Dapson in heterocyclic chemistry, part V: Synthesis, molecular docking and anticancer activity of some novel sulfonylbiscompounds carrying biologically active dihydropyridine, dihydroisoquinoline, 1,3-dithiolan, 1,3-dithian, acrylamide, pyrazole, pyrazolopyrimidineand benzochromenemoieties

Article abstract of DOI:10.1248/cpb.c12-00292

N,N′-(4,4′-Sulfonylbis(4,1-phenylene))bis(2-cyanoacetamid) 2 was utilized as a key intermediate for the synthesis of novel dihydropyridines 3, 4, 8, dihydroisoquinolines 5-7, dithiolan 10, dithian 11, acrylamide 12, benzochromenes 17 and 18 and chromenopyridones 19 and 20. Compound 2 was the starting material in the synthesis of the acrylamide derivative 14, the pyrazole derivative 15 and the pyrazolopyrimidine derivative 16. All the synthesized compounds were evaluated for their in vitro anticancer activity against human breast cancer cell line (MCF7). Compound 19 showed the best cytotoxic activity with IC₅₀ value 19.36 μM. In addition, molecular docking study of the synthesized compounds on the active sites of farnesyltransferase and arginine methyltransferase was performed in order to give a suggestion about the mechanism of action of their cytotoxic activity.

Full text of DOI:10.1248/cpb.c12-00292

August 2012
Regular Article
Chem. Pharm. Bull. 60(8) 1019–1028 (2012)
1019
Dapson in Heterocyclic Chemistry, Part V: Synthesis, Molecular
Docking and Anticancer Activity of Some Novel Sulfonylbiscompounds
Carrying Biologically Active Dihydropyridine, Dihydroisoquinoline,
1,3-Dithiolan, 1,3-Dithian, Acrylamide, Pyrazole, Pyrazolopyrimidine
and Benzochromenemoieties
,a
Mostafa Mohammed Ghorab,* Mansour Sulaiman Al-Said,a and Yassin Mohammed Nissanb
a Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud
b
University; P.O. Box 2457, Riyadh 11451, Saudi Arabia: and Pharmaceutical Chemistry Department, Faculty of
Pharmacy, Cairo University; Cairo 11562, Egypt. Received April 2, 2012; accepted May 1, 2012
N,N′-(4,4′-Sulfonylbis(4,1-phenylene))bis(2-cyanoacetamid) 2 was utilized as a key intermediate for the
synthesis of novel dihydropyridines 3, 4, 8, dihydroisoquinolines 5–7, dithiolan 10, dithian 11, acrylamide 12,
benzochromenes 17 and 18 and chromenopyridones 19 and 20. Compound 2 was the starting material in the
synthesis of the acrylamide derivative 14, the pyrazole derivative 15 and the pyrazolopyrimidine derivative
16. All the synthesized compounds were evaluated for their in vitro anticancer activity against human breast
cancer cell line (MCF7). Compound 19 showed the best cytotoxic activity with IC50 value 19.36µM. In addi-
tion, molecular docking study of the synthesized compounds on the active sites of farnesyltransferase and
arginine methyltransferase was performed in order to give a suggestion about the mechanism of action of
their cytotoxic activity.
Key words sulfone; pyridine; pyrazolopyrimidine; benzochromene; anticancer activity
A large number of sulfone derivatives have been found to cysteine in the C-terminal tetrapeptide sequence. Compound
exhibit a wide variety of pharmacological activities.1–8) Also, C showed the lowest IC50 as FTase inhibitor.18) Some other
diphenylsulfones and bisheterocyclic compounds are reported styryl heterocyclic sulfone derivatives D were interesting as
to have a broad spectrum of biological activities. Some are anticancer agents as they block the mitogen activated protein
endowed with antitumor,9) or antifungal properties.10) On the kinase (MAPK) cascade which phosphorylates a variety of
other hand, some pyridine and isoquinoline derivatives have proteins including several transcription factors, which translo-
various biological properties such as antimicrobial,11) antican- cate into the nucleus and activate gene transcription. Negative
cer activities.12–15)
Recent studies have proved the remarkable effect of Dapson events and will inhibit the proliferation of cancer cells.19,20)
1 on inhibiting cell growth in glioblastoma by acting as anti- On the preceding and as a part of our studies on new het-
regulation of this pathway could arrest the cascade of these
vascular endothelial growth factor (VEGF) and anti-angio- erocyclic compounds derived from Dapson as new potential
genic agent via depriving glioblastoma of neutrophil-mediated anticancer agents,21–24) we have synthesized a novel series of
growth promoting effects.16) Allantodapson A, a dapson deriv- dihydropyridines, dihydroisoquinolines, 1,3-dithiolan, 1,3-di-
ative showed high activity as anticancer through inhibition of thian, acrylamide, pyrazole, pyrazolopyrimidine and benzo-
arginine methyltranseferase (PRMT1) an enzyme which plays chromene derivatives to evaluate their anticancer activity.
an important role in hormone dependant cancers. A series of
acylated diarylsulfone derivatives were evaluated for the same
Results and Discussion
activity and compound B exihibited good activity as (PRMT1)
Chemistry The solvent-free reaction of arylamines
inhibitor.17) Some diarylsulfone derivatives bearing imidazole with ethyl cyanoacetate is well known to constitute one
ring were evaluated for their anticancer activity through their of the most widely used synthetic methods. Thermal fu-
action on inhibition of farnesyl-protein transferase (FTase), a sion of 4,4′-sulfonyldianiline (Dapson) 1 above its melting
zinc metalloenzyme which catalyzes the lipidation of a 3,4 point with ethyl-2-cyanoacetate yielded the corresponding
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: mmsghorab@yahoo.com
© 2012 The Pharmaceutical Society of Japan

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Vol. 60, No. 8
Reagents (a) ethylcyanoacetate, (b) acetylacetone or benzoylacetone, (c) benzylidenemalononitriles, (d) DMF/DMA.
