Design, synthesis, and biological evaluation of novel pyrimidine derivatives as CDK2 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2009.12.026

Novel derivatives of 2,4,5,6-tetrasubstituted pyrimidine cyclin-dependent kinase (CDK2) inhibitors was designed and synthesized. We built a library of proposed pyrimidine derivatives and by using pharmacophore and docking techniques we made our selections. We modified the proposed compounds due to the interaction of docked structures with the protein to achieve the best fit. The newly synthesized compounds showed potent and selective CDK2 inhibitory activities and inhibited in-vitro cellular proliferation in cultured human tumor cells. The design, synthesis and biological evaluation of these 2,4,5,6-tetrasubstituted pyrimidine derivatives are reported.

Full text of DOI:10.1016/j.ejmech.2009.12.026

European Journal of Medicinal Chemistry 45 (2010) 1158–1166
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, and biological evaluation of novel pyrimidine derivatives
as CDK2 inhibitors
*
Diaa A. Ibrahim , Amira M. El-Metwally
National Organization for Drug Control & Research, P.O. Box: 29, Cairo, Gizaa 112313, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel derivatives of 2,4,5,6-tetrasubstituted pyrimidine cyclin-dependent kinase (CDK2) inhibitors was
designed and synthesized. We built a library of proposed pyrimidine derivatives and by using phar-
macophore and docking techniques we made our selections. We modified the proposed compounds due
to the interaction of docked structures with the protein to achieve the best fit. The newly synthesized
compounds showed potent and selective CDK2 inhibitory activities and inhibited in-vitro cellular
proliferation in cultured human tumor cells. The design, synthesis and biological evaluation of these
2,4,5,6-tetrasubstituted pyrimidine derivatives are reported.
Received 9 October 2009
Received in revised form
13 November 2009
Accepted 10 December 2009
Available online 21 December 2009
Keywords:
Pharmacophore
Docking
Ó 2009 Elsevier Masson SAS. All rights reserved.
Pyrimidines
CDK2
Antitumor
1. Introduction
In our program to develop CDK2 inhibitors as anti-cancer
agents, we recently reported that pyrazolo[3,4-d]pyrimidines,
Cyclin-dependent kinases (CDKs) are a family of serine–threo-
nine kinases that play important roles as regulators of cell progres-
sion through consecutive phases of the cell cycle [1]. CDKs are
regulated by phosphorylation and activated by their association with
cyclins [2]. The precise regulation of CDK activity is essential for the
stepwise execution of the many processes required for cell growth
and division, including DNA replication and chromosome separation
[3]. Abnormal CDK control of the cell cycle has been strongly linked
to the molecular pathology of cancer [4]. Such revelations have led to
the investigation of small molecule CDK inhibitors (CDKIs) as
possible cancer therapeutics [5,6]. All CDK inhibitors, identified so
far, function by competing with ATP for binding to the catalytic site.
The design of inhibitors specific to a particular protein kinase was
originally thought of as an impossible task owing to the high degree
of homology shared by the ATP binding domains of these enzymes.
Actually, several CDKIs have entered clinical evaluation for the
treatment of cancer. These include flavopiridol [7], 7-hydroxystaur-
osporine (UCN-01) [8], roscovitine (CYC202) [9], and the amino-
thiazole compound (BMS-387032) [10]. Nowadays, the synthesis of
novel highly selective CDKs as candidates for CDK-target therapy in
cancer treatment is in high demand [11,12].
3,6-disubstituted [1,2,4]triazolo[3,4-b] [1,3,4]thiadiazole and pyr-
imido[4,5-d]pyrimidines analogs are novel anti-cancer inhibitors
and anti-proliferative agents [13,14]. To discover new different
CDK2 inhibitors with improved activity and selectivity, we have
designed, synthesized, and evaluated pyrimidine derivatives as
inhibitors of CDK2.
2. Rational and design
2.1. Structure preparation
The coordinate for the protein structure was obtained from the
RCSB Protein Data Bank (PDB) (2C6I) [15]. Protein Structure was
prepared using Discovery Studio (DS 2.0) software package [16].
The invalid or missing residues were added and the structures were
aligned using the protein structure alignment module. Hydrogen
atoms were added and the structure was minimized using
CHARMm force field to relax the backbone and to remove the
clashes. The protein was inspected visually for accuracy in the X2
dihedral angle of Asn and His residues and the X3 angle of Gln, and
rotated by 180ꢀ when needed to maximize hydrogen bonding. The
proper His tautomer was also manually selected to maximize
hydrogen bonding.
The proposed compounds were optimized by semiemperical
method (AM1) using Chem3D to eliminate bond length and bond
* Corresponding author. Tel.: þ202 33365599; fax: þ202 35855582.
E-mail address: dino1421@hotmail.com (D.A. Ibrahim).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.026

D.A. Ibrahim, A.M. El-Metwally / European Journal of Medicinal Chemistry 45 (2010) 1158–1166
1159
angle biases and saved to be used in the pharmacophore mapping
and docking steps.
2.2. Common features pharmacophore
The pharmacophore model was generated derived from 11
potent inhibitors (Fig. 1) of CDK2 by calculating the common
features. This model was made up of hydrophobic center, acceptor
atom associated to its protein donor site and donor atom (Fig. 2).
The interfeature distances were considered to 7.696, 8.684 and 8.24
Aꢀ for the distances between the acceptor and the hydrophobe, the
hydrophobe and the donor and the donor to the acceptor, respec-
tively. The association of the acceptor atom to the donor site in the
protein ensured the overall orientation of the molecules with
respect to the kinase. Only one angle constraint was used for the
hydrophobic and the acceptor atom features, thus allowing the
hydrophobic and the aromatic centers to cover the large domain in
the kinase active site, from the hydrophobic to the sugar pocket.
Using this pharmacophore model, we mapped our proposed
compounds which contain pyrimidine moiety in order to find the
promising compounds that are capable of binding to CDK2 with
a similar set of interactions (Fig. 3). Finally, we selected the proposed
compounds with high fit value for the docking step Table 1.
Fig. 2. Pharmacophore model, which derived from potential CDK2 inhibitors and used
in the pre-selection of the proposed compounds (hydrophobic center; HBA; HBD).
key hydrogen bond (with respect to the bi-dentate hydrogen
bonding of purine moiety) was formed in any of the top 10 solu-
tions and the final interaction could be described by either the
Olomoucine- or ATP-type binding model (Fig. 4).
