Synthesis, docking and in vitro anticancer evaluation of some new benzopyrone derivatives

Article abstract of DOI:10.1016/j.bioorg.2014.02.003

The synthesis of some new 3-alkyl-7-hydroxy-4-methyl-8-substituted-1H- benzopyran-2-ones, 6-alkyl-7-methyl-2-substituted amino-5H-pyrano[6,5-e] benzoxazol-5-ones, 7-alkyl-8-methyl-3-substituted-2,6-dihydropyrano[6,5-f]-1,4- benzoxazin-6-ones, 7,8-disubstituted-3-ethyl-4-methyl-1H-benzopyran-2-ones and 3-alkyl-4-methyl-7-substituted-1H-benzopyran-2-ones were described. Fourteen compounds were selected by National Cancer Institute (NCI), Bethesda, and evaluated for their in vitro anticancer activity in the full NCI 60 cell lines panel assay by a single dose test. Compounds 4a, 18a, 18b and 23a were found to be broad-spectrum antitumors showing effectiveness toward numerous cell lines that belong to different tumor subpanels. Furthermore, docking studies were undertaken to gain insight into the possible binding mode of these compounds with the binding site of the casein kinase II (CK2) enzyme which is involved in cell survival and proliferation through a number of downstream effectors.

Full text of DOI:10.1016/j.bioorg.2014.02.003

Bioorganic Chemistry 53 (2014) 50–66
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, docking and in vitro anticancer evaluation of some new
benzopyrone derivatives
b
Sohair L. El-Ansary a,b, Mohammed M. Hussein a,b, Doaa E. Abdel Rahman a, , Lina M.A. Abdel Ghany
⇑
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Misr University for Science and Technology, 6th October City, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 October 2013
Available online 19 February 2014
The synthesis of some new 3-alkyl-7-hydroxy-4-methyl-8-substituted-1H-benzopyran-2-ones,
6-alkyl-7-methyl-2-substituted amino-5H-pyrano[6,5-e] benzoxazol-5-ones, 7-alkyl-8-methyl-3-substi-
tuted-2,6-dihydropyrano[6,5-f]-1,4-benzoxazin-6-ones, 7,8-disubstituted-3-ethyl-4-methyl-1H-benzo-
pyran-2-ones and 3-alkyl-4-methyl-7-substituted-1H-benzopyran-2-ones were described. Fourteen
compounds were selected by National Cancer Institute (NCI), Bethesda, and evaluated for their in vitro
anticancer activity in the full NCI 60 cell lines panel assay by a single dose test. Compounds 4a, 18a,
18b and 23a were found to be broad-spectrum antitumors showing effectiveness toward numerous cell
lines that belong to different tumor subpanels. Furthermore, docking studies were undertaken to gain
insight into the possible binding mode of these compounds with the binding site of the casein kinase II
(CK2) enzyme which is involved in cell survival and proliferation through a number of downstream effectors.
Ó 2014 Elsevier Inc. All rights reserved.
Keywords:
Benzopyrones
Anticancer
Docking studies
Casein kinase II
1. Introduction
cer activities [19–22]. Warfarin A (Fig. 1) reduced metastases from
intestinal carcinomas to a great extent [23] and also used as an ad-
Cancer is a leading cause of death worldwide and accounted for
7.6 million deaths (around 13% of all deaths) in 2008 and deaths
from cancer worldwide are projected to continue to rise to over
13.1 million in 2030 [1]. Cancer cells develop a degree of autonomy
and grow uncontrollably disregarding the normal rules of cell
division, resulting in uncontrolled growth and proliferation. In fact,
almost 90% of cancer- related deaths are due to tumor spreading or
dissemination [2]. Several techniques involving surgery, radiation,
immunotherapy and chemotherapy were adopted for eradication
of cancerous cells. Unfortunately, no currently available anticancer
drugs would eradicate cancer cells without harming normal tissues
[3]. Accordingly, continued research is needed to develop new and
efficient antitumor agents.
Benzopyran-2-one comprises a group of natural compounds
found in a variety of plant sources. Benzopyran-2-ones are recog-
nized to possess a wide variety of biological activities against bac-
teria [4,5], fungi [6] and protozoa [7]. In addition, they are also
reported to possess anti-inflammatory [8], antioxidant [6,9],
antiallergic [10], antithrombotic [11], antiHIV [12], antidepressant
[13–15], photosensitizing [16,17], estrogenic like [18] and antican-
junct to the surgical treatment of malignant tumors [24].
The inhibition activity of benzopyran-2-one derivative B (Fig. 1)
against different cancer cell lines showed a high selectivity for HU-
VEC that can be potentially utilized as lead compound to develop
non toxic angiogenesis inhibitors and small molecular ligands to
target HUVEC [25].
In addition, daphnetin C (Fig. 1) was proven to act as tyrosine
kinase inhibitor. Daphnetin inhibited tyrosine kinase, epidermal
growth factor receptor, serine/threonine- specific protein kinase,
and protein kinase C in vitro [26]. Also, benzopyran-2-one deriva-
tive D (Fig. 1) was identified as a novel class of MEK 1 kinase inhib-
itors [27].
Furthermore, some heterocycles such as oxathiazolidine [28],
triazole [29], oxazole [30] and thiadiazole [31] were found to pos-
sess potential antitumor activity.
These findings have encouraged us to prepare compounds con-
taining the benzopyran-2-one nucleus substituted at 7-position
with different bioisosteric moieties as triazole, thiadiazole, thiazo-
lidinone, thiazole, hydrazone, oxathiazolidine, dihydropyrazole,
dihydropyrrole and dioxopyrrolidine. Also, 8-substituted deriva-
tives as chalcones, dihydropyridines, ureas and imidazolidinetri-
ones in addition to oxazolo and oxazinobenzopyran-2-one
derivatives were prepared. Fourteen compounds of the synthesized
compounds were selected by National Cancer Institute (NCI),
⇑
Corresponding author.
E-mail addresses: doaaezzat2004@yahoo.com, doaaezzat2004@cu.edu.eg
(D.E. Abdel Rahman).
http://dx.doi.org/10.1016/j.bioorg.2014.02.003
0045-2068/Ó 2014 Elsevier Inc. All rights reserved.

S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
51
Fig. 1. Anticancer, anti-angiogenic and kinase inhibitors benzopyrone derivatives.
Bethesda, MD, U.S.A., for in vitro one dose testing in the full NCI 60
cell lines panel assay. In addition, attempt to elucidate a molecular
target for activity was achieved via molecular docking of the pre-
pared compounds in the active site of casein kinase II enzyme
(CK2) using Molsoft ICM 3.4–8C program.
J = 8.4 Hz, H-20,60 Ar), 13.37 (s, 1H, OH). MS m/z (%): 296, M+ + 2
(16.49%). Anal. Calcd. for C17H14N2O3 (294.30): C, 69.38; H, 4.79;
N 9.52. Found: C, 69.44; H, 4.82; N, 9.60.
2.1.2.2. 3-Ethyl-7-hydroxy-4-methyl-8-phenylazo-2H-1-benzopyran-
2-one 2b. The crude product was crystallized from methanol. Yield
66%. mp 130–133 °C. IR t
max/cmꢀ1: 3460 (OH), 3047 (CH Ar), 2968,
2. Experimental
2910 (CH aliphatic), 1708 (C@O), 1629, 1593, 1550 (N@N, C@C). 1H
NMR (300 MHz, DMSO-d6) d ppm: 1.07 (t, 3H, CH2CH3), 2.42 (s, 3H,
CH3), 2.59 (q, 2H, CH2CH3), 6.99 (d, 1H, J = 9.0 Hz, H-6 Ar), 7.57–
7.67 (m, 3H, H-30,40,50 Ar), 7.83 (d, 1H, J = 9.3 Hz, H-5 Ar), 7.98 (d,
2H, J = 7.8 Hz, H-20,60 Ar), 13.37 (s, 1H, OH). MS m/z (%): 308, M+
(0.71%). Anal. Calcd. for C18H16N2O3 (308.33): C, 70.12; H, 5.23;
N, 9.09. Found: C, 70.14; H, 5.27; N, 9.14.
2.1. Chemistry
Melting points were determined by open capillary tube method
using Stuart SMP10 melting point apparatus and were uncorrected.
Microanalysis was carried out at The Regional Center for Mycology
and Biotechnology, Al-Azhar University. Infrared Spectra were re-
corded as potassium bromide discs on Schimadzu FT-IR 8400S
spectrophotometer (Shimadzu, Kyoto, Japan) and Bruker FT-IR
spectrophotometer and expressed in wave number tmax (cmꢀ1).
The 1H NMR spectra were recorded⁄on a Varian Mercury VX-300
NMR spectrometer at 300 MHz and JEOL-ECA500 NMR spectrom-
eter at 500 MHz in chloroform (CDCl3) or dimethylsulfoxide
(DMSO-d6). Chemical Shifts are quoted in d as parts per million
(ppm) downfield from tetramethylsilane (TMS) as internal stan-
dard and J values are reported in Hz. Mass spectra were performed
as EI at 70 eV on Hewlett Packard Varian (Varian, Polo, USA) and
Shimadzu Gas Chromatograph Mass spectrometer-QP 1000 EX
and direct inlet unit of Shimadzu GC/MS-QP5050A. TLC were car-
ried out using Macherey–Nagel Alugram Sil G/UV254 silica gel
plates with fluorescent indicator UV254 and acetonitrile:methanol
(9:1) as the eluting system and the spots were visualized at 366,
254 nm by UV Vilber Lourmat 77202 (Vilber, Marne La Vallee,
France).
2.1.3. General procedure for synthesis of 3-alkyl-8-amino-7-hydroxy-
4-methyl-2H-1-benzopyran-2-ones 3a,b (Scheme 1)
A solution of sodium dithionite (7 g, 0.04 mol) in water (30 ml)
was quickly added to a solution of the azo compound 2a,b
(0.01 mol) in 30% ammonium hydroxide solution (20 ml) and the
reaction mixture was refluxed for 15 min. After cooling, the crude
product was filtered off, washed and dried.
2.1.3.1.
3a. The crude product was crystallized from isopropanol. Yield
69%. mp 228–230 °C. IR
max/cmꢀ1: 3444, 3365 (NH2), 3199 (OH),
8-Amino-3,4-dimethyl-7-hydroxy-2H-1-benzopyran-2-one
t
2927, 2856 (CH aliphatic), 1680 (C@O), 1616, 1568, 1510 (NH,
C@C). 1H NMR (300 MHz, DMSO-d6) d ppm: 2.05 (s, 3H, CH3 at
C4), 2.30 (s, 3H, CH3 at C3), 6.67 (s, 2H, NH2), 6.89 (d, 1H,
J = 8.7 Hz, H-6 Ar), 7.59 (d, 1H, J = 8.7 Hz, H-5 Ar), 10.31 (s, 1H,
OH). MS m/z (%): 205, M+ (0.54%). Anal. Calcd. for C11H11NO3
(205.21): C, 64.38; H, 5.40; N, 6.83. Found: C, 64.37; H, 5.39; N,
6.91.
2.1.1. 3,4-Dimethyl-7-hydroxy-2H-1-benzopyran-2-one and 3-ethyl-
7-hydroxy-4-methyl-2H-1-benzopyran-2-one 1a,b
were prepared as reported in literature [32].
2.1.3.2. 8-Amino-3-ethyl-7-hydroxy-4-methyl-2H-1-benzopyran-2-
one 3b. The crude product was crystallized from isopropanol. Yield
2.1.2. General procedure for synthesis of 3-alkyl-7-hydroxy-4-methyl-
8-phenylazo-2H-1-benzopyran-2-ones 2a,b (Scheme 1)
58%. mp 176–178 °C. IR t
max/cmꢀ1: 3325, 3296 (NH2, OH), 3074
(CH Ar), 2970, 2872 (CH aliphatic), 1708 (C@O), 1620, 1610,
1602, 1566, 1510 (NH, C@C). 1H NMR (300 MHz, DMSO-d6) d
ppm: 1.02 (t, 3H, CH2CH3), 2.34 (s, 3H, CH3), 2.55 (q, 2H, CH2CH3),
3.80 (br s, 2H, NH2), 6.78 (d, 1H, J = 8.7 Hz, H-6 Ar), 7.59 (d, 1H,
J = 9.0 Hz, H-5 Ar), 9.98 (s, 1H, OH). MS m/z (%): 219, M+ (0.06%).
Anal. Calcd. for C12H13NO3 (219.24): C, 65.74; H, 5.98; N, 6.39.
Found: C, 65.78; H, 6.02; N, 6.46.
Phenyl diazonium chloride {freshly prepared by addition of so-
dium nitrite solution (4 g, 0.06 mol) in water (20 ml) to a mixture
of aniline (3.81 g, 0.041 mol) and hydrochloric acid (16 ml) drop-
wise while cooling in an ice bath 0–5 °C} was added slowly to a
cooled solution of compound 1a,b (0.041 mol) in 5% sodium
hydroxide solution (45 ml). The reaction mixture was stirred for
1 h, filtered, washed and dried.
2.1.2.1. 3,4-Dimethyl-7-hydroxy-8-phenylazo-2H-1-benzopyran-2-
2.1.4. General procedure for synthesis of 1-(3-alkyl-4-methyl-7-
hydroxy-2-oxo-2H-1-benzopyran-8-yl)-3-(4-(un)substituted phenyl)
ureas 4a–c (Scheme 1)
A mixture of the amino compound 3a,b (0.01 mol), the appro-
priate isocyanate (0.01 mol) in dichloromethane (5 ml) was re-
fluxed with stirring for 6 h. The obtained solid product was
filtered off, washed with ether and dried.
one 2a. The crude product was crystallized from methanol. Yield
50%. mp 140–141 °C. IR t
max/cmꢀ1: 3192 (OH), 2924, 2850 (CH ali-
phatic), 1670 (C@O), 1620, 1612, 1566, 1510 (N@N, C@C). 1H NMR
(300 MHz, DMSO-d6) d ppm: 2.10 (s, 3H, CH3 at C4), 2.38 (s, 3H,
CH3 at C3), 6.97 (d, 1H, J = 9.3 Hz, H-6 Ar), 7.60–7.67 (m, 3H,
H-30,40,50Ar), 7.82 (d, 1H, J = 9.3 Hz, H-5 Ar), 7.98 (d, 2H,