Chart 1
N,N′-(4,4′-sulfonylbis(4,1-phenylene))bis(2-cyanoacetamide) 2. bands at 3390, 3367cm−1 (2NH2) and 2160cm−1 (2C≡N). Its
IR spectrum of 2 revealed bands at 3448, 3363cm−1 (2NH), 1H-NMR spectrum in (DMSO-d6) displayed signals at 6.2ppm
2256cm−1 (2C≡N), 1701cm−1 (2C=O) and 1342, 1180cm−1 for NH2 group and 6.4ppm for CH of pyridone. IR spectrum
(SO2). 1H-NMR spectrum in (DMSO-d6) of 2 exhibited of 6 showed bands at 3425, 3260cm−1 (2NH2), 2187cm−1
1
signals at 4.0ppm due to CH2 group and 7.4–7.9ppm cor- (2C≡N) and 1373, 1153cm−1 (SO2) . Its H-NMR spectrum in
responding to aromatic protons. It is well known that many (DMSO-d6) displayed signals at 1.6ppm for CH3 group of pyr-
pyridine-2-one derivatives exhibited diverse biological activity idone and 2.4ppm corresponding to tolyl CH3. IR spectrum
such as anticancer activity.25) The present study was continued of 7 showed bands at 3464, 3350cm−1 (2NH2) and 2218cm−1
1
to report the reactivity of cyanoacetamide 2 towards certain (2C≡N). Its H-NMR spectrum in (DMSO-d6) displayed sig-
nucleophilic reagents. Thus, when compound 2 was refluxed nals at 1.9ppm due to CH3 groups and 6.5ppm due to CH2 of
with acetylacetone or benzoylacetone in ethanol containing piperonyl group.
a catalytic amount of pipredine cyclocondensation reaction
Treatment of compound 3 with dimethylformamide-di-
occurred and the corresponding 1,2-dihydropyridines 3 and methylacetal (DMF-DMA) in xylene produced the enaminone
4 were smoothly afforded, respectively. It can be postulated derivative 8. IR spectrum of 8 showed bands at 2950cm−1
that the reaction initially proceeds via a nucleophilic attack (CH, aliph.), 2198cm−1 (2C≡N) and 1377, 1161cm−1 (SO2).
to form aldol adducts 3A and 4A which in turn cyclized to 1H-NMR spectrum in (DMSO-d6) of 8 revealed signals at
adducts 3B and 4B then lost four molecules of water and af- 2.4ppm for N(CH3)2, doublet at 5.00, 5.10ppm due to CH=CH
forded the pyridine derivatives 3 and 4 (Chart 1). Compounds group and 6.3ppm corresponding to CH of pyridone.
3 and 4 were proved on the basis of elemental analysis,
Upon stirring the cyanoacetamide 2 with carbon disulfide
IR, 1H- and 13C-NMR. IR spectrum of 3 showed bands at in the presence of potassium hydroxide in dimethylformamide
2218cm−1 (2C≡N), 1695cm−1 (2C=O) and 1330, 1161cm−1 followed by cycloalkylation with 1,2-dibromoethane afforded
1
(SO2). Its H-NMR spectrum in (DMSO-d6) displayed signals the corresponding 1,3-dithiolan derivative 10. Also, stirring of
at 1.9ppm for CH3 groups, 6.2ppm due to CH of pyridine and compound 2 under the same reaction conditions with 1,3-di-
7.4–8.0ppm corresponding to aromatic protons. IR spectrum chloropropan-2-ol yielded the dithiolan derivative 11. Further-
of 4 showed bands at 2218cm−1 (2C≡N), 1655cm−1 (2C=O) more, reaction of compound 2 with carbon disulfide in the
1
and 1365, 1145cm−1 (SO2). Its H-NMR spectrum in (DMSO- presence of KOH and dimethylsulfate while stirring and cool-
d6) displayed signals at 1.6ppm corresponding to CH3 groups. ing afforded N,N′-(4,4′-sulfonylbis(4,1-phenylene))bis(2-cyano-
Application of Michael addition of 3 to benzylidenemalo- 3,3-bis(methylthio)acrylamide) 12 smoothly (Chart 2). The IR
nonitriles in the presence of piperidine furnished the cor- spectrum of compound 12 revealed bands at 3367cm−1 (NH)
1
responding 1,2-dihydroisoquinoline derivatives 5–7, respec- and 2179cm−1 (C≡N). H-NMR (DMSO-d6) spectrum of 12
tively. The structures of compounds 5–7 were verified on the revealed signals at 2.4ppm for SCH3 group and 7.0–7.9ppm
basis of analytical and spectral data. IR spectrum of 5 showed due to aromatic protons.

August 2012
1021
Reagents (a) KOH/CS2, (b) 1,2-dibromoethane, (c) 1,3-dichloropropan-2-ol, (d) dimethyl sulphate.
Chart 2
When compound 2 was left to react with phenyl isothiocya-
nate in the presence of dimethylsulfate in alkaline medium,
the corresponding methylated derivative 14 was afforded
through the formation of intermediate 13. The IR spectrum of
compound 14 revealed bands at 2191cm−1 (C≡N) and 1380,
1
1149cm−1 (SO2). H-NMR spectrum in (DMSO-d6) of 14 re-
vealed signals at 2.3ppm for SCH3 group, 8.8ppm for NH ani-
line and 10.1ppm for NHCO group. Cyclocondensation of the
acrylamide derivative 14 with hydrazine hydrate in refluxing
ethanol gave the pyrazole derivative 15. Presumably, forma-
tion of the aminopyrazole derivative 15 is assumed to proceed
via Michael addition of the hydrazinoamino group to the eth-
ylenic bond side chain in 14 with elimination of SCH3 group
followed by intramolecular cyclization at the cyano group.
Its IR spectrum revealed the presence of (NH, NH2) bands
at 3490, 3344, 3260cm−1and a band of (C=N) at 1589cm−1.
1H-NMR spectrum in (DMSO-d6) of 15 revealed signals at
6.00ppm corresponding to NH2 group and 11.20ppm due
to NH of pyrazole. When compound 15 was heated under
reflux with acetylacetone in glacial acetic acid, the cor-
responding pyrazolopyrimidine derivative 16 was obtained.
1H-NMR spectrum in (DMSO-d6) of 16 displayed signals at
2.3, 2.4ppm corresponding to CH3 groups, 8.9ppm for CH of
pyrimidines, 10.0ppm for NH anilino and 10.3ppm for NHCO
groups (Chart 3).
Furthermore, Perkin reaction was carried out by reacting
compound 2 with 2-hydroxyl-1-naphthaldehyde in acetic anhy-
dride in presence of sodium acetate to give the corresponding
chromene-2-one derivative 17, while reaction of 2 with the
same reagent in ammonium acetate yielded the chromen-2-im-
ino derivative 18. Compounds 17 and 18 were supported in
the basis of analytical and spectral data. Thus, the IR spectra
of compounds 17 and 18 showed the absence of (C≡N) bands.
Reagents (a) phenyl isothiocyanate/KOH/DMF, (b) dimethylsulfate, (c) hydra-
zine hydrate, (d) acetylacetone.
Chart 3
The chromene derivatives 17 and 18 were further reacted IR spectra of compounds 19 and 20 exhibited the presence of
with malononitrile in ammonium acetate furnished the cor- (C≡N) bands (Chart 4).
responding chromenopyridones 19 and 20, respectively. The
Molecular Docking The development of inhibitors of

1022
Vol. 60, No. 8
Reagents (a) 2-hydroxy-1-naphthaldehyde/acetic anhydride/sodium acetate, (b) 2-hydroxy-1-naphathaldehyde/ammonium acetate/ethanol, (c) ammonium acetate/malo-
nonitrile.