2.3. Docking
3. Chemistry
However, since we were interested in finding CDK2 inhibitors
representing novel chemotypes or at least chemotypes free of
intellectual property (IP) constrains, we opted to keep our docking
strategy unbiased. The newly proposed compounds which selected
from mapping to the pharmacophore models were docked into the
active site of CDK2 with Gold docking program [17] using Gold-
score function based on protein–ligand complexes. Ligand docking
poses were represented using a conventional chromosome repre-
sentation of translation, rotation and rotatable bond dihedral
angles. The overall orientation and internal conformation of the
compounds were searched with the GA, while the receptor site was
kept fixed. We considered a binding mode to be reasonable if the
We reported an improving method [18] for the synthesis of 6-
(2, 4-dihydroxyphenyl) – 2 – mercapto – 4 – methyl-N-aryl- l,6-
dihydropyrimidine-5-carboxamide (1a,b), as a key intermediate for
the synthesis of the proposed compounds, starting from acetoa-
cetanilids, 2,4-dihydroxybenzaldehyde and thiourea.
Pyrimidine derivatives (1a,b) were readily alkylated with benzyl
chloride in basic medium to afford the 2-benzylthio derivatives
(2a,b) in good yield which in turn reacted with different amines in
acetic acid to give the 2-aminoderivatives (3a–g) in good yields.
Also, compounds (2a,b) were oxidized with hydrogen peroxide to
give the sulfanyl derivatives (4a,b) as shown in Scheme 1.
NH
O
HN
Cl
O
N
N
N
N
HO
N
N
N
N
N
N
N
HN
N
N
H
H2 N
N
N
H
HN
N
N
HO
H
Olomoucine
NU2058
N201298
OH
Purvalanol A
H
N
O
O
H
N
N
NH2
HN
N
NH2
N
H
O
O
NH
N
N
N
N
N
HO
N
N
N
N
OH
Cl
N
N
HO
N
N
N
N
Purvalanol B
O
PU3
I17400
(R)-Roscovotine
O
N
N
O
H
N
N
O
S
NH
N
N
N
O
O
O
N
H
N
N
N
N
H2N
H
H
H
H
NH
LS51
1PU501
2PU501
Fig. 1. CDK2 inhibitors which used for building pharmacophore model.

1160
D.A. Ibrahim, A.M. El-Metwally / European Journal of Medicinal Chemistry 45 (2010) 1158–1166
family protein which has a proline directed serine/threonine kinase
activity, phosphorylatingserineorthreonineresidueaheadofproline.
Compounds were further evaluated for their cell division inhibitory
activitiesagainsttwohumantumorcelllines.Thesetumorcellsdivide
at least twice for 2-day period of the cell division inhibition test in the
absence of inhibitory compounds and the kinase activity of CDK2/
cyclinA is indispensable for the division of these cells [19,20].
In general, compounds 8a,b and 4a showed better CDK2 and cell
division inhibitory activities than the other compounds.
Compounds 8a,b were much more active than the reference
compounds (roscovitine and olomoucine) with IC50 ¼ 0.4 and 0.3
respectively, whereas compound 8a had no inhibitory activity on
cell division. Compounds 4b and 3g exhibited potent CDK2 inhib-
itory activity and reasonable cell division inhibitory activity. The
other compounds showed good to moderate CDK2 inhibitory
activities except compounds 3a and 5 which had no CDK2 inhibi-
tory activities. We study the correlation between docking scores
and CDK2 inhibitor activities and we found that there is a good
correlation between them (Fig. 5). Finally, most compounds did not
show any significant EGFR inhibitory activity, indicating good
selectivity in the situation of protein kinase inhibitors.
It seems that there is not a good correlationship between in-vitro
CDK2 inhibitory activity and in-vivo cell growth inhibition activity
among some tested compounds. For example, comparing compound
8a with 7a is more potent than 2a and 2b for CDK2 inhibition.
However, the degree of in-vivo cell growth inhibition is reversed
between them. Currently it is not clear for the reason. It may be due
to different abilities to internalize inside cells, or it may be resulted
from different inhibitory activities against some other cellular targets
within cells besides CDK2 that might be critical for cell growth. The
results demonstrate that structural variations at C-2 and C-5 of
pyrimidine derivatives might be led to potent inhibitors of CDK2, and
these information will be helpful for designing new inhibitors.
Fig. 3. Compound 8b mapped to the generated pharmacophore (Fit Value ¼ 2.993).
2-benzylthio derivative (2a,b) was reacted with hydrazine
hydrate to afford 2-hydrazinyl derivative (5), which reacted with
different aldehydes at room temperature to give hydrazone deriv-
atives (6a,b). We adopted three one-pot synthesis by the reaction of
compound (5) with ethyl acetoacetate and different aromatic
aldehydes to give the pyrazole derivatives (7a,b) in high yields.
Finally, we carried out another successful one-pot synthesis by
reaction of hydrazinyl derivative (5) with carbon disulphide, mono-
chloroacetic acid and aryl aldehydes in alcoholic potassium
hydroxide to form arylidene thiazolidinone derivatives (8a–c) in
moderate yields as depicted in Scheme 2.
5. Experimental
All melting points were uncorrected and determined by the
open capillary method using Gallenkamp melting point apparatus.
IR spectra ware recorded (KBr) on a Pye-Unicam SP-883 Perkin
Elmer spectrophotometer. 1H NMR spectra were recorded on
a Varian EM 400–600 MHZ spectrometer using DMSO-d6 as
a solvent and TMS as an internal reference, chemical shifts are
4. Biological results and discussion
CDK2 and EGFR activities of pyrimidine derivatives prepared
above were shown in Table 2, together with those of olomoucine,
roscovitineandPYK2104asreferencecompounds.CDK2isoneofCDK
expressed in d units (ppm). Mass spectra were recorded with a mass
spectrometer MS9 (AEI) 70 eV. All the analytical data were obtained
from Microanalytical Data Unit at Cairo University and Toledo
University and all the results were in an acceptable range.
Table 1
Docking Scores and Fit Values of the proposed pyrimidine derivatives.
Compounds
Docking Scorea
Fit value
5.1. Chemistry
2a
2b
3a
3b
3c
3d
3e
3f
3g
4a
4b
5
6a
6b
7a
7b
66.292
67.159
48.82
58.272
48.34
2.96
2.99
2.954
2.939
2.995
2.987
2.989
2.993
2.99
2.896
2.995
2.972
2.994
2.994
2.992
2.99
5.1.1. 6-(2,4-Dihydroxyphenyl)-2-mercapto-4-methyl-N-aryl-1,6-
dihydropyrimidine-5-carboxamide 1a,b
A mixture of acetoacetanilide (60 mmol), 2,4-dihydroxy benz-
aldehyde (50 mmol, 6.9 g), thiourea (50 mmol, 3.8 g), absolute
62.085
58.76
63.773
67.646
51.273
74.409
57.265
67.502
56.413
62.366
67.143
78.655
73.552
53.585
61.121
ethanol (20 ml) and conc. HCl (100 mL, Ca. 4 drops) was placed in
a 100 ml three-necked flask and heated at reflux (80 ꢀC inner temp.)
with stirring for 3 h. After solid Sodium bicarbonate (100 mg) was
added the reaction mixture was allowed to stand at 4 ꢀC for 5 h. The
resulting precipitate was filtered off, washed with water and
recrystallized from alcohol.
