Synthesis and docking studies of novel benzopyran-2-ones with anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2010.05.050

Novel series of 7-substituted-benzopyran-2-ones was synthesized by incorporating heterocyclic rings as oxadiazole, triazole, pyrazole or pyrazolin-5-one to benzopyran-2-one nucleus at p-7 via methylene-oxy or acetoxy linker. In-vitro anticancer activity w

Full text of DOI:10.1016/j.ejmech.2010.05.050

European Journal of Medicinal Chemistry 45 (2010) 3950e3959
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and docking studies of novel benzopyran-2-ones with
anticancer activity
,
b
c
Magda M.F. Ismail a , Heba S. Rateb , Mohammad M.M. Hussein
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), 11754 Al-Azhar University, Cairo, Egypt
b Department of Pharmaceutical and Medicinal Chemistry, Pharmacy College, Misr University for Science and Technology, Cairo, Egypt
c Department of Pharmaceutical Chemistry; Faculty of Pharmacy, Cairo University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel series of 7-substituted-benzopyran-2-ones was synthesized by incorporating heterocyclic rings as
oxadiazole, triazole, pyrazole or pyrazolin-5-one to benzopyran-2-one nucleus at p-7 via methylene-oxy
or acetoxy linker. In-vitro anticancer activity was evaluated for these hybrids; twelve compounds were
selected by National Cancer Institute for anticancer screening. Among them, compound 9a exhibited
Received 14 March 2010
Received in revised form
23 May 2010
Accepted 25 May 2010
Available online 1 June 2010
broad spectrum antitumor activity showing full panel median growth inhibition (GI50) ¼ 5.46
mM.
According to docking results using Molsoft ICM 3.4-8c program, the target compounds may act through
inhibition of topoismerase 1, where camptothecin is used as ligand.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Benzopyran-2-one
Synthesis
Docking
Anticancer activity
1. Introduction
In the present study, we have aimed to synthesize novel gei-
parvarin analogs by incorporating benzopyran-2-one and
Improved understanding of cancer biology at the molecular
level has led to a shift in treatment paradigm from the traditional
cytotoxics toward more rationally designed targeted therapies.
Geiparvarin [1,2] (Fig. 1) a naturally occurring product bearing
the benzopyran-2-one (coumarin) [3e6] residue possesses signif-
icant inhibitory activity against variety of cell lines including
sarcoma 180, Lewis lung carcinoma P-388, lymphocytic leukemia
and Walker 256 carcinosarcoma.
A series of geiparvarin analogs modified on the unsaturated
alkenyloxy bridge, where a H-atom replaced the 30-Me group, were
synthesized and evaluated against a panel of human tumor cell
lines [7,8]. These compounds demonstrated a stronger increase in
growth inhibitory activity when compared to the parent
compound, geiparvarin. In particular, the activity increased even
further in the series of demethylated compounds when a Me
substituent in the coumarin moiety is introduced [8]. Moreover;
5-membered heterocyclic residues (triazole/pyrazole/pyrazolin-5-
one/oxzdiazole) via eOCH2e/eCOCH2Oe bridge in a single molecule
and investigate the anticancer activity of the resultant compounds.
Additionally, docking of the new compounds into human DNA
topoisomerase I (Top1) [15] complexed with its bound inhibitor
Camptothecin (Cpt) using Molsoft [16] ICM 3.4-8c program was
performed in order to predict the affinity and orientation of the
these compounds to the active site. Top1 is an essential enzyme
participating to all those processes associated with separation of
DNA strands. It manages superhelical tensions through the tran-
sient breakage of one strand of duplex DNA, followed by the
unwinding of supercoiled DNA [17].
2. Results and discussion
2.1. Chemistry
a
variety of 7-[(1,5-dialkyl-1H-1,2,4-triazolyl) methoxy (and
The designed compounds are synthesized starting from
7-hydroxy-4-and/-8-substituted-benzopyran-2-ones byits reaction
with ethylchloroacetate to give the corresponding esters, 1aec.
Hydrazinolysis of the latter afforded the acid hydrazides 2aec;
which were condensed with carbon disulphide, to furnish the target
compounds, 5-mercapto-1,3,4-oxadiazoles, 3aec. Methylation of
3a,c with CH3I/anhyrous K2CO3 produced the corresponding
5-methyl-thio oxadiazoles 4a,b.
methyl)] benzopyran-2-ones were synthesized and showed anti-
cancer (breast, lung, CNS cancers) properties in vitro [9] Besides, it
has been well documented that pyrazole [10,11], pyrazolin-5-one
[12] and oxadiazole [13,14], have cytotoxic activity.
* Corresponding author. Tel.: þ20 2 22713531.
E-mail address: magdafathi_111@hotmail.com (M.M.F. Ismail).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.050

M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 3950e3959
3951
2.2. Pharmacological screening
2.2.1. Anticancer screening
Development Therapeutic Program (DTP), Division of Cancer
Treatment and Diagnosis (DCTD), National Cancer Institute (NCI),
Bethesda, Maryland, USA, has adopted an in-vitro anticancer
screening.
Fig. 1. Geiparvarin.
Twelve compounds (3aec, 4a,b, 6a,b, 7a, 8aec and 9a) were
selected by NCI to be evaluated. The screening is a two-stage
process, beginning with the evaluation of all compounds against
the 60 cell lines representing cancers of lung, colon, brain, ovary,
breast, prostate, kidney, leukemia, and melanoma [18] at a single
dose of 10 mM (Table 1).
3-Methyl-1-(2-(8-methyl-4-phenyl-2H-1-benzopyran-2-one-7-
yloxy)acetyl)-1,2-dihydropyrazol-5-one (9a) was passed on to
On the other hand, reaction of the key intermediates, 2aec with
benzylisothiocyanate gave the thiosemi-carbazides, 5aec which
were cyclized using sodium hydroxide to give 5-mercapto-1,3,4-
triazoles, 6aec. Moreover, refluxing the intermediates, 2aec with
benzoic acid/phosphorous oxychloride furnished the 5-aryl/
phenyl-1,3,4-oxadiazoles, 7aee (Scheme 1). In addition, more
target compounds were obtained by cyclocondensation of 2aec
with active-methylene compounds such as acetylacetone, ethyl-
acetoacetate or diethylmalonate to afford the corresponding pyr-
azoles 8aec, pyrazolin-5-ones, 9aec and pyrazolidinediones, 10a,
b respectively (Scheme 2).
further evaluation at five concentration levels (0.01e100
mM)
[18e20]. It exhibited broad spectrum anticancer activity against
all tested sub-panel tumor cell lines, with GI50 full panel mean-
graph mid-point (MG-MID) ¼ 5.46
mM (Table 2), TGI full panel
Scheme 1. Reagents and conditions: a; NH2NH2$H2O/absolute EtOH/ref. 2 h. b; CS2/ethanolic KOH/ref. 48 h. c; CH3I/anhydrous K2CO3/dry acetone/ref. d; PhCH2NCS/absolute EtOH/
ref. 6 h. e; 2 N NaOH/ref.3 h. f; R2C6H5COOH/POCl3/ref. 2 h.

3952
M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 3950e3959
Scheme 2. Reagents and conditions: a; CH3COCH2COCH3/AcOH/ref.6 h. b; CH3COCH2COOEt/AcOH/ref. 6 h. c; EtOCOCH2COOEt/NaOEt/ref. 2 h.
MG-MID ¼ 29.16
m
M and LC50 full panel MG-MID ¼ 60.87
m
M
than methoxy-linker (4b, 6a, 6b, 7a). Moreover, 9a bearing the
higher lipophilic substituents at p-4 (Ph) and p-8 (Me) displayed
the highest cytotoxicity when compared to other analogs 9b, 9c
which are not selected by NCI for preliminary cytotoxic
screening.
(Table 2).
With regard to sensitivity against individual cell lines, it
exhibited a super sensitivity profile toward Leukemia CCRF-CEM
and HL-60(TB) with GI50 value lying in the nanomolar range
(GI50 < 0.04
exhibited high activity against Colon Cancer HTC-116, Melanoma
LOX IMVI, Ovarian Cancer OVCAR-4 (GI50 ¼ 1.94, 1.99 and 1.95 M)
and (TGI ¼ 3.76, 3.88 and 4.73 M) respectively. It also showed high
sensitivity against CNS cancer and Prostate Cancer with GI50 of 2.58,
2.38 M and TGI values of 8.85 and 5.75 M, respectively. The LC50
(cytotoxicity) values were >100 M for most tested cell lines.
