65 (d, J ¼ 8.7 Hz, 1H, ArH); 6.84 (d, J ¼ 8.7 Hz,1H,
ArH); 7.14 (d, J ¼ 8.7 Hz, 2H, ArH); 7.29 (d, J ¼ 8.7 Hz, 2H, ArH); 8.38
(s, 1H, N]CH) ppm; Anal. Calcd. for C20H17ClN2O3 (368.82): C 65.13,
H 4.65, N 7.60; Found: C 65.28, H 4.66, N 7.80%.
4.1.2.2. (ZE) (RS)-Ethyl N-4-(4-chlorophenyl)-3-cyano-7-ethoxy-4H-
chromen-2-ylformimidate (3b). Crystallized from methanol. Yield
61%. mp 135e136 ꢁC. IR (KBr): 3067 (CH arom.), 2981 (CH aliph.),
4.1.4.1. (RS)-12-(4-Chlorophenyl)-9-methoxy-12H-chromeno[3,2-e]
[1,2,4]triazolo[1,5-c]pyrimidine (5a). Yield 86%. mp 215e216 ꢁC. IR
(KBr): 3073 (CH arom.), 2932 (CH aliph.), 1626 (C]N) cmꢀ1 1H
;
2208 (CN), 1619 (C]N) cmꢀ1; 1H NMR (CDCl3):
d 1.28e1.36 (m, 6H,
NMR (CDCl3):
d 3.84 (s, 3H, OCH3); 5.67 (s, 1H, C12H); 6.73 (d,
2CH2CH3); 3.94 (q, 2H, OCH2CH3); 4.35 (q, 2H, N]CHOCH2CH3);
4.71 (s, 1H, C4H); 6.49 (s, 1H, ArH); 6.54 (d, J ¼ 8.4 Hz, 1H, ArH); 6.74
(d, J ¼ 8.4 Hz, 1H, ArH); 7.06 (d, J ¼ 8.4 Hz, 2H, ArH); 7.20 (d,
J ¼ 8.4 Hz, 2H, ArH); 8.30 (s, 1H, N]CH) ppm; 13C NMR (CDCl3):
J ¼ 8.4 Hz, 1H, ArH); 6.85 (s, 1H, ArH); 7.06 (d, J ¼ 8.4 Hz, 1H, ArH);
7.23 (d, J ¼ 8.7 Hz, 2H, ArH); 7.27 (d, J ¼ 8.7 Hz, 2H, ArH); 8.28 (s, 1H,
C2H); 9.14 (s, 1H, C5H) ppm; Anal. Calcd. for C19H13ClN4O2 (364.79):
C 62.56, H 3.59, N 15.36; Found: C 62.70, H 3.40, N 15.56%.
d
14.3, 14.4, 32.2, 62.9, 65.2, 62.8, 95.0, 103.3, 108.9, 123.3, 129.4,
129.7, 129.8, 131.5, 141.3, 156.5, 157.0, 157.3, 163.6; EIMS: m/z
(%) ¼ 384 (M þ 2 , 7.31), 382 (M , 20.40), 271 (Me
C6H4Cl , 100); Anal. Calcd. for C21H19ClN2O3 (382.85): C 65.88,
4.1.4.2. (RS)-12-(4-Chlorophenyl)-9-ethoxy-12H-chromeno[3,2-e]
[1,2,4]triazolo[1,5-c]pyrimidine (5b). Yield 81%. mp 177e178 ꢁC. IR
(KBr): 3077 (CH arom.), 2979 (CH aliph.), 1626 (C]N) cmꢀ1 1H
;
H 5.00, N 7.32; Found: C 66.09, H 4.80, N 7.12%.
NMR (CDCl3):
d
1.44 (t, J ¼ 7.2 Hz, 3H, CH2CH3); 4.06 (q, J ¼ 7.2 Hz,
2H, CH2CH3); 5.67 (s,1H, C12H); 6.72 (d, J ¼ 8.4 Hz,1H, ArH); 6.84 (s,
1H, ArH); 7.05 (d, J ¼ 8.4 Hz, 1H, ArH); 7.24 (d, J ¼ 8.1 Hz, 2H, ArH);
7.29 (d, J ¼ 8.1 Hz, 2H, ArH); 8.28 (s,1H, C2H); 9.14 (s, 1H, C5H) ppm;
4.1.3. General procedure for the preparation of compounds 4aed
A solution of 3a and 3b (0.01 mol) and either methylamine or
hydrazine hydrate (99%, 0.01 mol) in absolute ethanol (30 mL) was
stirred at room temperature for 1 h. The solid formed was filtered
and crystallized from benzene to give 4aed.