5.1.2. 6-(2,4-Dihydroxyphenyl)-2-mercapto-4-methyl-N-(4-
methylphenyl)-1,6-dihydropyrimidine-5-carboxamide 1a
6-(2,4-Dihydroxyphenyl)-2-mercapto-4-methyl-N-(4-methylp-
henyl)-1,6-dihydropyrimidine-5-carboxamide 1a was obtained as
orange crystals (86% yield); mp (ꢀC) 180–183; MS m/z 370 (M þ H)þ;
8a
8b
8c
2.991
2.993
2.98
Ligand-2C6I
2.696
a
The RMSD of the re-docking the native ligand is 0.91.

D.A. Ibrahim, A.M. El-Metwally / European Journal of Medicinal Chemistry 45 (2010) 1158–1166
1161
Fig. 4. Hydrogen bonding Interactions of compound 8b with the active site.
13C NMR (DMSO-d6) (
d
ppm) 18.8, 22.5, 45.3, 103.6, 110.3, 115.2,
orange crystals (90% yield); mp (ꢀC) 123–125; MS m/z 460 (M þ H)þ;
120.7, 121.5, 130.2, 133.3, 135.6, 140.1, 147.4, 155.5, 157.8, 160.1, 170.1;
1H NMR (DMSO-d6) 2.2 (s, 3H, CH3-phenyl), 2.7 (s, 3H, CH3-Pyrim-
idine), 4.8 (s, 1H, Pyrimidine-H6), 6.1 (s, 1H, H3-dihydroxyphenyl),
6.2–6.8 (d, 2H, j ¼ 8.1 Hz, H5,6; dihydroxyphenyl), 7.1–7.4 (dd, 4H,
j ¼ 8.4 Hz), 9.2 (s, lH, NH-Pyrimidine), 9.8 (s, 1H, NH-amide), 10.1–
102 (br, 2H, 2OH), 12.1 (s, 1H, SH). Anal. Calcd for C19H19N3O3S: C,
61.77; H, 5.18; N, 11.37. Found: C, 60.9; H, 4.8; N, 10.9.
1H NMR (DMSO-d6) (
d ppm) 2.1 (s, 3H, CH3-phenyl), 2.7 (s, 3H, CH3-
Pyrimidine), 4.7 (s,1H, Pyrimidine-H6),4.6 (s, 2H, SCH2), 6.1 (s,1H, H3-
dihydroxy-phenyl), 6.2–6.8 (d, 2H,, j ¼ 7.9 Hz, H5,6; dihydrox-
yphenyl), 7.2–7.5(m, 9H, Ar), 9.2(s, lH, NH-Pyrimidine), 9.8(s,1H, NH-
amide),10.1–10.3 (br, 2H, 2 OH). Anal. Calcd forC26H23N3O3S:C, 67.95;
H, 5.48; N, 9.14. Found: C, 67.1; H, 4.9; N, 8.9.
5.1.6. 2-(Benzylthio)-6-(2,4-dihydroxyphenyl)-4-methyl-N-(3-
nitrophenyl)-l,6-dihydro-pyrimidine-5-carboxamide 2b
5.1.3. 6-(2,4-Dihydroxyphenyl)-2-mercapto-4-methyl-N-(3-nitrophe
nyl)-1,6-dihydropyrimidine-5-carboxamide 1b
2-(Benzylthio)-6-(2,4-dihydroxyphenyl)-4-methyl-N-(3-nitro-
phenyl)-l,6-dihydro-pyrimidine-5-carboxamide 2b was obtained as
orange crystals (95% yield); mp(ꢀC)140–142; MS m/z 490 (M þ H)þ;
6-(2,4-Dihydroxyphenyl)-2-mercapto-4-methyl-N-(3-nitro-
phenyl)-1,6-dihydropyrimidine-5-carboxamide 1b was obtained
as yellow crystals (88% yield); mp (ꢀC) 168–170; MS m/z 402
13C NMR (DMSO-d6) (
d ppm) 22.5, 35.1, 45.3,101.6,103.2,110.3,115.2,
(Mþ2); 1H NMR (DMSO-d6) (
d
ppm) 2.5 (s, 3H, CH3-Pyrimidine),
118.7, 122.5, 129.2, 132.3, 134.6, 135.9, 140.1, 143.4, 147.1, 150.5, 151.1,
5.1 (s, 1H, Pyrimidine-H6), 6.1 (s, 1H, H3-dihydroxyphenyl), 6.2–
6.8 (d, 2H, j ¼ 7.8 Hz, H5,6; dihydroxyphenyl), 7.8–8.0 (m, 3H,
Ar), 8.3 (s, 1H, Ar), 9.5 (s, lH, NH-Pyrimidine), 9.6 (s, 1H, NH-
amide), 10.5–10.6 (br, 2H, 2OH), 12.5 (s, 1H, SH). Anal. Calcd for
C18H16N4O5S: C, 53.99; H, 4.03; N,13.99. Found: C, 54.5; H, 4.6; N,14.6.
158.3, 160.1, 163.2, 166.4, 171.1; 1H NMR (DMSO-d6) (
d
ppm) 2.9 (s,
3H, CH3-Pyrimidine) 4.8 (s, 1H, Pyrimidine-H6),4.5 (s, 2H, SCH2), 6.1
(s, 1H, H3; dihydroxyphenyl), 6.2–6.8 (d, 2H, j ¼ 8.1 Hz, H5,6; dihy-
droxyphenyl), 7.2–7.5 (m, 5H, j ¼ 7.2 Hz), 7.7–8.1 (m, 3H, H4,5,6; 3-
nitrophenyl), 8.5 (s, 1H, H2; 3-nitrophenyl),, 9.1 (s, lH, NH-Pyrimi-
dine), 9.9 (s, 1H, NH-amide), 10.0–10.2 (br, 2H, 2 OH). Anal. Calcd for
C25H20N4O5S: C, 61.21; H, 4.52; N,11.42. Found:C, 60.5; H, 4.9; N,11.9.