The ratio obtained by dividing the compounds’ full panel MG-
MID ( M) by its individual sub-panel MG-MID ( M) is considered as
a measure of compound selectivity. Ratios between 3 and 6 refer to
moderate selectivity, ratios >6 indicate high selectivity toward the
corresponding cell line, while compounds meeting neither of these
criteria are rated non-selective [15]. Compound 9a proved to be non-
selective with a broad spectrum of activity against the nine tumor
subpanelstested withratiosrangingbetween0.51 and2.29 (Table 2).
5-Methyl thioether of 1,3,4-oxadiazole, 4b showed good activity
against CNS Cancer (SF-295) with growth % ¼ 21.89. Moreover,
5-phenyl-1,3,4-oxadiazole, 7a elicited anticancer activity against
Leukemia (RPMI-8226) with growth % ¼ 47.45.
mM and TGI of 0.57 and 2.62 mM respectively). It also
2.3. Docking studies
m
m
To further rationalize the observed antitumor activity of the new
compounds, a flexible ligandereceptor docking investigation was
undertaken using “Internal coordinate Mechanics (Molsoft ICM 3.5-
0a)”.
Molecular modeling docking studies are performed and ICM
score values [21e23] combined with hydrogen bonds formed with
the surrounding amino acid residues help to predict the correct
binding geometry for each binder at the active site. In order to
compare the binding affinity of the of synthesized benzopyran-2-
ones, we docked them into the empty binding site of the experi-
mentally known crystal structure of the human DNA top-
m
m
m
m
m
oisomerase
I (top 1, 70 kDa) in complex with the poison
Camptothecin (Cpt, ligand) and covalent complex with A 22 Base
Pair DNA Duplex (1T8I) [15] as a target for anticancer compounds.
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 the
receptor and internal conformational energy of the ligand, during
this process a stack of alternative low energy conformations is
saved (Table 3).
Human top I is a multidomain enzyme that contains two highly
conserved globular domains (the core and the COOH-terminal
domain) that are crucial for catalytic activity, and two other regions
(NH2-terminal and linker) that are not strictly required for its
catalytic and relaxation functions. The phosphate of the tyrosi-
neeDNA phosphodiester bond makes close interactions with the
guanidinium groups of Arg488 and Arg590. Both arginines are
implicated in the reaction mechanism of the enzyme [24]. Arg488
5-Mercapto-4-benzyl-1,2,4-triazoles, 6a,b elicited activity
against 6 panels of cancer cell lines. They displayed high antitumor
activities against Leukemia, Non-Small Cell Lung Cancer, CNS
Cancer and Melanoma; 6a showed further activity against Colon
and Prostate Cancers; while further activities against Ovarian and
Breast Cancers were elicited by 6b.
In brief, compound 9a, which is a hybrid of pyrazolin-5-one
linked to benzopyran-2-one nucleus via acetoxy-linker proved to
be the most potent cytotoxic agent, therefore, pyrazolin-5-one
synergizes the activity of benzopyran-2-one rather than that for
1,3,4-oxadiazoles (4b, 7a)/1,3,4-triazoles (6a, 6b). On the other
hand, the length of the acetoxy-linker (9a) favors the activity

M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 3950e3959
3953
Table 1
The mean growth percent, delta value and the growth percent of the synthesized compounds, 3e9.
Cpd No
NSC-Number
Mean growth percent
Delta
Panel
Subpanel cell lines (growth %)
3a
3b
3c
4a
4b
7a
6a
749093/1
749094/1
749095/1
749105/1
749106/1
749109/1
749098/1
101.00
101.83
101.73
95.34
86.39
94.74
84.81
21.08
24.55
27.18
28.88
64.50
47.29
53.31
All panelsa
_
_
_
_
All panelsa
All panelsa
All panelsa
CNS cancer
Leukemia
Leukemia
SF-295 (21.89)
RPMI-8226 (47.45)
MOLT-4 (49.01), RPMI-8226 (31.50), K-562 (53.60).
Non-small cell Lung cancer
Colon cancer
CNS cancer
Prostate cancer
Melanoma
NCI-H522 (56.62)
HT29 (57.15).
SF-295 (45.78)
PC-3 (56.56)
UACC-62 (59.87).
6b
749100/1
82.61
45.34
Leukemia
HL-60(TB) (59.74), MOLT-4 (58.74), RPMI-8226
(37.27), K-562 (46.91), SR (57.60).
Non-small cell Lung cancer
CNS cancer
EKVX (57.70)
SF-295 (53.03)
Melanoma
SK-MEL-2 (53.36), UACC-62 (59.70).
Ovarian cancer
Breast cancer
OVCAR-4 (48.66),
T-47D (50.70)
_
_
8a
8b
8c
9a
749090/1
749091/1
749092/1
749096/1
102.17
104.56
102.35
ꢀ20.25
19.04
21.07
18.81
72.70
All panelsa
All panelsa
All panelsa
_
Non-small cell Lung cancer
A549/ATCC (0.69), EKVX (ꢀ33.60), HOP-62 (ꢀ18.64),
HOP-92(-64.41), NCI-H322M (ꢀ24.66), NCI-H460 (10.90),
NCI-H522 (ꢀ39.18), NCI-H226 (0.36), NCI-H23 (1.72).
COLO 205 (6.21), HCC-2998 (17.35), HCT-116 (ꢀ65.87),
Colon cancer
Breast cancer
Ovarian cancer
Leukemia
HCT-15(38.32), HT29 (ꢀ44.22), KM12 (ꢀ11.98), SW-620 (ꢀ28.31).
MCF7 (ꢀ14.43), MDA-MB-468 (ꢀ8.13), MDA-MB-231/ATCC
(ꢀ29.98), HS 578T (ꢀ4.17), BT-549 (ꢀ65.85), T-47D (ꢀ1.54)
IGROVI (ꢀ17.54), OVCAR-3 (ꢀ61.15), OVCAR-4 (ꢀ42.92), OVCAR-5
(10.22), OVCAR-8 (ꢀ30.48), SK-OV-3 (15.91).
CCRF-CEM (13.46), HL-60(TB) (ꢀ66.59), MOLT-4 (22.70), RPMI-8226
(ꢀ18.20), K-562 (ꢀ24.06), SR (18.53).
Renal cancer
Melanoma
786-0 (ꢀ58.84), A498 (ꢀ3.79), ACHN (41.59), SN12C (ꢀ69.30),
RXF-393 (ꢀ36.95), TK-10 (ꢀ33.43), UO-31 (18.57).
LOX IMVI (ꢀ61.79), M14 (17.58), MALME-3M (ꢀ8.61), MDA-MB-435
(ꢀ39.57), SK-MEL-2 (ꢀ12.23), SK-MEL-28 (ꢀ47.17), SK-MEL-5
(ꢀ31.48), UACC-257 (6.57), UACC-62 (ꢀ27.27).
Prostate cancer
CNS cancer
PC-3 (ꢀ92.95), DU-145 (ꢀ56.27).
SF-268 (2.94), SF-295 (ꢀ77.21), SF-539 (ꢀ78.24), SNB-19 (33.65),
SNB-75 (ꢀ35.21), U251 (ꢀ72.36).
a
Inactive.
makes non-bonded interaction with thymine nucleotide in minor
groove (T16); Lys532 forms a hydrogen bond with thymine nucle-
otide (T10), another hydrogen bond formed between Thr718 and
Guanine (G12). Asp533 forms non-bonded interaction with
backbone sugar of Adenine (A114) whereas Tyr723 forms interac-
tion with backbone sugar of Thymine (T10).
As shown in Table 3, Cpt (ligand) reveals ICM score of ꢀ54.51
and forms eight H bonds with Arg488, Gly531, Arg590, Asn631 and
Ala486 (Fig. 2); and Geiparvarin reveals ICM score of ꢀ79.36; and
forms seven H bonds with Arg488, Arg488, Arg590, Arg590,
Arg590, Arg590 and Cys630 (Table 3).
Table 2
GI50, TGI, LC50 of sub-panel tumor cell lines, full panel MG-MID and selectivity ratio
of compound 9a.