13C NMR (CDCl3):
d 29.8, 56.0, 107.2, 111.6, 118.5, 127.6, 129.1, 129.6,
129.9, 130.8, 132.0, 144.1, 147.2, 147.9, 155.5, 160.8; EIMS: m/z
(%) ¼ 380 (M þ 2 , 8.27), 378 (M , 22.04), 267 (Me
C6H4Cl , 100); Anal. Calcd. for C20H15ClN4O2 (378.82): C 63.41,
4.1.3.1. (ZE) (RS)-5-(4-Chlorophenyl) 8-methoxy-3-methyl-3H-chro-
H 3.99, N 14.79; Found: C 63.30, H 3.80, N 14.49%.
meno[2,3-d]pyrimidin-4(5H)-imine (4a). Yield 95%. mp 12ꢀ81e
129 ꢁC. IR (KBr): 3221 (NH), 2966 (CH aliph.), 1643 (C]N) cm
;
1H NMR (CDCl3):
1H, C5H); 6.59e7.28 (m, 7H, ArH þ 1H, NH, D2O exchangeable);
7.79 (s, 1H, C2H) ppm; EIMS: m/z (%) ¼ 355 (M þ 2 , 10.41), 353
(M , 28.27), 338 (MeCH3 , 29.34), 105 (MeC12H11ClN3O
d 3.40 (s, 3H, NeCH3); 3.77 (s, 3H, OCH3); 4.86 (s,
4.1.5. General procedure for the preparation of compounds 5c and
5d
A mixture of 4b and 4d (0.001 mol) and acetic anhydride (10 mL)
was heated under reflux for 6 h. The precipitated solid formed after
cooling were filtered, washed with ethanol, dried and recrystallized
to afford compounds 5c and 5d.
,
100); Anal. Calcd. for C19H16ClN3O2 (353.81): C 64.50, H 4.56, N
11.88; Found: C 64.63, H 4.49, N 11.58%.

M.M. Kandeel et al. / European Journal of Medicinal Chemistry 59 (2013) 183e193
189
Cl
Cl
CN
CN
NH
..
N2H4
NHNH2
RO
O
N
OC2H5
RO
O
OC2H5
7a and 7b
- H2NN=CHOC2H5
R = CH
7a
7b
3
R = C H
2
5
Cl
CN
R = CH
2a
2b
3
R = C H
2
5
RO
O
2b
NH2
and
2a
Fig. 2. The suggested mechanism for the reaction of compounds 3a and 3b with excess hydrazine hydrate.
4.1.5.1. (RS)-12-(4-Chlorophenyl)-2-methyl-9-methoxy-12H-chro-
meno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5c). Crystallized from
methanol. Yield 85%. mp 231e232 ꢁC. IR (KBr): 3061 (CH arom.),
19.72), 267 (MeC6H4Cl
(378.82): C 63.41, H 3.99, N 14.79; Found: C 63.50, H 4.10, N 14.70%.
, 100); Anal. Calcd. for C20H15ClN4O2
2960 (CH aliph.), 1628 (C]N) cmꢀ1; 1H NMR (CDCl3):
d
2.57 (s, 3H,
4.1.5.2. (RS)-12-(4-Chlorophenyl)-9-ethoxy-2-methyl-12H-chromeno
[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (5d). Crystallized from meth-
anol: chloroform (1:1). Yield 89%. mp 214e215 ꢁC. IR (KBr): 3060
C2CH3); 3.84 (s, 3H, OCH3); 5.65 (s, 1H, C12H); 6.73 (d, J ¼ 8.7 Hz,
1H, ArH); 6.85 (s, 1H, ArH); 7.07 (d, J ¼ 8.7 Hz, 1H, ArH); 7.24 (d,
J ¼ 8.4 Hz, 2H, ArH); 7.31 (d, J ¼ 8.4 Hz, 2H, ArH); 9.01 (s, 1H,
(CH arom.), 2981 (CH aliph.), 1626 (C]N) cmꢀ1 1H NMR (CDCl3):
;
C5H) ppm; EIMS: m/z (%) ¼ 380 (M þ 2
, 7.06), 378 (M
,
d
1.43 (t, J ¼ 7.2 Hz, 3H, CH2CH3); 2.55 (s, 3H, C2CH3); 4.05 (q,
NC
COOC2H5
NH
Cl
HN
N
H2N
NH
Cl
N
N
H5C2O
N
- C2H5OH
+
O
O
H5C2OOC
CN
and
4b
4d
OR
R = CH
4b
4d
Cl
3
OR
Cl
R = C H
2
5
N
CN
H5C2OOC
N
_
N
- C2H5OOC-CH-CN
NH
- H+
N
O
5b
OR
N
N
5a
and
R = CH
5a
3
N
O
OR
R = C H
5b
2
5
Fig. 3. The suggested mechanism for the reaction of 4b and 4d with ethyl ethoxymethylene cyanoacetate.

190
M.M. Kandeel et al. / European Journal of Medicinal Chemistry 59 (2013) 183e193
Cl
CH3
N
N
O
..
NH
H2N
N
N
..
NH
N
O
CH3
CH
O
3
- H2O
Cl
O
O
CH
3
+
OR
4d
and
4b
RO
R = CH
4b
_
R = C H3
4d
2
5
- CH2COCH3
- H+
Cl
H3C
N
N
N
N
O
OR
and
5c
5d
R = CH
5c
5d
3
R = C H
2
5
Fig. 4. The suggested mechanism for the reaction of 4b and 4d with acetyl acetone.
J ¼ 7.2 Hz, 2H, CH2eCH3); 5.63 (s, 1H, C12H); 6.71 (d, J ¼ 8.4 Hz, 1H,
ArH); 6.83 (s, 1H, ArH); 7.05 (d, J ¼ 8.4 Hz, 1H, ArH); 7.21 (d,
J ¼ 8.4 Hz, 2H, ArH); 7.29 (d, J ¼ 8.4 Hz, 2H, ArH); 9.00 (s, 1H,
281 (MeC6H4Cl
64.21, H 4.36, N 14.26; Found: C 64.10, H 4.50, N 13.96%.