5.1.4. 2-(Benzylthio)-6-(2,4-dihydroxyphenyl)-4-methyl-N-aryl-
l,6-dihydro-pyrimidine-5-carboxamide 2a,b
Sodium hydroxide (0.024 mol) and compound 1 (0.024 mol)
were dissolved in 100 ml of water (pH of solution 7–8), and
a solution of benzyl chloride (0.024 mol) and alkali (0.024 mol) in
100 ml of water was added dropwise. The reaction mixture was
stirred for 3 h at 60 ꢀC. After cooling, the mixture was acidified with
10% HCl to pH 1–2. The resulting orange precipitate was filtered off,
washed with cold water, and dried.
5.1.7. 2-(Arylamino)-6-(2,4-dihydroxyphenyl)-4-methyl-N-aryl-l,6-
dihydropyrimi-dine-5-carboxamide 3a–g
A mixture of compound 2 (0.01 mol) and the appropriate amine
(0.015 mol) in glacial acetic acid (25 ml) was refluxed for 6–8 h.
After cooling, the reaction mixture was poured onto ice-water
(200 ml). The resulting precipitate was filtered off, washed well
with water and recrystallized from DMF–H2O mixture or alcohol.
5.1.5. 2-(Benzylthio)-6-(2,4-dihydroxyphenyl)-4-methyl-N-(4-
methylphenyl)-l,6-dihydro-pyrimidine-5-carboxamide 2a
5.1.8. 2-(Benzylamino)-6-(2,4-dihydroxyphenyl)-4-methyl-N-(4-
methylphenyl)-l,6-dihydropyrimi-dine-5-carboxamide 3a
2-(Benzylthio)-6-(2,4-dihydroxyphenyl)-4-methyl-N-(4-methyl-
phenyl)-l,6-dihydro-pyrimidine-5-carboxamide 2a was obtained as
2-(Benzylamino)-6-(2,4-dihydroxyphenyl)-4-methyl-N-(4-methyl-
phenyl)-l,6-dihydropyrimi-dine-5-carboxamide 3a was obtained as

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D.A. Ibrahim, A.M. El-Metwally / European Journal of Medicinal Chemistry 45 (2010) 1158–1166
Scheme 1. Reagent and condition: (i) EtOH, HCL, reflux; (ii) NaOH, EtOH, PhzCl; (iii) RNH2, ACOH, reflux; (iv) H2O2, ACOH.
yellow crystals (80% yield); mp (ꢀC) 155–157; MS m/z 443 (M þ H)þ;
5.1.10. 2-(Benzylamino)-6-(2,4-dihydroxyphenyl)-4-methyl-N-(3-
nitrophenyl)-l,6-dihydro-pyrimi-dine-5-carboxamide 3c
1H NMR (DMSO-d6) (
d ppm) 2.3 (s, 3H, CH3-phenyl), 2.8 (s, 3H, CH3-
Pyrimidine), 3.8 (s, 2H, N–CH2), 4.9 (s, 1H, Pyrimidine-H6), 6.1 (s,
1H, H3, dihydroxyphenyl), 6.3–6.8 (d, 2H, j ¼ 7.8 Hz, H5,6; dihy-
droxyphenyl), 7.2–7.5 (m, 9H, Ar), 9.2 (s, lH, NH-Pyrimidine), 9.7 (s,
1H, NH-benzyl), 9.9 (s, 1H, NH-amide), 10.0–10.2 (br, 2H, 2 OH).
Anal. Calcd for C26H24N4O3: C, 70.57; H, 5.92; N, 12.66. Found: C,
69.8; H, 4.9; N, 12.9.
2-(Benzylamino)-6-(2,4-dihydroxyphenyl)-4-methyl-N-(3-nitr-
ophenyl)-l,6-dihydro-pyrimi-dine-5-carboxamide 3c was obtained
as orange crystals (75% yield); mp (ꢀC) 180–183; MS m/z 474
(M þ H)þ; 1H NMR (DMSO-d6) (
d ppm) 2.6 (s,3H,CH3-Pyrimidine),
3.6(s, 2H, N–CH2), 4.9 (s, 1H, Pyrimidine-H6), 6.5 (s, 1H, H3; dihy-
droxyphenyl), 6.6–6.7 (d, 2H,, j ¼ 8.3 Hz, H5,6; dihydroxyphenyl),
7.2–7.4 (m, 5H, Ar), 7.9–8.1 (m, 3H, H4,5,6; m-nitrophenyl), 8.6 (s,
1H, H2; m-nitrophenyl), 10.2 (s, 1H, NH-amide), 10.3–10.5 (br, 2H, 2
OH). Anal. Calcd for C25H21N5O5: C, 63.42; H, 4.90; N, 14.79. Found:
C, 63.9; H, 4.1; N, 13.9.
5.1.9. 2-(2-Chloro-4-nitrophenylamino)-6-(2,4-dihydroxyphenyl)-
4-methyl-N-(4-methylphenyl)-l,6-dihydropyrimidine-5-
carboxamide 3b
2-(2-Chloro-4-nitrophenylamino)-6-(2,4-dihydroxyphenyl)-4-
methyl-N-(4-methylphenyl)-l,6-dihydropyrimidine-5-carbox-
amide 3b was obtained as yellow crystals (77% yield); mp (ꢀC) 188–
5.1.11. 2-(2-Chloro-4-nitrophenylamino)-6-(2,4-dihydroxyphenyl)-
4-methyl-N-(3-nitrophenyl)-l,6-dihydropyrimidine-5-carboxamide
3d
2-(2-Chloro-4-nitrophenylamino)-6-(2,4-dihydroxyphenyl)-4-
methyl-N-(3-nitrophenyl)-l,6-dihydropyrimidine-5-carboxamide
3d was obtained as yellow crystals (69% yield); mp(ꢀC) 207–209;
190; MS m/z 507.5 (M þ H)þ; 1H NMR (DMSO-d6) (
d ppm) 2.1 (s, 3H,
CH3-phenyl), 2.4 (s, 3H, CH3-Pyrimidine), 5.0 (s, 1H, Pyrimidine-
H6), 6.1 (s, 1H, H3-dihydroxyphenyl), 6.3–6.5 (d, 2H, j ¼ 8.1 Hz,
H5,6; dihydroxyphenyl), 7–7.2 (d, 2H, j ¼ 9.5 Hz, H2,3,5,6; 4-
methylphenylamino), 7.1–7.4 (m, 4H, Ar), 7.5–7.6 (d, 1H, j ¼ 9.5 Hz,
H6; 2-chloro-4-nitrophenyl), 7.9–8 (d, 1H, j ¼ 9.5 Hz, H5; 2-chloro-
4-nitrophenyl ring) 8.2 (s, 1H, H3, 2-chloro-4-nitrophenyl ring), 9.0
(s, 1H, NH-Pyrimidine), 9.5 (s, 1H, NH, 2-chloro-4-nitrophenyl ring),
9.9 (s, 1H, NH-amide), 10.2–10.4 (br, 2H, 2 OH). Anal. Calcd for
C25H20CIN5O5: C, 59.12; H, 4.37; N, 13.79. Found: C, 59.8; H, 4.1; N,
12.9.