52
S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
Scheme 1. Reagents and conditions: (i) phenyl diazonium chloride, 5% NaOH, r.t., 1 h, (ii) sodium dithionite, 30% NH4OH, reflux, 15 min., (iii) appropriate isocyanate, CH2Cl2,
reflux, 6 h, (iv) oxalyl chloride, benzene, 60–65 °C, 8–10 h, (v) appropriate isothiocyanate, ethanol, triethylamine, reflux, 15 h, and (vi) appropriate phenacyl bromide, sodium
ethoxide, ethanol, reflux, 2 h.
2.1.4.1. 1-(3,4-Dimethyl-7-hydroxy-2-oxo-2H-1-benzopyran-8-yl)-3-
from isopropanol. Yield 57%. mp 197–198 °C. IR t
max/cmꢀ1: 3300,
phenylurea 4a. The crude product was crystallized from isopropa-
3230 (NH, OH), 2950, 2860 (CH aliphatic) 1697, 1676 (2C@O),
1614, 1560, 1535, 1512 (NH, C@C). 1H NMR (300 MHz, DMSO-d6)
d ppm: 1.01 (t, 3H, CH2CH3), 2.34 (s, 3H, CH3), 2.52 (q, 2H, CH2CH3),
5.22 (s, 1H, NH), 6.53 (d, 1H, J = 8.7 Hz, H-6 Ar), 6.77 (d, 1H,
J = 8.4 Hz, H-5 Ar), 7.31 (d, 2H, J = 9.0 Hz, H-30,50 Ar), 7.48 (d, 2H,
J = 9.0 Hz, H-20,60 Ar), 8.84 (s, 1H, NH), 10.36 (s, 1H, OH). MS m/z
(%): 374, M+ + 1 (11.76%). Anal. Calcd. for C19H17ClN2O4 (373.80):
C, 61.21; H, 4.60; Cl, 9.51; N, 7.51. Found: C, 61.24; H, 4.67; Cl,
9.62; N, 7.56.
nol. Yield 76%. mp 312–314 °C. IR t
max/cmꢀ1: 3300, 3230 (NH, OH),
3057 (CH Ar), 2965, 2875 (CH aliphatic), 1697, 1685 (2C@O), 1622,
1608, 1564, 1539, 1512 (NH, C@C). 1H NMR (300 MHz, DMSO-d6) d
ppm: 2.04 (s, 3H, CH3 at C4), 2.32 (s, 3H, CH3 at C3), 6.78 (d, 1H,
J = 8.1 Hz, H-6 Ar), 6.96 (t, 1H, H-40 Ar), 7.27 (t, 2H, H-30,50 Ar),
7.44 (d, 1H, J = 7.8 Hz, H-5 Ar), 7.59 (d, 2H, J = 9.0 Hz, H-20,60 Ar),
8.66 (s, 1H, NH), 10.37 (s, 2H, NH, OH). MS m/z (%): 324, M+
(0.08%). Anal. Calcd. for C18H16N2O4 (324.33): C, 66.66; H, 4.97;
N, 8.64. Found: C, 66.72; H, 5.03; N, 8.73.
2.1.5. General procedure for synthesis of 1-(3-ethyl-7-hydroxy-4-
methyl-2-oxo-2H-1-benzopyran-8-yl)-3-(4-(un)substituted phenyl)
imidazolidin-2,4,5-triones 5a,b (Scheme 1)
To a solution of urea derivatives 4a,b (0.001 mol) in dry ben-
zene (5 ml), oxalyl chloride (0.25 g, 0.002 mol) was added drop-
wise while stirring. The reaction mixture was heated at 60–65 °C
for 8–10 h, cooled, filtered and washed with ether.
2.1.4.2. 1-(3-Ethyl-7-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-8-
yl)-3-phenylurea 4b. The crude product was crystallized from iso-
propanol. Yield 88%. mp 159–160 °C. IR
t
max/cmꢀ1: 3263, 3136
(2NH, OH), 3082 (CH Ar), 2966, 2873 (CH aliphatic), 1743, 1678
(2C@O), 1604, 1554, 1500 (NH, C@C). 1H NMR (300 MHz, DMSO-
d6) d ppm: 1.01 (t, 3H, CH2CH3), 2.33 (s, 3H, CH3), 2.52 (q, 2H, CH2-
CH3), 4.99 (s, 1H, NH), 6.47 (d, 1H, J = 7.5 Hz, H-6 Ar), 6.77 (d, 1H,
J = 8.7 Hz, H-5 Ar), 6.98 (t, 2H, H-30,50 Ar), 7.30 (t, 1H, H-40 Ar),
7.58 (d, 2H, J = 8.7 Hz, H-20,60 Ar), 8.65 (s, 1H, NH), 10.37 (s, 1H,
OH). MS m/z (%): 338, M+ (0.05%). Anal. Calcd. for C19H18N2O4
(338.38): C, 67.44; H, 5.36; N, 8.28. Found: C, 67.48; H, 5.34; N,
8.36.
2.1.5.1. 1-(3-Ethyl-7-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-8-
yl)-3-phenylimidazolidin-2,4,5-trione 5a. The crude product was
crystallized from ethanol. Yield 62%. mp 205–208 °C. IR tmax
/
cmꢀ1: 3267 (OH), 3082 (CH Ar), 2974, 2873 (CH aliphatic), 1747,
1685 (4C@O), 1600, 1550, 1500 (C@C). 1H NMR (300 MHz,
DMSO-d6) d ppm: 1.03 (t, 3H, CH2CH3), 2.36 (s, 3H, CH3), 2.56 (q,
2H, CH2CH3), 6.53 (d, 1H, J = 7.2 Hz, H-6 Ar), 6.97 (d, 1H,
J = 7.2 Hz, H-5 Ar), 7.25–7.61 (m, 3H, H-30,40,50 Ar), 7.84 (d, 2H,
2.1.4.3. 1-(3-Ethyl-7-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-8-
yl)-3-(4-chlorophenyl)urea 4c. The crude product was crystallized

S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
53
J = 9.0 Hz, H-20,60 Ar), 10.37 (s, 1H, OH). MS m/z (%): 394, M+ + 2
(0.04%). Anal. Calcd. for C21H16N2O6 (392.36): C, 64.28; H, 4.11;
N, 7.14. Found: C, 64.31; H, 4.10; N, 7.19.
centrated then left to cool. The formed precipitate was filtered,
washed and dried.
2.1.7.1. 7,8-Dimethyl-3-phenyl-2,6-dihydropyrano[6,5-f]-1,4-benzox-
2.1.5.2.
3-(4-Chlorophenyl)-1-(3-ethyl-7-hydroxy-4-methyl-2-oxo-
azin-6-one 7a. The crude product was crystallized from isopropa-
t
max/cmꢀ1: 2927, 2852 (CH
2H-1-benzopyran-8-yl)imidazolidin-2,4,5-trione 5b. The crude prod-
uct was crystallized from ethanol. Yield 89%. mp 230–232 °C. IR
nol. Yield 66%. mp 366–367 °C. IR
aliphatic), 1680 (C@O), 1616, 1566, 1510 (C@N, C@C). 1H NMR
(300 MHz, DMSO-d6) d ppm: 2.07 (s, 3H, CH3 at C4), 2.37 (s, 3H,
CH3 at C3), 5.73 (s, 2H, CH2 Oxazine), 6.99–7.05 (m, 2H, H-6,40
Ar), 7.58 (t, 2H, H-30,50 Ar), 7.70 (d, 1H, J = 8.7 Hz, H-5 Ar), 8.04
(d, 2H, J = 8.4 Hz, H-20,60 Ar). MS m/z (%): 305, M+ (57.44%). Anal.
Calcd. for C19H15NO3 (305.33): C, 74.74; H, 4.95; N, 4.59. Found:
C, 74.81; H, 5.02; N, 4.67.
t
max/cmꢀ1: 3284 (OH), 3053 (CH Ar), 2966, 2873 (CH aliphatic),
1759, 1708, 1689 (4C@O), 1614, 1597, 1543, 1504 (C@C). 1H
NMR (300 MHz, DMSO-d6) d ppm: 1.02 (t, 3H, CH2CH3), 2.34 (s,
3H, CH3), 2.52 (q, 2H, CH2CH3), 6.78 (d, 1H, J = 8.7, H-6 Ar), 7.31
(d, 1H, J = 9.0 Hz, H-5 Ar), 7.47 (d, 2H, J = 8.7 Hz, H-30,50 Ar), 7.59
(d, 2H, J = 8.7 Hz, H-20,60 Ar), 10.39 (s, 1H, OH). MS m/z (%): 426,
M+ (0.03%). Anal. Calcd. for C21H15ClN2O6 (426.81): C, 59.10; H,
3.54; Cl, 8.31; N, 6.56. Found: C, 59.13; H, 3.59; Cl, 8.39; N, 6.62.
2.1.7.2. 7,8-Dimethyl-3-(4-methylphenyl)-4,6-dihydropyrano[6,5-f]-
1,4-benzoxazin-6-one 7b. The crude product was crystallized from
isopropanol. Yield 55%. mp 267–269 °C. IR t
max/cmꢀ1: 3258 (NH),
2.1.6. General procedure for synthesis of 6,7-dimethyl-2-substituted
amino-5H-pyrano[6,5-e] benzoxazol-5-ones 6a–c (Scheme 1)
To a solution of amino compound 3b (2.05 g, 0.01 mol) in etha-
nol (75 ml) containing few drops of triethylamine, the appropriate
isothiocyanate derivative (0.01 mol) was added. The solution was
refluxed for 15 h or till the evolution of hydrogen sulfide gas
ceases. The solvent was distilled under reduced pressure and the
residue was crystallized from the appropriate solvent.
3034 (CH Ar), 2969, 2873 (CH aliphatic), 1702 (C@O), 1609, 1581,
1569, 1505 (NH, C@C). ⁄1H NMR (500 MHz, DMSO-d6) d ppm:
2.03 (s, 3H, CH3 at C4), 2.31 (s, 3H, CH3 at C3), 2.36 (s, 3H, CH3 at
C40), 5.64 (s, 1H, CH Oxazine), 6.63 (d, 1H, J = 6.0 Hz, H-6 Ar),
6.74 (d, 2H, J = 6.9 Hz, H-30,50 Ar), 7.35 (d, 1H, J = 7.7 Hz, H-5 Ar),
7.55 (d, 2H, J = 8.4 Hz, H-20,60 Ar), 10.33 (s, 1H, NH). MS m/z (%):
321, M+ + 2 (0.03%). Anal. Calcd. for C20H17NO3 (319.35): C,
75.22; H, 5.37; N, 4.39. Found: C, 75.19; H, 5.41; N, 4.52.
2.1.6.1. 6,7-Dimethyl-2-ethylamino-5H-pyrano[6,5-e] benzoxazol-5-
one 6a. The crude product was crystallized from ethanol. Yield
50%. mp 199–203 °C. IR
2.1.7.3.
benzoxazin-6-one 7c. The crude product was crystallized from
isopropanol. Yield 71%. mp 130–133 °C. IR
max/cmꢀ1: 2927 (CH
7-Ethyl-8-methyl-3-phenyl-2,6-dihydropyrano[6,5-f]-1,4-
t
max/cmꢀ1: 3263 (NH), 3062 (CH Ar),
2966, 2873 (CH aliphatic), 1678 (C@O), 1604, 1554, 1500 (C@N,
NH, C@C). 1H NMR (300 MHz, DMSO-d6) d ppm: 1.21 (t, 3H, CH2-
CH3), 2.04 (s, 3H, CH3 at C4), 2.32 (s, 3H, CH3 at C3), 3.39 (q, 2H,
CH2CH3), 6.77 (d, 1H, J = 8.7 Hz, H-6 Ar), 7.59 (d, 1H, J = 9.0 Hz, H-
5 Ar), 8.16 (t, 1H, NH). MS m/z (%): 258, M+ (21.41)%. Anal. Calcd.
for C14H14N2O3 (258.27): C, 65.11; H, 5.46; N, 10.85. Found: C,
65.17; H, 5.50; N, 11.04.
t
aliphatic), 1678 (C@O), 1616, 1562, 1508 (C@N, C@C). ⁄1H NMR
(500 MHz, CDCl3) d ppm: 1.11 (t, 3H, CH2CH3), 2.35 (s, 3H, CH3),
2.64 (q, 2H, CH2CH3), 5.35 (s, 2H, CH2 Oxazine), 6.75 (d, 1H,
J = 8.4 Hz, H-6 Ar), 6.91 (d, 2H, J = 8.4 Hz, H-5 Ar), 7.49 (t, 2H,
H-30,50 Ar), 7.63 (t, 1H, H-40Ar), 7.97 (d, 2H, J = 7.7 Hz, H-20,60 Ar).
MS m/z (%): 319, M+ (50.00%). Anal. Calcd. for C20H17NO3
(319.35): C, 75.22; H, 5.37; N, 4.39. Found: C, 75.21; H, 5.39; N,
4.51.
2.1.6.2. 2-Allylamino-6,7-dimethyl-5H-pyrano[6,5-e] benzoxazol-5-
one 6b. The crude product was crystallized from isopropanol. Yield
45%. mp 278–281 °C. IR t
max/cmꢀ1: 3267 (NH), 3062 (CH Ar), 2970,
2.1.7.4.
ano[6,5-f]-1,4-benzoxazin-6-one 7d. The crude product was crystal-
lized from isopropanol. Yield 88%. mp 110–112 °C. IR
max/cmꢀ1
7-Ethyl-8-methyl-3-(4-methylphenyl)-2,6-dihydropyr-
2830 (CH aliphatic), 1690 (C@O), 1600, 1550, 1500 (C@N, NH,
C@C). 1H NMR (300 MHz, DMSO-d6) d ppm: 2.11 (s, 3H, CH3 at
C4), 2.41 (s, 3H, CH3 at C3), 3.97–4.02 (m, 2H, CH2CH@CH2),
5.12–5.31 (m, 2H, CH2CH@CH2), 5.92–6.31 (m, 1H, CH2CH@CH2),
7.37 (d, 1H, J = 8.7 Hz, H-6 Ar), 7.41 (d, 1H, J = 8.4 Hz, H-5 Ar),
8.40 (t, 1H, NH). MS m/z (%): 270, M+ (100%). Anal. Calcd. for
t
:
2927, 2854 (CH aliphatic), 1678 (C@O), 1616, 1566, 1512 (C@N,
C@C). 1H NMR (300 MHz, DMSO-d6) d ppm: 1.03 (t, 3H, CH2CH3),
2.38 (s, 3H, CH3 at C4), 2.40 (s, 3H, CH3 at C40), 2.50 (q, 2H, CH2CH3),
5.68 (s, 2H, CH2 Oxazine), 6.99 (d, 1H, J = 9.0 Hz, H-6 Ar), 7.38 (d,
2H, J = 7.8 Hz, H-30,50 Ar), 7.69 (d, 1H, J = 8.7 Hz, H-5 Ar), 7.93 (d,
2H, J = 8.4 Hz, H-20,60 Ar). MS m/z (%): 333, M+ (0.06%). Anal. Calcd.
for C21H19NO3 (333.38): C, 75.66; H, 5.74; N, 4.20. Found: C, 75.69;
H, 5.72; N, 4.28.
C15H14N2O3 (270.28): C, 66.66; H, 5.22; N, 10.36. Found: C, 66.71;
H, 5.24; N, 10.49.
2.1.6.3. 2-Benzylamino-6,7-dimethyl-5H-pyrano[6,5-e] benzoxazol-5-
one 6c. The crude product was crystallized from ethanol. Yield 33%.
mp 241–243 °C. IR
t
max/cmꢀ1: 3398 (NH), 2954, 2854 (CH ali-
phatic), 1701 (C@O), 1612, 1566, 1508 (C@N, NH, C@C). 1H NMR
(300 MHz, DMSO-d6) d ppm: 2.05 (s, 3H, CH3 at C4), 2.33 (s, 3H,
CH3 at C3), 4.57 (d, 2H, J = 6.3 Hz, CH2C6H5), 6.78 (d, 1H,
J = 8.7 Hz, H-6 Ar), 7.33–7.42 (m, 5H, Ar-H), 7.60 (d, 1H,
J = 8.7 Hz, H-5 Ar), 8.75 (t, 1H, NH). MS m/z (%): 320, M+ (0.25%).
Anal. Calcd. for C19H16N2O3 (320.34): C, 71.24; H, 5.03; N, 8.74.
Found: C, 71.31; H, 5.06; N, 8.85.
2.1.7.5. 3-(4-Bromophenyl)-7-ethyl-8-methyl-2,6-dihydropyrano[6,5-
f]-1,4-benzoxazin-6-one 7e. The crude product was crystallized
from isopropanol. Yield 65%. mp 146–149 °C. IR t
max/cmꢀ1: 3072
(CH Ar), 2926 (CH aliphatic), 1699 (C@O), 1618, 1608, 1566, 1510
(C@N, C@C). 1H NMR (300 MHz, DMSO-d6) d ppm: 1.03 (t, 3H, CH2-
CH3), 2.38 (s, 3H, CH3), 2.52 (q, 2H, CH2CH3), 5.70 (s, 2H, CH2 Oxa-
zine), 7.00 (d, 1H, J = 8.7 Hz, H-6 Ar), 7.69 (d, 1H, J = 8.7 Hz, H-5 Ar),
7.80 (d, 2H, J = 8.7 Hz, H-30,50 Ar), 7.96 (d, 2H, J = 8.1 Hz, H-20,60 Ar).
MS m/z (%): 398, M+ (0.11%). Anal. Calcd. for C20H16BrNO3 (398.25):
C, 60.32; H, 4.05; Br, 20.06; N, 3.52. Found: C, 60.38; H, 4.11; Br,
20.12; N, 3.63.
2.1.7. General procedure for synthesis of 7-alkyl-8-methyl-3-(4-(un)
substituted phenyl)-2,6-dihydropyrano[6,5-f]-1,4-benzoxazin-6-ones
7a–e (Scheme 1)
To a solution of compound 3a,b (0.01 mol) and sodium ethoxide
(0.01 mol) in ethanol (50 ml), the appropriate phenacyl bromide
derivative (0.02 mol) was added and the solution was refluxed
for 2 h. The reaction mixture was filtered and the filtrate was con-
2.1.8. (3-Ethyl-4-methyl-2-oxo-2H-1-benzopyran-7-yl)acetate 8
(Scheme 2)
was prepared as reported in literature [33].