Chart 4
farnesyltransferase (FTIs) has been pursued with the initial present investigations the authors have performed molecular
aim of targeting aberrant rat sarcoma (RAS) function in can- docking of the synthesized compounds on the active sites of
cer.26,27) As a critical step in the processing of RAS, inhibition both farnesyltransferase and PRMT1 which may lead to an
of farnesylation alone may be sufficient to abrogate the cell understanding of their effect as antitumor agents. These two
signaling and transforming function of RAS in cancer. Vari- enzymes were selected for docking based on the activity of
ous tumors, including those of colon, bladder, lung, prostate several diaryl sulfone derivatives as PRMT1 inhibitors17) and
and pancreas showed dose-dependent growth inhibition and as FTIs.18)
prophylactic administration of farnesyltransferase inhibitors
Molecular Docking on the Active Site of Farnesyltrans-
daily delayed both tumor onset and growth.28) Blocking by ferase The protein data bank file (PDB ID: 3E30) was se-
FTIs of farnesylation and RAS processing in transformed lected for this purpose. The file contains farnesyltransferase
cells prevents many of the morphological changes associated enzyme co-crystallized with a sulfone ligand. All docking
with neoplastic growth.29) Likewise, signaling pathways such procedures were achieved by MOE (Molecular Operating En-
as the proto-oncogene serine/threonine protein kinase (RAF)/ vironment) software 10.2008 provided by chemical computing
MEKK/MAPK cascade, which are activated in H-RAS- group, Canada. Docking on the active site of farnesyltrans-
transformed fibroblasts, were down regulated after exposure to ferase enzyme was performed for all synthesized compounds
FTI. Thus, there was cytoplasmic accumulation of soluble and 2–8, 10–12, 14–20.
inactive unprocessed proto-oncogene serine serine/therionine
Docking protocol was verified by redocking of the co-
protein kinare (RAS)/RAF complexes.30) Although FTIs were crystallized ligand in the vicinity of the active site of the
originally conceived as targeting mutant or aberrant RAS enzyme with energy score (S)=−25.6345kcal/mol and root
function in cancer, the observation that a broad range of hu- mean standard deviation (RMSD)=2.8268 (Fig. 1). The sul-
man cancer cells are susceptible to FTI therapy independently fone ligand interacts with the active site of farnesyltransferase
of RAS mutation status led to the search for other critical pre- by four interactions: Try B361 with a hydrogen bond of 2.95Å
nylated proteins that may be targets for FTI therapy.31)
and arene-arene interaction, Trp B102 with a hydrogen bond
On the other hand, epigenetic modifications, such as chang- of 2.83Å and Zn with the lone pair of imidazole nitrogen. All
es in DNA methylation patterns and histone alterations, play synthesized compounds were fit to the active site of farnesyl-
important roles in cancer.32,33) Given the prevalence of PRMT1 transferase enzyme with good energy scores (S) suggesting
substrates in the cell, it is highly probable that PRMT1 and activity as farnesyltransferase inhibitors. Energy scores (S)
arginine methylation is linked to many diseases.34) PRMT1 is and amino acid interactions for all synthesized compounds
thought to contribute to as much as 85% of all cellular PRMT were listed in Table 1. Compound 11 showed the best energy
activity.35) The PRMT1 mRNA is found in all embryonic and score (S)=−39.3306 kcal/mol and interacted with Lys B356
adult tissues examined demonstrating the widespread im- with a hydrogen bond of 2.95Å, Lys A164 with a hydrogen
portance of this enzyme in cellular functions.36) The activity bond of 2.82Å and with Zn through its OH of the 1,3-dthiolan
of PRMT1 can be regulated in several fashions. PRMT1 is ring (Fig. 2). 3D interaction of compound 19 with the amino
present in both the cytoplasm and the nucleus, and is highly acid of active site of the enzyme as well as Zn+2 is illustrated
mobile between these compartments.37)
Thus, the present investigation is concerned with the syn-
in Fig. 3.
Molecular Docking on the Active Site of Arginine Meth-
thesis of novel anticancer agents and trying to understand yltransferase (PRMT1) The protein data bank file (PDB
their mechanism of action. In order to perform the aim of the ID: 3Q7E) was selected for this purpose. The file contains

August 2012
1023
Fig. 1. Co-crystallized Sulfone Ligand on the Active Site of Farnesyl-
transferase
arginine methyltransferase co-crystallized with its ligand (S-
adenosyl methionine). All docking procedures were achieved
by MOE (Molecular Operating Environment) software
10.2008 provided by chemical computing group, Canada.
Docking on the active site of arginine methyltransferase
enzyme was performed for all synthesized compounds 2–8,
10–12, 14–20.
Fig. 2. Compound 11 on the Active Site of Farnesyltransferase
Docking protocol was verified by redocking of the co-crys-
tallized ligand in the vicinity of the active site of the enzyme
with energy score (S)=−18.5932kcal/mol and root mean stan-
dard deviation (RMSD)=0.3523 The ligand interacts with the
active site of arginine methyltransferase by five interactions:
Val 128 with a hydrogen bond of 3.00Å, Arg 54 with a hy-
drogen bond of 2.64Å, Gly 78 with a hydrogen bond of 1.81Å
and Glu 100 with two hydrogen bonds of 181, 186Å (Fig. 4).
All synthesized compounds were fit to the active site of argi-
nine methyltransferase enzyme with good energy scores (S)
suggesting activity as arginine methyltransferase inhibitors
for most of the synthesized compounds. Energy scores (S) and
amino acid interactions for all synthesized compounds were
listed in Table 2. Compound 16 showed the best energy score
(S)=−27.9591kcal/mol and interacted with Glu 100 with a hy-
drogen bond of 1.97Å, Lys 127 and Arg 327 with arene cation Fig. 3. Compound 19 on the Active Site of Farnesyltransferase
interactions (Fig. 5). 3D interaction of compound 19 with the
amino acid of active site of the enzyme (Fig. 6).
In Vitro Antitumor Activity The newly synthesized in the range of 38.42 to 51.95µM. The promising results of
compounds were evaluated for their in vitro cytotoxic activity cytotoxic activity of the synthesized compounds especially
against human breast cancer cell line (MCF7). Doxorubicin compound 19 which was the most active compound with IC50
which is one of the most effective anticancer agents was used value 19.36µM urge more investigations for their mechanism
as the reference drug in this study. The relationship between of action. The trial in the present investigation to predict an
surviving fraction and drug concentration was plotted to assumption on the mechanism of action of the synthesized
obtain the survival curve of breast cancer cell line (MCF7). compounds were conducted through molecular docking on the
The response parameter calculated was the IC50 value, which active site of two enzymes based on the similarities between
corresponds to the concentration required for 50% inhibition the synthesized compounds and the enzyme inhibitors of these
of cell viability. Table 3 shows the in vitro cytotoxic activity enzymes.
of the synthesized compounds where all compounds exhibited
significant activity compared to the reference drug.