MS m/z 539.5 (M þ H)þ; 1H NMR (DMSO-d6) (
d ppm) 2.4 (s, 3H,
CH3-Pyrimidine), 4.8 (s, 1H, Pyrimidine-H6), 6.3 (s, 1H, H3, dihy-
droxyphenyl), 6.2–6.4 (d, 2H, j ¼ 8.0 Hz, H5,6; dihydroxyphenyl),
7.3–7.4 (d, 1H, H6, j ¼ 9.6 Hz, H6; 2-chloro-4-nitrophenyl), 7.6–7.9
(m, 3H, H4,5,6; m-nitrophenyl), 7.8–7.9 (d, 1H, H5, j ¼ 9.6 Hz, H5; 2-
chloro-4-nitrophenyl), 8.2 (s, 1H, H3, 2-chloro-4-nitrophenyl ring),
8.5 (s, 1H, H2; m-nitrophenyl), 9.0 (s, lH, NH-Pyrimidine), 9.9 (s, 1H,

D.A. Ibrahim, A.M. El-Metwally / European Journal of Medicinal Chemistry 45 (2010) 1158–1166
1163
Scheme 2. Reagent and condition: (i) NH2NH2, reflux; (ii) R1CHO1, DMF, RT; (iii) EAA, R1CHO1KOH, reflux; (iv) CS2, CICH2COOH, R1CHO, KOH.
NH, 2-chloro-4-nitrophenyl ring), 10.5 (s, 1H, NH-amide), 10.1–10.3
(br, 2H, 2 OH). Anal. Calcd for C24H17CIN6O7: C, 53.49; H, 3.55; N,
15.59. Found: C, 53.8; H, 4.0; N, 15.9.
5.1.12. 6-(2,4-Dihydroxyphenyl)-4-methyl-N-(3-nitrophenyl)-2-(4-
(N-pyrimidin-2-yl-sulfamoyl)-phenylamino)-l,6-
dihydropyrimidine-5-carboxamide 3e
6-(2,4-Dihydroxyphenyl)-4-methyl-N-(3-nitrophenyl)-2-(4-(N-
pyrimidin-2-yl-sulfamoyl)-phenylamino)-l,6-dihydropyrimidine-
5-carboxamide 3e was obtained as yellowisþh white crystals (55%
yield); mp (ꢀC) 162–164; MS m/z 617 (M þ H) ; 1H NMR (DMSO-d6)
Table 2
CDK2 and cell division inhibitory activities of the newly synthesized pyrimidine
derivatives.
Compounds
IC50
(
m
M)
GI50 (mM)
(d ppm) 2.5(s,3H,CH3 Pyrimidine), 4.9 (s, 1H, Pyrimidine-H6), 6.5 (s,
CDK2
EGFR
A431a
HCT116b
1H, H3; dihydroxyphenyl), 6.6–6.8 (d, 2H, j ¼ 7.8 Hz, H5,6;
2a
2b
3a
3b
3c
3d
3e
3f
3g
4a
4b
5
6a
6b
7a
7b
25.4
24.6
>100
44.9
46.8
8.1
57.5
11.5
0.9
48.3
0.9
>100
3.8
22.9
10.3
3.1
0.4
0.3
28.2
7.0
0.5
–
–
93.5
55.6
23.5
60.9
>100
>100
88.3
79.8
>100
>100
>100
47.9
>100
>100
>100
>100
99.3
80.6
>100
–
20.77
>100
42.67
31.86
35.60
3.94
69.22
3.94
2.94
32.02
28.85
24.28
2.91
19.14
15.84
37.08
28.88
2.78
24.28
–
14.5
28.98
66.71
28.24
>100
10.88
77.55
8.99
16.6
23.45
10.88
47.68
8.89
38.19
>100
26.46
>100
4.50
16.61
–
1000
100
10
1
8a
8b
8c
Olomoucine
Rescovitine
PYK2104
Doxorubicine
0.1
–
–
–
0.18
–
–
0.13
45
50
55
60
65
70
75
80
0.0008
–
Docking Scores
a
A341, human vulvar epidermoid carcinoma cell line.
HCT116,human colon epitherial carcinoma cell line.
Fig. 5. Correlation between Docking scores and IC50 of the newly synthesized
compounds.
b

1164
D.A. Ibrahim, A.M. El-Metwally / European Journal of Medicinal Chemistry 45 (2010) 1158–1166
dihydroxyphenyl), 6.9 (t, 1H, j ¼ 5.0 Hz), 7.0–7.4 (dd, 4H, j ¼ 7.7 Hz,
H2,3,5,6; Ar), 7.9–8.1 (m, 3H, H4,5,6; m- nitrophenyl), 8.4 (d, 2H,
j ¼ 5.0 Hz, H4,6 pyrimidine), 8.6 (s, 1H, H2; m-nitrophenyl), 9.6 (s,
1H, NH-Pyrimidine), 10.2 (s, 1H, NH-amide), 10.3–10.5 (br, 2H, 2
OH), 12.3 (s,1H, NH-sulpha). Anal. Calcd for C28H22N8O7S: C, 54.54;
H, 3.92; N, 18.17. Found: C, 53.9; H, 4.1; N, 17.9.
crystals (88% yield); mp (ꢀC) 170–173; MS m/z 520 (Mþ); 1H NMR
(DMSO-d6) ( ppm) 2.4 (s, 3H, CH3-Pyrimidine), 5.1 (s, 2H, SCH2),
6.4 (s, 1H, H3; dihydroxyphenyl), 6.6–7.0 (d, 2H, j ¼ 7.9 Hz, H5,6;
dihydroxyphenyl), 7.2–7.4 (m, 5H, Ar), 7.8–8.0 (m, 3H, H4,5,6; 3-
nitrophenyl), 8.8 (s, 1H, H2; 3-nitrophenyl), 10.1 (s, 1H, NH-amide),
10.4–10.5 (br, 2H, 2 OH). Anal. Calcd for C25H20N4O7S: C, 57.69; H,
3.87; N, 10.76. Found: C, 56.9; H, 3.1; N, 10.9.