All the target compounds elicited higher binding affinities (ICM
scores of ranges from ꢀ61.68 to ꢀ83.66) to the active site of top 1
than that for ligand.
Subpanel tumor
cell linesa
GI50
MG-MID
(
m
M)
TGI (
MG-MID
m
M)
LC50
MG-MID
(
m
M)
Selectivity
ratio
I
II
III
IV
V
VI
VII
VIII
3.44
3.69
8.98
2.58
4.65
8.53
10.72
2.38
4.23
5.46b
50.75
37.40
32.64
8.85
43.21
28.73
21.40
5.75
93.97
65.07
64.52
27.66
72.42
52.03
55.23
29.45
87.50
60.87d
1.58
1.48
0.61
2.11
1.17
0.64
0.51
2.29
1.29
2-Mercapto-oxadiazoles, 3aec are biologically inactive; they
have high ICM scores of ranges from ꢀ61.68 to ꢀ66.78 but they
bind incorrectly in the active site. 5-Methyl thioether of the latter,
4a,b bind correctly to hinge region catalytic residues of top 1
(Arg488, Lys532 and Asn631) in the active site with 5H bonds;
however compound is 4b showed activity probably due to it is
more stable (ICM ¼ ꢀ73.89) than 4a (ICM ¼ ꢀ64.97). Analogously,
5-aryl/phenyl-1,3,4-oxadiazoles, 7aee have ICM scores of ranges
from ꢀ68.89 to ꢀ77.97, but the only biologically active one is
compound 7a as it binds with the important amino acids: Arg488,
Lys532, Asn631 and Thr718 by 7H bonds.
IX
33.71
Full panel MG-MID
29.16c
a
I, Leukemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V,
melanoma; VI, ovarian cancer; VII, renal cancer; VIII, prostate cancer; IX, breast
cancer.
b
GI50
(
m
M) full panel mean-graph mid-point (MG-MID) ¼ The average sensitivity
1,3,4-Triazoles, 6aec have ICM scores of ranges from ꢀ81.41 to
ꢀ83.66; where both 6a,b showed stability and affinity to the active
site of Top 1 e.g. 6a binds with 6H bonds to Lys532, Asp533, Ser534,
Tyr723; the counterpart, 6b binds by 7H bonds to Arg488 and
of all cell lines toward the test agent.
c
TGI (
m
M) full panel mean-graph mid-point (MG-MID) ¼ The average sensitivity
of all cell lines toward the test agent.
d
LC50 (mM) full panel mean-graph mid-point (MG-MID).

3954
M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 3950e3959
Table 3 (continued)
Table 3
ICM Scores of Camptothecin, Geiparvarin, the compounds, and hydrogen bonds
formed with amino acid residues and their lengths.
Compounds
ICM scores No. of
Involved
Atom of Length of
H-bonds group
ligand
H-bond Å
Compounds
ICM scores No. of
Involved
Atom of Length of
of
involved
H-bonds group
ligand
H-bond Å
amino acid
of
involved
8a
ꢀ62.49
3
Lys493/Hz2
Thr50/Hn
Thr50/Hg1
Val502/Hn
Asp533/Hn
Lys374/Hz
Arg375/Hh11 O-2
Asn419/Hn O-2
Arg488/Hh12 O-3
Arg488/Hh12 N-2
Arg488/Hh21 O-3
O-2
O-2
O-2
O-2
O-3
N-2
2.58
2.28
1.97
2.78
2.31
2.39
2.69
1.64
1.86
2.69
2.65
1.48
2.50
2.74
2.54
1.53
1.95
2.21
2.76
2.05
2.75
2.47
2.14
1.36
2.12
amino acid
Camptothecin ꢀ54.51
8
Arg488/Hh11 O-4
Arg488/Hh12 N-1
Arg488/Hh21 N-1
2.77
2.36
1.78
2.79
2.31
2.56
2.03
2.07
8b
8c
ꢀ67.97
ꢀ69.93
2
3
Gly531/Hn
O-2
Asn631/Hd21 O-2
Asn631/Hd22 O-2
Arg590/Hh22 O-4
9a
ꢀ68.53
6
Ala 486/O
H16
Geiparvarin
ꢀ79.36
ꢀ61.68
7
4
Arg488/Hh12 O-2
Arg488/Hh12 O-4
Arg590/Hh21 O-2
Arg590/Hh22 O-3
Arg590/Hh22 O-1
Arg590/Hh22 O-2
1.80
1.81
1.57
2.75
1.60
2.53
1.39
2.31
2.62
1.98
1.91
1.49
2.04
2.38
1.79
1.79
2.66
2.78
2.25
2.79
1.91
2.44
Lys532/Hn
Asn631/Hd21 N-2
O-4
Thr718/Hg1
Asn491/Hn
Asn491/Hn
Asn352/Hd21 O-4
Asn352/Hd21 O-5
Asn352/Hd22 O-5
Lys374/Hz2
Tyr426/Hn
Gly214/Hn
Arg434/Hh21 O-4
Arg434/Hh22 O-4
O-2
O-4
O-5
9b
9c
ꢀ69.35
ꢀ75.37
2
3
Cys630/O
H5
3a
Asn352/Hd21 O-4
Glu356/Hn
Lys374/Hz2
Lys354/O
N-2
O-2
H14
H9
10a
10b
ꢀ66.15
ꢀ64.88
2
4
O-2
O-5
O-3
3b
3c
ꢀ61.90
ꢀ66.78
2
4
Arg364/O
Ala499/Hn
Glu356/Hn
Gln421/He21 O-4
Lys425/Hz1
Lys425/Hz3
N-2
O-2
Ile215/O
H20
N-1
N-1
Arg590 which are implicated in the reaction mechanism of top 1.;
however 6c binds to a different amino acids (Table 3).
4a
4b
6a
ꢀ64.97
ꢀ73.89
ꢀ81.41
5
5
6
Arg488/Hh11 N-2
Arg488/Hh12 O-3
Arg488/Hh12 O-4
Lys532/Hn
Asn631/Hd21 O-4
3,5- Dimethylpyrazoles, 8aec as well as pyrazolidine-3,5-dio-
nes, 10a,b lack affinity to the active site of top 1 (Table 3).
Pyrazolin-5-one, 9a binds (ICM ¼ ꢀ68.53) to hinge region
catalytic residues Arg488, Lys532, Asn631 with five H bonds and
another H-bond with Thr718, one of residues of the carboxyl-
terminal domain of the enzyme (Fig. 3). In addition, it binds to both
of Thr718 that impedes the interaction with guanine (G12), Ala486
by hydrophobic interaction as well as Tyr723. 9a develops dis-
turbing interactions with these important residues. On the other
hand; 9b,c do not bind with the characteristic amino acids for
enzyme inhibition (Table 3). These interactions and the corre-
sponding induced fit in the active site conformation are compared
with the one occurring with Cpt.
N-2
Arg488/Hh11 N-2
Arg488/Hh12 O-3
Lys532/Hn
Lys532/Hn
2.80
2.66
2.73
1.89
2.50
O-3
N-2
Asn631/Hd21 O-4
Lys532/Hz2
Lys532/Hz2
Asp533/Hn
Ser534/Hn
Ser534/Hg
Tyr723/O-3
N-2
N-3
O-2
O-2
O-2
H14
2.74
1.99
2.28
1.98
2.46
2.63
6b
ꢀ82.01
7
Arg488/Hh12 O-1
Arg488/Hh12 N-3
Arg488/Hh21 N-2
Arg488/Hh21 N-3
Arg488/Hh22 N-2
Arg488/Hh22 N-3
Arg590/Hh22 O-2
2.53
2.78
1.53
1.24
1.73
2.53
2.35
3. Experimental
3.1. Chemistry
6c
7a
ꢀ83.66
ꢀ73.29
4
7
Lys493/Hz1
Lys493/Hz1
Ala499/Hn
His367/Nd1
N-2
N-3
N-2
H16
2.49
1.83
2.34
2.35
All melting points are uncorrected and determined by the open
capillary method using Gallencamp melting point apparatus (MFB-
595-010 M). Microanalysis is carried out at the microanalytical
center, Faculty of Science, Cairo University and the microanalytical
center, Faculty of Science, Assiut University. Infrared spectra are
determined using Shimadzu Infrared Spectrometer (IR-435). 1H
NMR spectra are carried out using A JEOL FX-200 MHz NMR spec-
trometer and A JEOL FX-300 MHz NMR spectrometer for 1H NMR.