54
S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
Scheme 2. Reagents and conditions: (i) acetic anhydride reflux, 2 h, (ii) anhydrous AlCl3, 125–155 °C, 2 h, (iii) malononitrile, benzaldehyde, ammonium acetate, ethanol,
reflux, 8 h, (iv) benzaldehyde, 2.5% NaOH, ethanol, r.t., 3 h, and (v) benzoyl chloride, 160–170 °C, 1.5 h.
Scheme 3. Reagents and conditions: (i) ethyl chloroacetate, anhydrous K2CO3, acetone, reflux, 24 h, (ii) hydrazine hydrate, ethanol, reflux, 24 h, (iii) appropriate
isothiocyanate, ethanol, reflux, 12 h, (iv) appropriate phenacyl bromide, ethanol/chloroform 1:3, reflux, 3 h, (v) chloroacetic acid, anhydrous sodium acetate, glacial acetic
acid, reflux, 4 h, (vi) conc. H2SO4, r.t. overnight, and (vii) 2N NaOH, reflux, 2 h.
2.1.9. 8-Acetyl-3-ethyl-7-hydroxy-4-methyl-2H-1-benzopyran-2-one
9 (Scheme 2)
was refluxed and stirred for 8 h. The reaction mixture was cooled,
the formed solid precipitate was filtered and dried. The crude prod-
uct was crystallized from ethanol. Yield 92%. mp 151–152 °C. IR
was prepared as reported in literature [33].
t
max/cmꢀ1: 3442 broad (2NH, OH), 3030 (CH Ar), 2933, 2835 (CH
aliphatic), 2210 (CN), 1716 (C@O), 1620, 1597, 1546 (C@N, NH,
C@C). 1H NMR (300 MHz, DMSO-d6) d ppm: 1.01 (t, 3H, CH2CH3),
2.31 (s, 3H, CH3), 2.52 (q, 2H, CH2CH3), 6.78 (s, 1H, C5-H dihydro-
pyridine), 6.93 (d, 1H, J = 8.7 Hz, H-6 Ar), 7.24–8.20 (m, 6H, Ar-H,
H-5 Ar), 8.26 (s, 1H, NH exchanged with D2O), 8.90 (s, 1H, NH ex-
2.1.10. 6-(3-Ethyl-7-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-8-yl)-
2-imino-4-phenyl-1,2-dihydropyridine-3-carbonitrile 10 (Scheme 2)
A mixture comprised of acetyl compound 9 (2.46 g, 0.01 mol),
malononitrile (0.66 g, 0.01 mol), ammonium acetate (6.16 g,
0.08 mol) and benzaldehyde (1.06 g, 0.01 mol) in ethanol (50 ml)

S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
55
changed with D2O), 12.60 (s, 1H, OH exchanged with D2O) MS m/z
(%): 397, M+ (12.69%). Anal. Calcd. for C24H19 N3O3 (397.43): C,
72.53; H, 4.82; N, 10.57. Found: C, 72.51; H, 4.88; N, 10.71.
NH2), 3062 (CH Ar), 2962, 2870 (CH aliphatic), 1695, (2 C@O),
1625, 1612, 1568, 1510, (NH, C@C). ⁄1H NMR (500 MHz, DMSO-
d6) d ppm: 1.00 (t, 3H, CH2CH3), 2.34 (s, 3H, CH3), 2.51 (q, 2H, CH2-
CH3), 4.32 (s, 2H, NH2), 4.56 (s, 2H, OCH2), 6.89 (s, 1H, H-8 Ar), 6.94
(d, 1H, J = 8.4 Hz, H-6 Ar), 7.66 (d, 1H, J = 8.4 Hz, H-5 Ar), 9.37 (s,
1H, NH). MS m/z (%): 276, M+ (36.51%). Anal. Calcd. for
2.1.11. 8-Cinnamoyl-3-ethyl-7-hydroxy-4-methyl-2H-1-benzopyran-
2-one 11 (Scheme 2)
A solution of benzaldehyde (1.06 g, 0.01 mol) in ethanol (30 ml)
was added while stirring and cooling to a solution of the acetyl
compound 9 (2.46 g, 0.01 mol) in 2.5% solution of sodium hydrox-
ide (12 ml). The reaction mixture was stirred at room temperature
for 3 h and left overnight. It was poured onto ice and the separated
precipitate was filtered and dried. It was crystallized from ethanol.
C14H16N2O4 (276.29): C, 60.86; H, 5.84; N, 10.14. Found: C, 61.59;
H, 5.85; N, 9.35.
2.1.15. General procedure for synthesis of 1-(3,4-dimethyl-2-oxo-2H-
1-benzopyran-7-yloxy)acetyl-4-substituted thiosemicarbazides 15a,b
(Scheme 3)
To a solution of compound 14a (2.62 g, 0.01 mol) in ethanol
(30 ml), the appropriate substituted isothiocyanate (0.01 mol)
was added and the mixture was refluxed while stirring for 12 h.
The separated solid was filtered, washed with diethyl ether and
dried.
Yield 89%. mp 187–189 °C. IR t
max/cmꢀ1: 3446 (OH), 3032 (CH Ar),
2966, 2873 (CH aliphatic), 1716, 1678 (2C@O), 1618, 1604, 1564
(C@C). 1H NMR (300 MHz, DMSO-d6) d ppm: 1.04 (t, 3H, CH2CH3),
2.39 (s, 3H, CH3), 2.86 (q, 2H, CH2CH3), 6.93 (d, 1H, J = 9.0 Hz, H-6
Ar), 7.08–7.99 (m, 8H, CH@CH, Ar-H, H-5 Ar), 10.85 (s, 1H, OH).
MS m/z (%): 334, M+ (41.97%). Anal. Calcd. for C21H18O4 (334.37):
C, 75.43; H, 5.43. Found: C, 75.47; H, 5.42.
2.1.15.1. 1-(3,4-Dimethyl-2-oxo-2H-1-benzopyran-7-yloxy)acetyl-4-
ethylthiosemicarbazide 15a. The crude product was crystallized
from ethanol. Yield 76%. mp 221–223 °C. IR
2.1.12. (8-Acetyl-3-ethyl-4-methyl-2-oxo-2H-1-benzopyran-7-yl)
benzoate 12 (Scheme 2)
t
max/cmꢀ1: 3392,
3365, 3246 (3NH), 3061 (CH Ar), 2980, 2873 (CH aliphatic), 1708,
1691 (2C@O), 1627, 1600, 1568, 1548 (NH, C@C), 1282 (C@S). 1H
NMR (300 MHz, DMSO-d6) d ppm: 1.06 (t, 3H, CH2CH3), 2.08 (s,
3H, CH3 at C4), 2.37 (s, 3H, CH3 at C3), 3.45 (q, 2H, CH2CH3), 4.70
(s, 2H, OCH2), 6.63 (s, 1H, NH exchanged with D2O), 6.97 (s, 1H,
H-8 Ar), 7.01 (d, 1H, J = 8.4 Hz, H-6 Ar), 7.18 (s, 1H, NH, exchanged
with D2O), 7.72 (d, 1H, J = 9.0 Hz, H-5 Ar), 8.03 (t, 1H, NH ex-
changed with D2O). MS m/z (%): 349, M+ (0.58%). Anal. Calcd. for
A mixture of compound 9 (2.62 g, 0.01 mol) and benzoyl chlo-
ride (2.81 g, 0.02 mol) was refluxed for 1.5 h at 160–170 °C in an
oil bath. The mixture was poured onto ice-water. The obtained
sticky mass was suspended in the least amount of ethanol and fil-
tered off. The residue was washed several times with petroleum
ether and dried. It was crystallized from isopropanol. Yield 95%.
mp 195–196 °C. IR t
max/cmꢀ1: 3085 (CH Ar), 2970, 2887 (CH ali-
phatic), 1723 (3C@O), 1594 (C@C). 1H NMR (300 MHz, DMSO-d6)
d ppm: 1.08 (t, 3H, CH2CH3), 2.47 (s, 3H, CH3), 2.58 (s, 3H, COCH3),
2.62 (q, 2H, CH2CH3), 7.42 (d, 1H, J = 8.7 Hz, H-6 Ar), 7.63 (t, 2H, H-
30,50 Ar), 7.78 (t, 1H, H-40 Ar), 7.98 (d, 1H, J = 8.7 Hz, H-5 Ar), 8.08 (d,
2H, J = 8.4 Hz, H-20,60Ar). MS m/z (%): 350, M+ (0.07%). Anal. Calcd.
for C21H18O5 (350.36): C, 71.99; H, 5.18. Found: C, 72.08; H, 5.18.
C16H19N3O4S (349.40): C, 55.00; H, 5.48; N, 12.03; S, 9.18. Found:
C, 55.05; H, 5.52; N, 11.98; S, 9.22.
2.1.15.2. 1-(3,4-Dimethyl-2-oxo-2H-1-benzopyran-7-yloxy)acetyl-4-
phenylthiosemicarbazide 15b. The crude product was crystallized
from ethanol. Yield 88%. mp 200–204 °C. IR
t
max/cmꢀ1: 3332,
3192, 3126 (3NH), 3070 (CH Ar), 2927, 2856 (CH aliphatic), 1740,
1710 (2C@O), 1606, 1546 (NH, C@C), 1251 (C@S). 1H NMR
(300 MHz, DMSO-d6) d ppm: 2.06 (s, 3H, CH3 at C4), 2.33 (s, 3H,
CH3 at C3), 4.75 (s, 2H, OCH2), 6.90–7.74 (m, 8H, Ar-H), 10.26 (s,
1H, NH), 11.00 (s, 1H, NH), 14.08 (s, 1H, NH). MS m/z (%): 397,
M+ (16.48%). Anal. Calcd. for C20H19N3O4S (397.45): C, 60.44; H,
4.82; N, 10.57; S, 8.07. Found: C, 60.48; H, 4.86; N, 10.65; S, 8.04.
2.1.13. General procedure for synthesis of ethyl (3-alkyl-4-methyl-2-
oxo-2H-1-benzopyran-7-yloxy)acetates 13a [34], b (Scheme 3)
A mixture of the hydroxy coumarin derivatives 1a,b (0.1 mol),
anhydrous potassium carbonate (2.76 g, 0.2 mol) and ethyl chloro-
acetate (14.64 g, 0.12 mol) in dry acetone (50 ml) was refluxed
with continuous stirring for 24 h. After completion of the reaction,
reaction mixture was cooled, filtered and washed with acetone.
The washing and filtrate was evaporated and residue obtained
was dried.
2.1.16. General procedure for synthesis of 2-(3,4-dimethyl-2-oxo-2H-
1-benzopyran-7-yloxyl)-N-[2-(substituted imino)-4-(4-
(un)substituted phenyl)thiazol-3(2H)-yl] acetamides 16a–c
(Scheme 3)
A mixture of compound 15a,b (0.002 mol) and the appropriate
phenacyl bromide (0.002 mol) in ethanol/chloroform 1:3 mixture
(100 ml) was refluxed for 3 h, concentrated, left to cool, filtered,
washed with water and dried.
2.1.13.1.
yloxy)acetate 13b. The crude product was crystallized from etha-
nol. Yield 71%. mp 99–101 °C. IR
max/cmꢀ1: 3030 (CH Ar), 2970,
Ethyl
(3-ethyl-4-methyl-2-oxo-2H-1-benzopyran-7-
t
2873 (CH aliphatic), 1757, 1707 (2C@O), 1608, 1566, 1508 (C@C).
1H NMR (300 MHz, DMSO-d6) d ppm: 1.03 (t, 3H, CH2CH3), 1.21
(t, 3H, OCH2CH3), 2.38 (s, 3H, CH3), 2.55 (q, 2H, CH2CH3), 4.18 (q,
2H, OCH2CH3), 4.90 (s, 2H, OCH2), 6.95 (d, 2H, J = 9.6 Hz, H-6, 8
Ar), 7.69 (d, 1H, J = 8.3 Hz, H-5 Ar). MS m/z (%): 290, M+ (0.14%).
Anal. Calcd. for C16H18O5 (290.31): C 66.19; H 6.25. Found: C
66.21; H 6.28.
2.1.16.1.
2-(3,4-Dimethyl-2-oxo-2H-1-benzopyran-7-yloxy)-N-(2-
ethylimino-4-phenylthiazol-3(2H)-yl)- acetamide 16a. The crude
product was crystallized from glacial acetic acid. Yield 56%. mp
160–162 °C. IR t
max/cmꢀ1: 3435 (NH), 3061 (CH Ar), 2943, 2895
2.1.14. General procedure for synthesis of (3-alkyl-4-methyl-2-oxo-
2H-1-benzopyran-7-yloxy)acetic acid hydrazides 14a [34], b (Scheme 3)
A solution of 14a,b (0.01 mol) and hydrazine hydrate 99% (1 g,
0.02 mol) in absolute ethanol (10 ml) was refluxed for 2 h. The pre-
cipitate 14a or 14b was filtered and dried.
(CH aliphatic), 1730, 1708 (2C@O), 1620, 1571, 1506 (C@N, NH,
C@C). 1H NMR (300 MHz, DMSO-d6) d ppm: 1.31 (t, 3H, CH2CH3),
2.08 (s, 3H, CH3 at C4), 2.36 (s, 3H, CH3 at C3), 4.07 (q, 2H, CH2CH3),
4.98 (s, 1H, NH, exchanged with D2O), 5.40 (s, 2H, OCH2), 7.04 (d,
1H, J = 9.0 Hz, H-6 Ar), 7.14 (s, 2H, H-8 Ar, C5-H thiazoline), 7.55
(t, 1H, H-40 Ar), 7.65–7.74 (m, 3H, H-5 Ar, H-30,50 Ar), 8.01 (d, 2H,
J = 8.1 Hz, H-20,60 Ar). MS m/z (%): 449, M+ (2.35%). Anal. Calcd.
for C24H23N3O4S (449.52): C, 64.13; H, 5.16; N, 9.35; S, 7.13. Found:
C, 64.21; H, 5.14; N, 9.47; S, 7.22.
2.1.14.1. (3-Ethyl-4-methyl-2-oxo-2H-1-benzopyran-7-yloxy)acetic
acid hydrazide 14b. The crude product was crystallized from acetic
acid. Yield 90%. mp 175–177 °C. IR t
max/cmꢀ1: 3446, 3213 (NH,