d
5.1.13. 6-(2,4-Dihydroxyphenyl)-2-(4-(N-(4,6-dimethylpyrimidin-
2-yl)sulfamoyl)phenylamino)-4-methyl-N-(3-nitrophenyl)-l,6-
dihydropyrimidine-5-carboxamide 3f
5.1.18. 6-(2,4-Dihydroxyphenyl)-2-hydrazinyl-4-methyl-N-(3-
nitrophenyl)-l,6-dihydro-pyrimidine-5-carboxamide 5
6-(2,4-Dihydroxyphenyl)-2-(4-(N-(4,6-dimethylpyrimidin-2-yl)su
lfamoyl)phenylamino)-4-methyl-N-(3-nitrophenyl)-l,6-dihydropyri
midine-5-carboxamide 3f was obtained as þyellowish white crystals
A mixture of thioester 2 (0.01 mol, 4.9 g) and hydrazine hydrate
85% (0.03 mol, 1.5 g) in 2-propanol (50 ml) was refluxed for 3 h. After
cooling, the precipitate was filtered off, recrystallized from alcohol,
and dried in a vacuum over P2O5 to give the target compound as
green crystals. Yield (85%); mp(ꢀC)157–160; MS m/z 400 (Mþ2H)þ;
(50% yield); mp (ꢀC) 132–134; MS m/z644(M ); 13C NMR(DMSO-d6)(
d
ppm) 19.8, 20.4, 40.6,100.3,106.1,110.6,115.2,117.2,120.1,122.3,125.2,
129.3, 132.1, 135.5, 140.2, 142.6, 145.1, 148.6, 150.1, 153.2, 155.8, 159.6,
1H NMR (DMSO-d6) (
d ppm) 2.7 (s,3H,CH3-Pyrimidine), 4.8 (s, 1H,
160.2, 162.4,166.3, 170.7; 1H NMR (DMSO-d6) (
d
ppm) 2.1 (s, 6H, 2CH3-
Pyrimidine-H6), 6.2 (s, 1H, H3, dihydroxyphenyl), 6.3–6.6 (d, 2H,
j ¼ 7.9 Hz, H5,6; dihydroxyphenyl), 7.4–7.9 (m, 3H, H4,5,6; m-
nitrophenyl), 8–8.2 (t, 1H, NH-hydrazinyl), 8.2–8.3 (d, 2H, NH2-
hydrazinyl), 8.5 (s, 1H, H2; m-nitrophenyl), 9.2 (s, lH, NH-Pyrimi-
dine),10.0 (s, 1H, NH-amide), 10.2–10.4 (br, 2H, 2 OH). Anal. Calcd for
C18H16N6O5: C, 54.27; H, 4.55; N, 21.10. Found: C, 53.8; H, 3.8; N, 20.9.
pyrimidine), 2.4 (s, 3H, CH3-Pyrimidine), 4.9 (s,1H, Pyrimidine-H6), 6.4
(s, 1H, H3; dihydroxyphenyl), 6.5–6.7 (d, 2H, j ¼ 8.0 Hz, H5,6; dihy-
droxyphenyl), 7.0 (s, 1H, H5 pyrimidine), 7.0–7.4 (dd, 4H, j ¼ 7.8 Hz,
H2,3,5,6; Ar), 7.9–8.1 (m,3H, H4,5,6; m-nitrophenyl), 8.6 (s, 1H, H2; m-
nitrophenyl), 9.5 (s, 1H, NH-Pyrimidine), 9.9 (s, 1H, NH-amide), 10.1–
10.3 (br, 2H, 2 OH),12.1 (s,1H, NH-sulpha). Anal. Calcd for C30H26N8O7S:
C, 55.89; H, 4.38; N, 17.38. Found: C, 54.9; H, 4.1; N, 17.9.
5.1.19. 6-(2,4-Dihydroxyphenyl)-2-(2-(arylmethylene)hydrazinyl)-
4-methyl-N-(3-nitrophenyl)-l,6-dihydropyrimidine-5-carboxamide
6a,b
5.1.14. 6-(2,4-Dihydroxyphenyl)-4-methyl-2-(4-(N-(5-
methylisoxazol-3-yl)sulfamoyl)phenylamino)-N-(3-nitrophenyl)-
1,2,5,6-tetrahydropyrimidine-5-carboxamide 3g
A mixture of hydrazinyl compound 5 (3.33 mmol, 1.33 g) and the
appropriate aldehyde (9.99 mmol) in DMF (20 ml) was stirred at
room temperature for 10 h. After the reaction was completed, the
solution was evaporated under reduced pressure and the residue
was triturated with water to give crystals, which were collected by
filtration and recrystallized from alcohol.
6-(2,4-Dihydroxyphenyl)-4-methyl-2-(4-(N-(5-methylisoxazol-
3-yl)sulfamoyl)phenylamino)-N-(3-nitrophenyl)-1,2,5,6-tetrahy-
dropyrimidine-5-carboxamide 3g was obtained as yellþow crystals
(65% yield); mp (ꢀC) 132–134; MS m/z 621 (M þ H) ; 1H NMR
(DMSO-d6) (d ppm) 2.2 (s, 3H, CH3-isoxazole), 2.3 (s, 3H, CH3-
Pyrimidine), 4.9 (s,1H, Pyrimidine-H6), 6.1 (s,1H, H4-isoxazole), 6.2
(s, 1H, H3; dihydroxyphenyl), 6.6–6.8 (d, 2H, j ¼ 8.0 Hz, H5,6;
dihydroxyphenyl),7.0–7.4 (dd, 4H, j ¼ 7.7 Hz, H2,3,5,6; Ar), 7.9–8.1
(m,3H, H4,5,6; m-nitrophenyl), 8.6 (s, 1H, H2; m-nitrophenyl), 9.7
(s, 1H, NH-Pyrimidine), 9.9 (s, 1H, NH-amide), 10.1–10.2 (br, 2H, 2
OH), 11.6 (s, 1H, NH-sulpha). Anal. Calcd for C28H25N7O8S: C, 54.28;
H, 4.07; N, 15.82. Found: C, 54.9; H, 3.2; N, 15.9.