Mass spectra are carried out using Finnigan MAT, SSQ 7000 mass
spectrometer at 70 eV. TLC is carried out using Art.5735, Kieselgel 60
F245 sheets and spots are visualized by UV- 365, 254 nm. The starting
materials,1b,c and the intermediates, 2a,b were prepared according
the reported procedures [25,26].
Arg488/ Hh12 O-3
Arg488/Hh21 O-4
Lys532/Hn
Lys532/Hn
2.16
2.76
2.11
2.59
2.65
2.45
2.62
N-1
N-2
Asn631/Hd21 O-4
Thr718/Hg1
Thr718/Hg1
Lys218/Hz1
Lys218/Hz3
O-1
O-2
N-2
O-1
7b
ꢀ70.08
2
2.05
2.59
7c
ꢀ69.97
ꢀ77.97
1
Lys443/Hz3
O-2
1.58
7d
5
Asn491/Hn
Asp533/Hn
Ser534/Hn
Ser534/Hg
Ser534/Hg
N-1
O-1
O-1
O-2
O-1
2.60
2.78
2.62
1.42
2.67
3.1.1. Ethyl (8-methyl-4-phenyl-2H-1-benzopyran-2-one-7-yloxy)-
acetate (1a)
A mixture of 7-hydroxy-8-methyl-4-phenyl-2H-1-benzopyran-
2-ones (0.1 mol), anhydrous potassium carbonate (2.76 g, 0.2 mol)
7e
ꢀ68.89
2
Gly717/Hn
Lys720/Hz3
O-2
O-1
2.33
2.72

M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 3950e3959
3955
and ethylchloroacetate (14.64 g, 0.12 mol) in dry acetone (200 ml)
was refluxed with continuous stirring for 24 h, cool then filter,
wash with acetone. The combined filtrate and wash was concen-
trated. The remained residue was filtered then crystallized from
isopropyl alcohol. Yield: 100%; m.p. 151e153 ꢁC; IR (KBr cmꢀ1):
1745, 1709 (C]O of
DMSO, ppm):
on C8), 4.2 (q, 2H, CH2eCH3, J ¼ 7.2 Hz), 4.9 (s, 2H, OeCH2), 6.2 (s,
1H, C3eH), 6.9 (d, 1H, C6eH, J ¼ 8.6 Hz), 7.2 (d,1H, C5eH,
J ¼ 8.6 Hz), 7.5 (s, 5H, PheH). Anal. Calcd for C20H18O5, C: 71.00; H:
5.36; found: C: 71.22, H: 5.12.
a
-pyrone and OC]O); 1H NMR (200 MHz,
¼ 1.2 (t, 3H, CH2eCH3, J ¼ 7.2 Hz), 2.2 (s, 3H, CH3,
d
d
3.1.2. (8-Methyl-4-propyl-2H-1-benzopyran-2-one-7-yloxy)-acetic
acid hydrazide (2c)
A mixture of ethyl (4,8-disubstituted-2H-1-benzopyran-2-one-7-
yloxy)-acetate (1aec) (0.01 mol) and hydrazine hydrate 99% (1 ml,
0.02 mol) in absolute ethanol (30 ml) was refluxed for 2 h. The
precipitate was filtered off, washed with water and crystallized from
dil.aceticacid. Yield:97%;m.p. 207e209ꢁC,IR(KBrcmꢀ1): 3337, 3308,
3208 (NH2, NH), 1714 (C]O,
(300 MHz, CDCl3):
a
-pyrone), 1675 (C]O, amide). 1H NMR
¼ 1.05 (t, 3H, CH3eCH2eCH2, J ¼ 7.5 Hz), 1.7 (m,
d
2H, CH3eCH2eCH2, J ¼ 7.5 Hz), 2.7 (t, 2H, CH3eCH2eCH2, J ¼ 7.5 Hz),
2.4 (s, 3H, CH3 at C8), 4.0 (broad s, 2H, NH2 disappeared upon
deuteration), 4.7 (s, 2H, OeCH2), 6.2 (s, 1H, C3eH), 6.8 (d, 1H, C6eH,
J ¼ 9 Hz), 7.5(d, 1H, C5eH, J ¼ 9 Hz), 7.6 (s, 1H, NH disappeared upon
deuteration). MS (m/z, %): Mþ (290, 26.4). Anal. Calcd for C15H18N2O4,
C: 62.06; H: 6.25; N: 9.65, found: C: 61.55, H: 6.78; N: 9.34.
3.1.3. 7-[(5-Mercapto-1,3,4-oxadiazol-2-yl)methoxy]-4,8-
disubstituted-2H-1-benzopyran-2-ones (3aec)
To a solution of KOH (0.56 g, 0.01 mol) in ethanol (50 ml) the
acid hydrazide 2aec (0.01 mol) and carbon disulphide (1 g,
0.013 mol) were added. The mixture was refluxed for 48 h while
stirring, the solvent was then removed in vacuo, the residue was
dissolved in water and acidified with diluted HCl. The precipitate
was washed with water and crystallized from ethanol.
Fig. 2. Binding mode of camptothecin in the active site of human DNA topoisomerase 1.
3.1.4. 7-[(5-Mercapto-1,3,4-oxadiazol-2-yl)methoxy]-8-methyl-4-
phenyl-2H-1-benzopyran-2-one (3a)
Yield: 98%; m.p. 232e235 ꢁC, IR (KBr cmꢀ1): 3150 (NH), 1710 (C]O
of a dppm): d2.2 (s, 3H, CH3 on C8),
-pyrone); 1HNMR(200MHz, DMSO,
4.8 (s, 2H, OeCH2), 6.2 (s, 1H, C3eH), 6.3 (d, 1H, C6eH, J ¼ 9 Hz), 6.9 (d,
1H, C5eH, J¼ 9 Hz), 7.1e7.5 (m, 5H, PheHs),10.2 (s,1H, NH disappeared
upon deuteration); MS (m/z, %): Mþ (366, 0.76). Anal. Calcd for
C19H14N2O4S, C: 62.29; H: 3.85; N: 7.65, found: C: 61.75, H: 4.42; N: 7.23.
3.1.5. 7-[(5-Mercapto-1,3,4-oxadiazol-2-yl)methoxy]-4-propyl-2H-
1-benzopyran-2-one (3b)
Yield: 96%; m.p.197e199 ꢁC, IR (KBr cmꢀ1): 3210 (NH),1676 (C]
O of a d ppm): d 0.97 (t, 3H,
-pyrone); 1H NMR (200 MHz, DMSO,
CH3eCH2eCH2, J ¼ 7.4 Hz), 1.6 (m, 2H, CH3eCH2eCH2, J ¼ 7.4 Hz),
2.7 (t, 2H, CH3eCH2eCH2, J ¼ 7.4 Hz), 5.4 (s, 2H, OeCH2), 6.2 (s, 1H,
C3eH), 7.1 (d, 1H, C6eH, J ¼ 8.8 Hz), 7.2 (s, 1H, C8eH), 7.8 (d, 1H,
C5eH, J ¼ 8.8 Hz), 10.3 (s, 1H, NH disappeared upon deuteration);
MS (m/z, %): Mþ (318, 70.5). Anal. Calcd for C15H14N2O4S, C: 56.59;
H: 4.43; N: 8.80, found: C: 56.20, H: 4.75; N: 8.33.
3.1.6. 7-[(5-Mercapto-1,3,4-oxadiazol-2-yl)methoxy]-4-propyl-2H-
1-benzopyran-2-one (3c)
Yield: 97%; m.p. 252e255 ꢁC, IR (KBr): 3306 (NH), 1709 (C]O of
a
-pyrone); 1H NMR (300 MHz, CDCl3,
d ppm): d 1.03 (t, 3H,
CH3eCH2eCH2, J ¼ 7.5 Hz), 1.7 (m, 2H, CH3eCH2eCH2, J ¼ 7.5 Hz),
2.3 (s, 3H, CH3 at benzopyronyl C8), 2.7 (t, 2H, CH3eCH2eCH2,
J ¼ 7.5 Hz), 4.7 (s, 2H, OCH2), 6.2 (s, 1H, benzopyronyl C3eH), 6.8 (d,
1H, benzopyronyl C6eH, J ¼ 8.7 Hz) 7.5 (d, 1H, benzopyronyl C5eH,
Fig. 3. Binding mode of 9a in the active site of human DNA topoisomerase 1.