The results of molecular docking and biological activ-
ity may suggest better activity of the synthesized compounds
All the synthesized compounds showed better cytotoxic as farnesyltransferasse (Table 1) inhibitor than as arginine
activity than Doxorubicin especially the benzochromene de- methyltransferase inhibitors (Table 2) as good energy scores
rivatives 17–20 which showed IC50 values 34.95, 34.53, 19.36 and amino acid interactions were better in the case of farne-
and 35.45µM, respectively. Compounds 2–7 showed cytotoxic syltransferase also good interaction with Zn+2 was observed
activity with IC50 values in the range of 37.04 to 57.69µM. On with most of the synthesized compounds. However, we could
the other hand, compound 8 showed better cytotoxic activ- not define a relationship between the cytotoxic activity and
ity than compounds 2–7 with IC50 value 30.70µM. Finally the docking results which may suggest other mechanisms of
compounds 10–16 showed cytotoxic activity with IC50 values action for the synthesized compounds. On the other hand,

1024
Vol. 60, No. 8
Table 1. Binding Scores and Amino Acid Interactions of the Docked Compounds on the Active Site of Farnesyltransferase (FT)
Compound No.
S (kcal/mol)
Amino acid interactions
Leu B295, Lys B294
H bond length (Å)
Interaction with Zn
2
3
−22.2685
−25.1744
3.37, 2.76
No interaction
CN
Trp B102, Arg B202, Tyr A166,
Tyr B251
3.16, 2.89, 2.87, 2.97
4
5
−30.8654
−29.8040
−25.7866
−30.7866
−23.3447
−23.3689
−39.3306
−29.2271
−27.9248
−27.2643
−36.9713
−26.5360
−20.2098
−23.8669
−35.0407
Gln A187
Arg B202
3.00
2.73
CN
SO2
6
Lys A164, Ser B357
Arg B281, Lys B284
Arg B202, Lys B294
Arg B202, Arg B291, Lys B294
Lys A164, Lys B356
His B361
2.62, 2.72
3.21, 2.87
3.10, 2.60
2.57, 2.93, 2.68
2.82, 2.95
3.17
No interaction
No interaction
No interaction
No interaction
OH
7
8
10
11
12
14
15
16
17
18
19
20
CN
Arg B281, Lys B294
Leu B295, Lys B363, Asp B352
Arg B202, Arg B352
Lys A164, Lys B294, Asp B352
Arg B281, Lys B294
Lys A164
2.75, 2.95
1.87–2.12, 2.53, 1.65
2.55, 1.29
2.44, 2.72, 1.45
2.6, 3.3
No interaction
No interaction
NHCO
NHCO
No interaction
C=O
2.54
Lys A164, Arg B202
3.25, 2.47–2.7
C=NH
Fig. 5. : Compound 16 on the Active Site of Arginine Methyltransfer-
ase (PRMT1)
Fig. 4. Co-crystallized S-Adenosyl Methionine Ligand on the Active
Site of Arginine Methyltransferase (PRMT1)
the promising cytotoxic results of the synthesized compounds
make them a good trial to discover new anticancer agents and
urge the researchers to seek for more derivatives of diarylsul-
fone derivatives.
Experimental
Chemistry Melting points (°C, uncorrected) were de-
termined in open capillaries on a Gallenkemp melting point
apparatus (Sanyo Gallenkemp, Southborough, U.K.) and were
uncorrected. Precoated silica gel plates (silica gel 0.25mm, 60
G F254; Merck, Germany) were used for thin layer chromatog-
raphy, dichloromethane–methanol (9.5:0.5) mixture was used
as a developing solvent system and the spots were visualized
by UV light and/or iodine. Infra-red spectra were recorded in
KBr discs using IR-470 Shimadzu spectrometer (Shimadzu,
Tokyo, Japan). NMR spectra (in DMSO-d6) were recorded
Fig. 6. Compound 19 on the Active Site of Arginine Methyltransferase
(PRMT1)
on Bruker AC-300 Ultra Shield NMR spectrometer (Bruker, designed as follows: s, singlet; d, doublet; t, triplet; m, multi-
Flawil, Switzerland, δ ppm) at 300MHz using tetramethylsi- plet. Elemental analyses were performed on Carlo Erba 1108
lane (TMS) as internal standard and peak multiplicities are Elemental Analyzer (Heraeus, Hanau, Germany).

August 2012
1025
(s, 2H, 2NH exchangeable). 13C-NMR (DMSO-d6): 24.4(2),
115.6(2), 119.2(2), 119.3(2), 128.1(2), 129.2(2), 137.8(2), 142.7(2),
162.2(2). Anal. Calcd for C18H14N4O4S (382.39): C, 56.54; H,
3.69; N, 14.65. Found: C, 56.81; H, 3.84; N, 14.29.
Table 2. Binding Scores and Amino Acid Interactions of the Docked
Compounds on the Active Site of Arginine Methyltransferase (PRMT1)
Amino acid
Compound No.
S (kcal/mol)
H bond length (Å)
interactions
General Procedure for Synthesis of Compounds 3 and
Equimolar amounts of compound 2 (3.82g, 0.01mol) and
2
3
4
5
6
7
−20.0584
−17.4225
−23.2276
−18.4486
−14.9319
−23.3465
Lys 127, His 293
Lys 127
2.65, 2.81
2.67
4
acetylacetone (2.00g, 0.02mol) or benzoylacetone (3.24g,
0.02mol) were refluxed in ethanol (50mL) containing piperi-
dine (0.5mL) for 5h. After trituration with ethanol, the solid
obtained was filtered and crystallized from dioxane to give 3
and 4, respectively.