5.1.20. 6-(2,4-Dihydroxyphenyl)-2-(2-(furan-2-
ylmethylene)hydrazinyl)-4-methyl-N-(3-nitro-phenyl)-l,6-
dihydropyrimidine-5-carboxamide 6a
6-(2,4-Dihydroxyphenyl)-2-(2-(furan-2-ylmethylene)hy-
drazinyl)-4-methyl-N-(3-nitro-phenyl)-l,6-dihydropyrimidine-5-
carboxamide 6a was obtained as greenish yþellow crystals (86%
yield); mp (ꢀC) 180–182; MS m/z 477 (M þ H) ; 13C NMR (DMSO-
d6) (d ppm) 20.1, 40.3, 96.9, 104.1, 110.6, 112.2, 115.4, 120.3, 122.7,
5.1.15. 2-(Benzylsulfonyl)-4-(2,4-dihydroxyphenyl)-6-methyl-N-
arylpyrimidine-5-carboxamide 4a,b
125.1, 129.5, 130.2, 133.3, 135.9, 140.2, 145.1, 150.2, 153.1, 155.4,
159.6, 162.2, 165.1, 170.1; 1H NMR (DMSO-d6) (
d
ppm) 2.4 (s, 3H,
A mixture of compound 2 (5.49 mmol), hydrogen peroxide 30%
(1 ml) and glacial acetic acid (30 ml) was stirred at room temper-
ature for 1 h and stirring was continued for another 3 h at 70 ꢀC.
After removal of acetic acid and water under reduced pressure, the
residue was washed well with water, filtered off, recrystallized
from alcohol and dried.
CH3-Pyrimidine), 5.1 (s, 1H, Pyrimidine-H6), 6.1 (s, 1H, H3, dihy-
droxyphenyl), 6.2–6.4 (d, 2H, j ¼ 8.0 Hz, H5,6; dihydroxyphenyl),
6.5–7.3 (m, 3H, furan ring), 7.5–8.0 (m, 3H, H4.5.6; m-nitrophenyl),
8.1 (s, 1H, N]CH), 8.5 (s, 1H, H2; m-nitrophenyl), 9.6 (s, lH, NH-
Pyrimidine), 10.1 (s, 1H, NH-amide), 10.4–10.6 (br, 2H, 2 OH), 11.1 (s,
1H, NH-hydrazinyl). Anal. Calcd for C23H18N6O6: C, 57.98; H, 4.23; N,
17.64. Found: C, 58.1; H, 4.8; N, 18.5.
5.1.16. 2-(Benzylsulfonyl)-4-(2,4-dihydroxyphenyl)-6-methyl-N(4-
methylphenyl)pyrimidine-5-carboxamide 4a
5.1.21. 6-(2,4-Dihydroxyphenyl)-4-methyl-N-(3-nitrophenyl)-2-(2-
(thiophen-2-ylmethylene)-hydrazinyl)-l,6-dihydropyrimidine-5-
carboxamide 6b
2-(Benzylsulfonyl)-4-(2,4-dihydroxyphenyl)-6-methyl-N(4-
methylphenyl)pyrimidine-5-carboxamide 4a was obtained as
yellow crystals (91% yield); mp (ꢀC) 160–162; MS m/z 490 (M þ H)þ;
6-(2,4-Dihydroxyphenyl)-4-methyl-N-(3-nitrophenyl)-2-(2-(thi-
ophen-2-ylmethylene)-hydrazinyl)-l,6-dihydropyrimidine-5-car-
boxamide 6b was obtained as grþeenish yellow crystals (83% yield);
1H NMR (DMSO-d6) (
d ppm) 2.2 (s, 3H, CH3-phenyl), 2.4 (s,3H,CH3-
Pyrimidine), 5.3 (s,2H,SCH2), 6.4 (s, 1H, H3-dihydroxyphenyl), 6.5–
7.0 (d, 2H, j ¼ 8.0 Hz, H5,6; dihydroxyphenyl), 7.2–7.7 (m, 9H, Ar),
10.3 (s, 1H, NH-amide), 10.3–10.5 (br, 2H, 2 OH). Anal. Calcd for
C26H23N3O5S: C, 63.79; H, 4.74; N, 8.58. Found: C, 64.1; H, 4.1; N, 7.9.
mp (ꢀC) 172–175; MS m/z 492 (M ); 1H NMR (DMSO-d6) (
d ppm) 2.7
(s, 3H, CH3-Pyrimidine), 4.9 (s, 1H, Pyrimidine-H6), 6.3 (s, 1H, H3,
dihydroxyphenyl), 6.4–6.6 (d, 2H, j ¼ 8.0 Hz, H5,6; dihydrox-
yphenyl), 7.2–7.5 (m, 3H, thiophen ring), 7.6–8.1 (m, 3H, H4,5,6; m-
nitrophenyl), 8.5 (s, 1H, N]CH), 8.7 (s, 1H, H2; m-nitrophenyl), 9.8
(s, lH, NH-Pyrimidine), 9.9 (s, 1H, NH-amide), 10.1–10.3 (br, 2H, 2
OH), 11.3 (s, 1H, NH-hydrazinyl). Anal. Calcd for C23H18N6O5S: C,
56.09; H, 4.09; N, 17.06. Found: C, 57.3; H, 4.8; N, 17.5.
5.1.17. 2-(Benzylsulfonyl)-4-(2,4-dihydroxyphenyl)-6-methyl-N-(3-
nitrophenyl)pyrimidine-5-carboxamide 4b
2-(Benzylsulfonyl)-4-(2,4-dihydroxyphenyl)-6-methyl-N-(3-
nitrophenyl)pyrimidine-5-carboxamide 4b was obtained as yellow

D.A. Ibrahim, A.M. El-Metwally / European Journal of Medicinal Chemistry 45 (2010) 1158–1166
1165
5.1.22. 6-(2,4-Dihydroxyphenyl)-2-(4-(arylmethylene)-3-methyl-5-
oxo-4,5-dihydro-1H-pyrazol-1-yl) -4-methyl-N-(3-nitrophenyl)-
1,6-dihydropyrimidine-5-carboxamide 7a,b
(DMSO-d6) (d ppm) 2.4 (s, 3H, CH3-Pyrimidine), 4.8 (s, 1H, Pyrimi-
dine-H6), 6.0 (s,1H, H3, dihydroxyphenyl), 6.2–6.6 (d, 2H, j ¼ 8.1 Hz,
H5,6; dihydroxyphenyl), 7.0 (s,1H, ¼ CH methylene), 7.4–7.8 (m, 3H,
furan ring), 7.9–8.2 (m, 3H, H4,5,6; m-nitrophenyl), 8.6 (s, 1H, H2;
m-nitrophenyl), 9.8 (s, lH, NH-Pyrimidine), 9.9 (s, 1H, NH-amide),
10.0–10.2 (br, 2H, 2 OH), 11.2 (s, 1H, NH 3-aminothiazolidine). Anal.
Calcd for C26H20N6O7S2: C, 52.70; H, 3.40; N,14.18. Found: C, 53.5; H,
4.1; N, 15.0.