3956
M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 3950e3959
J ¼ 8.7 Hz), 7.7 (s, 1H, NH disappeared upon deuteration); MS (m/z,
%): Mþ (332, 2.6). Anal. Calcd for C16H16N2O4S, C: 57.82; H: 4.85; N:
8.43, found: C: 57.12, H: 4.70; N: 8.96.
3.3.3. N-Benzyl-N1((8-methyl-4-propyl-2H-benzopyran-2-one-7-
yloxy)acetyl)-thiosemicarbazide (5c)
Yield: 92%; m.p. 214e216 ꢁC, IR (KBr cmꢀ1): 3336e3286 (3NH),
1697 (C]O of a
-pyrone), 1605 (C]O, amide). 1H NMR (200 MHz,
3.2. General procedure for preparation of 7-[(5-methylthio-1,3,4-
oxadiazol-2-yl)methoxy]-4,8-disubstituted-2H-1-benzopyran-2-
ones (4a,b)
DMSO): 0.97 (t, 3H, eCH2CH2CH3, J ¼ 7.2 Hz), 1.7 (m, 2H, eCH2CH2CH3,
J ¼ 7.2 Hz) 2.3(s, 3H, CH3 at C8 of benzopyrone), 2.7 (t, 2H,
eCH2CH2CH3, J ¼ 7.2 Hz), 4.8 (s, 4H, OeCH2 and eCH2eC6H5), 6.2 (s,
1H, benzopyronyl C3eH), 6.9 (d, 1H, benzopyronyl C6eH, J ¼ 8.4 Hz),
7.3 (m, 5H, CH2eC6H5), 7.6(d, 1H, benzopyronyl C5eH, J ¼ 8.4 Hz), 8.6,
9.4, 10.1(3s, 3H, 3NH disappeared upon deuteration). MS m/z (%): Mþ
(439, 0.18%), base peak (218,100%). Anal. Calcd for C23H25N3O4S, C:
62.85; H: 5.37; N: 9.56, found: C: 62.17, H: 5.80, N: 9.93.
A mixture of 3 (0.01 mol), methyl iodide (2.8 g, 0.02 mol) and
anhydrous potassium carbonate (2.8 g, 0.02 mol) in dry acetone
(100 ml) was refluxed and stirred for 24 h. The product was filtered
while hot, then the solvent was distilled off and the residue was
crystallized from ethanol.
3.3.4. 7-[(5-Mercapto-4-benzyl-1,2,4-triazol-3-yl)methoxy]-4,8-
disubstituted-2H-1-benzopyran-2-ones (6aec)
3.2.1. 7-[(5-Methylthio-1,3,4-oxadiazol-2-yl)methoxy]-8-methyl-4-
phenyl-2H-1-benzopyran-2-one (4a)
A mixture of 5aec (0.005 mol) and 2 N NaOH (10 ml) was
refluxed and stirred for 3 h, cooled, acidified with HCl and the
separated solid was crystallized from ethanol/water.
Yield: 89%; m.p. 155e157 ꢁC. IR (KBr cmꢀ1): 1719 (C]O of
a-
pyrone); 1H NMR (200 MHz, DMSO,
d ppm): d 2.3 (s, 3H, CH3 at C8),
2.6 (s, 3H, SCH3), 4.8 (s, 2H, OeCH2), 6.3 (s, 1H, C3eH), 6.9 (d, 1H,
C6eH, J ¼ 8.8 Hz), 7.2 (d, 1H, C5eH, J ¼ 8.8 Hz), 7.5 (m, 5H, PheH);
MS (m/z, %): Mþ (380, 74.6). Anal. Calcd for C20H16N2O4S, C: 63.15;
H: 4.24; N: 7.36, found: C: 62.66, H: 4.13, N: 7.17.
3.3.5. 7-[(5-Mercapto-4-benzyl-1,2,4-triazol-3-yl)methoxy]-8-
methyl-4-phenyl-2H-1-benzopyran-2-one (6a)
Yield: 99%; m.p. 250e251 ꢁC, IR (KBr cmꢀ1): 1722 (C]O,
a-
pyrone); 1H NMR (200 MHz, DMSO,
d ppm): d 2.5 (s, 3H, CH3, C8),
3.2.2. 7-[(5-Methylthio-1,3,4-oxadiazol-2-yl)methoxy]-8-methyl-
2.8 (s, 1H, SH), 5.2 (s, 2H, eCH2eC6H5), 5.3 (s, 2H, OeCH2), 6.2 (s, 1H,
C3eH), 7.0e7.6 (m, 12H, C5, C6eH, CH2eC6H5 þ PheH); MS: (m/z,
%) ¼ (455, 10.9). Anal. Calcd for C26H21N3O3S, C: 68.55; H: 4.65; N:
9.22, found: C: 68.14, H: 4.82, N: 9.32.
4-propyl-2H-1-benzopyran-2-one (4b)
Yield: 91%; m.p. 160e163 ꢁC, IR (KBr cmꢀ1): 1709 (C]O of
a-
pyrone); 1H NMR (300 MHz, CDCl3,
d ppm): d 1.05 (t, 3H,
CH3eCH2eCH2,J¼ 7.5 Hz), 1.7 (m, 2H, CH3eCH2eCH2, J¼ 7.5 Hz), 2.3 (s,
3H, CH3 at C8), 2.72 (t, 2H, CH3eCH2eCH2, J ¼ 7.5 Hz), 2.76 (s,3H, SCH3),
5.3 (s, 2H, OeCH2), 6.2 (s,1H, C3eH), 7.0 (d, 1H, C6eH, J ¼ 9 Hz), 7.5 (d,
1H, C5eH, J ¼ 9 Hz). MS (m/z, %): Mþ (346, 100). Anal. Calcd for
C17H18N2O4S, C: 58.94; H: 5.24; N: 8.09, found: C: 58.63, H: 5.12, N: 7.80.
3.3.6. 7-[(5-Mercapto-4-benzyl-1,2,4-triazol-3-yl)-methoxy]-4-
propyl-2H-1-benzopyran-2-one (6b)
Yield: 96%; m.p. 186e188 ꢁC, IR (KBr cmꢀ1): 1730 (C]O,
a-
pyrone); 1H NMR (200 MHz, DMSO,
d ppm): d 0.96 (t, 3H,
eCH2CH2eCH3, J ¼ 7.0 Hz), 1.6 (m, 2H, eCH2CH2CH3, J ¼ 7.0 Hz), 2.7
(t, 2H, eCH2CH2eCH3, J ¼ 7.0 Hz) 5.2 (s, 2H, eCH2eC6H5), 5.3 (s, 2H,
OeCH2), 6.2 (s, 1H, C3eH), 6.7 (d, 1H, C6eH, J ¼ 8.6 Hz), 7.2 (m, 5H,
CH2eC6H5), 7.7 (d,1H C5eH, J ¼ 8.6 Hz); MS (m/z, %): Mþ (407, 0.01).
Anal. Calcd for C22H21N3O3S, C: 64.85; H: 5.19; N: 10.31, found: C:
64.25, H: 5.97, N: 10.26.
3.3. General procedure for preparation of N-substituted-N1((8-
methyl-4-phenyl-2H-benzopyran-2-one-7-yloxy)acetyl)-thiosemi-
carbazides (5aec)
To a solution of the acid hydrazide 2a,b (0.01 mol) in hot
absolute ethanol (30 ml) the appropriate substituted isothiocyanate
(0.01 mol) was added, the mixture was refluxed while stirring for
6 h. The separated solid was washed with ethanol and crystallized
from ethanol/chloroform.