Lys 127, His 293
His 45
3.02, 3.10
3.02
Asn 326, Arg 327 3.06, 2.88–2.92
Arg 327, Asn 157, 2.55–2.57, 2.88,
Lys 127
3.03
1,1′-(4,4′-Sulfonylbis(4,1-phenylene))bis(4,6-dimethyl-2-oxo-
1,2-dihydropyridine-3-carbonitrile) (3): Yield 89%, mp
358.1°C. IR: νmax/cm−1 3074 (CH arom.), 2940, 2843 (CH
8
10
11
12
14
15
−18.1201
−22.1917
−11.6688
−17.2712
−16.3269
−18.4808
Val 128
3.19
Lys 127
2.94
Thr 158, His 127
Lys 127, Arg 3.27
2.85, 2.44
2.45, 2.95
1
aliph.), 2218 (2C≡N), 1655 (2C=O), 1330, 1161 (SO2) . H-
NMR (DMSO-d6, D2O) δ: 1.9 (s, 12H, 4CH3), 6.2 (s, 2H, 2CH
of pyridines), 7.4–8.0 (m, 8H, Ar-H). 13C-NMR (DMSO-d6):
20.6(2), 21.4(2), 109.1(2), 113.0(2), 115.6(2), 124.3(4), 129.7(4),
140.6(2), 142.9(2), 143.9(2), 153.9(2), 106.2(2). Anal. Calcd for
C28H22N4O4S (510.56): C, 65.87; H, 4.34; N, 10.97. Found: C,
66.13; H, 4.21; N, 11.26
Arg 127, Glu 136 2.40–3.11, 1.60
Glu 100, Glu 130, 1.38–1.74, 3.20,
Lys 127
2.55
16
17
18
19
−27.9591
−11.4933
−18.6764
−24.5515
Glu 100
1.97
Lys 127, Arg 327
Arg 327, Glu 129
2.40, 2.57
2.41, 1.72
Asn 157, Glu 153,
Lys 127
2.88, 1.89,
2.58
1,1′-(4,4′-Sulfonylbis(4,1-phenylene))bis(4-methyl-2-oxo-6-
phenyl-1,2-dihydropyridine-3-carbonitrile) (4): Yield 79%,
mp 238.2°C. IR: νmax/cm−1 3055 (CH arom.), 2935, 2858 (CH
20
−19.2200
Lys 127, His 45
2.49, 3.03
1
aliph.), 2218 (2C≡N), 1655 (2C=O), 1365, 1145 (SO2) . H-
N,N′-(4,4′-Sulfonylbis(4,1-phenylene))bis(2-cyanoacet- NMR (DMSO-d6, D2O) δ: 1.6 (s, 6H, 2CH3), 6.2 (s, 2H, 2CH
amid) (2) A mixture of Dapson 1 (2.48g, 0.01mol) and ex- of pyridones), 6.5–8.0 (m, 18H, Ar-H). 13C-NMR (DMSO-d6):
cess ethyl cyanoacetate was fused at 220°C in an oil bath for 15.6(2), 91.2(2), 115.4(2), 116.2(2), 124.3(2), 126.9(4), 127.9(2),
2h. Excess ethyl cyanoacetate was evaporated under vacuum. 128.7(4), 129.3(4), 130.6(2), 135.5(2), 140.7(2), 144.0(2),
The solid product remained was triturated with diethylether 159.2(2), 160.8(2). Anal. Calcd for C38H26N4O4S (634.70): C,
(100mL) then filtered. The solid obtained was crystallized 71.91; H, 4.13; N, 8.83. Found: C, 72.20; H, 4.41; N, 8.66.
from ethanol to give 2. Yield 92%, mp 137.5°C. IR: νmax/cm−1
General Procedure for Synthesis of Compounds 5–7
A
3448, 3363 (2NH), 3062 (CH arom.), 2960, 2931 (CH aliph.), mixture of compound 3 (5.1g, 0.01mol) and benzylidenemalo-
1
2256 (C≡N), 1701 (2C=O), 1342, 1180 (SO2). H-NMR (DM- nonitrile (0.02mol) in ethanol (50mL) containing piperidine
SO-d6, D2O) δ: 4.0 (s, 4H, 2CH2), 7.4–7.9 (m, 8H, Ar-H), 10.7 (0.5mL) was refluxed for 6h. The reaction mixture was cooled
Table 3. In Vitro Anticancer Screening of the Synthesized Compounds against Human Breastcell Line (MCF7)
Compound concentration (µM)
Compound
10µM
25µM
50µM
100µM
IC50 (µM)
Surviving fraction (mean S.E.)a)
Doxorubicin
0.721 0.020
0.727 0.134
0.59 0.089
0.773 0.188
0.817 0.023
0.797 0.021
0.547 0.129
0.531 0.053
0.722 0.040
0.791 0.050
0.827 0.050
0.822 0.006
0.748 0.160
0.574 0.077
0.676 0.095
0.563 0.079
0.370 0.037
0.540 0.092
0.546 0.020
0.427 0.055
0.499 0.140
0.547 0.031
0.567 0.058
0.470 0.101
0.327 0.076
0.237 0.009
0.533 0.075
0.537 0.025
0.615 0.012
0.496 0.090
0.427 0.085
0.274 0.045
0.318 0.069
0.238 0.140
0.237 0.057
0.375 0.066
0.461 0.010
0.307 0.029
0.400 0.078
0.387 0.016
0.333 0.012
0.213 0.059
0.298 0.067
0.283 0.019
0.329 0.051
0.336 0.020
0.359 0.051
0.245 0.059
0.224 0.004
0.371 0.047
0.189 0.011
0.401 0.056
0.209 0.005
0.242 0.015
0.494 0.030
0.317 0.021
0.380 0.107
0.380 0.044
0.307 0.025
0.340 0.053
0.323 0.087
0.281 0.026
0.297 0.022
0.307 0.013
0.190 0.041
0.297 0.033
0.268 0.036
0.309 0.076
0.249 0.022
0.239 0.070
0.240 0.034
0.305 0.013
71.80
46.58
50.65
57.69
52.95
47.30
37.04
30.70
48.82
51.95
49.82
47.47
41.79
38.42
34.95
34.52
19.36
35.45
2
3
4
5
6
7
8
10
11
12
14
15
16
17
18
19
20
a) Each value is the mean of three values standard error.

1026
Vol. 60, No. 8
and poured on crushed ice and acidified by dil HCl. The solid mixture was cooled at 0°C in an ice bath, then carbon di-
product was filtered off and crystallized from dioxane to give sulfide (1.52g, 0.02mol) was added slowly over the course
compounds 5–7, respectively.
of 10min. After complete addition, stirring of the reaction
2,2′-(4,4′-Sulfonylbis(4,1-phenylene))bis(8-amino-3-methyl- mixture was continued for additional 3h. dibromoethane
1-oxo-6-phenyl-1,2-dihydroisoquinoline-7-carbonitrile)
(5): (1.88g, 0.02mol) or 1,3-dichloropropan-2-ol (2.58g, 0.02mol)
Yield 68%, mp 174.5°C. IR: νmax/cm−1 3390, 3367 (2NH2), or dimethylsulfate (2.52g, 0.02mol) was added to the reac-
3060 (CH arom.), 2939, 2858 (CH aliph.), 2160 (2C≡N), tion mixture while cooling (−15°C) and stirring for 2h. then
1
1654 (2C=O), 1360, 1153 (SO2). H-NMR (DMSO-d6, D2O) poured into crushed ice, the resulting precipitate was filtered
δ: 1.9 (s, 6H, 2CH3), 6.2 (s, 2H, 2NH2, exchangeable), 6.4 (s, off and crystallized from ethanol to give compounds 10–12,
2H, 2CH of pyridones), 7.3–7.9 (m, 20H, Ar-H). 13C-NMR respectively.
(DMSO-d6, D2O): 20.7(2), 92.9(2), 114.6(2), 115.7(2), 119.6(2),
N,N′-(4,4′-Sulfonylbis(4,1-phenylene))bis(2-cyano-2-(1,3-
122.8(4), 127.9(2), 128.3(4), 129.1(4), 130.1(4), 133.6(2), dithiolan-2-ylidene)acetamide) (10): Yield 74%, mp 218.1°C.