, 100); Anal. Calcd. for C21H17ClN4O2 (392.85): C
C5H) ppm; 13C NMR (CDCl3):
d
14.2, 29.8, 65.3, 107.3, 110.6, 118.2,
4.1.6. General procedure for the preparation of compounds 5eeh
A mixture of 4b and 4d (0.001 mol) and either diethyl oxalate or
ethyl cyanoacetate (0.001 mol) in absolute ethanol (15 mL) was
127.2,129.4,129.8,130.0,131.3,142.1,147.8,153.5,154.8,157.3,160.4,
171.6; EIMS: m/z (%) ¼ 394 (M þ 2
, 8.34), 392 (M
, 22.62),
CN
NH
Cl
..
O
..
NH
N
H2N
NH
N
O
N
N
- C2H5OH
Cl
COOC2H5
O
NC
+
OR
RO
4b and 4d
- H2O
Cl
N
R = C H
4d
2
5
R = CH
4b
3
N
N
N
R = CH
5g
3
R = C H
5h
OR
2
5
N
O
5g and 5h
Fig. 5. The suggested mechanism for the reaction of 4b and 4d with ethyl cyanoacetate.

M.M. Kandeel et al. / European Journal of Medicinal Chemistry 59 (2013) 183e193
191
Table 1
Cl
H CO
Results of in vitro cytotoxic activity of reference
drug and the tested compounds on human breast
tumor cell line (MCF-7).
Cl
Cpd no
IC50 mM
N
H
N
Colchicine
0.013
0.007
0.008
0.011
0.033
0.007
0.039
0.011
0.029
0.008
0.008
N
N
N
4a
4b
5a
5c
5f
5g
5h
6d
7c
7f
H
N
N
H
N
O
OR
O
OCH
7c and 7f
7c R = CH
3
4b
7f R = C2H
5
Fig. 7. Structures of compounds 4b, 7c and 7f.
IC50: concentration of a drug that is required for
50% inhibition.
J ¼ 8.4 Hz, 2H, ArH); 7.26 (d, J ¼ 8.4 Hz, 2H, ArH); 9.08 (s, 1H,
C5H) ppm; 13C NMR (CDCl3):
18.4, 29.5, 56.0, 107.4, 110.5, 114.9,
118.2, 127.9, 129.0, 129.4, 129.8, 130.4, 131.3, 141.1, 147.8, 153.5, 155.2,
160.5, 171.6; EIMS: m/z (%) ¼ 405 (M þ 2 , 9.51), 403 (M
25.87), 292 (MeC6H4Cl , 100); Anal. Calcd. for C21H14ClN5O2
heated under reflux for 4 h. After cooling, the solid formed was
collected and crystallized from the appropriate solvent to afford
compounds 5eeh.
d
,
4.1.6.1. (RS)-Ethyl 12-(4-chlorophenyl)-9-methoxy-12H-chromeno[3,2-
e][1,2,4]triazolo[1,5-c]pyrimidine-2-carboxylate (5e). Crystallized from
a mixture of methanol: benzene (3:1). Yield 75%. mp 229e230 ꢁC. IR
(KBr): 3067 (CH arom.), 2956 (CH aliph.), 1741 (C]O), 1626 (C]N)
(403.82): C 62.46, H 3.49, N 17.34; Found: C 62.51, H 3.59, N 17.06%.
4.1.6.4. (RS)-2-(12-(4-Chlorophenyl)-9-ethoxy-12H-chromeno[3,2-e]
[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)acetonitrile (5h).
cmꢀ1; 1H NMR (DMSO-d6):
d
1.32 (t, J ¼ 7.2 Hz, 3H, CH2CH3); 3.78 (s,
Crystallized from benzene Yield 78%. mp 218e219 ꢁC. IR (KBr):
3H, OCH3); 4.38 (q, J ¼ 7.2 Hz, 2H, CH2CH3); 5.81 (s, 1H, C12H); 6.77e
7.34 (m, 7H, ArH); 9.75 (s, 1H, C5H) ppm; EIMS: m/z (%) ¼ 438
3060 (CH arom.), 2982 (CH aliph.), 2251 (CN), 1628 (C]N) cmꢀ1; 1H
NMR (CDCl3):
d
1.44 (t, J ¼ 7.2 Hz, 3H, CH2CH3); 4.00 (s, 2H, CH2CN);
(M þ 2
, 11.99), 436 (M
, 31.30), 325 (MeC6H4Cl
, 100);
4.08 (q, J ¼ 7.2 Hz, 2H, CH2eCH3); 5.63 (s, 1H, C12H); 6.73 (d,
J ¼ 8.4 Hz, 1H, ArH); 6.84 (s, 1H, ArH); 7.05 (d, J ¼ 8.4 Hz, 1H, ArH);
7.21 (d, J ¼ 8.7 Hz, 2H, ArH); 7.29 (d, J ¼ 8.7 Hz, 2H, ArH); 9.08 (s, 1H,
C5H) ppm; Anal. Calcd. for C22H16ClN5O2 (417.85): C 63.24, H 3.86, N
16.76; Found: C 63.23, H 4.09, N 16.53%.
Anal. Calcd. for C22H17ClN4O4 (436.85): C 60.49, H 3.92, N 12.83;
Found: C 60.69, H 4.10, N 12.86%.