3.3.7. 7-[(5-Mercapto-4-benzyl-1,2,4-triazol-3-yl)-methoxy]-8-
methyl-4-propyl-2H-1-benzopyran-2-one (6c)
Yield: 97%, m.p. 187e189 ꢁC; IR (cmꢀ1): 1712 (C]O, -pyrone); 1H
a
NMR(DMSO);
d
1.0 (t, 3H, eCH2CH2eCH3, J ¼ 7.2 Hz), 1.7 (m, 2H,
3.3.1. N-Benzyl-N1((8-methyl-4-phenyl-2H-benzopyran-2-one-7-
yloxy)acetyl)-thiosemicarbazide (5a)
eCH2CH2CH3, J ¼ 7.2 Hz), 2.5 (s, 3H, CH3, on benzopyronyl C8), 2.7 (t,
2H, eCH2CH2eCH3, J ¼ 7.2 Hz) 5.2 (s, 2H, eCH2eC6H5), 5.3 (s, 2H,
OeCH2),6.2(s,1H,benzopyronylC3eH), 7.0e7.3 (m, 6H, benzopyronyl,
C6eH and 5H, PheHs), 7.6 (d,1H, benzopyronyl, C5eH, J ¼ 9 Hz). MS m/
z(%): Mþ (421, 2.9%), basepeak(91,100%). Anal. Calcdfor C23H23N3O3S,
C: 65.54; H: 5.50; N: 9.97, found: C: 65.33, H: 5.37, N: 9.51.
Yield: 78%; m.p. 230e232 ꢁC, IR (KBr cmꢀ1): 3338, 3261, 3218
(3NH), 1698 (C]O of
(200 MHz, DMSO, ppm):
a
-pyrone), 1602 (C]O, amide); 1H NMR
d
d 2.3 (s, 3H, CH3 at C8), 4.8 (s, 2H,
CH2eC6H5), 4.9 (s, 2H, OeCH2) 6.2 (s, 1H, C3eH), 6.9 (d, 1H, C6eH,
J ¼ 8.8 Hz), 7.1e7.5 (m, 11H, C5eH, CH2eC6H5, PheH), 8.6, 9.4, 10.1
(3s, 3H, 3NH disappeared upon deuteration). MS (m/z, %): Mþ (473,
0.37). Anal. Calcd for C26H23N3O4S, C: 65.95; H: 4.90; N: 8.87, found:
C: 65.21, H: 4.68, N: 8.98.
3.3.8. 7-{[5-(Aryl)-1,3,4-oxadiazol-2-yl] methoxy}-4,8-
disubstituted-2H-1-benzopyran-2-ones (7aee)
A mixture of the acid hydrazide 1aec (0.005 mol) and benzoic
acid or p-methoxy benzoic acid (0.005 mol) in phosphorous oxy-
chloride (5 ml) was heated under reflux temperature for 2 h, cooled
down and slowly added to ice/water, the precipitated solid was
filtered off, washed with water and crystallized from DMF/H2O.
3.3.2. N-Benzyl-N1((4-propyl-2H-benzopyran-2-one-7-yloxy)
acetyl)-thiosemicarbazide (5b)
Yield: 90%; m.p. 180e183 ꢁC, IR (KBr cmꢀ1): 3403, 3365, 3186
(3NH), 1730 (C]O of
(200 MHz, DMSO, ppm):
a
-pyrone), 1612 (C]O, amide); 1H NMR
d
d
0.97 (t, 3H, eCH2CH2CH3, J ¼ 7.2 Hz),
3.3.9. 7-{[5-(Phenyl)-1,3,4-oxadiazol-2-yl]methoxy}-8-methyl-4-
1.7 (m, 2H, eCH2CH2CH3, J ¼ 7.2 Hz), 2.8 (t, 2H, eCH2CH2CH3,
J ¼ 7.2 Hz), 4.8 (s, 4H, OeCH2 and eCH2eC6H5), 6.2 (s, 1H, C3eH),
7.0e7.2 (m, 6H, C6eH, CH2eC6H5), 7.7 (d, 1H, C5eH, J ¼ 9.4 Hz), 8.6,
9.4, 10.2 (3s, 3H, 3NH disappeared upon deuteration); MS (m/z, %):
Mþ (425, 1.25). Anal. Calcd for C22H23N3O4S, C: 62.10; H: 5.45; N:
9.88, found: C: 62.73, H: 5.57, N: 9.40.
phenyl-2H-1-benzopyran-2-one, 7a
Yield: 95%, m.p. 127e130 ꢁC; IR (cmꢀ1) 1710 (C]O of
a
-pyrone).
1H NMR (DMSO);
d 2.2(s, 3H, CH3 at benzopyronyl C8), 4.9 (s, 2H,
OeCH2), 6.2 (s, 1H, benzopyronyl C3eH), 6.9(d, 1H, benzopyronyl
C6eH, J ¼ 9 Hz), 7.2(d,1H, benzopyronyl C5eH, J ¼ 9 Hz), 7.4e7.6 (m,
10H, PheH). MS m/z (%): Mþþ1 (411, 0.02%), base peak (352, 100%).

M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 3950e3959
3957
Anal. Calcd for C25H18N2O4, C: 73.16; H: 4.42; N: 6.83, found: C:
3.3.17. 7-[2-(3,5-Dimethyl-1H-pyrazole-1-yl)-2-oxoethoxy]-8-
72.87, H: 4.67; N: 6.76.
methyl-4-propyl-2H-1-benzopyran-2-one, 8c
Yield: 76%, m.p. 220e221 ꢁC; IR (cmꢀ1) 1774 (CO of
a
-pyrone),
0.97 (t, 3H, CH3eCH2eCH2,
J ¼ 7.2 Hz), 1.6 (m, 2H, CH3eCH2eCH2, J ¼ 7.2 Hz), 2.2 (s, 9H, 3CH3),
2.7 (t, 2H, CH3eCH2eCH2, 7.2 Hz), 4.8 (s, 3H,
3.3.10. 7-{[5-(Phenyl)-1,3,4-oxadiazol-2-yl]methoxy}-4-propyl-2H-
1663 (CO of amide). 1H NMR (DMSO);
d
1-benzopyran-2-one, 7b
Yield: 100%, m.p. 205e207 ꢁC; IR (cmꢀ1) 1709 (C]O of
a
-
J
¼
pyrone). 1H NMR (DMSO);
d
0.97 (t, 3H, CH3eCH2eCH2), 1.2 (m, 2H,
OeCH2 þ pyrazolyleCH), 6.2 (s,1H, benzopyronyl C3eH), 6.9(d, 1H,
benzopyronyl C6eH, J ¼ 8.4 Hz), 7.6 (d, 1H, benzopyronyl C5eH,
J ¼ 8.4 Hz). MS m/z (%) Mþ (354, 1.27%), base peak (189, 100%). Anal.
Calcd for C20H22N2O4, C: 67.78; H: 6.26; N: 7.90, found: C: 67.80, H:
6.55, N: 7.54.
CH3eCH2eCH2), 1.6 (t, 2H, CH3eCH2eCH2), 4.8 (s, 2H, OeCH2), 6.2
(s, 1H, benzopyronyl C3eH), 6.9e8.0 (m, 8H, benzopyronyl C8, C5,
C6eH, þPheHs). MS m/z (%): Mþ (362,1.95%), base peak (135,100%).
Anal. Calcd for C21H18N2O4, C: 69.60; H: 5.01; N: 7.73, found: C:
69.15; H: 5.45; N: 7.29.
3.3.18. 3-Methyl-1-(2-(4,8-disubstituted-2H-1-benzopyran-2-one-
7-yloxy)acetyl)-1,2-dihydropyrazol-5-ones (9aec)
3.3.11. 7-{[5-(Phenyl)-1,3,4-oxadiazol-2-yl]methoxy}-8-methyl-4-
propyl-2H-1-benzopyran-2-one, 7c
A mixture of the acid hydrazide 2aec (0.1 mol) and ethyl-
acetoacetate (26 ml, 0.2 mol) in glacial acetic acid (30 ml) was
refluxed for 6 h, the precipitated solid was filtered off and crys-
tallized from DMF/H2O.
Yield: 84%, m.p. 157e160 ꢁC; IR (cmꢀ1) 1722 (C]O of
a-pyrone).