56
S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
2.1.16.2.
N-[4-(4-bromophenyl)-2-ethyliminothiazol-3(2H)-yl]-2-
2.1.18. General procedure for synthesis of 7-(5-Substituted amino-1,3,
4-thiadiazol-2-yl)methoxy-3,4-dimethyl-2H-1-benzopyran-2-one 18a,b
(Scheme 3)
A solution of thiosemicarbazides 15a,b (0.01 mol) in concen-
trated sulfuric acid (10 ml) was cooled and allowed to stand over-
night. The reaction mixture was cooled then poured onto ice-water
and neutralized with ammonium hydroxide solution to pH 7. The
produced precipitate was filtered off, washed and dried.
(3,4-dimethyl-2-oxo-2H-1-benzopyran-7-yloxy)acetamide 16b. The
crude product was crystallized from glacial acetic acid. Yield 46%.
mp 140–142 °C. IR
t
max/cmꢀ1: 3431 (NH), 3070 (CH Ar), 2976,
2872 (CH aliphatic), 1708, 1695 (2C@O), 1612, 1583, 1568, 1508
(C@N, NH, C@C). ⁄1H NMR (500 MHz, DMSO-d6) d ppm: 1.18 (t,
3H, CH2CH3), 2.04 (s, 3H, CH3 at C4), 2.32 (s, 3H, CH3 at C3), 3.99
(q, 2H, CH2CH3), 4.93 (s, 1H, NH), 5.39 (s, 2H, OCH2), 7.00 (s, 2H,
H-8 Ar, C5-H thiazoline), 7.10 (d, 1H, J = 6.9 Hz, H-6 Ar), 7.68 (d,
2H, J = 9.2 Hz, H-20,60 Ar), 7.73 (d, 1H, J = 6.1 Hz, H-5 Ar), 7.97 (d,
2H, J = 9.2 Hz, H-30,50 Ar). MS m/z (%): 528, M+ (4.51%). Anal. Calcd.
for C24H22BrN3O4S (528.42): C, 54.55; H, 4.20; Br, 15.12; N, 7.95; S,
6.07. Found: C, 54.58; H, 4.27; Br, 15.23; N, 8.11; S, 6.10.
2.1.18.1.
dimethyl-2H-1-benzopyran-2-one 18a. The crude product was crys-
tallized from glacial acetic acid. Yield 91%. mp 247–249 °C. IR tmax
7-(5-Ethylamino-1,3,4-thiadiazol-2-yl)methoxy-3,4-
/
cmꢀ1: 3157 (NH), 3030 (CH Ar), 1708 (C@O), 1660, 1612, 1566
(C@N, NH, C@C). ⁄1H NMR (500 MHz, DMSO-d6) d ppm: 1.22 (t,
3H, CH2CH3), 2.04 (s, 3H, CH3 at C4), 2.33 (s, 3H, CH3 at C3), 4.00
(q, 2H, CH2CH3), 5.33 (s, 2H, OCH2), 7.01 (d, 1H, J = 10.7 Hz, H-6
Ar), 7.10 (s, 1H, H-8 Ar), 7.70 (d, 1H, J = 9.2 Hz, H-5 Ar), 13.87 (s,
1H, NH). MS m/z (%): 331, M+ (78.89%). Anal. Calcd. for C16H17N3O3-
S (331.39): C, 57.99; H, 5.17; N, 12.68; S, 9.68. Found: C, 58.06; H,
5.22; N, 12.83; S, 9.64.
2.1.16.3. N-[4-(4-bromophenyl)-2-phenyliminothiazol-3(2H)-yl]-2-
(3,4-dimethyl-2-oxo-2H-1-benzopyran-7-yloxy)acetamide 16c. The
crude product was crystallized from glacial acetic acid. Yield 32%.
mp 180–182 °C. IR
t
max/cmꢀ1: 3406 (NH), 3059 (CH Ar), 2924,
2852 (CH aliphatic), 1710 (2C@O), 1664, 1612, 1587, 1566, 1548
(C@N, NH, C@C). 1H NMR (300 MHz, DMSO-d6) d ppm: 2.06 (s,
3H, CH3 at C4), 2.34 (s, 3H, CH3 at C3), 4.94 (s, 1H, NH), 5.20 (s,
2H, OCH2), 6.74 (s, 1H, H-8 Ar), 7.06 (d, 1H, J = 8.4 Hz, H-6 Ar),
7.09–7.47 (m, 11H, Ar H, H-5 Ar, C5-H thiazoline). MS m/z (%):
578, M++2 (0.02%). Anal. Calcd. for C28H22BrN3O4S (576.46): C,
58.34; H, 3.85; Br, 13.86; N, 7.29. Found: C, 58.41; H, 3.93; Br,
14.01; N, 7.35.
2.1.18.2.
dimethyl-2H-1-benzopyran-2-one 18b. The crude product was crys-
tallized from glacial acetic acid. Yield 96%. mp 305–306 °C. IR tmax
7-(5-Phenylamino-1,3,4-thiadiazol-2-yl)methoxy-3,4-
/
cmꢀ1: 3305 (NH), 3041 (CH Ar), 2924, 2854 (CH aliphatic), 1693
(C@O), 1606, 1573, 1504 (C@N, NH, C@C). 1H NMR (300 MHz,
DMSO-d6) d ppm: 2.08 (s, 3H, CH3 at C4), 2.37 (s, 3H, CH3 at C3),
5.53 (s, 2H, OCH2), 7.05–7.58 (m, 7H, H-6, 8 Ar, Ar-H), 7.74 (d,
1H, J = 8.7 Hz, H-5 Ar), 10.50 (s, 1H, NH). MS m/z (%): 379, M+
(64.41%). Anal. Calcd. for C20H17N3O3S (379.43): C, 63.31; H, 4.52;
N, 11.07; S, 8.45. Found: C, 63.35; H, 4.56; N, 11.13; S, 8.53.
2.1.17. General procedure for synthesis of N0-(4-Oxo-3-substituted
thiazolidin-2-yl)-2-(3,4-dimethyl-2-oxo-2H-1-benzopyran-7-
yloxy)acetohydrazides 17a,b (Scheme 3)
To a suspension of substituted thiosemicarbazide derivatives
15a,b (0.01 mol) in glacial acetic acid (5 ml), anhydrous sodium
acetate (1.64 g, 0.02 mol) and chloroacetic acid (1.89 g, 0.02 mol)
were added. The reaction mixture was refluxed for 4 h then cooled,
diluted with water and allowed to stand overnight. The product
was filtered off, washed and dried.
2.1.19. General procedure for synthesis of 7-(4-Substituted-5-thioxo-
1,2,4-triazol-3-yl)methoxy-3,4-dimethyl-2H-1-benzopyran-2-ones
19a,b (Scheme 3)
A solution of compound 15a,b (0.04 mol) in 2 N sodium hydrox-
ide (12 ml) was refluxed for 2 h. The reaction mixture was cooled
and acidified with 10% hydrochloric acid to pH 5. The produced
precipitate was filtered, washed and dried.
2.1.17.1. N0-(3-Ethyl-4-oxothiazolidin-2-yl)-2-(3,4-dimethyl-2-oxo-
2H-1-benzopyran-7-yloxy)acetohydrazide 17a. The crude product
was crystallized from DMF/ H2O. Yield 87%. mp 232–235 °C. IR
2.1.19.1.
dimethyl-2H-1-benzopyran-2-one 19a. The crude product was crys-
tallized from methanol. Yield 69%. mp 198–200 °C. IR
max/cmꢀ1
7-(4-Ethyl-5-thioxo-1,2,4-triazol-3-yl)methoxy-3,4-
t
max/cmꢀ1: 3431 (NH), 3047 (CH Ar), 2980, 2875 (CH aliphatic),
t
:
1735, 1722, 1703 (3C@O), 1624, 1595, 1570, 1539 (C@N, NH,
C@C). ⁄1H NMR (500 MHz, DMSO-d6) d ppm: 1.25 (t, 3H, CH2CH3),
2.04 (s, 3H, CH3 at C4), 2.33 (s, 3H, CH3 at C3), 3.65 (q, 2H, CH2CH3),
4.71 (s, 2H, CH2 thiazolidine), 5.38 (s, 2H, OCH2), 7.01 (d, 1H, J = 8.4,
H-6 Ar), 7.11 (s, 1H, H-8 Ar), 7.60 (d, 1H, J = 8.3 Hz, H-5 Ar), 10.53
(s, 1H, NH exchanged with D2O). MS m/z (%): 389, M+ (79.95%).
Anal. Calcd. for C18H19N3O5S (389.43): C, 55.52; H, 4.92; N, 10.79;
S, 8.23. Found: C, 55.57; H, 4.88; N, 10.92; S, 8.19.
3390 (NH), 3057 (CH Ar), 2920, 2850 (CH aliphatic), 1689 (C@O),
1612, 1573, 1506 (C@N, NH, C@C), 1274 (C@S). ⁄1H NMR
(500 MHz, DMSO-d6) d ppm: 1.20 (t, 3H, CH2CH3), 2.02 (s, 3H,
CH3 at C4), 2.30 (s, 3H, CH3 at C3), 4.06 (q, 2H, CH2CH3), 4.70 (br
s, 1H, NH), 5.30 (s, 2H, OCH2), 7.00 (d, 1H, J = 8.4 Hz, H-6 Ar),
7.09 (s, 1H, H-8 Ar), 7.67 (d, 1H, J = 8.4 Hz, H-5 Ar). MS m/z (%):
331, M+ (0.80%). Anal. Calcd. for C16H17N3O3S (331.39): C, 57.99;
H, 5.17; N, 12.68; S, 9.68. Found: C, 58.04; H, 5.19; N, 12.79; S, 9.74.
2.1.17.2. N0-(4-Oxo-3-phenylthiazolidin-2-yl)-2-(3,4-dimethyl-2-oxo-
2H-1-benzopyran-7-yloxy)acetohydrazide 17b. The crude product
was crystallized from DMF/ H2O. Yield 83%. mp 347–350 °C. IR
2.1.19.2.
dimethyl-2H-1-benzopyran-2-one 19b. The crude product was crys-
tallized from methanol. Yield 38%. mp 191–194 °C. IR
max/cmꢀ1
7-(4-Phenyl-5-thioxo-1,2,4-triazol-3-yl)methoxy-3,4-
t
:
t
max/cmꢀ1: 3192 (NH), 3070 (CH Ar), 2916, 2862 (CH aliphatic),
3415 (NH), 3039 (CH Ar), 2924, 2856 (CH aliphatic), 1700 (C@O),
1643, 1602, 1568, 1502 (C@N, NH, C@C), 1286 (C@S). 1H NMR
(300 MHz, DMSO-d6) d ppm: 2.05 (s, 3H, CH3 at C4), 2.33 (s, 3H,
CH3 at C3), 5.09 (s, 2H, OCH2), 6.80 (d, 1H, J = 8.7 Hz, H-6 Ar),
6.90 (s, 1H, H-8 Ar), 7.43–7.55 (m, 5H, Ar-H), 7.64 (d, 1H,
J = 8.7 Hz, H-5 Ar), 14.06 (s, 1H, NH exchanged with D2O). MS m/
z (%): 380, M+ + 1 (57.34%). Anal. Calcd. for C20H17N3O3S (379.43):
C, 63.31; H, 4.52; N, 11.07; S, 8.45. Found: C, 63.36; H, 4.57; N,
11.18; S, 8.51.
1703, 1687, 1680 (3C@O), 1624, 1606, 1566 (C@N, NH, C@C). 1H
NMR (300 MHz, DMSO-d6) d ppm: 2.06 (s, 3H, CH3 at C4), 2.37 (s,
3H, CH3 at C3), 4.72 (s, 2H, CH2 thiazolidine), 5.19 (s, 2H, OCH2),
6.84 (d, 1H, J = 9.0 Hz, H-6 Ar), 6.94 (s, 1H, H-8 Ar), 7.01 (d, 1H,
J = 9.0 Hz, H-5 Ar), 7.48–7.73 (m, 5H, Ar-H), 10.16 (s, 1H, NH ex-
changed with D2O). MS m/z (%): 439, M+ + 2 (0.01%). Anal. Calcd.
C22H19N3O5S for (437.47): C, 60.40; H, 4.38; N, 9.61; S, 7.33. Found:
C, 60.38; H, 4.43; N, 9.70; S, 7.41.

S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
57
2.1.20. 4-Benzyl-1-(3,4-dimethyl-2-oxo-2H-1-benzopyran-7-yloxy-
acetyl)semicarbazide 20 (Scheme 4)
cmꢀ1: 3427 (NH), 2932 (CH aliphatic), 1685 (2C@O), 1612 (C@C).
1H NMR (300 MHz, DMSO-d6) d ppm: 2.07 (s, 3H, CH3 at C4),
2.36 (s, 3H, CH3 at C3), 3.80 (s, 3H, OCH3), 5.24 (s, 2H, OCH2),
6.93 (d, 1H, J = 6.9 Hz, H-6 Ar), 7.00 (d, 2H, J = 8.4 Hz, H-30,50 Ar),
7.62–7.74 (m, 4H, H-5, 8 Ar, H-20,60 Ar), 8.27 (s, 1H, N@CH),
11.49 (s, 1H, NH, exchanged with D2O). MS m/z (%): 380, M+
(73.18%). Anal. Calcd. for C21H20N2O5 (380.39): C, 66.31; H, 5.30;
N, 7.36. Found: C, 66.37; H, 5.34; N, 7.49.
A mixture of 14a (2.62 g, 0.01 mol) and benzyl isocyanate
(1.59 g, 0.01 mol) in dichloroethane was stirred and refluxed for
3 h. The formed precipitate was filtered, washed with diethylether
and dried. The crude product was crystallized from isopropanol.
Yield 98%. mp 272–273 °C. IR
t
max/cmꢀ1: 3460, 3327, 3294 (3
NH), 3057 (CH Ar), 2912, 2877 (CH aliphatic), 1722, 1693
(3C@O), 1649, 1624, 1566, 1537, 1500 (NH, C@C). 1H NMR
(300 MHz, DMSO-d6) d ppm: 2.07 (s, 3H, CH3 at C4), 2.36 (s, 3H,
CH3 at C3), 4.24 (d, 2H, J = 6.0 Hz, CH2C6H5), 4.69 (s, 2H, OCH2),
6.96–7.28 (m, 7H, H-6, 8 Ar, Ar-H), 7.71 (d, 1H, J = 8.7 Hz, H-5
Ar), 7.95 (s, 1H, NH), 8.14 (s, 1H, NH), 9.90 (s, 1H, NH). MS m/z
(%): 395, M+ (1.42%). Anal. Calcd. for C21H21N3O5 (395.41): C,
63.79; H, 5.35; N, 10.63. Found: C, 63.81; H, 5.39; N, 10.76.
2.1.22.2. N0-(4-Chlorobenzylidene)-2-(3-ethyl-4-methyl-2-oxo-2H-1-
benzopyran-7-yloxy)acetohydrazide 22b. The crude product was
crystallized from ethanol. Yield 53%. mp 252–254 °C. IR tmax
/
cmꢀ1: 3446 (NH), 3030 (CH Ar), 2976, 2875 (CH aliphatic), 1732,
1716 (2C@O), 1624, 1610, 1595, 1558, 1541, 1505 (NH, C@C). 1H
NMR (300 MHz, DMSO-d6) d ppm: 1.04 (t, 3H, CH2CH3), 2.39 (s,
3H, CH3), 2.56 (q, 2H, CH2CH3), 5.27 (s, 2H, OCH2), 6.94 (s, 1H, H-
8), 6.98 (d, 1H, J = 8.4 Hz, H-6 Ar), 7.50 (d, 1H, J = 8.4 Hz, H-5 Ar),
7.58 (d, 2H, J = 8.4 Hz, H-30,50 Ar), 7.90 (d, 2H, J = 8.7 Hz, H-20,60
Ar), 8.01 (s, 1H, N@CH), 11.70 (s, 1H, NH). MS m/z (%): 398, M+
(0.39%). Anal. Calcd. for C21H19ClN2O4 (398.84): C, 63.24; H, 4.80;
N, 7.02. Found: C, 63.25; H, 4.83; N, 7.14.
2.1.21. 3-Benzyl-1-(3,4-dimethyl-2-oxo-2H-1-benzopyran-7-yl-oxy)
acetamidoimidazolidin-2,4,5-trione 21 (Scheme 4)
To a solution of compound 20 (3.95 g, 0.001 mol) in benzene
(5 ml), oxalyl chloride (0.25 g, 0.002 mol) was added dropwise
while stirring, then refluxed at 60–65 °C for 2 h. The solvent was
distilled under reduced pressure and the obtained residue was
dried. The crude product was crystallized from ethanol. Yield
88%. mp 207–208 °C. IR
t
max/cmꢀ1: 3415 (NH), 3084 (CH Ar),
2.1.23. General procedure for synthesis of 1-[2-(3-Alkyl-4-methyl-2-
oxo-2H-1-benzopyran-7-yloxy)acetyl]-3-methyl-1,2-dihydropyrazol-
5-one 23a [13], b (Scheme 4)
A mixture of the acid hydrazide 14a,b (0.01 mol) and ethyl ace-
toacetate (2.6 g, 0.02 mol) in glacial acetic acid (5 ml) was refluxed
for 6 h then diluted with water. The precipitated solid 23a,b was
filtered and dried.
2972, 2873 (CH aliphatic), 1749, 1728, 1707, 1699 (5C@O), 1620,
1597, 1558 (NH, C@C). 1H NMR (300 MHz, DMSO-d6) d ppm: 2.07
(s, 3H, CH3 at C4), 2.37 (s, 3H, CH3 at C3), 4.78 (s, 2H, CH2C6H5),
4.96 (s, 2H, OCH2), 6.98 (s, 1H, H-8 Ar), 7.01 (d, 1H, J = 8.7 Hz, H-
6 Ar), 7.32–7.36 (m, 5H, Ar-H), 7.74 (d, 1H, J = 9.0 Hz, H-5 Ar),
11.44 (s, 1H, NH). MS m/z (%): 449, M+ (17.70%). Anal. Calcd. for
C23H19 N3O7 (449.41): C, 61.47; H, 4.26; N, 9.35. Found: C, 61.49;
H, 4.31; N, 9.43.
2.1.23.1.
1-[2-(3-Ethyl-4-methyl-2-oxo-2H-1-benzopyran-7-yloxy)
2.1.22. General procedure for synthesis of 2-(3-Alkyl-4-methyl-2-oxo-
2H-1-benzopyran-7-yloxy)-N0-(4-substituted benzylidene)acetohydrazides
22a,b (Scheme 4)
acetyl]-3-methyl-1,2-dihydropyrazol-5-one 23b. The crude product
was crystallized from glacial acetic acid. Yield 35%. mp 178–
179 °C. IR t
max/cmꢀ1: 3257 (NH), 3034 (CH Ar), 2987, 2856 (CH ali-
A solution of the acid hydrazides 14a,b (0.01 mol) and the
appropriate aromatic aldehyde (0.01 mol) in glacial acetic acid
(20 ml) was refluxed for 6 h. The solvent was distilled under
vacuum.
phatic), 1747, 1732, 1705 (3C@O), 1612, 1570, 1543, 1500 (NH,
C@C). 1H NMR (300 MHz, DMSO-d6) d ppm: 1.05 (t, 3H, CH2CH3),
1.87 (s, 3H, CH3 at C3 pyrazolone), 2.38 (s, 3H, CH3 at C4), 2.56
(q, 2H, CH2CH3), 4.71 (s, 2H, OCH2), 4.79 (s, 1H, CH pyrazolone),
6.94 (d, 1H, J = 8.7 Hz, H-6 Ar), 7.01 (s, 1H, H-8 Ar), 7.69 (d, 1H,
J = 8.7 Hz, H-5 Ar), 10.08 (s, 1H, NH). MS m/z (%): 343, M+ + 1
(5.41%). Anal. Calcd. for C18H18N2O5 (342.35): C, 63.15; H, 5.30;
N, 8.18. Found: C, 63.19; H, 5.28; N, 8.29.
2.1.22.1.
methoxybenzylidene)acetohydrazide 22a. The crude product was
crystallized from ethanol. Yield 69%. mp 225–226 °C. IR tmax
2-(3,4-Dimethyl-2-oxo-2H-1-benzopyran-7-yloxy)-N0-(4-
/
Scheme 4. Reagents and conditions: (i) benzyl isocyanate, dichloroethane, reflux, 3 h, (ii) oxalyl chloride, benzene, reflux, 2 h, (iii) appropriate aromatic aldehyde, glacial
acetic acid, reflux, 6 h, (iv) ethyl acetoacetata, glacial acetic acid, reflux, 6 h, and (v) appropriate cyclic acid anhydride, glacial acetic acid, reflux, 9 h.