4.1.6.2. (RS)-Ethyl 12-(4-chlorophenyl)-9-ethoxy-12H-chromeno[3,2-
e][1,2,4]triazolo[1,5-c]pyrimidine-2-carboxylate (5f).
Crystallized from a mixture of methanol: benzene (3:1). Yield 60%.
mp 279e280 ꢁC. IR (KBr): 3063 (CH arom.), 2983 (CH aliph.), 1745
4.1.7. General procedure for the preparation of compounds 6aed
To a solution of 4b and 4d (0.004 mol) in dioxane (15 mL) was
added the appropriate acid chloride (0.004 mol), followed by the
addition of triethylamine (0.40 g, 0.004 mol). The reaction mixture
was stirred at room temperature for 24 h then poured into ice-cold
water. The solid product that formed was filtered, washed with
water, dried and crystallized from a mixture of ethanol: chloroform
(3:1) to give 6aed.
(C]O), 1627 (C]N) cmꢀ1 1H NMR (CDCl3):
; d 1.42e1.50 (m, 6H, 2
CH3); 4.06 (q, J ¼ 7.2 Hz, 2H, OCH2eCH3); 4.55 (q, J ¼ 7.2 Hz, 2H,
COOCH2CH3); 5.78 (s, 1H, C12H); 6.73e7.36 (m, 7H, ArH); 9.18 (s,
1H, C5H) ppm; 13C NMR (CDCl3):
d 13.6, 14.3, 29.8, 59.1, 56.1, 107.3,
110.6, 118.7, 127.2, 129.4, 129.8, 130.0, 131.3, 141.1, 150.5, 151.9, 153.5,
154.8, 157.3, 167.0, 171.6; Anal. Calcd. for C23H19ClN4O4 (450.87): C
61.27, H 4.25, N 12.43; Found: C 61.13, H 4.28, N 12.61%.
4.1.7.1. (RS)-2-(Chloromethyl)-12-(4-chlorophenyl)-9-methoxy-12H-
chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (6a). Yield 80%. mp
200e201 ꢁC. IR (KBr): 3056 (CH arom.), 2959 (CH aliph.), 1628 (C]
4.1.6.3. (RS)-2-(12-(4-Chlorophenyl)-9-methoxy-12H-chromeno[3,2-e]
[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)acetonitrile (5g). Crystallized from
benzene. Yield 80%. mp 207e208 ꢁC. IR (KBr): 3072 (CH arom.), 2966
N) cmꢀ1 1H NMR (CDCl3):
; d 3.85 (s, 3H, OCH3); 4.73 (s, 2H, CH2);
(CH aliph.), 2259 (CN), 1630 (C]N) cmꢀ1; 1H NMR (CDCl3):
d 3.85 (s,
5.68 (s, 1H, C12H); 6.75 (d, J ¼ 8.7 Hz, 1H, ArH); 6.86 (s, 1H, ArH);
3H, OCH3); 4.00 (s, 2H, CH2CN); 5.64 (s, 1H, C12H); 6.75 (d, J ¼ 8.7 Hz,
7.07 (d, J ¼ 8.7 Hz, 1H, ArH); 7.23 (d, J ¼ 8.4 Hz, 2H, ArH); 7.27 (d,
1H, ArH); 6.86 (s, 1H, ArH); 7.06 (d, J ¼ 8.7 Hz, 1H, ArH); 7.21 (d,
Cl
Cl
Cl
Cl
N
H C OOC
5
2
H
N
N
H
N
N
N
N
N
N
H
N
N
N
N
H
OC H
H
O
N
2
5
N
O
OC H
2
5
OCH
O
3
N
O
OCH
3
4a
5f
5a
5h
Fig. 8. Structure of compound 5a and 5h.
Fig. 6. Structures of compounds 4a and 5f.

192
M.M. Kandeel et al. / European Journal of Medicinal Chemistry 59 (2013) 183e193
Cl
C23H20Cl2N4O2(455.34): C 60.67, H 4.43, N 12.30; Found: C 60.51, H
4.51, N 12.51%.
Cl
4.1.8. General procedure for the preparation of compounds 7aef
To a mixture of 4b and 4d (0.004 mol) and the respective ary-
lidene malononitrile (0.004 mol) in absolute ethanol (25 mL),
piperidine (0.2 mL) was added. The reaction mixture was heated
under reflux for 2 h. The precipitate that formed was collected by
filtration while hot and crystallized from a mixture of methanol:
chloroform (3:1) to afford 7aef.
N
N
N
OC H
O
N
2
5
6d
Fig. 9. Structure of compound 6d.
4.1.8.1. (RS)-12-(4-Chlorophenyl)-9-methoxy-2-phenyl-12H-chro-
meno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7a). Yield 75%. mp
264e265 ꢁC. IR (KBr): 3058 (CH arom.), 2964 (CH aliph.), 1627 (C]
J ¼ 8.4 Hz, 2H, ArH); 9.07 (s, 1H, C5H) ppm; 13C NMR (CDCl3):
d 29.8,
45.1, 56.2, 107.2, 110.6, 118.5, 127.7, 129.4, 129.6, 129.8, 130.9, 132.0,
141.1, 147.8, 153.5, 155.4, 160.4, 171.6; Anal. Calcd. for C20H14Cl2N4O2
(413.26): C 58.13, H 3.41, N 13.56; Found: C 58.05, H 3.71, N 13.91%.