1H NMR (CDCl3);
d
1.05 (t, 3H, CH3, CH3eCH2eCH2, J ¼ 7.5 Hz),1.3 (m,
2H, CH3eCH2eCH2, J ¼ 7.5 Hz),1.7 (t, 2H, CH3eCH2eCH2, J ¼ 7.5 Hz),
2.4 (s, 3H, CH3 at benzopyronyl C8), 4.8 (s, 2H, OeCH2), 6.2 (s, 1H,
benzopyronyl C3eH), 6.7 (d, 1H, benzopyronyl C6eH, J ¼ 9 Hz), 7.3
(m, 5H, PheHs), 7.4 (d,1H, benzopyronyl C5eH, J ¼ 9 Hz). MS m/z (%):
3.3.19. 3-Methyl-1-(2-(8-methyl-4-phenyl-2H-1-benzopyran-2-
one-7-yloxy)acetyl)-1,2-dihydro- pyrazol-5-one (9a)
M
þ (376, 47.17%), base peak (221, 100%). Anal. Calcd for C22H20N2O4,
Yield: 84%; m.p. 309e311 ꢁC, IR (KBr cmꢀ1): 3318 (NH),1718 (C]
C: 70.20; H: 5.36; N: 7.44, found: C: 69.85, H: 5.98; N: 7.89.
O of
2.3 (s, 6H, 2CH3), 4.8 (s, 2H, OeCH2) 5.0 (s, IH, CHepyrazolone), 6.2
a d ppm):
-pyrone), 1616 (NC]O); 1H NMR (200 MHz, DMSO,
d
3.3.12. 7-{[5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl]methoxy}-4-
(s,1H, C3eH), 6.9 (d,1H, C6eH, J ¼9.4 Hz), 7.2 (d,1H, C5eH, J ¼ 9.4 Hz),
7.5 (m, 5H, AreH),10.2 (s,1H, NH disappeared upon deuteration). MS
(m/z, %): Mþ (390, 0.39). Anal. Calcd for C22H18N2O5, C: 67.69; H:
4.28; N: 7.18, found: C: 67.34, H: 4.55; N: 7.12.
propyl-2H-1-benzopyran-2-one, 7d
Yield: 99%, m.p. 228e230 ꢁC; IR (cmꢀ1) 1718 (C]O of
a
-pyrone).
1H NMR (DMSO);
d 0.97 (t, 3H, CH3eCH2eCH2), 1.2 (m, 2H,
CH3eCH2eCH2), 1.6 (t, 2H, CH3eCH2eCH2), 2.3 (s, 3H, CH3 at ben-
zopyronyl C8), 3.8 (s, 3H, OeCH3), 4.8 (s, 2H, OeCH2), 6.2 (s,1H,
benzopyronyl C3eH), 6.6e7.9 (m, 7H, benzopyronyl C8, C5, C6eH,
þPheHs). MS m/z (%): Mþ (392, 10.62%), base peak (135, 100%).
Anal. Calcd for C22H20N2O5, C: 67.34; H: 5.41; N: 7.14, found: C:
67.93, H: 5.55; N: 7.73.
3.3.20. 3-Methyl-1-(2-(4-propyl-2H-1-benzopyran-2-one-7-yloxy)
acetyl)-1,2-dihydropyrazol-5-one (9b)
Yield: 90%; m.p. 229e231 ꢁC, IR (KBr cmꢀ1): 3319 (NH),1712 (C]
O of
ppm):
a
-pyrone), 1648,1617 (2NC]O); 1H NMR (200 MHz, DMSO,
d
d
0.97 (t, 3H, CH3eCH2eCH2, J ¼ 7.2 Hz), 1.6 (m, 2H,
CH3eCH2eCH2, J ¼ 7.2 Hz), 2.1 (s, 3H, eCH3 onpyrazolone), 2.7 (t, 2H,
CH3eCH2eCH2, J ¼ 7.2 Hz), 4.8 (s, 2H, OeCH2) 5.0 (s, IH, CHepyr-
azolone), 6.2 (s, 1H, C3eH), 6.8e7.0 (m, 2H, C6, C8eH), 7.2 (d, 1H,
C5eH, J ¼ 8.6 Hz),10.3 (s,1H, NH disappeared upon deuteration). MS:
(m/z, %) Mþ (342, 0.99). Anal. Calcd for C18H18N2O5, C: 63.15; H: 5.30;
N: 8.18, found: C: 63.82, H: 5.62; N: 7.86.
3.3.13. 7-{[5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl]methoxy}8-
methyl-4-propyl-2H-1-benzopyran -2-one, 7e
Yield: 88%, m.p. 137e140 ꢁC; IR (cmꢀ1) 1718 (C]O of
a-
pyrone). 1H NMR (DMSO);
d 0.97 (t, 3H, CH3eCH2eCH2,
J ¼ 7.6 Hz), 1.2(m, 2H, CH3eCH2eCH2, J ¼ 7.6 Hz), 1.6 (t, 2H,
CH3eCH2eCH2, J ¼ 7.6 Hz), 2.2 (s, 3H, at benzopyronyl C8), 3.8 (s,
3H, OeCH3), 4.8 (s, 2H, OeCH2), 6.2 (s, 1H, benzopyronyl C3eH),
6.8e7.9 (m, 6H, benzopyronyl C5, C6eH, þPheH). Anal. Calcd for
C23H22N2O5, C: 67.97; H: 5.46; N: 6.89, found: C: 67.53, H: 5.95; N:
7.14.
3.3.21. 3-Methyl-1-(2-(8-methyl-4-propyl-2H-1-benzopyran-2-
one-7-yloxy)acetyl)-1,2-dihydro-pyrazol-5-one (9c)
Yield: 92%; m.p. 303e306 ꢁC IR (cmꢀ1): 3164 (NH), 1717 (C]O of
a
-pyrone), 1611 (2NC]O). 1H NMR (DMSO);
d 0.97 (t, 3H,
CH3eCH2eCH2, J ¼ 7.2 Hz), 1.6 (m, 2H, CH3eCH2eCH2, J ¼ 7.2 Hz),
2.3 (s, 3H, CH3 at C8 of benzopyrone), 2.5 (s, 3H, eCH3 on pyr-
azolone), 2.7 (t, 2H, CH3eCH2eCH2, J ¼ 7.2 Hz), 4.8 (s, 3H,
OeCH2 þ CH of pyrazolone), 6.2 (s, 1H, benzopyronyl C3eH), 7.0 (d,
1H, benzopyronyl C6eH, J ¼ 9 Hz), 7.6 (d, 1H, benzopyronyl C5eH,
J ¼ 9 Hz), 10.2 (s, 1H, NH disappeared upon deuteration). MS m/z
(%): Mþ (356, 0.31%), base peak (218, 100%). Anal. Calcd for
C19H20N2O5, C: 64.04; H: 5.66; N: 7.86, found: C: 64.01, H: 6.17; N:
7.49.
3.3.14. 7-[2-(3,5-Dimethyl-1H-pyrazole-1-yl)-2-oxoethoxy]-4,8-
disubstituted-2H-1-benzopyran-2-ones (8aec)
A mixture of the acid hydrazide 1aec (0.01 mol) and acetyla-
cetone (2 ml, 0.02 mol) in acetic acid (10 ml) was refluxed for 6 h,
the formed solid was filtered off and crystallized from ethyl alcohol.
3.3.15. 7-[2-(3,5-Dimethyl-1H-pyrazole-1-yl)-2-oxoethoxy]-8-
methyl-4-phenyl-2H-1-benzopyran-2-one, 8a
Yield: 86%, m.p. 240e242 ꢁC; IR (cmꢀ1) 1774 (C]O of
a-pyrone),
1664(C]O of amide). MS m/z (%): Mþ (388, 0.04%), base peak (276,
100%). Anal. Calcd for C23H20N2O4, C: 71.12; H: 5.19; N: 7.21, found:
C: 69.88, H: 5.56, N: 7.27.
3.3.22. 1-{[(4,8-Disubstituted-2H-1-benzopyran-2-one-7-yl)oxy]
acetyl} pyrazolidine-3,5-diones (10a,b)
To a solution of sodium ethoxide (made of 0.198 g of sodium in
20 ml of absolute ethanol) was added a freshly distilled dieth-
ylmalonate (0.68 g, 0.0424 mol) followed by addition of the acid
hydrazide, 2 (0.028 mol). The reaction mixture was refluxed for 2 h,
the residue was further heated at 150e155 ꢁC for 5 h, it was dis-
solved in water, filtered off, acidified with 10% HCl. The precipitate
was filtered off, washed with cold water then crystallized from
ethanol.