58
S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
2.1.24. General procedure for synthesis of N-(2,5-Dioxo-2,5-dihydro-
1H-pyrrol-1-yl)-2-(3-ethyl-4-methyl-2-oxo-2H-1-benzopyran-7-yloxy)
acetamide 24a and N-(2,5-Dioxopyrrolidin-1-yl)-2-(3-ethyl-4-
methyl-2-oxo-2H-1-benzopyran-7-yloxy)acetamide 24b (Scheme 4)
A mixture of compound (14b) (0.01 mol) and the appropriate
cyclic acid anhydride (0.01 mol) in glacial acetic acid (10 ml) was
refluxed for 9 h. The solvent was concentrated then poured onto
ice-water, the precipitated product was filtered, dried and crystal-
lized from isopropanol.
temperature. After staining, unbound dye was removed by wash-
ing five times with 1% acetic acid and the plates were air dried.
Bound stain was subsequently solubilized with 10 mM trizma
base, and the absorbance was read on an automated plate reader
at a wavelength of 515 nm [35–38].
Using the seven absorbance measurements [time zero, (Tz),
control growth, (C), and test growth in the presence of drug at
the 10ꢀ5 M concentration level (Ti)], the percentage growth was
calculated. Percentage growth inhibition was calculated as:
½ðTi ꢀ TzÞ=ðC ꢀ TzÞꢁ ꢂ 100 for concentrations for which Ti P Tz
½ðTi ꢀ TzÞ=Tzꢁ ꢂ 100 for concentrations for which Ti < Tz
2.1.24.1.
N-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-(3-ethyl-4-
methyl-2-oxo-2H-1-benzopyran-7-yloxy)acetamide 24a. The crude
product was crystallized from isopropanol. Yield 29%. mp 208–
210 °C. IR t
max/cmꢀ1: 3446 (NH), 3093 (CH Ar), 2968, 2873 (CH ali-
phatic), 1751, 1703, 1691, 1678 (4C@O), 1622, 1604, 1568, 1506
(NH, C@C). 1H NMR (300 MHz, DMSO-d6) d ppm: 1.04 (t, 3H, CH2-
CH3), 2.40 (s, 3H, CH3), 2.57 (q, 2H, CH2CH3), 4.90 (s, 2H, OCH2),
6.99–7.05 (m, 3H, CH@CH, H-6 Ar), 7.20 (s, 1H, H-8), 7.74 (d, 1H,
J = 9.0 Hz, H-5 Ar), 10.80 (s,1H, NH). MS m/z (%): 356, M+ (7.59%).
Anal. Calcd. for C18H16N2O6 (356.33): C, 60.67; H, 4.53; N, 7.86.
Found: C, 60.70; H, 4.52; N, 7.98.
Mean graph is the mean of presenting the in vitro test results to
emphasize differential effects of test compounds on various human
tumor cell lines. It plots the growth relative to no drug control and
relative to time zero number of cells. The mean is the average of
growth across the tested cell lines, while delta is the maximum dif-
ference from the mean.
2.3. Molecular docking
2.1.24.2.
2H-1-benzopyran-7-yloxy)acetamide 24b. The crude product was
crystallized from isopropanol. Yield 69%. mp 238–239 °C. IR tmax
N-(2,5-Dioxopyrrolidin-1-yl)-2-(3-ethyl-4-methyl-2-oxo-
Docking studies of fourteen screened compounds (4a–c, 5a, 6c,
7a–e, 10, 18a,b, 23a) compounds performed by Molsoft ICM 3.4-8C
program.
/
cmꢀ1: 3446 (NH), 3070 (CH Ar), 2981, 2833 (CH aliphatic), 1707,
1691, 1676 (4C@O), 1602, 1583, 1558, 1525 (NH, C@C). 1H NMR
(300 MHz, DMSO-d6) d ppm:1.04 (t, 3H, CH2CH3), 2.39 (s, 3H,
CH3), 2.57 (q, 2H, CH2CH3), 2.80 (t, 4H, CH2CH2), 4.88 (s, 2H,
OCH2), 6.99 (s, 1H, H-8 Ar), 7.03 (d, 1H, J = 8.7 Hz, H-6 Ar), 7.74
(d, 1H, J = 8.7 Hz, H-5 Ar), 10.80 (s, 1H, NH). MS m/z (%): 358, M+
(11.72%). Anal. Calcd. for C18H18N2O6 (358.35): C, 60.33; H, 5.06;
N, 7.82. Found: C, 60.39; H, 5.10; N, 7.97.
2.3.1. Preparation of the target enzyme
Convert PDB file into an ICM object:
The X-ray crystal structure of the enzyme with coumarin ligand
DBC, 3,8-dibromo-7-hydroxy-4-methylchromen-2-one, (PDB code:
2QC6) [39] was obtained from the protein data bank in PDB format.
This conversion involves addition of hydrogen bonds, assignment
of atom types, and charges from the residue templates. Click on
MolMechanics/convert/protein, and then delete water molecules.
To perform ICM small molecule docking:
2.2. Antitumor screening
The human tumor cell lines of the cancer screening panel were
grown in RPMI 1640 medium containing 5% fetal bovine serum and
2 mM L-glutamine. For a typical screening experiment, cells were
Setup Docking Project:
inoculated into 96 well microtiter plates in 100 ll at plating densi-
(1) Set Project Name: Click on Docking/set project name, press
OK.
ties ranging from 5000 to 40,000 cells/well depending on the dou-
bling time of individual cell lines. After cell inoculation, the
microtiter plates were incubated at 37 °C, 5% CO2, 95% air and
100% relative humidity for 24 h prior to addition of experimental
drugs.
After 24 h, two plates of each cell line were fixed in situ with tri-
chloroacetic acid (TCA), to represent a measurement of the cell
population for each cell line at the time of drug addition (Tz).
Experimental drugs were solubilized in dimethyl sulfoxide (DMSO)
at 400-fold the desired final maximum test concentration and
stored frozen prior to use. At the time of drug addition, an aliquot
of frozen concentrate was thawed and diluted to twice the desired
final test concentration (10ꢀ5 M) with complete medium contain-
(2) Setup the Receptor: Click on Docking/Receptor setup, enter
the receptor molecule in the receptor molecule data entry
box (a_⁄) will do, then click on identify the binding sites but-
ton to identify the potential ligand binding pockets, press
OK. After the receptor setup is complete, the program nor-
mally displays the receptor with selected binding site resi-
dues highlighted in yellow Xstick presentation.
(3) Review and Adjust Binding Site: ICM makes a box around the
ligand binding site based on the information entered in the
receptor setup section. The position of the box encompasses
the residue expected to be involved in ligand binding. Click
on the menu Docking/Review/Adjust ligand/Box.
(4) Make Receptor Maps: The step now is to construct energy
maps of the environment within the docking box. Click on
menu Docking/Make Receptor Maps, select the resolution
of the map by entering a value into the grid cell size data
entry box which is 0.5, this step takes few min.
ing 50
were added to the appropriate microtiter wells already containing
100 of medium, resulting in the required final drug
lg/ml gentamicin. Aliquots of 100 ll of these drug dilutions
ll
concentrations.
Following drug addition, the plates were incubated for an addi-
tional 48 h at 37 °C, 5% CO2, 95% air, and 100% relative humidity.
For adherent cells, the assay was terminated by the addition of cold
TCA. Cells were fixed in situ by the gentle addition of 50
ll of cold
2.3.2. Preparation of compounds for docking
50% (w/v) TCA (final concentration, 10% TCA) and incubated for
60 min. at 4 °C. The supernatant was discarded, and the plates
were washed five times with tap water and air dried. Sulforhoda-
The target compounds stated earlier were built using Chem-
Draw ultra version 9.0.3 and their energy were minimized through
Chem3D ultra version 9.0.3/MOPAC, Jop Type: Minimum RMS Gra-
dient of 0.010 kcal/mol and RMS distance of 0.1 Å, and saved as
MDL MolFile (⁄mol).
mine B (SRB) solution (100
ll) at 0.4% (w/v) in 1% acetic acid was
added to each well, and plates were incubated for 10 min. at room

S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
59
2.3.3. Docking running
NH bands and an additional C@O bands at 1759–1685. 1H NMR
spectra revealed the disappearance of signals corresponding to
the 2 NH protons of the urea group. MS spectra showed their
molecular ion peaks.
Use interactive docking to dock one ligand at a time. Click on
menu Docking/Interactive docking/Mol Table Ligand, use the drop
down arrow to find the table of ligand and/or compounds to be
docked, and then enter the thoroughness which represents the
length of simulation. Generally 1 is a reasonable value, select Calc.
ICM Score, then select Display run which display the ligand sam-
pling the energy in the ligand binding project.
Reaction of amino group of o-aminophenol 3b with appropriate
isothiocyanates in ethanol formed thioureas which were spontane-
ously cyclized with neighboring OH giving the 2-substituted amin-
oxazole compounds 6a–c (Scheme 1) in good yields with the
evolution of hydrogen sulfide gas [42,43]. The termination of the
reaction was detected and monitored by lead acetate paper in
addition to TLC. In addition to the elemental microanalyses, IR
spectra showed disappearance of bands corresponding to NH2
and OH group and appearance of band at 3398–3263 cmꢀ1 corre-
sponding to the newly formed NH group. 1H NMR showed triplet
signal at d = 8.16–8.75 ppm corresponding to NH group with the
disappearance of signal corresponding to NH2 and OH. Appearance
of triplet signal at 1.21 ppm and a quartet signal at 3.39 ppm were
assigned for ethyl protons in spectra of compound 6a. 1H NMR of
compound 6b showed three multiples at 3.79–4.02, 5.12–5.31
and 5.92–6.31 ppm assigned to allyl protons. Compound 6c re-
vealed a singlet at d = 4.56 ppm and a multiplet at 7.33–7.42 ppm
corresponding to CH2 and the phenyl protons of the benzyl moiety,
respectively.
2.3.4. Display the result
⁄Click Docking/Browse/Stack Conformations.
⁄ICM stochastic global optimization algorithm attempts to find
the global minimum of the energy function that include five grid
potentials describing interaction of the flexible ligand with
receptor and internal conformational energy of the ligand, during
this process a stack of alternative low energy conformations is
saved.
3. Results and discussion
3.1. Chemistry
The procedure for the preparation of target compounds 4a–c–
7a–e, 10–12, and 15a,b–24a,b were summarized in Schemes 1–4.
The starting compounds 1a,b were prepared as reported in litera-
ture [32].
1,4-Benzoxazines 7a–e were achieved starting from o-amino-
phenol 3b via reaction with appropriate phenacyl bromide deriva-
tive in presence of sodium ethoxide (Scheme 1). The structures of
7a–e were proved by the elemental analyses and spectral data. IR
spectra revealed disappearance of bands corresponding to OH
and NH2 group except for only spectra of compound 7b which
showed band at 3258 corresponding to NH group which confirmed
the formation of oxazine ring. 1H NMR spectra of 7a,c–e revealed
disappearance of signals corresponding to OH, NH and appearance
of a singlet signal at d = 5.35–5.73 ppm corresponding to two pro-
tons of CH2 of oxazine ring, added aromatic protons at d = 6.99–
8.04 ppm corresponding to the phenyl or p-substituted phenyl
ring. 1H-NMR spectrum of 7b showed three singlet signals at
d = 2.36, 5.64 and 10.33 ppm assigned to protons of methyl group
at C-40, CH oxazine and NH, respectively in addition to two dou-
blets at 6.74 and 7.55 ppm assigned to aromatic protons of p-
substituted phenyl ring. MS spectra showed their molecular ion
peaks.
The key intermediates, 8-aminobenzopyrone 3a,b (Scheme 1)
were prepared in two steps from starting compounds 1a,b by the
formation of 8-azo derivatives 2a,b followed by its reduction.
Azo coupling reaction [40] followed by reduction [41] was chosen
instead of nitration followed by reduction due to the facile work up
and purity of the products. Compounds 2a,b were obtained
through diazo-coupling of 3-alkyl-7-hydroxy-4-methyl-2H-1-
benzopyran-2-one 1a,b by dropwise addition of freshly prepared
phenyl diazonium chloride. The structures of 2a,b were confirmed
by microanalyses and spectral data. 1H NMR spectra revealed the
disappearance of singlet signal corresponding to H-8 and appear-
ance of multiplet signal at d 7.57–7.67 ppm corresponding to the
H-30,40,50 and a doublet signal at 7.98 ppm assigned to H-20,60 of
added phenyl protons. Mass spectra revealed their molecular ion
peaks. 8-Amino compounds 3a,b were synthesized by reducing
the azo derivatives 2a,b using sodium dithionite in ammonia for
15 min. The structures of 3a,b were confirmed by elemental anal-
yses and spectral data. IR spectra showed 2 bands at 3444–
3296 cmꢀ1 corresponding to NH2 group. 1H NMR spectrum re-
vealed the appearance of a singlet signal at d 3.80–6.67 ppm corre-
sponding to NH2 and the disappearance of multiplet signals
corresponding to the aromatic protons of the phenyl azo moiety.
MS spectra showed their molecular ion peaks at 205 for 3a and
at 219 for 3b.
The key intermediate 8-acetyl-7-hydroxybenzopyrone
9
(Scheme 2) [33] were prepared starting from compound 1b in
two steps by formation of acetate ester compound 8 followed by
Fries rearrangement reaction. Fries rearrangement catalyzed with
Lewis acid such as AlCl3 [44] is the preferred method for rearrange-
ment of phenyl esters into o-ketophenols.
One pot reaction of acetyl derivative 9, malononitrile and benz-
aldehyde in presence of ammonium acetate yielded 2-iminodihy-
dropyridine-3-carbonitrile derivative 10 (Scheme 2). The
structure of compound 10 was confirmed by the elemental analysis
and spectral data. IR spectrum showed broad band at 3442 cmꢀ1
corresponding to 2NH and OH groups and a band at 2210 cmꢀ1 as-
signed to the nitrile group. 1H NMR showed appearance of singlet
signal at d = 6.78 ppm assigned to C5-H of dihydropyridine, multi-
plet signal at d = 7.24–8.20 ppm corresponding to the added phe-
nyl protons and two singlet signals at d = 8.26 and 8.90 ppm that
were exchanged with D2O corresponding to two NH groups. MS
spectrum showed molecular ion peak at 397.
Base catalyzed Claisen–Schmidt condensation of 8-acetyl deriv-
ative 9 with benzaldehyde in ethanol in presence of sodium
hydroxide afforded chalcone derivative 11 (Scheme 2). The struc-
ture of 11 was elucidated by analytical and spectral data. 1H
NMR revealed disappearance of methyl protons of acetyl group
and appearance of multiplet signal at d = 7.08–7.99 ppm corre-
sponding CH@CH and phenyl protons.
Reaction of amino compounds 3a,b with different isocyanate
derivatives in dichloromethane yielded the corresponding substi-
tuted ureas 4a–c (Scheme 1). The structures of 4a–c were eluci-
dated with the aid of elemental analyses and spectral data. IR
spectra revealed bands at 3300–3136 cmꢀ1 corresponding to 2
NH in addition to 2 carbonyl bands at 1743–1676 cmꢀ1 1H NMR
.
showed 2 singlet signals for compound 4a and 3 singlet signals
for compounds 4b,c at d 4.99–10.37 ppm assigned to 2 NH and
OH, in addition to signals at 6.98–7.59 ppm corresponding to addi-
tional aromatic protons. MS spectrum revealed their molecular ion
peaks.
Cyclization of substituted ureas 4b,c with oxalyl chloride in dry
benzene provided the imidazolidin-2,4,5-trione derivatives 5a,b
(Scheme 1). The structures of 5a,b were deduced by microanalyti-
cal and spectral data. The IR spectra revealed the disappearance of