N) cmꢀ1; 1H NMR (CDCl3):
d
3.85 (s, 3H, OCH3); 5.75 (s, 1H, C12H);
6.73e8.26 (m, 12H, ArH); 9.09 (s, 1H, C5H) ppm; Anal. Calcd. for
25H17ClN4O2 (440.88): C 68.11, H 3.89, N 12.71; Found: C 68.07, H
C
3.88, N 13.04%.
4.1.7.2. (RS)-12-(4-Chlorophenyl)-2-(chloropropyl)-9-methoxy-12H-
chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (6b). Yield 81%. mp
175e176 ꢁC. IR (KBr): 3057 (CH arom.), 2951 (CH aliph.), 1628 (C]
4.1.8.2. (RS)-2,12-bis-(4-Chlorophenyl)-9-methoxy-12H-chromeno
[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7b). Yield 82%. mp 280e
281 ꢁC. IR (KBr): 3078 (CH arom.), 2970 (CH aliph.), 1625 (C]N)
N) cmꢀ1; 1H NMR (CDCl3):
d
2.22 (pentet, J ¼ 7.5 Hz, 2H, CH2); 3.07
(t, J ¼ 7.5 Hz, 2H, CH2); 3.66 (t, J ¼ 7.5 Hz, 2H, CH2); 3.84 (s, 3H,
OCH3); 5.67 (s, 1H, C12H); 6.75 (d, J ¼ 8.4 Hz, 1H, ArH); 6.85 (s, 1H,
ArH); 7.07 (d, J ¼ 8.4 Hz, 1H, ArH); 7.19 (d, J ¼ 8.7 Hz, 2H, ArH); 7.29
(d, J ¼ 8.7 Hz, 2H, ArH); 9.01 (s, 1H, C5H) ppm; Anal. Calcd. for
cmꢀ1; 1H NMR (CDCl3)
d: 3.85 (s, 3H, OCH3); 5.72 (s, 1H, C12H); 6.74
(d, J ¼ 8.7 Hz, 1H, ArH); 6.86 (s, 1H, ArH); 7.08 (d, J ¼ 8.7 Hz, 1H,
ArH); 7.21 (d, J ¼ 8.4 Hz, 2H, ArH); 7.36 (d, J ¼ 8.4 Hz, 2H, ArH); 7.48
(d, J ¼ 8.4 Hz, 2H, ArH); 8.20 (d, J ¼ 8.4 Hz, 2H, ArH); 9.08 (s, 1H,
C
22H18Cl2N4O2 (441.31): C 59.88, H 4.11, N 12.70; Found: C 60.09, H
C5H) ppm; 13C NMR (CDCl3)
d: 29.8, 56.0, 107.3, 110.5, 118.2, 127.9,
4.25, N 12.50%.
128.4, 129.4, 129.8, 130.4, 131.2, 133.8, 134.7, 141.1, 148.0, 153.5,
155.2, 160.5, 171.6; Anal. Calcd. for C25H16Cl2N4O2 (475.33): C 63.17,
H 3.39, N 11.79; Found: C 63.44, H 3.59, N 11.94%.
4.1.7.3. (RS)-2-(Chloromethyl)-12-(4-chlorophenyl)-9-ethoxy-12H-
chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (6c). Yield 87%. mp
197e198 ꢁC. IR (KBr): 3059 (CH arom.), 2980 (CH aliph.), 1628 (C]
N) cmꢀ1; 1H NMR (CDCl3):
d
1.44 (t, J ¼ 7.2 Hz, 3H, CH2CH3); 4.06 (q,
4.1.8.3. (RS)-12-(4-Chlorophenyl)-9-methoxy-2-(4-methoxyphenyl)-
12H-chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7c). Yield 69%.
mp 256e257 ꢁC. IR (KBr): 3060 (CH arom.), 2933 (CH aliph.), 1619
J ¼ 7.2 Hz, 2H, CH2eCH3); 4.73 (s, 2H, CH2Cl); 5.67 (s, 1H, C12H);
6.73 (d, J ¼ 8.4 Hz, 1H, ArH); 6.84 (s, 1H, ArH); 7.05 (d, J ¼ 8.4 Hz, 1H,
ArH); 7.20 (d, J ¼ 8.7 Hz, 2H, ArH); 7.29 (d, J ¼ 8.7 Hz, 2H, ArH); 9.06
(C]N) cmꢀ1 1H NMR (CDCl3):
; d 3.85 (s, 3H, OCH3); 3.89 (s, 3H,
OCH3); 5.77 (s, 1H, C12H); 6.76 (d, J ¼ 9 Hz, 1H, ArH); 6.87 (s, 1H,
ArH); 7.02 (d, J ¼ 9 Hz,1H, ArH); 7.07 (d, J ¼ 8.4 Hz, 2H, ArH); 7.21 (d,
J ¼ 8.4 Hz, 2H, ArH); 7.37 (d, J ¼ 8.7 Hz, 2H, ArH); 8.21 (d, J ¼ 8.7 Hz,
2H, ArH); 9.06 (s, 1H, C5H) ppm; Anal. Calcd. for C26H19ClN4O3
(470.91): C 66.31, H 4.07, N 11.90; Found: C 66.20, H 4.20, N 12.20%.