134.8(2), 137.1(2), 138.6(2), 140.9(2), 143.8(2), 151.8(2), IR: νmax/cm−1 3448 (2NH), 2203 (2C≡N), 1652 (2C=O),
1
153.9(2). Anal. Calcd for C46H32N6O4S (764.85): C, 72.24; H, 1315, 1184 (SO2). H-NMR (DMSO-d6, D2O) δ: 3.5–3.6 (m,
4.22; N, 10.99. Found: C, 72.11; H, 4.50; N, 10.68.
8H, 4CH2), 7.5–8.0 (m, 8H, Ar-H) 10.2 (s, 2H, 2NH, ex-
2,2′-(4,4′-Sulfonylbis(4,1-phenylene))bis(8-amino-3-methyl- changeable). 13C-NMR (DMSO-d6): 38.9(4), 89.4(2), 116.3(2),
1-oxo-6-p-tolyl-1,2-dihydroisoquinoline-7-carbonitrile)
(6): 123.5(4), 129.1(4), 135.9(2), 142.1(2), 162.9(2), 183.8(2). Anal.
Yield 81%, mp 227.3°C. IR: νmax/cm−1 3425, 3260 (2NH2), Calcd for C24H18N4O4S5 (586.75): C, 49.13; H, 3.09; N, 9.55.
2931, 2870 (CH aliph.), 2187 (2C≡N), 1655 (2C=O), 1373, Found: C, 49.40; H, 3.32; N, 9.31.
1
1153 (SO2) . H-NMR (DMSO-d6, D2O) δ: 1.6 (s, 6H, 2CH3
N,N′-(4,4′-Sulfonylbis(4,1-phenylene))bis(2-cyano-2-(5-
pyridones), 2.4 (s, 6H, 2CH3 tolyls), 6.1 (s, 4H, 2NH2, ex- hydroxy-1,3-dithian-2-ylidene)acetamide) (11): Yield 78%,
changeable), 6.4 (s, 2H, 2CH pyridones), 7.2–7.9 (m, 18H, mp 213.6°C. IR: νmax/cm−1 3433 (2OH), 3385 (2NH), 2202
Ar-H). 13C-NMR (DMSO-d6): 21.6(2), 23.1(2), 94.2(2), 99.1(2), (2C≡N),1675 (2C=O), 1350, 1184 (SO2). 1H-NMR (DMSO-
115.9(2), 118.2(2), 119.1(2), 125.1(4), 127.1(4), 128.6(4), 129.9(4), d6, D2O) δ: 3.44, 3.45 (2d, 8H, 4CH2, J=7.6, 7.4Hz), 4.2
132.9(2), 137.6(2), 138.1(2), 139.8(2), 140.6(2), 142.3(2), (m, 4H, 2CH+2OH) 7.5–7.9 (m, 8H, Ar-H) 10.5 (s, 2H,
143.8(2), 148.3(2), 154.8(2). Anal. Calcd for C48H36N6O4S 2NH, exchangeable). 13C-NMR (DMSO-d6): 37.6(4), 78.0(2),
(792.90): C, 72.71; H, 4.58; N, 10.60. Found: C, 72.99; H, 4.38; 78.9(2), 115.5(2), 123.6(4), 129.1(4), 136.9(2), 143.6(2), 162.2(2),
N, 10.29.
2,2′-(4,4′-Sulfonylbis(4,1-phenylene))bis(8-amino-6- 3.43; N, 8.66 Found: C, 48.61; H, 3.18; N, 8.42.
(benzo[d][1,3]dioxol-5-yl)-3-methyl-1-oxo-1,2-dihydroisoquin N,N′-(4,4′-Sulfonylbis(4,1-phenylene))bis(2-cyano-3,3-
183.8(2). Anal. Calcd for C26H22N4O6S5 (646.80): C, 48.28; H,
oline-7-carbonitrile) (7): Yield 66%, mp >350°C. IR: νmax
/
bis(methylthio)acrylamide) (12): Yield 69%, mp 113.6°C.
cm−1 3464, 3350 (2NH2), 3074 (CH arom.), 2218 (2C≡N), IR: νmax/cm−1 3367 (2NH), 3065 (CH arom.), 2920, 2870
1
1655 (2C=O), 1330, 1161 (SO2) . H-NMR (DMSO-d6, D2O) (CH aliph.), 2179 (2C≡N), 1655 (2C=O), 1390, 1149 (SO2).
δ: 1.9 (s, 6H, 2CH3), 6.2 (s, 4H, 2NH2, exchangeable), 6.4 (s, 1H-NMR (DMSO-d6, D2O) δ: 2.4 (s, 12H, 4SCH3), 7.0–7.9
2H, 2CH pyridones), 6.5 (s, 4H, 2CH2 pipernonyl), 6.6–8.0 (m, (m, 8H, Ar-H) 13.5 (s, 2H, 2NH, exchangeable). 13C-NMR
16H, Ar-H). 13C-NMR (DMSO-d6): 21.4(2), 93.8(2), 98.8(2), (DMSO-d6, D2O): 17.80(4), 17.75(4), 95.0(2), 115.7(2), 123.3(4),
102.7(2), 113.0(2), 115.1(2), 115.9(2), 116.6(2), 117.3(2), 120.7(2), 129.2(4), 138.1(2), 144.1(2), 164.0(2), 178.9(2). Anal. Calcd for
122.1(4), 129.3(4), 129.9(2), 137.7(2), 138.6(2), 139.2(2), C24H22N4O4S5 (590.78): C, 48.79; H, 3.75; N9.48. Found: C,
142.8(2), 145.2(2), 149.1(2), 150.8(2), 152.6(2), 153.9(2). Anal. 48.92; H, 3.44; N 9.20
Calcd for C48H32N6O8S (852.87): C, 67.60; H, 3.78; N, 9.85.
Found: C, 67.98; H, 3.43; N, 9.64.