60
S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
Acylation of 8-acetyl-7-hydroxybenzopyrone 9 with benzoyl
2-(substituted imino)-4-(substituted phenyl)thiazoline derivatives
16a–c (Scheme 3). The structures of compounds 16a–c were con-
firmed with elemental analyses and spectral data. IR spectra re-
chloride provided compound 12 (Scheme 2). Negative FeCl3 test
indicated the absence of the hydroxyl group and verified the pro-
duced ester. The structure of 12 was confirmed by elemental anal-
ysis which was in accordance with the spectral data. IR spectrum
showed disappearance of band corresponding to OH group. 1H
NMR showed disappearance of signal assigned to OH group and
presence of two triplets and one doublet signals at d = 7.63, 7.78
and 8.08 ppm corresponding to the added phenyl ring protons.
MS spectra showed molecular ion peak at 350.
vealed disappearance of
a
band at 1282–1251 cmꢀ1
corresponding to C@S and only one band at 3435–3406 cmꢀ1 cor-
responding to one NH group. 1H NMR revealed a singlet signal at
d = 4.93–4.98 ppm assigned to one NH group. In addition, a singlet
signal at d = 7.00–7.14 ppm assigned to C5-H thiazoline and H-8 Ar
protons for compounds 16a,b while in compound 16c, a multiplet
signal at d = 7.09–7.47 ppm assigned to C5-H thiazoline proton and
10 aromatic protons. MS spectra showed their molecular ion peaks.
2-Substituted iminothiazolidin-4-ones 17a,b were obtained by
the reaction of acylthiosemicarbazides 15a,b with chloroacetic
acid, anhydrous sodium acetate in glacial acetic acid (Scheme 3).
The form (I) not (II) was assigned to the resulting thiazolidin-4-
one (Fig. 2). This was attributed to previous literature that reported
cyclization of 1-acyl-4-substituted thiosemicarbazide with ethyl
bromoacetate yielded 2-substituted iminothiazolidin-4-one in case
of aliphatic substituted thiosemicarbazide and 1,3,4-oxadiazole
derivatives in case of aryl substituted thiosemicarbazide [45].
The structures of 17a,b were elucidated by microanalyses and
spectral data. IR spectra showed band at 3431–3192 cmꢀ1 corre-
sponding to NH group and three bands at 1735–1680 cmꢀ1 as-
signed to 3 C@O of formed thiazolidin-4-one. 1H NMR revealed
two singlet signals at d = 4.71–4.72 ppm corresponding to CH2
group of the thiazolidinone moiety and 10.16–10.53 ppm ex-
changed with D2O assigned to NH. MS spectra showed their molec-
ular ion peaks.
The key intermediates (3-alkyl-4-methyl-2-oxo-2H-1-benzopy-
ran-7-yloxy) acetic acid hydrazides 14a [34], b were synthesized
from
ethyl
(3-alkyl-4-methyl-2-oxo-2H-1-benzopyran-7-
yloxy)acetate 13a [34], b starting from hydroxycoumarin deriva-
tives 1a,b by reaction with ethyl chloroacetate in presence of anhy-
drous potassium carbonate followed by hydrazinolysis (Scheme 3).
The structure of 13b was confirmed by IR spectrum that revealed
the disappearance of band corresponding to OH group and appear-
ance of two bands at 1757, 1707 cmꢀ1 corresponding to 2 C@O
groups due to additional ester group. 1H NMR elicited the appear-
ance of a singlet signal at d = 4.90 ppm corresponding to OCH2CO
protons, in addition to triplet and a quartet signals at d = 1.21
and 4.18 ppm, respectively corresponding to ethyl protons of ester
moiety. MS spectrum showed the molecular ion peak at 290. In
addition, structure of 14b was deduced by IR spectrum that
showed bands at 3446, 3213 cmꢀ1 corresponding to NH and NH2
groups. 1H NMR spectrum revealed 2 singlet signals at 4.32 and
9.37 ppm corresponding to NH2 and NH protons, respectively and
disappearance of the triplet and quartet signals corresponding to
ethyl protons of ester moiety.
Acylthiosemicarbazides 15a,b were achieved via reaction of
acid hydrazide 14a with the appropriate isothiocyanate in ethanol
(Scheme 3). The structure of compounds 15a,b were elucidated by
their elemental analyses and spectral data. IR spectra showed three
bands at 3392–3126 cmꢀ1 corresponding to 3NH, a band at 1282–
1251 cmꢀ1 corresponding to C@S. 1H NMR spectrum revealed three
signals at d = 6.63–14.08 ppm corresponding to the 3NH groups.
Appearance of a triplet signal at d = 1.06 ppm and a quartet signal
at d = 3.45 ppm corresponding to the ethyl protons in 15a spec-
trum and a multiplet at d = 6.90–7.74 ppm corresponding to the
phenyl protons in compound 15b spectra. Ms. showed their molec-
ular ion peaks at 349 for 15a and at 397 for 15b.
Cyclization of acylthiosemicarbazides 15a,b with concentrated
sulfuric acid on cold yielded 1,3,4-thiadiazole derivatives 18a,b
(Scheme 3). The structures of 18a,b were deduced by elemental
analyses and spectral data. IR spectra showed disappearance of
C@S band at 1282–1251 cmꢀ1 and only one NH band at 3305–
3157 cmꢀ1 1H NMR revealed one singlet signal at d = 10.50–
.
13.87 ppm corresponding to NH proton. MS spectra showed their
molecular ion peaks at 331 and 379 for 18a and 18b, respectively.
4-Substituted-1,2,4-triazol-5-thione derivatives 19a,b were ob-
tained upon reaction of acylthiosemicarbazide derivatives 15a,b
with a 2 N NaOH (Scheme 3). The structures of 19a,b were con-
firmed by the elemental analyses and spectral data. IR spectra re-
vealed a band at 3415–3390 cmꢀ1 corresponding to NH group
and a band at 1286–1274 cmꢀ1 corresponding to C@S. 1H NMR of
19a revealed a broad singlet signal at 4.70 ppm corresponding to
NH proton while compound 19b showed a singlet signal at
Cyclization of acylthiosemicarbazides 15a,b with substituted
phenacyl bromide derivatives in ethanol/chloroform mixture gave
Fig. 2. The proposed structure of compounds 17a,b.

S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
61
d = 14.06 ppm exchanged with D2O corresponding to one NH
proton.
mean graph of the percent growth of the treated cells and pre-
sented as percentage growth inhibition (GI%). The obtained results
of the tested benzopyrone analogs showed distinctive potential
pattern of selectivity, as well as broad-spectrum antitumor activity
(Table 1).
4-Benzyl-1-acylsemicarbazide 20 was achieved via refluxing
acid hydrazide 14a with benzyl isocyanate in dichloroethane
(Scheme 4). The structure of 20 was confirmed by elemental anal-
ysis and spectral data. IR spectrum showed bands at 3460–
3294 cmꢀ1 corresponding to 3NH. 1H NMR spectrum revealed a
singlet signal at d = 4.23 ppm and a multiplet signal at 6.96–
7.28 ppm corresponding to CH2 and phenyl protons of the benzyl
moiety in addition to three a singlet signals at 7.95, 8.14 and
9.90 ppm corresponding to 3NH protons. MS spectrum showed
the molecular ion peak at 395.
Cyclization of acylsemicarbazide 20 using oxalyl chloride in dry
benzene produced imidazolidin-2,4,5-trione 21 (Scheme 4). The
structure of 21 was proved by microanalysis and spectral data. IR
spectrum showed a band at 3415 cmꢀ1 corresponding to NH group
and bands at 1749–1699 cmꢀ1 corresponding to 5C@O groups. 1H
NMR spectrum revealed a singlet signal at d = 11.44 ppm corre-
sponding to one NH group. MS spectrum showed molecular ion
peak at 449.
Regarding the activity towards individual cell lines, urea deriv-
ative as compound 4a showed a promising activity towards most
of the cell lines, compared to compounds 4b,c. It revealed a potent
activity against the leukemia cell line RPMI-8226 with GI value of
69.50%, while recording a good overall activity against the other
leukemia subpanels CCRF-CEM, HL-60 (TB) and K-562, with GI val-
ues of 34.32, 41.78 and 49.57%, respectively. It also exhibited activ-
ity towards the non-small cell lung cancer HOP-92 and NCI-H522
with GI values of 38.58 and 37.45%, respectively. Moreover it re-
vealed activity against the colon subpanel KM12 with GI values
of 37.30%, the CNS cell line SF-295 and U251 with GI value of
45.07 and 31.24%, respectively. The melanoma cell lines LOX IMVI,
MALME-3M, SK-MEI-5 and UACC-62 with GI values of 31.09, 47.65,
35.35 and 52.02%, respectively, while it showed activity against the
ovarian subpanel OVCAR-3, renal cell lines ACHN and SN12C, pros-
tate PC-3 and the breast T-47D and MDA-MB-468 with GI values of
55.39, 39.40, 32.73, 51.41, 38.34 and 56.53% respectively.
On the other hand, compound 5a showed activity towards the
leukemia cell lines SR with GI values of 35.68% while compound
6c exhibited activity towards renal cancer subpanel UO-31 with
GI value of 32.71%. The oxazole ring had reported selective toxicity
against renal cell line UO-31, which may account for 6c effect on
renal cancer [46].
Concerning pyranobenzoxazines 7a–e, they revealed a similar
pattern of antitumor activity. Compound 7a showed GI values
22.62, 27.55 and 20.24% against non-small cell lung cancer HOP-
92, colon HCT-15 and prostate PC-3, respectively. Compound 7c
showed GI values 25.04 and 20.10% against leukemia subpanel
SR and renal UO-31, respectively. Compound 7d showed activity
towards the renal cancer subpanel A498 with GI values of 21.69%
while compound 7e exhibited activity towards non-small cell lung
cancer HOP-92 with GI value of 25.07%.
Dihydropyridine carbonitrile compound 10 revealed a consider-
able broad antitumor activity against most of the cell lines tested.
It showed activity against leukemia cancer HL-60 (TB), MOLT-4, SR,
CNS cancer SNB-75, melanoma SK-MEL-5, renal A498, CAKI-1, UO-
31, prostate PC-3, breast MDA-MB-231/ATCC and T-47D with GI
values ranging from 20 to 30%. Good activity was reported against
the non-small cell lung cancer HOP-92 with GI value of 53.04%.
This effect on HOP-92 may be due to the pyridine ring which is re-
ported to have a selective effect on non-small cell lung cancer [47]
while broad antitumor activity against various cell lines may be
due to the presence of the pyridine carbonitrile that was reported
to possess a cytotoxic effect against different types of cancers [48].
Thiadiazole compounds 18a,b showed excellent activity on leu-
kemia cancer where 18a and 18b caused GI values of 56.58 and
71.00% against RPMI-8226, respectively while revealed good activ-
ity upon K-562 cell lines with GI values of 34.27% and 40.43%,
respectively. This may be due to the thiadiazole ring that is re-
ported to have an excellent inhibitory effect upon leukemia [49].
Compound 18a showed moderate activity against ovarian cancer
OVCAR-3, prostate PC-3 and breast cancer MDA-MB-468 with GI
value of ꢃ30–45%. In addition, 18a showed activity upon non small
cell lung cancer HOP-92 and NCI-H522, CNS cancer SF-295, mela-
noma MALME-3M and UACC-62 and breast T-47D with GI value
of ꢃ20–30%. Also compound 18b elicited a high activity upon
CNS cancer SF-295, melanoma MALME-3M, ovarian OVAR-3 and
breast cancer MDA-MB-468 with GI value of 50.46%, 56.89%,
58.37% and 55.31%, respectively. 18b revealed GI values of ꢃ30–
45% against leukemia K-562, colon KM12, melanoma SK-MEL-5
and UACC-62, renal ACHN, prostate PC-3 and breast cancer
Benzylidene acetohydrazide derivatives 22a,b were achieved
via refluxing acid hydrazide 14a,b with appropriate aromatic alde-
hyde in glacial acetic acid (Scheme 4). The structures of 22a,b were
confirmed with elemental analyses and spectral data. IR spectrum
elicited a band at 3446–3427 cmꢀ1 corresponding to NH group. 1H
NMR displayed a singlet at d = 8.01–8.27 ppm corresponding to
benzylidene proton and another singlet at 11.49–11.70 ppm as-
signed to NH. MS spectra revealed their molecular ion peaks.
Condensation of acid hydrazide 14a,b with ethyl acetoacetate in
glacial acetic acid afforded the corresponding pyrazol-5-one deriv-
atives 23a [13], b (Scheme 4). The structure of 23b was deduced
from microanalytical and spectral data. IR spectra displayed NH
band at 3257 cmꢀ1 and 3C@O bands at 1747, 1732, 1705 cmꢀ1
.
1H NMR spectra showed three singlet signals at 1.87 ppm assigned
to CH3 at C3 pyrazolone, 4.79 ppm corresponding to CH pyrazolone
and 10.08 ppm exchanged with D2O assigned to NH.
Upon reacting acid hydrazide 14b with cyclic anhydrides in gla-
cial acetic acid the corresponding 2,5-dihydro-1H-pyrrol-2,5-dione
24a or pyrrolidin-2,5-dione 24b was obtained (Scheme 4). The IR
spectra of 24a,b showed bands at 1751–1676 cmꢀ1 assigned to
4C@O groups. 1H NMR spectra of 24a,b displayed disappearance
of NH2 and NH singlet signals at d = 4.32 and 9.37 ppm of parent
acid hydrazide 14b and presence of a singlet signal at 10.80 ppm
corresponding to NH group. 1H NMR spectrum of 24a showed pres-
ence of multiplet signal at 6.99–7.05 ppm assigned to CH@CH pro-
tons while 1H NMR spectrum of 24b revealed a triplet signal at
d = 2.80 ppm corresponding to four protons of CH2CH2. MS spectra
revealed the molecular ion peaks 356 and 358, respectively.
3.2. Antitumor screening
3.2.1. Preliminary in vitro antitumor screening
Fourteen newly synthesized compounds (4a–c, 5a, 6c, 7a–e, 10,
18a,b, 23a) were selected by National Cancer Institute (NCI) Devel-
opmental Therapeutic Program (http://www.dtp.nci.nih.gov),
Bethesda, MD, U.S.A. The synthesized compounds were subjected
to the NCI’s disease-oriented human cell lines screening assay to
be evaluated for their in vitro antitumor activity. The anticancer as-
says were performed in accordance with the protocol of the Drug
Evaluation Branch, NCI, Bethesda [35–37]. A single dose (10 lM)
of the test compounds were used in the full NCI 60 cell lines panel
assay which includes nine tumor subpanels namely: leukemia,
non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate
and breast cancer cells. A 48 h drug exposure protocol was used
and sulforhodamine B (SRB) protein assay was applied to estimate
the cell viability and growth [38]. The results were reported as