(s, 1H, C5H) ppm; EIMS: m/z (%) ¼ 431 (M þ 4
, 9.96), 429
, 39.64), 315 (MeC6H4Cl , 100);
(M þ 2
, 25.76), 427 (M
Anal. Calcd. for C21H16Cl2N4O2 (427.28): C 59.03, H 3.77, N 13.11;
Found: C 59.09, H 3.89, N 13.02%.
4.1.7.4. (RS)-12-(4-Chlorophenyl)-2-(chloropropyl)-9-ethoxy-12H-
chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (6d). Yield 88%. mp
171e172 ꢁC. IR (KBr): 3071 (CH arom.), 2974 (CH aliph.),1628 (C]N)
4.1.8.4. (RS)-12-(4-Chlorophenyl)-9-ethoxy-2-phenyl-12H-chromeno
[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7d). Yield 87%. mp 219e
220 ꢁC. IR (KBr): 3058 (CH arom.), 2982 (CH aliph.), 1625 (C]N)
cmꢀ1
;
1H NMR (CDCl3):
d
1.44 (t, J ¼ 7.2 Hz, 3H, CH2CH3); 2.25
cmꢀ1
;
1H NMR (CDCl3):
d
1.45 (t, J ¼ 7.2 Hz, 3H, CH2CH3); 4.06 (q,
J ¼ 7.2 Hz, 2H, CH2eCH3); 5.74 (s, 1H, C12H); 6.71e8.28 (m, 12H,
ArH); 9.08 (s, 1H, C5H) ppm; 13C NMR (CDCl3)
: 14.3, 29.8, 56.0,
(pentet, J ¼ 7.2 Hz, 2H, CH2); 3.06 (t, J ¼ 7.2 Hz, 2H, CH2); 3.66 (t,
J ¼ 7.2 Hz, 2H, CH2); 4.06 (q, J ¼ 7.2 Hz, 2H, CH2eCH3); 5.65 (s, 1H,
C12H); 6.72 (d, J ¼ 8.4 Hz, 1H, ArH); 6.84 (s, 1H, ArH); 7.05 (d,
J ¼ 8.4 Hz, 1H, ArH); 7.22 (d, J ¼ 8.7 Hz, 2H, ArH); 7.30 (d, J ¼ 8.7 Hz,
d
107.3, 110.8,118.2,127.0, 127.9, 128.4,129.4,129.8, 130.4,131.2, 136.7,
141.1, 153.5, 154.8, 155.2, 157.3, 160.6, 171.6; Anal. Calcd. for
2H, ArH); 9.01 (s, 1H, C5H) ppm; 13C NMR (CDCl3):
d 14.3, 29.6, 56.1,
107.1, 111.6, 118.2, 127.0, 127.5, 128.4, 129.0, 129.4129.8, 130.0, 131.3,
136.9, 141.1, 148.3, 153.9, 154.8, 157.7, 171.6; Anal. Calcd. for
C
26H19ClN4O2 (454.91): C 68.65, H 4.21, N 12.32; Found: C 68.61, H
4.30, N 12.60%.
Cl
Cl
4.1.8.5. (RS)-2,12-bis-(4-Chlorophenyl)-9-ethoxy-12H-chromeno
[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7e). Yield 88%. mp 264e
265 ꢁC. IR (KBr): 3067 (CH arom.), 2983 (CH aliph.), 1627 (C]N)
N
H
cmꢀ1
;
1H NMR (CDCl3):
d
1.45 (t, J ¼ 7.2 Hz, 3H, CH2CH3); 4.06 (q,
H
N
N
H
J ¼ 7.2 Hz, 2H, CH2eCH3); 5.76 (s, 1H, C12H); 6.74 (d, J ¼ 8.4 Hz, 1H,
ArH); 6.85 (s, 1H, ArH); 7.08 (d, J ¼ 8.4 Hz, 1H, ArH); 7.22 (d,
J ¼ 8.4 Hz, 2H, ArH); 7.35 (d, J ¼ 8.4 Hz, 2H, ArH); 7.48 (d, J ¼ 8.4 Hz,
2H, ArH); 8.21 (d, J ¼ 8.4 Hz, 2H, ArH); 9.09 (s, 1H, C5H) ppm; EIMS:
N
N
N
N
OCH
N
O
OCH
N
O
m/z (%) ¼ 493 (M þ 4
10.56), 488 (M ꢀ 1
, 1.05), 491(M þ 2
, 6.86), 489 (M
,
, 17.68), 459 (MeC2H5
, 100); Anal. Calcd.
5g
5c
for C26H18Cl2N4O2 (489.35): C 63.81, H 3.71, N 11.45; Found: C 64.02,
H 3.92, N 11.66%.
Fig. 10. Structure of compounds 5c and 5g.

M.M. Kandeel et al. / European Journal of Medicinal Chemistry 59 (2013) 183e193
193
with microtubule network of HBL100 cells and binding to tubulin, Biochemistry
45 (2006) 9210e9218.
4.1.8.6. (RS)-12-(4-Chlorophenyl)-9-ethoxy-2-(4-methoxyphenyl)-
12H-chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7f). Yield 73%.