(2Z,2′Z)-N,N′-(4,4′-Sulfonylbis(4,1-phenylene))bis(2-
cyano-3-(methylthio)-3-(phenylamino)acrylamide) (14) To
(E)-1,1′-(4,4′-Sulfonylbis(4,1-phenylene))bis(4-((E)- a suspension of potassium hydroxide (1.12g, 0.02mol) in dry
2-(dimethylamino)vinyl)-6-methyl-2-oxo-1,2-dihydro- dimethylformamide (20mL) cyanoacetamide derivative
2
pyridine-3-carbonitrile) (8) To a solution of compound (3.82g, 0.01mol) was added during stirring, phenylisothio-
3 (5.1g, 0.01mol) in xylene (50mL), DMF-DMA (2.30g, cyanate (2.70g, 0.02mol) was dropped slowly to the reaction
0.02mol) was added. The reaction mixture was refluxed mixture. After complete addition, stirring of the reaction
for 6h, cooled and the solid obtained was crystallized from mixture was continued for 7h, and dimethylsulfate (2.52g,
ethanol to give 8. Yield 65%, mp >350°C. IR: νmax/cm−1 0.02mol) was added. The reaction mixture was stirred for
3100 (CH arom.), 2920, 2870 (CH aliph.), 2198 (2C≡N), 1654 2h. then poured into crushed ice water. The resulting pre-
1
(2C=O), 1377, 1161 (SO2). H-NMR (DMSO-d6, D2O) δ: 1.8 cipitate was filtered off, dried and crystallized from metha-
(s, 6H, 2CH3 pyridones), 2.4 (s, 12H, 4N(CH3)2), 5.0, 5.1 (2d, nol to give 14. Yield 76%, mp 103.5°C. IR: νmax/cm−1 3332,
4H, 2CH=CH, J=7.3, 7.6Hz), 6.3 (s, 2H, 2CH pyridones), 3290 (4NH), 3060 (CH arom.), 2940, 2860 (CH aliph.), 2191
1
7.5–7.9 (m, 8H, Ar-H). 13C-NMR (DMSO-d6): 21.6(2), 44.8(4), (2C≡N),1654 (2C=O), 1380, 1149 (SO2). H-NMR (DMSO-d6,
99.9(2), 108.2(2), 109.1(2), 116.7(2), 119.9(4), 128.6(4), 130.9(2), D2O) δ: 2.3 (s, 6H, 2SCH3), 6.7–7.9 (m, 18H, Ar-H), 8.8 (s, 2H,
131.8(2), 132.9(2), 148.2(2), 155.4(2), 177.1(2). Anal. Calcd for 2NH anilino, exchangeable). 13C-NMR (DMSO-d6): 16.4(2),
C34H32N6O4S (620.72): C, 65.79; H, 5.20; N, 13.54. Found: C, 79.2(2), 117.9(2), 118.4(4), 120.4(2), 124.4(4), 128.7(4), 129.8(4),
65.90; H, 5.11; N, 13.20.
139.4(2), 143.0(2), 144.2(2), 167.8(2), 179.6(2). Anal. Calcd for
General Procedure for Synthesis of Compounds 10–12 C34H28N6O4S3 (680.82): C, 59.98; H, 4.15; N, 12.34. Found: C,
To a stirred suspension of finely powdered potassium hy- 60.11; H, 4.41; N, 12.01
droxide (1.12g, 0.02mol) in dry dimethylformamide (20mL),
N,N′-(4,4′-Sulfonylbis(4,1-phenylene))bis(5-amino-3-
cyanoacetamide 2 (3.82g, 0.01mol) was added. The resulted (phenylamino)-1H-pyrazole-4-carboxamide) (15) A mix-

August 2012
1027
ture of compound 14 (6.80g, 0.01mol) and hydrazine hydrate 0.02mol) in ethanol (50mL) were refluxed for 5h. The solid
(1.00g, 0.02mol) in ethanol (50mL) was refluxed for 5h, and obtained was separated by filteration and crystallized from
allowed to cool. The solid product obtained was filtered and ethanol to give 19 and 20, respectively.
crystallized from dioxane. Yield 71%, mp 245°C. IR: νmax
/
3,3′-(4,4′-Sulfonylbis(4,1-phenylene))bis(2-amino-4,5-
cm−1 3490, 3344, 3260 (NH, NH2), 1654 (2C=O), 1589 (2C= dioxo-4,5-dihydro-3H-benzo[f ]chromeno[3,4-c]pyridine-1-
1
N), 1340, 1145 (SO2). H-NMR (DMSO-d6, D2O) δ: 6.0 (s, 4H, carbonitrile) (19): Yield 75%, mp 348°C. IR: νmax/cm−1 3460,
2NH2, exchangeable), 7.2–7.9 (m, 18H, Ar-H), 8.5 (s, 2H, 2NH 3336, 3232 (2NH2), 2199 (2C≡N), 1690, 1645 (4C=O), 1356,
1
anilino, exchangeable) 9.1 (s, 2H, 2NHCO, exchangeable). 13C- 1180 (SO2). H-NMR (DMSO-d6, D2O) δ: 6.3 (s, 4H, 2NH2,
NMR (DMSO-d6, D2O): 87.9(2), 117.9(4), 118.1(2), 119.5(4), exchangeable), 7.3–7.9 (m, 20H, Ar-H). 13C-NMR (DMSO-d6):
128.1(4), 128.8(4), 136.6(2), 142.9(2), 143.1(2), 150.6(2), 75.1(2), 116.4(2), 116.6(2), 117.1(2), 117.3(2), 118.8(2), 123.0(4),
152.8(2), 163.1(2). Anal. Calcd for C32H28N10O4S (648.69): C, 124.6(2), 126.5(2), 128.4(4), 128.5(2), 129.9(2), 130.7(2),
59.25; H, 4.35; N, 21.59. Found: C, 59.00; H, 4.62; N, 21.31.
131.5(2), 135.6(2), 142.9(2), 153.2(2), 157.8(2), 159.4(2),
N,N′-(4,4′-Sulfonylbis(4,1-phenylene))bis(5,7-dimethyl-2- 162.3(2), 169.0(2). Anal. Calcd for C46H24N6O8S (820.78): C,
(phenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide) 67.31; H, 2.95; N, 10.24. Found: C, 67.02; H, 3.08; N, 10.11.
(16) A mixture of compound 15 (6.48g, 0.01mol) and
3,3′-(4,4′-Sulfonylbis(4,1-phenylene))bis(2-amino-5-imino-
acetylacetone (2.00g, 0.02mol) in glacial acetic acid (30mL) 4-oxo-4,5-dihydro-3H-benzo[f]chromeno[3,4-c]pyridine-1-
was refluxed for 5h. then left to cool. The obtained solid carbonitrile) (20): Yield 78%, mp 246.4°C. IR: νmax/cm−1 3448,
was filtered and crystalized from acetic acid. Yield 69%, mp 3348, 3217 (NH, NH2), 2214 (2C≡N), 1716 (2C=O), 1334,
1
286.5–287°C. IR: νmax/cm−1 3448, 3313, (4NH), 3055 (CH 1145 (SO2). H-NMR (DMSO-d6, D2O) δ: 6.6 (s, 4H, 2NH2,
arom.), 2924, 2860 (CH aliph.), 1670 (2C=O), 1385, 1149 exchangeable), 7.2–8.2 (m, 20H, Ar-H), 9.3 (s, 2H, 2NH, ex-
1
(SO2). H-NMR (DMSO-d6, D2O) δ: 2.3, 2.4 (2s, 12H, 4CH3), changeable). 13C-NMR (DMSO-d6): 64.4(2), 113.2(2), 116.3(2),
6.9–7.9 (m, 18H, Ar-H), 8.9 (s, 2H, 2CH pyrimidines),10.0 (s, 117.0(2), 119.6(2), 122.4(2), 123.1(4), 124.6(2), 126.6(2),
2H, 2NH anilino, exchangeable), 10.3 (s, 2H, 2NHCO, ex- 128.8(4), 128.9(2), 129.8(2), 130.4(2), 131.1(2), 135.6(2),
changeable). 13C-NMR (DMSO-d6): 24.1(2), 86.0(2), 109.3(2), 138.0(2), 152.6(2), 155.8(2), 156.8(2), 160.2(2), 166.4(2) Anal.