3.3.16. 7-[2-(3,5-Dimethyl-1H-pyrazole-1-yl)-2-oxoethoxy]-4-
propyl-2H-1-benzopyran-2-one, 8b
Yield: 78%, m.p. 180e182 ꢁC; IR (cmꢀ1) 1764 (C]O of
a-pyrone),
1664(C]O of amide). MS m/z (%): Mþ (341, 9.85%), base peak (45,
100%). Anal. Calcd for C19H20N2O4, C: 67.05; H: 5.92; N: 8.23, found:
C: 67.54, H: 5.50, N: 7.94.

3958
M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 3950e3959
ꢂ The GI50 value (growth inhibitory activity) corresponds to the
concentration of the compounds causing 50% decrease in net
cell growth,
ꢂ The TGI value (cytostatic activity) is the concentration of the
compounds resulting in total growth inhibition,
ꢂ The LC50 value (cytotoxic activity) is the concentration of the
compounds causing net 50% loss of initial cells at the end of the
incubation period (48 h).
3.3.23. 1-{[(8-Methyl-4-phenyl-2H-1-benzopyran-2-one-7-yl)oxy]
acetyl}pyrazolidine-3,5-dione (10a)
Yield: 87%; m.p. 170e173 ꢁC; IR (cmꢀ1): 3420 (OH), 1724 (C]O
of a d 2.3 (s, 3H, CH3 on
-pyrone), 1672 (3NC]O). 1H NMR (DMSO);
C8 of benzopyranone), 2.4 (s, 2H, CH2 of pyrazolone), 4.8 (s,4H,
OeCH2), 6.2 (s, 1H, benzopyronyl C3eH), 6.9 (d, 1H, benzopyranonyl
C6eH, J ¼ 9 Hz), 7.2 (d, 1H, benzopyronyl C5eH, J ¼ 9 Hz), 7.5 (m, 5H,
PheHs),10.0 (s,1H, OH, disappeared upon deuteration). MS m/z (%):
Mþ (392, 0.2%), base peak (236, 100%). Anal. Calcd for C21H16N2O6,
C: 64.28; H: 4.11; N: 7.14, found: C: 64.52, H: 4.43; N: 7.12.
Subpanel and full panel mean-graph mid-point values (MG-
MID) for certain agents are the average of individual GI50, TGI, or
LC50 values of all cell lines in the sub-panel or the full panel,
respectively.
The NCI antitumor drug discovery program was designed to
distinguish between broad spectrum antitumor compounds and
tumor or subpanel-selective agents.
3.3.24. 1-{[(8-Methyl-4-propyl-2H-1-benzopyran-2-one-7-yl)oxy]
acetyl}pyrazolidine-3,5-dione (10b)
Yield: 84%; m.p. 208e210 ꢁC; IR (cmꢀ1): 3400 (OH),1772 (C]O of
a
-pyrone), 1662 (3NC]O). 1H NMR (CDCl3);
d 1.04 (t, 3H,
CH3eCH2eCH2, J ¼ 7.5 Hz),1.7 (m, 2H, CH3eCH2eCH2, J ¼ 7.5 Hz), 2.7
(t, 2H, CH3eCH2eCH2 J ¼ 7.5 Hz), 2.3 (s, 3H, CH3 on C8 of benzopyr-
anone), 2.4 (s, 2H, CH2 of pyrazolone), 4.8 (s, 2H, OeCH2), 6.2 (s, 1H,
benzopyronyl C3eH), 6.7 (d, 1H, benzopyranonyl C6eH, J ¼ 8.7 Hz),
7.5(d,1H, benzopyronyl C5eH, J ¼ 8.7 Hz). Anal. Calcd for C18H18N2O6,
C: 60.33; H: 5.06; N: 7.82, found: C: 60.96, H: 5.31; N: 7.83.
3.5. Docking studies
All docking studies were performed using “Internal coordinate
Mechanics (Molsoft ICM 3.5-0a)”.
3.4. Antitumor screening
3.5.1. Generation of ligand and enzyme structures
The crystal structure of the human DNA topoisomerase I (1T8I)
was retrieved from the Protein Data Bank (http://www.pdb.org/
pdb/explore/explore.do?structureId¼1T8I).
3.4.1. Methodology of the in vitro cancer screen
The human tumor cell lines of the cancer screening panel are
grown in RPMI 1640 medium containing 5% fetal bovine serum
All bound waters ligands and cofactors were removed from the
protein. The amino acids of the binding site where defined using
data in pdbsum http://www.ebi.ac.uk/thornton-srv/databases/cgi-
bin/pdbsum/GetPage.pl.
and 2 mM
L
-glutamine. For a typical screening experiment, cells
l at plating
are inoculated into 96 well microtiter plates in 100
m
densities ranging from 5000 to 40,000 cells/well depending on
the doubling time of individual cell lines. After cell inoculation,
the microtiter plates are 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 are fixed in situ with TCA,
to represent a measurement of the cell population for each cell line
at the time of drug addition. Experimental drugs are solubilized in
dimethylsulfoxide 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 is thawed and diluted to
twice the desired final maximum test concentration with complete
3.5.2. Preparation of small molecule
A set of 7-substituted-benzopyran-2-ones, was designed to bind
with human DNA topoisomerase I, ChemDraw 3D structures were
constructed using ChemDraw 3D ultra 9.0 software [Cambridge
Soft Corporation, USA (2004)], and then they were energetically
minimized by using MOPAC (semi-empirical quantum mechanics)
with MM2, Jop Type with 100 iterations and minimum RMS
gradient of 0.01, and saved as MDL MolFile (*.mol).
3.5.3. Docking using Molsoft ICM 3.4-8C program [27,28]
3.5.3.1. Convert our PDB file 1T8I into an ICM object. This conversion
involves addition of hydrogen bonds, assignment of atoms types,
and charges from the residue templates. Click on MolMechanics/
Convert/Protein, and then delete water molecules.
medium containing 50
or ½ log serial dilutions are made to provide a total of five drug
concentrations plus control. Aliquots of 100 l of these different
drug dilutions are added to the appropriate microtiter wells
mg/ml gentamycin. Additional four, 10-fold
m
already containing 100
drug concentrations.
ml of medium, resulting in the required final
3.5.3.2. To perform ICM small molecule docking.
Following drug addition, the plates are incubated for an addi-
tional 48 h at 37 ꢁC, 5% CO2, 95% air, and 100% relative humidity. For
adherent cells, the assay is terminated by the addition of cold TCA.
(a) Setup docking project:
(i) Set Project Name: Click on Docking/Set project name, press
OK.
Cells are fixed in situ by the gentle addition of 50
v) TCA (final concentration, 10% TCA) and incubated for 60 min at
ꢁC. The supernatant is discarded, and the plates are washed five
times with tap water and air dried. Sulforhodamine B (SRB) solution
(100 l) at 0.4% (w/v) in 1% acetic acid is added to each well, and
ml of cold 50% (w/
(ii) 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
button to identify the potential ligand binding pockets,
press OK. After the receptor setup is complete, the program
normally displays the receptor with selected binding site
residues highlighted in yellow xstick presentation.
(iii) 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 residues expected to be involved in ligand binding. Click
on the menu Docking/Review/Adjust ligand/Box.
4
m
plates are incubated for 10 min at room temperature. After staining,
unbound dye is removed by washing five times with 1% acetic acid
and the plates are air dried. Bound stain is subsequently solubilized
with 10 mM trizma base, and the absorbance is read on an auto-
mated plate reader at a wavelength of 515 nm. For suspension cells,
the methodology is the same except that the assay is terminated by
fixing settled cells at the bottom of the wells by gently adding 50
of 80% TCA (final concentration, 16% TCA).
ml
Three response parameters (GI50, TGI, and LC50) were calculated
for each cell line.
(vi) Make receptor maps: The step now is to construct energy
maps of the environment within the docking box. Click on

M.M.F. Ismail et al. / European Journal of Medicinal Chemistry 45 (2010) 3950e3959
3959
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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 minutes.
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(b) Start docking simulation: 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 we wish to dock, and then enter the
thoroughness which represent the length of simulation.
Generally 1 is reasonable value, select Calc ICM Score, then
select Display run which display the ligand sampling the
energy in the ligand binding project.
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grid potentials describing interaction of the flexible ligand with the
receptor and internal conformational energy of the ligand, during
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