62
S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
Table 1
Growth inhibition percent of tested compounds against 60 different cell lines.
Panel/cell line
Test compounds and growth inhibition percent of cell line
4a
4b
4c
5a
6c
7a
7b
7c
7d
7e
10
18a
18b
23a
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
34.32
41.78
49.57
24.27
69.50
21.76
nt
–
–
–
–
nt
–
–
–
–
nt
16.58
–
12.12
–
35.69
nt
–
–
–
–
nt
13.71
–
–
nt
–
–
–
–
nt
nt
–
–
–
12.48
12.30
nt
nt
20.83
–
23.61
18.34
23.88
13.42
18.20
34.27
–
56.58
–
27.41
19.42
40.43
–
71.00
–
27.69
28.91
42.61
14.39
74.04
–
11.33
14.72
11.36
–
10.92
11.60
11.25
–
–
–
–
–
15.66
–
25.04
19.88
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
25.75
nt
–
–
nt
–
–
nt
–
–
nt
–
–
nt
14.79
–
nt
–
–
nt
–
–
nt
–
–
–
–
–
nt
–
10.73
19.87
–
–
nt
–
nt
nt
–
13.41
nt
–
HOP-92
38.58
–
13.22
14.18
27.42
37.45
–
21.61
–
–
–
–
nt
20.07
–
–
–
–
nt
13.23
–
–
–
–
nt
22.62
–
–
15.26
–
nt
12.90
–
–
–
–
nt
18.46
–
–
–
–
nt
–
–
25.07
–
12.35
–
–
nt
53.04
12.62
14.67
–
13.22
12.60
28.67
–
–
–
–
–
–
–
47.63
13.77
13.74
–
10.43
35.06
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
14.43
–
–
–
–
12.35
–
–
–
–
10.68
27.45
28.31
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
11.84
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10.25
–
14.14
–
22.32
12.69
–
18.91
–
21.17
–
20.99
30.29
–
–
–
26.99
15.26
28.65
37.30
21.28
19.27
–
–
10.46
–
18.83
–
34.96
24.81
15.03
27.55
–
–
–
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
11.08
45.07
–
13.10
14.63
31.24
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
nt
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
nt
–
–
–
–
11.67
–
–
22.91
nt
13.39
–
–
25.72
–
27.34
–
–
10.79
15.12
50.46
–
12.52
–
39.73
–
13.93
–
18.84
–
25.21
nt
11.16
–
nt
13.48
–
21.35
nt
10.52
nt
nt
nt
25.80
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
31.09
47.65
28.88
18.35
21.84
18.82
35.35
20.10
52.02
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
nt
–
–
–
–
–
–
–
–
nt
–
–
–
–
–
–
–
–
nt
–
–
–
–
–
–
–
–
nt
–
–
–
–
–
–
–
–
nt
–
–
–
–
–
–
–
–
nt
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
nt
–
18.21
–
–
–
–
–
28.56
–
13.36
–
13.12
56.89
27.27
22.21
–
22.19
32.52
nt
–
26.89
11.48
–
–
–
18.61
–
21.97
49.40
20.32
–
–
–
32.53
12.43
28.42
39.83
Ovarian cancer
IGROV1
22.76
55.39
24.32
–
21.97
–
–
–
nt
–
–
–
–
–
–
nt
–
–
–
–
–
–
nt
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
nt
–
–
–
–
–
–
nt
–
–
–
–
–
–
–
–
–
–
–
–
–
nt
–
–
15.77
58.37
16.31
–
–
–
–
–
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
33.31
–
–
19.68
–
–
49.50
18.56
–
18.56
–
–
13.84
–
–
–
Renal cancer
786-0
–
16.01
–
–
–
–
–
–
19.18
–
–
–
–
–
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
–
10.22
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
20.1
21.69
–
–
–
–
–
–
23.37
10.38
23.77
–
–
–
–
–
22.69
27.73
–
39.40
12.69
–
32.73
26.67
26.86
–
–
–
–
–
–
–
–
–
–
–
–
15.63
–
–
–
34.84
–
–
19.66
–
17.41
12.28
–
–
–
17.49
–
–
–
–
–
19.18
13.06
27.89
14.06
16.70
–
UO-31
19.03
32.71
19.53
17.38
12.47
29.88
10.51
Prostate cancer
PC-3
DU-145
51.41
–
–
–
14.25
–
10.71
–
10.23
–
20.24
–
11.14
–
11.38
–
–
–
–
–
20.70
–
39.41
–
44.70
–
51.25
–
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
16.55
28.25
–
13.48
26.21
–
–
–
–
–
22.03
–
–
22.26
–
–
nt
27.89
56.98
10.59
–
–
–
–
19.29
38.34
56.53
12.08
–
–
–
11.16
–
10.19
–
24.05
41.28
32.63
55.31
MDA-MB-468
–, GI < 10%; nt, not tested.

S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
63
MDA-MB-468. In addition, 18b showed activity upon leukemia
CCRF-CEM, non small cell lung cancer NCI-H522, colon HCT-116
and HT-29, melanoma M14, MDA-MB-435, SK-MEL-28 and breast
MCF-7 with GI value of ꢃ20–30%. Overall, 18b seemed to have
improved activity compared to 18a.
in a tetrameric form composed by two catalytic units, CK2
a
with
three possible isoforms and two regulatory units, CK2b [57]. In
the majority of the case, the catalytic unit has demonstrated to
phosphorylate a particular consensus sequence different from
other protein kinase known. This is composed by 4 aminoacids:
Ser-X-X-Acidic, where the acidic residues can be Glu, Asp, pSer or
pTyr [58]. On the other hand the b units are able to stabilize the
tetrameric complex and at the same time to enhance and to mod-
ulate the activity thanks to a crucial role in substrates recruitment
[59,60].
The binding affinities of the ligand was evaluated with energy
score ICM (kcal/mol). The compound which revealed the highest
binding affinity, minimum dock score, is the one forming the most
stable ligand enzyme complex. Number and length of the hydrogen
bond were used to assess the binding models. The results of the
docking studies, ICM scores, and involved amino acids interacted
ligand moieties and hydrogen bond length for each compound
and the reference native ligand are listed in Table 2, Fig. 3–5.
Analysis of docking results revealed that:
Dihydropyrazol-5-one compound 23a revealed the most potent
effect upon various cell lines with the highest effect on leukemia
cancer K-562 and RPMI-8226 with GI values of 42.61% and
74.04%, respectively. GI values of 47.63% and 35.06% was also ob-
served on non small cell lung cancer HOP-92 and NCI-H522, GI val-
ues of 34.96% and 24.81% on colon cancer HT29, KM12, 39.73% and
25.80% on CNS cancer SF-259, U251, respectively. Compound 23a
also showed activity on melanoma subpanel MALME-3M, SK-
MEL-5 and UACC-62 with GI values of 49.40%, 32.53% and
28.42%, respectively. At last, it revealed GI value of 49.50%,
51.25% and 56.98% on ovarian cancer OVCAR-3, prostate PC-3
and breast MDA-MB-468 cell lines respectively. This was attrib-
uted to presence of the dihydropyrazole ring that was reported
to have broad spectrum antitumor effect over various types of can-
cers [50,51].
Close examination of the data presented in Table 1, revealed
that compounds 4a, 18a, 18b and 23a were the most active mem-
bers of this study, showing effectiveness toward numerous cell
lines belong to different tumor subpanels. They exhibited high GI
values against leukemia cell line RPMI-8226, CNS cell line SF-
295, melanoma cell line MALME-3M, ovarian subpanel OVCAR-3,
prostate cell line PC-3 and breast cell line MDA-MB-468. Com-
pound 10 revealed a considerable broad antitumor activity against
most of the cell lines tested. On the other hand, compounds 5a, 7a,
7c, 7d and 7e possessed moderate antitumor activity; while com-
pounds 4b, 4c, 6c and 7b were the least active antitumors in the
present investigation.
(1) The inhibitor DBC, 3,8-dibromo-7-hydroxy-4-methylchro-
men-2-one, nearly fits in the active site of CK2 and has
ICM score (ꢀ53.11 kcal/mol, Table 2), and form two hydro-
gen bonds between O of the 7-OH group with Lys 68 and
Asp 175 of distance 2.16 ÅA and 2.63 ÅA respectively (Fig. 3).
(2) For urea derivatives 4a–c (dock scores, ꢀ66.61 to
ꢀ79.17 kcal/mol) a high negative score was estimated to
the 3-methyl substituent, compound 4a, the most active
compound in the series, while the other two derivatives with
3-ethyl substituent that revealed a weak antitumor activity
were found to have less negative dock scores.
0
0
Imidazolidin-2,4,5-trione, compound 5a revealed a high dock
score of (ꢀ76.13 kcal/mol), this value was not correlated to the
activity of 5a that had a moderate antitumor activity.
Concerning the oxazole derivative 6c the docking scores was
(ꢀ66.67 kcal/mol) which is consistent with the weak antitumor
activity it had over various cell lines.
The oxazine compounds 7a–e had different ICM scores in the
range between (ꢀ47.79 to ꢀ69.20 kcal/mol) and this was corre-
lated to the antitumor screening where compounds 7a,c–e re-
vealed moderate activity while 7b with the lowest dock score
(ꢀ47.79 kcal/mol) exhibited an overall weak activity against vari-
ous cell lines.
Pyridine carbonitrile derivative 10 (dock score, ꢀ73.83 kcal/
mol), this high dock score was consistent with the considerable
activity it revealed in the one dose assay.
In case of thiadiazole derivatives 18a,b, both had high ICM
scores of ꢀ83.22 and ꢀ85.31 kcal/mol respectively. These scores
are correlated to the high antitumor activity observed with com-
pound 18b having a better score and a better biological activity
compared to 18a.
3.2.2. Structure–activity correlation
Examination of the bioactivity results revealed that combina-
tion of benzopyrone scaffold with dihydropyrazole ring, compound
23a or thiadiazole ring compounds 18a,b afforded broad antitumor
activity with high GI values. This was attributed to previous knowl-
edge of antitumor activity of both dihydropyrazole and thiadiazole
rings. In addition, selective effect of pyridine ring on non-small cell
lung cancer was expressed by high GI value of compound 10
against non small cell lung cancer HOP-92.
Disubstituted urea derivative with 3-methyl or 3-ethyl substi-
tuted benzopyrone and aryl substituent varied in activity. Substitu-
tion of benzopyrone scaffold with methyl group at C3, compound
4a, exhibited broad antitumor activity with high GI values while
substitution of with ethyl group at C3 lowered activity, compounds
4b,c. Cyclization of aryl substituted urea derivative, compound 4b,
to imidazolidin-2,4,5-trione, compound 5a, slightly improved
activity.
Fused ring system of benzopyrone with oxazine ring, com-
pounds 7a,c–e, exhibited moderate activity while in case of oxa-
zole ring, compound 6c, activity was low.
Finally compound 23a had ICM score of ꢀ80.25 kcal/mol. The
high score of 23a is related to the potent antitumor activity it
revealed.
3.3. Molecular docking
(3) Inspection of the binding mode also demonstrated that all
compounds show from one to six hydrogen bonds with the
enzyme active site residue. Tyr 307, Arg 278, Leu 249, Asp
175, Arg 172, Lys 170, Asn 118, Lys 68, Ser 51, Tyr 50 and
Asp 14 are the amino acid residues involved in these interac-
tions. In common with DBC, CK2 inhibitor, most of the
docked compound, that exhibited some kind of activity,
interacted with at least one amino acid residues Asp 175
and Lys 68.
Casein Kinase II enzyme (CK2) represents one of most excep-
tional protein Kinase. Several experimental data support the CK2
importance for cancer transformation and development. CK2 has
been found to be over expressed in tumors in the head and neck
[52], in prostate [53], in the kidney [54], in mammary gland [55]
and lung [56]. The final effect of CK2 seems linked to an action
on both oncogenes and tumor suppressor proteins. It promotes a
number of proto-oncogenic products stimulating cell proliferation
and differentiation and inhibiting apoptosis. In humans, CK2 exists