117.1(4), 118.9(2), 121.0(4), 128.5(4), 128.8(4), 135.0(2), 139.7(2), Calcd for C46H26N8O6S (818.81): C, 67.47; H, 3.20; N, 13.68.
142.7(2), 143.6(2), 146.4(2), 162.1(2), 169.1(2), 171.9(2). Anal. Found: C, 67.76; H, 3.35; N, 13.30.
Calcd for C42H36N10O4S (776.86): C, 64.93; H, 4.67; N, 18.03.
Found: C, 65.06; H, 4.49; N, 17.94.
Molecular Docking All the molecular modeling studies
were carried out on an Intel Pentium 1.6GHz processor, 512
N,N′-(4,4′-Sulfonylbis(4,1-phenylene))bis(3-oxo-3H- MB memory with Windows XP operating system using Mo-
benzo[f]chromene-2-carboxamide) (17) To a solution of lecular Operating Environment (MOE, 10.2008) software. All
2 (3.82g, 0.01mol) in acetic anhydride (30mL), 2-hydroxyl- the minimizations were performed with MOE until a RMSD
1-nphathaldehyde (3.44g, 0.02mol) and fused sodium acetate gradient of 0.05kcalmol−1Å−1 with MMFF94X forcefield and
(1.60g, 0.02mol) were added. The reaction mixture was re- the partial charges were automatically calculated. The X-ray
fluxed for 2h, cooled and the solid obtained was crystallized crystallographic structure of franesyltransferase and arginine
from ethanol. Yield 59%, mp 126.3°C. IR: νmax/cm−1 3425 methyltransferase (PRMT1) complexes with their ligands
(2NH), 3093 (CH arom.), 1766, 1720 (4C=O), 1369, 1190 (PDB ID: 3E30, 3Q7E) were obtained from the protein data
1
(SO2). H-NMR (DMSO-d6, D2O) δ: 7.2–8.0 (m, 20H, Ar-H), bank. The enzymes were prepared for docking studies where:
8.4 (s, 2H, 2CH chromene), 10.3 (s, 2H, 2NH, exchangeable). (i) Ligand molecule was removed from the enzyme active site.
13C-NMR (DMSO-d6): 118.2(2), 119.6(2), 120.8(2), 122.1(2), (ii) Hydrogen atoms were added to the structure with their
125.1(4), 125.9(2), 127.3(2), 128.6(4), 128.7(2), 128.8(2), standard geometry. (iii) MOE Alpha Site Finder was used for
130.5(2), 130.7(2), 131.6(2), 140.5(2), 144.4(2), 146.9(2), the active sites search in the enzyme structure and dummy
168.4(2), 169.0(2). Anal. Calcd for C40H24N2O8S (692.69): C, atoms were created from the obtained alpha spheres. (iv) The
69.36; H, 3.49; N, 4.04. Found: C, 69.78; H, 3.33; N, 3.91.
N,N′-(4,4′-Sulfonylbis(4,1-phenylene))bis(3-imino-3H- zymes interactions at the active site.
benzo[f]chromene-2-carboxamide) (18) mixture of Biological Screening. In Vitro Antitumor Activity Hu-
(3.82g, 0.01mol), 2-hydroxyl-1-nphathaldehyde (3.44g, man tumor breast cell line (MCF7) was used in this study.
obtained model was then used in predicting the ligand en-
A
2
0.02mol) and anhydrous ammonium acetate (2.30g, 0.02mol) The cytotoxic activity was measured in vitro for the newly
in ethanol (50mL) was refluxed for 3h. The solid product was synthesized compounds using the Sulfo-Rhodamine-B stain
filtered and crystallized from dioxane. Yield 71%, mp 276.9°C. (SRB) assay using the method of Skehan et al.38) The in vitro
IR: νmax/cm−13383, 3294 (4NH), 3072 (CH arom.), 1685 (2C= anticancer screening was done by the pharmacology unit at
O), 1620 (2C=N), 1395, 1149 (SO2). 1H-NMR (DMSO-d6, the National Cancer Institute, Cairo University.
D2O) δ: 6.1 (s, 2H, 2CH chromene), 6.6–8.1 (m, 20H, Ar-H),
Cells were plated in 96-multiwell plate (104 cells/well) for
9.2 (s, 2H, 2NHCO exchangeable), 13.1 (s, 2H, 2NH imino, 24h before treatment with the compound(s) to allow attach-
exchangeable). 13C-NMR (DMSO-d6): 115.6(2), 118.6(2), ment of cell to the wall of the plate. The tested compounds
119.5(2), 121.7(4), 125.8(2), 127.9(4), 128.7(4), 128.8(2),129.2(2), were dissolved in dimethylsulfoxide. Different concentrations
129.6(2), 134.8(2), 137.3(2), 137.8(2), 141.7(2), 153.4(2), 155.7(2), of the compound under test (10, 25, 50, 100µM) were added
160.3(2), 161.0(2). Anal. Calcd for C40H26N4O6S (690.72): C, to the cell monolayer. Triplicate wells were prepared for each
69.55; H, 3.79; N, 8.11. Found: C, 69.84; H, 3.66; N, 8.07.
individual concentration. Monolayer cells were incubated
General Procedure for Synthesis of Compounds 19 and with the compound(s) for 48h at 37°C and in atmosphere of
20 Equimolar amounts of compound 17 or 18, malononitrile 5% CO2. After 48h, cells were fixed, washed and stained
(1.32g, 0.02mol) and anhydrous ammonium acetate (2.20g, for 30min with 0.4% (w/v) SRB dissolved in 1% acetic acid.

1028
Vol. 60, No. 8
3066–3077 (2010).
Excess unbound dye was removed by four washes with 1%
acetic acid and attached stain was recovered with Tris ethyl-
enediaminetetraacetic acid (EDTA) buffer. Color intensity was
measured in an enzyme-linked immunosorbent assay (ELISA)
reader. The relation between surviving fraction and drug
concentration is plotted to get the survival curve for breast
tumor cell line after the specified time. The molar concentra-
tion required for 50% inhibition of cell viability (IC50) was
calculated and compared to the reference drug doxorubicin
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each tested compound and the reference drug in concentra-
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viving fraction of cells in control test (0µM) was considered
as 100% viable cells. Concentrations of the tested compounds
and the reference drug were plotted against surviving frac-
tions of the cells using Microsoft Excel (2007). A trend line
was drawn for each curve and a corresponding equation was
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