[10] W. Kemnitzer, J. Drewe, S. Jiang, H. Zhang, Y. Wang, J. Zhao, S. Jia, J. Herich,
D. Labreque, R. Storer, K. Meerovitch, D. Bouffard, R. Rej, R. Denis, Ch. Blais,
S. Lamothe, G. Attardo, H. Gourdeau, B. Tseng, Sh. Kasibhatla, S.X. Cai,
Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using
a cell- and Caspase-based high-throughput screening assay. 1. Structuree
activity relationships of the 4-aryl group, J. Med. Chem. 47 (2004) 6299.
[11] A. Afantitis, G. Melagraki, H. Sarimveis, P.A. Koutentis, J. Markopoulos,
O. Igglessi-Markopoulou, A novel QSAR model for predicting induction of
apoptosis by 4-aryl-4H-chromenes, Bioorg. Med. Chem. 14 (2006) 6686e
6694.
mp 174e175 ꢁC. IR (KBr): 2969 (CH aliph.), 1608 (C]N) cmꢀ1 1H
;
NMR (CDCl3):
d
1.45 (t, J ¼ 7.2 Hz, 3H, CH2CH3); 3.89 (s, 3H, OCH3);
4.07 (q, J ¼ 7.2 Hz, 2H, CH2eCH3); 5.72 (s, 1H, C12H); 6.74e7.27 (m,
7H, ArH); 7.36 (d, J ¼ 8.7 Hz, 2H, ArH); 8.20 (d, J ¼ 8.7 Hz, 2H, ArH);
9.05 (s, 1H, C5H) ppm; Anal. Calcd. for C27H21ClN4O3 (484.93): C
66.87, H 4.36, N 11.55; Found: C 66.92, H 4.72, N 11.19%.
[12] S. Sciabola, E. Carosati, L. Cucurull-Sanchez, M. Baronic, R. Mannholdd, Novel
TOPP descriptors in 3D-QSAR analysis of apoptosis inducing 4-aryl-4H-chro-
menes: comparison versus other 2D- and 3D-descriptors, Bioorg. Med. Chem.
15 (2007) 6450e6462.
[13] M.H. Fatemi, S. Gharaghani, A novel QSAR model for prediction of apoptosis-
inducing activity of 4-aryl-4H-chromenes based on support vector machine,
Bioorg. Med. Chem. 15 (2007) 7746e7754.
[14] M. Gao, M. Wang, K.D. Miller, G.D. Hutchins, Q.H. Zheng, Synthesis of carbon-
11-labeled 4-aryl-4H-chromenes as new PET agents for imaging of apoptosis
in cancer, Appl. Radiat. Isot. 68 (2010) 110e116.
[15] W. Kemnitzer, Sh. Kasibhatla, S. Jiang, H. Zhang, J. Zhao, Sh. Jia, L. Xu,
C. Crogan-Grundy, R. Denis, N. Barriault, L. Vaillancourt, S. Charron, J. Dodd,
G. Attardo, D. Labrecque, S. Lamothe, H. Gourdeau, B. Tseng, J. Drewea,
S.X. Caia, Discovery of 4-aryl-4H-chromenes as a new series of apoptosis
inducers using a cell- and caspase-based high-throughput screening assay. 2.
Structureeactivity relationships of the 7- and 5-, 6-, 8-positions, Bioorg. Med.
Chem. Lett. 15 (2005) 4745e4751.
4.2. Antitumor activity
The breast tumor cell line was obtained frozen in liquid nitrogen
(ꢀ180 ꢁC) from the American Type Culture Collection (ATCC) and
was maintained at the National Cancer Institute, Cairo, Egypt, by
serial sub culturing. Colchicine was used in this experiment as
a positive control. The tested compounds were dissolved in 20%
DMSO in concentration 1 mg/mL.
Serial dilutions were made reaching final concentration of the
compounds to 0, 5, 12.5, 25, 50 mg/mL. All chemicals used in this
study are of high analytical grade. They either obtained from
(SigmaeAlderich or Biorad).
[16] W. Kemnitzer, S. Jiang, Y. Wang, Sh. Kasibhatla, C. Crogan-Grundy, M. Bubenik,
D. Labrecque, R. Denis, S. Lamothe, G. Attardo, H. Gourdeau, B. Tseng,
J. Drewea, S.X. Caia, Discovery of 4-aryl-4H-chromenes as a new series of
apoptosis inducers using a cell- and caspase-based high throughput screening
assay. 4. structureeactivity relationships of N-alkyl substituted pyrrole fused
at the 7, 8-positions, J. Med. Chem. 51 (2008) 417e423.
[17] W. Kemnitzer, S. Jiang, Y. Wang, Sh. Kasibhatla, C. Crogan-Grundy, M. Bubenik,
D. Labrecque, R. Denis, S. Lamothe, G. Attardo, H. Gourdeau, B. Tseng,
J. Drewea, S.X. Caia, Discovery of 4-aryl-4H-chromenes as a new series of
apoptosis inducers using a cell- and caspase-based HTS assay. Part 5: modi-
fications of the 2- and 3-positions, Bioorg. Med. Chem. Lett. 18 (2008) 603e
607.
4.2.1. Measurement of potential cytotoxic activity
The cytotoxic activity was measured in vitro on human breast
tumor cell line (MCF-7) using Sulforhodamine-B stain (SRB) assay
applying the method of Skehan et al. [25]. The results of in vitro
cytotoxic activity experiments are presented in (Table 1).
References
[1] K. Odlo, J. Fournier-Dit-Chabert, S. Ducki, O.A.B.S.M. Gani, I. Sylte, T.V. Hansen,
1,2,3-Triazole analogs of combretastatin A-4 as potential microtubule-binding
agents, Bioorg. Med. Chem. 18 (2010) 6874e6885.