64
S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
Table 2
Docking results.
Compound
ICM scores (kcal/mol)
No of H bonds
Involved group of amino acid
Atom of ligand involved
Length of H-bond (Å)
DBC
ꢀ53.11
2
Lys 68ꢄ ꢄ ꢄHH
Asp175ꢄ ꢄ ꢄHH
O of 7-OH
O of 7-OH
2.16
2.63
4a
ꢀ79.17
5
Ser 51ꢄ ꢄ ꢄHH
Ser 51ꢄ ꢄ ꢄO
O of CO of urea
H of NH
O of 7-OH
O of CO of pyrone
O of 7-OH
1.94
2.29
2.22
2.65
2.68
Lys 68ꢄ ꢄ ꢄHH
Lys 68ꢄ ꢄ ꢄHH
Asp 175ꢄ ꢄ ꢄHH
4b
4c
ꢀ67.28
ꢀ66.61
4
3
Leu 249ꢄ ꢄ ꢄO
Leu 249ꢄ ꢄ ꢄO
Leu 249ꢄ ꢄ ꢄO
Arg 278ꢄ ꢄ ꢄHH
Asp 14ꢄ ꢄ ꢄHH
Asp 14ꢄ ꢄ ꢄO
Asp 14ꢄ ꢄ ꢄO
H of NH
H of NH
H of NH
O of CO of urea
2.55
1.38
2.20
2.31
O of 7-OH
H of NH
H of NH
2.36
2.06
2.34
5a
6c
7a
ꢀ76.13
ꢀ66.67
ꢀ55.49
1
1
3
Asn 118ꢄ ꢄ ꢄHH
Tyr 307ꢄ ꢄ ꢄHH
Lys 170ꢄ ꢄ ꢄHH
Lys 170ꢄ ꢄ ꢄHH
Arg 172ꢄ ꢄ ꢄHH
O of imidazolidintrione
O of oxazole
1.52
2.21
O of oxazine
O of CO of pyrone
O of CO of pyrone
2.15
2.52
2.13
7b
ꢀ47.79
3
Arg 278ꢄ ꢄ ꢄHH
Arg 278ꢄ ꢄ ꢄHH
Arg 278ꢄ ꢄ ꢄHH
O of CO of pyrone
O of oxazine
O of CO of pyrone
1.65
2.37
2.39
7c
7d
7e
10
ꢀ66.18
ꢀ69.20
ꢀ62.15
ꢀ73.83
1
1
1
5
Lys 68ꢄ ꢄ ꢄHH
Lys 68ꢄ ꢄ ꢄHH
Lys 68ꢄ ꢄ ꢄHH
Ser 51ꢄ ꢄ ꢄHH
Ser 51ꢄ ꢄ ꢄHH
Lys 68ꢄ ꢄ ꢄHH
Lys 68ꢄ ꢄ ꢄHH
Asp 175ꢄ ꢄ ꢄHH
O of oxazine
O of oxazine
O of oxazine
2.32
2.20
2.29
N of Pyridine
N of Pyridine
N of Pyridine
O of 7-OH
1.85
2.59
2.74
2.31
2.58
O of 7-OH
18a
ꢀ83.22
4
Ser 51ꢄ ꢄ ꢄHH
Lys 68ꢄ ꢄ ꢄHH
Lys 68ꢄ ꢄ ꢄHH
Asp 175ꢄ ꢄ ꢄHH
N-3 of Thiadiazole
O of CO of pyrone
O of OCH2
2.14
2.40
2.17
2.66
O of CO of pyrone
18b
23a
ꢀ85.31
ꢀ80.25
2
6
Asn 118ꢄ ꢄ ꢄHH
Asp 175ꢄ ꢄ ꢄO
O of CO of pyrone
H of NH
2.54
2.10
Tyr 50ꢄ ꢄ ꢄHH
Ser 51ꢄ ꢄ ꢄHH
Lys 68ꢄ ꢄ ꢄHH
Lys 68ꢄ ꢄ ꢄHH
Asp 175ꢄ ꢄ ꢄHH
Asp 175ꢄ ꢄ ꢄO
O of Pyrazolone
O of Pyrazolone
O of CO of pyrone
O of pyrone
O of CO of pyrone
H of NH
2.26
2.47
2.36
2.22
2.55
2.75
Fig. 4. Binding mode of 18b into the binding site of CK2 enzyme.
between Lys 68 and O of 7-OH and O of CO of pyrone, one hydrogen
bond between Asp 175 with O of 7-OH with a distance of 1.94,
2.29, 2.22, 2.65 and 2.68 Å respectively.
Fig. 3. Binding mode of DBC into the binding site of CK2 enzyme.
For example, binding mode of 4a into the active site of the en-
zyme mediated five hydrogen bonds, two hydrogen bonds between
Ser 51 and O of CO of urea and H of NH, two hydrogen bonds
The binding mode of 10 show five hydrogen bonds, two hydro-
gen bonds between Ser 51 with N of pyridine moiety, two hydro-
gen bonds between Lys 68 with N of pyridine and O of 7-OH and

S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
65
Appendix A. Supplementary material
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bioorg.
2014.02.003.
References
[1] World Health Organization (WHO). Factsheets, Cancer, 2012 <http://
www.who.int/mediacentre/factsheets/fs297/en/>.
[2] M. Hejmadi, Introduction to cancer biology, 2010, pp. 6.
[3] B.A. Chabner, J.M. Collins (Eds.), Cancer Chemotherapy: Principles and Practice,
Lippincott, Philadelphia, 1990. pp. 314–328.
[4] T. Ojala, S. Remes, P. Haansuu, H. Vuorela, R. Hiltunen, K. Haahtela, P. Vourela, J.
Ethnopharmacol. 73 (2000) 299–305.
[5] S.M. De Souza, M.F. Delle, A. Smania Jr., Z. Naturforsch C 60 (2005) 693–700.
Fig. 5. Binding mode of 23a into the binding site of CK2 enzyme.
ˇ
ˇ ´
[6] B. Šarkanj, M. Molnar, M. Cacic, L. Gille, Food Chem. 13 (2013) 8146–8149.
[7] J.T. Pierson, A. Dumètre, S. Hutter, F. Delmas, M. Laget, J.P. Finet, N. Azas, S.
Combes, Eur. J. Med. Chem. 45 (2010) 864–869.
[8] P. Selvam, N. Ramlakshmi, G. Uma, S.A. Kumar, A. Umamasheswari, RASAYAN J.
Chem. 3 (2010) 275–280.
[9] I. Kostova, S. Bhatia, P. Grigorov, S. Balkansky, V.S. Parmar, A.K. Prasad, L. Saso,
Curr. Med. Chem. 18 (2011) 3929–3951.
[10] E. Gonsior, G. Schultze-Werninghaus, B. Wüthrich, Int. J. Clin. Pharmacol.
Biopharm. 17 (1979) 283–290.
[11] B.L. Thumber, V.G. Vasoya, T.R. Desai, Y.T. Naliapara, K.V. Shah, P.R. Tirgar,
Pharmacol. Newslett. 2 (2011) 1010–1017.
finally one hydrogen bond between Asp 175 and O of 7-OH with a
distances of 1.85, 2.59, 2.74, 2.31 and 2.58 Å, respectively.
Compound 18b formed two hydrogen bonds, one hydrogen
bond between Asn 118 with O of CO of pyrone and another one be-
tween Asp 175 with H of NH with a distance of 2.54 and 2.10 Å,
respectively (Fig. 4).
Finally the binding mode of 23a into the active site of CK2 en-
zyme revealed six hydrogen bonds, one hydrogen bond between
Tyr 50 and O of pyrazolone, one between Ser 51 and O of pyrazo-
lone, two hydrogen bonds between Lys 68 with O of CO of pyrone
and O of pyrone moiety, and another two between Asp 175 with O
of CO of pyrone and H of NH with a distance of 2.26, 2.47, 2.36,
2.22, 2.55 and 2.75 Å, respectively (Fig. 5).
[12] C. Spino, M. Dodier, S. Sotheeswaran, Bioorg. Med. Chem. Lett. 8 (1998) 3475–
3478.
[13] O.M. Abdelhafez, K.M. Amin, H.I. Ali, M.M. Abdalla, R.Z. Batran, Neurochem. Int.
62 (2013) 198–209.
[14] O.M. Abdelhafez, K.M. Amin, H.I. Ali, M.M. Abdalla, R.Z. Batran, J. Med. Chem.
55 (2012) 10424–10436.
[15] J.C. Capra, M.P. Cunha, D.G. Machado, A.D.E. Zomkowski, B.G. Mendes, A.R.S.
Santos, M.G. Pizzolatti, A.L.S. Rodrigues, Eur. J. Pharmacol. 643 (2010) 232–
238.
[16] A.M. El-Mofty, J. Royal Egypt. M.A. 31 (1948) 651.
[17] S.C. Shim, K.H. Chae, Photochem. Photobiol. 30 (2008) 349–353.
[18] S.M. Boué, C.H. Carter, K.C. Ehrlich, T.E. Cleveland, J. Agric. Food Chem. 48
(2000) 2167–2172.
4. Conclusion
[19] A. Lacy, R. O’Kennedy, Studies on coumarins and coumarin-related compounds
to determine their therapeutic role in the treatment of cancer, Curr. Pharm.
Des. 10 (2004) 3797–3811.
[20] X.Y. Huang, Z.J. Shan, H.L. Zhai, L. Su, X.Y. Zhang, Chem. Biol. Drug Des. 78
(2011) 651–658.
[21] I. Kostova, Expert Opin. Drug Dis. 2 (2007) 1605–1618.
[22] R.N. Gacche, S.G. Jadhav, J. Exp. Clin. Med. 4 (2012) 165–169.
[23] R.C.N. Williamson, P.J. Lyndon, A.J.C. Tudway, Br. J. Cancer 42 (1980) 85–94.
[24] J.J. Ryan, A.S. Ketcham, H. Wexler, Cancer Res. 29 (1969) 2191–2194.
[25] S. Lee, K. Sivakumar, W-S. Woon-Seob Shin, F. Xie, Q. Wang, Bioorg. Med.
Chem. Lett. 16 (2006) 4596–4599.
New benzopyrone derivatives comprised of substituted-1H-
benzopyran-2-ones, substituted amino-5H-pyrano[6,5-e]ben-
zooxazol-5-ones and substituted-2,6-dihydropyrano[6,5-f]-1,4-
benzoxazin-6-ones were synthesized. Fourteen compounds were
selected by National Cancer Institute (NCI), Bethesda, and evalu-
ated for their in vitro anticancer activity in the full NCI 60 cell lines
panel assay by a single dose test (10 lM). Results showed that,
combination of benzopyrone scaffold with dihydropyrazole ring
(compound 23a) or thiadiazole ring (compounds 18a,b) afforded
broad antitumor activity toward various cell lines that belong to
different tumor subpanels while combination with pyridine ring
(compound 10) expressed selective effect on non-small cell lung
cancer. In addition, disubstituted urea derivatives varied in activity
(compound 4a showed broad-spectrum antitumor activity) and
cyclization of disubstituted urea derivative slightly improved
activity (cyclized compound 5a compared with urea derivative
4b). Moreover, fused ring system of benzopyrone with oxazine ring
exhibited moderate activity while in case of oxazole ring activity
was low. Docking studies of biological evaluated compounds were
performed with casein kinase II (CK2) enzyme in order to gain in-
sight into their possible binding mode. Although a correlation be-
tween dock score and observed in vitro antitumor activity
expressed as GI value by the compounds was routinely observed,
most of the docked compounds shared some binding interactions
with CK2 similar to those of the native ligand inhibitor. This sug-
gests that these compounds might possibly act as CK2 inhibitors,
and this may contribute at least in part to their antitumor activity.
[26] E.B. Yang, Y.N. Zhao, K. Zhang, P. Mack, Biochem. Biophys. Res. Commun. 260
(1999) 682–685.
[27] S. Han, V. Zhou, S. Pan, Y. Liu, M. Hornsby, D. McMullan, H.E. Klock, J. Haugan,
S.A. Lesley, N. Gray, J. Caldwell, X.Ju. Gu, Bioorg. Med. Chem. Lett. 15 (2005)
5467–5473.
[28] P. Bilbao, O.M. Del, V.A. Alonso, B. Castro, J. Bilbao, T. Palomares, Melanoma
Res. 12 (2002) 17–26.
[29] S. Kim, M. Cho, T. Lee, S. Lee, H.-Y. Min, S.K. Lee, Bioorg. Med. Chem. Lett. 17
(2007) 4584–4587.
[30] D. Kumar, N.M. Kumar, S. Sundaree, E.O. Johnson, K. Shah, Eur. J. Med. Chem.
45 (2010) 1244–1249.
[31] D. Sunil, A.M. Isloor, P. Shetty, Der Pharm. Chem. 1 (2009) 19–26.
[32] F.W. Canter, F.H. Curd, A. Robertson, J. Chem. Soc. (Resumed) (1931) 1255–
1265.
[33] A. Kathuria, N. Priya, K. Chand, P. Singh, A. Gupta, S. Jalal, S. Gupta, H.G. Raj, S.K.
Sharma, Bioorg. Med. Chem. 20 (2012) 1624–1638.
[34] U. Karl, B. Rendenbach-Muller, Ger. Offen, 1996, DE 4435250 A1 19960411.
[35] M.R. Grever, S.A. Schepartz, B.A. Chabner, Semin. Oncol. 19 (1992) 622–638.
[36] A. Monks, D. Scudiero, P. Skehan, J. Natl. Cancer Inst. 83 (1991) 757–766.
[37] M.R. Boyd, K.D. Paull, Drug Dev. Res. 34 (1995) 91–109.
[38] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.R.
Warren, H. Bokesch, S. Kenney, M.R. Boyd, J. Natl. Cancer Inst. 82 (1990) 1107–
1112.
[39] A. Chillin, R. Battistutta, A. Bortolato, G. Cozza, S. Zanatta, G. Poletto, M.
Mazzorana, G. Zagotto, E. Uriarte, A. Guiotto, L.A. Pinna, F. Meggio, S. Moro, J.
Med. Chem. 51 (2008) 752–759.
[40] H. Abu-Shady, S.T. Hassib, A.N. Mikhel, S. El-Ansary, Egypt J. Pharm. Sci. 29
(1988) 471–483.
[41] H.A. Abu Shady, S.L. El-Ansary, D.A. Abou El-Ella, N.A.H. Farag, Bull. Fac. Pharm.
Cairo Univ. 42 (2004) 31–42.
[42] K.A. Amin, M.M. Hussein, T.A. El-Kersh, Egypt J. Pharm. Sci. 28 (1987) 171–181.
[43] M.M. Hussein, A.M. Ahmedy, Az. J. Pharm. Sci. 10 (1992) 126–135.
Acknowledgment
Thanks are due to the NCI, Bethesda, MD, for performing the
antitumor testing of the synthesized compounds.

66
S.L. El-Ansary et al. / Bioorganic Chemistry 53 (2014) 50–66
[44] V.F. Traven, N.Y. Podhaluzina, A.V. Vasilyev, A.V. Manaev, ARKIVOC xi (2000)
931–938.
[53] S. Yenice, A.T. Davis, S.A. Goueli, A. Akdas, C. Limas, K. Ahmed, Prostate 24
(1994) 11–16.
[45] N. Cesur, Z. Cesur, A. Gursoy, Arch. Pharm. (weinheim) 325 (1992) 623–624.
[46] W.M. Abdou, M.A.I. Salem, R.F. Barghash, ARKIVOC Xv (2007) 45–60.
[47] S.A. Rostom, G.S. Hassan, H.I. El-Subbagh, Arch. Pharm. 342 (2009) 584–590.
[48] A.-G.E. Amr, A.M. Mohamed, S.F. Mohamed, N.A. Abel-Hafez, A.E.-F. Hammam,
Bioorg. Med. Chem. 14 (2006) 5481–5488.
[49] M.N. Noolvi, H.M. Patel, N. Singh, A.K. Gadad, S.S. Cameorta, A. Badiger, Eur. J.
Med. Chem. 46 (2011) 4411–4418.
[50] X.H. Liu, H.F. Liu, J. Chen, Y. Yang, B.A. Song, L.S. Bai, J.X. Liu, H.L. Zhu, X.B. Qi,
Bioorg. Med. Chem. Lett. 20 (2010) 5705–5708.
[51] M.M.F. Ismail, H.S. Rateb, M.M.M. Hussein, Eur. J. Med. Chem. 45 (2010) 3950–
3959.
[52] R.A. Faust, S. Tawfic, A.T. Davis, L.A. Bubash, K. Ahmed, Head Neck 22 (2000)
341–346.
[54] G. Stalter, S. Siemer, E. Becht, M. Ziegler, K. Remberger, O.G. Issinger, Biochem.
Biophys. Res. Commun. 202 (1994) 141–147.
[55] E. Landesman-Bollag, R. Romieu-Mourez, D.H. Song, G.E. Sonenshein, R.D.
Cardif, D.C. Seldin, Oncogene 20 (2001) 3247–3257.
[56] M. Daya-Makin, J.S. Sanghera, T.L. Mogentale, M. Lipp, J. Parchomchuk, J.C.
Hogg, S.L. Pelech, Cancer Res. 54 (1994) 2262–2268.
[57] R.D. Gietz, K.C. Graham, D.W. Litchfield, J. Biol. Chem. 270 (1995) 13017–
13021.
[58] L.A. Pinna, F. Meggio, Progr. Cell Cycle Res. 3 (1997) 77–97.
[59] K.C. Graham, D.W. Litchfield, J. Biol. Chem. 275 (2002) 5003–5010.
[60] F. Meggio, B. Boldyref, O. Marin, L.A. Pinna, O.G. Issinger, Eur. J. Biochem. 204
(1992) 293–297.