[18] W. Kemnitzer, S. Jiang, H. Zhang, Sh. Kasibhatla, C. Crogan-Grundy, Ch. Blais,
G. Attardo, R. Denis, S. Lamoth, H. Gourdeau, B. Tseng, J. Drewe, S.X. Cai,
Discovery of 4-aryl-2-oxo-2H-chromenes as
inducers using a cell- and caspase-based high-throughput screening assay,
Bioorg. Med. Chem. Lett. 18 (2008) 5571e5575.
a new series of apoptosis
[2] D. Raffa, M.C. Edler, G. Daidone, B. Maggio, M. Merickech, S. Plescia,
D. Schillaci, R. Bai, E. Hamel, Synthesis, cytotoxicity, and inhibitory effects on
tubulin polymerization of
a
new 3-heterocyclo substituted 2-
[19] S.Y. Liao, L. Qian, T.F. Miao, Y. Shen, K.C. Zheng, 3D-QSAR Studies of substituted
4-aryl/heteroaryl-4H-chromenes as apoptosis inducers using CoMFA and
CoMSIA, J. Theor. Comput. Chem. 8 (2009) 143e155.
[20] N. Zhang, S. Ayral-Kaloustian, Th. Nguyen, R. Hernandez, C. Beyer, 2-
Cyanoaminopyrimidines as a class of antitumor agents that promote tubulin
polymerization, Bioorg. Med. Chem. Lett. 17 (2007) 3003e3005.
[21] F. Montel, C. Lamberth, P.M.J. Jung, First synthesis of 7-amido-[1,2,4]triazolo
[1,5-a]pyrimidines using halogenemetal exchange, Tetrahedron 64 (2008)
6372e6376.
[22] N.D. Kim, E.-S. Park, Y.H. Kim, S.K. Moon, S.S. Lee, S.K. Ahn, D.-Y. Yu, K.T. No, K.-
H. Kim, Structure-based virtual screening of novel tubulin inhibitors and their
characterization as anti-mitotic agents, Bioorg. Med. Chem. 18 (2010) 7092e
7100.
styrylquinazolinone, Eur. J. Med. Chem. 39 (2004) 299e304.
[3] M. Botta, S. Forli, M. Magnani, F. Manetti, Molecular modeling approaches to
study the binding mode on tubulin of microtubule destabilizing and stabi-
lizing agents, Top. Curr. Chem. 286 (2009) 279e328.
[4] M. Medarde, A. Ramos, E. Caballero, R. Pelàez-Lamamié de Clairac, J.L. Lòpez,
D.G. Gràvalos, A. San Feliciano, Synthesis and neoplastic activity of com-
bretastatin analogues: heterocombretastatins, Eur. J. Med. Chem. 33 (1998)
71e77.
[5] I.A. Saleh, M. Areej, M.A. Ahmed, M.H. Mostafa, N. Eman, M.G. Mostafa,
Synthesis and biological evaluation of 2-amino-7,7-dimethyl-substituted-5-
oxo-1-(3,4,5-trimethoxy)-1,4,5,6,7,8-hexahydro-quinoline-3-carbonitrile
derivatives as potential cytotoxic agents, Bioorg. Med. Chem. Lett. 19 (2009)
6939e6942.
[23] F.F. Abdel-Latif, Heterocycles synthesis through reactions of nucleophiles with
acrylonitriles: part XI-A convenient one-pot synthesis of 4H-chromenes,
Indian J. Chem. 25B (1990) 664e666.
[24] A.H. Bedair, N.A. El-Hady, M.S. Abd El-Latif, A.H. Fakery, A.M. El-Agrody, 4-
Hydroxycoumarin in heterocyclic synthesis Part III. Synthesis of some new
[6] G.R. Pettit, M.P. Grealish, D.L. Herald, M.R. Boyd, E. Hamel, R.K. Pettit, Anti-
neoplastic Agents. 443. Synthesis of the cancer cell growth inhibitor
hydroxyphenstatin and its sodium diphosphate prodrug, J. Med. Chem. 43
(2000) 2731e2737.
[7] M.N. Islam, N.I. Magdy, Microtubulin binding sites as target for developing
anticancer agents, Mini Rev. Med. Chem. 4 (2004) 1077e1104.
[8] R.M. Lee, D.A. Gewirtz, Colchicine site inhibitors of microtubule integrity as
vascular disrupting agents, Drug Dev. Res. 69 (2008) 352e358.
[9] C. Rappl, P. Barbier, V. Bourgarel-Rey, C. Gregoire, R. Gilli, M. Carre, S. Combes, J.-
P. Finet, V. Peyrot, Interaction of 4-arylcoumarin analogues of combretastatins
pyrano[2,3-d]pyrimidine-2-substituted[1,2,4]triazolo[1,5-c]pyrimidine
pyrimido[1,6-b][1,2,4]triazine derivatives, Farmaco 55 (2000) 708e714.
and
[25] A. Monk, D.A. Scudiero, P. Skehan, R.H. Shoemaker, K.D. Paull, DTP, DCTD
Tumor repository a catalog of in vitro cell lines, transplantable animal and
human tumors and micro assays, J. Natl. Cancer Inst. 83 (1991) 757e766.