A mixture of hydrazinyl compound 5 (3.33 mmol, 1.33 g) and
ethyl acetoacetate (4 mmol, 0.5 g) was heated on a boiling water
bath for 20 min. After cooling a mixture of appropriate aldehyde
(3.4 mmol) and KOH (8 mmol, 0.45 g) in absolute ethanol (30 ml)
was added and the reaction mixture was refluxed for 4 h. The
reaction mixture was neutralized with glacial acetic acid, cooled to
0
ꢀC, filtered off and recrystallized from alcohol.
5.1.27. 6-(2,4-Dihydroxyphenyl)-4-methyl-N-(3-nitrophenyl)-2-(4-
oxo-5-(thiophen-2-ylmethylene)-2-thioxothiazolidin-3-ylamino)-
1,6-dihydropyrimidine-5-carboxamide 8b
5.1.23. 6-(2,4-Dihydroxyphenyl)-2-(4-(furan-2-ylmethylene)-3-
methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-N-(3-
nitrophenyl)-1,6-dihydropyrimidine-5-carboxamide 7a
6-(2,4-Dihydroxyphenyl)-4-methyl-N-(3-nitrophenyl)-2-(4-
oxo-5-(thiophen-2-ylmethylene)-2-thioxothiazolidin-3-ylamino)-
1,6-dihydropyrimidine-5-carboxamide 8b was obtained as yellow
crystals (48% yield); mp (ꢀC) 255–257; MS m/z 609 (M þ H)þ; 1H
6-(2,4-Dihydroxyphenyl)-2-(4-(furan-2-ylmethylene)-3-methyl-
5-oxo-4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-N-(3-nitrophenyl)-
1,6-dihydropyrimidine-5-carboxamide 7a was obtainedþas yellow
crystals (55%yield); mp (ꢀC) 208–210; MS m/z 543 (M þ H) ; 1H NMR
NMR (DMSO-d6) (d ppm) 2.3 (s, 3H, CH3-Pyrimidine), 4.9 (s, 1H,
Pyrimidine-H6), 6.1 (s, 1H, H3, dihydroxyphenyl), 6.3–6.6 (d, 2H,
j ¼ 8.0 Hz, H5,6; dihydroxyphenyl), 7.3 (s, 1H, ¼ CH methylene),
7.5–8.0 (m, 3H, thiophene ring), 8.1–8.3 (m, 3H, H4,5,6; m-nitro-
phenyl), 8.5 (s, 1H, H2; m-nitrophenyl), 9.7 (s, lH, NH-Pyrimidine),
10.2 (s, 1H, NH-amide), 10.4–10.6 (br, 2H, 2 OH), 11.3 (s, 1H, NH 3-
aminothiazolidine). Anal. Calcd for C26H20N6O6S3: C, 51.31; H, 3.31;
N, 13.81. Found: C, 51.5; H, 4.0; N, 14.0.
(DMSO-d6) (d ppm) 2.1 (s, 3H, CH3-pyrazole), 2.4 (s, 3H, CH3-Pyrim-
idine), 4.9 (s, 1H, Pyrimidine-H6), 6.2 (s, 1H, H3, dihydroxyphenyl),
6.3–6.5 (d, 2H, j ¼ 8.1 Hz, H5,6; dihydroxyphenyl), 6.9 (s, 1H, ¼ CH
methylene), 7.1–7.8 (m, 3H, furan ring), 7.8–8.0 (m, 3H, H4,5,6; m-
nitrophenyl), 8.5 (s, 1H, H2; m-nitrophenyl), 9.5 (s, lH, NH-Pyrimi-
dine),10.1 (s,1H, NH-amide),10.3–10.5 (br, 2H, 2 OH). Anal. Calcd for
C27H22N6O7: C, 59.78; H, 4.09; N,15.49. Found: C, 58.9; H, 4.6; N,15.5.
5.1.28. 2-(5-Benzylidene-4-oxo-2-thioxothiazolidin-3-ylamino)-6-
(2,4-dihydroxyphenyl)-4-methyl-N-(3-nitrophenyl)-1,6-
dihydropyrimidine-5-carboxamide 8c
5.1.24. 6-(2,4-Dihydroxyphenyl)-4-methyl-2-(3-methyl-5-oxo-4-
(thiophen-2-ylmethylene)-4,5-dihydro-1H-pyrazol-1-yl)-N-(3-
nitrophenyl)-1,6-dihydropyrimidine-5-carboxamide 7b
6-(2,4-Dihydroxyphenyl)-4-methyl-2-(3-methyl-5-oxo-4-(thio-
phen-2-ylmethylene)-4,5-dihydro-1H-pyrazol-1-yl)-N-(3-nitro-
phenyl)-1,6-dihydropyrimidine-5-carboxamide 7b was obtaiþned as
yellow crystals (52% yield); mp(ꢀC) 220–223; MS m/z 558 (M ); 13C
2-(5-Benzylidene-4-oxo-2-thioxothiazolidin-3-ylamino)-6-(2,4-
dihydroxyphenyl)-4-methyl-N-(3-nitrophenyl)-1,6-dihydropyr-
imidine-5-carboxamide 8c was obtained as pale yellowcrystals (51%
yield); mp (ꢀC) 270–272; MS m/z 602 (Mþ); 13C NMR (DMSO-d6) (
d
ppm) 20.6, 40.2, 100.6, 102.3, 105.6, 110.1, 115.3, 120.2, 124.5, 126.3,
129.8, 131.5, 135.3, 136.9, 139.2, 141.1, 143.3, 145.6, 147.5, 149.7, 155.1,
NMR (DMSO-d6) (d ppm) 15.1, 22.3, 46.3, 105.1, 110.1, 114.2, 116.9,
120.8,124.1,125.6, 130.1,134.9, 136.1,138.2, 139.8, 140.1,142.2,145.2,
157.3, 159.6, 161.3, 165.4, 175.6; 1H NMR (DMSO-d6) (
d ppm) 2.5 (s,
148.6, 150.4, 152.7, 155.8, 158.7, 160.2, 162.1, 165.3, 169.2; 1H NMR
3H, CH3-Pyrimidine), 4.8 (s, 1H, Pyrimidine-H6), 6.2 (s, 1H, H3,
dihydroxyphenyl), 6.4–6.7 (d, 2H, j ¼ 8.0 Hz, H5,6; dihydrox-
yphenyl), 7.3–7.6 (m, 5H, Ar), 7.7 (s,1H,]CH methylene), 8.0–8.3 (m,
3H, H4,5,6; m-nitrophenyl), 8.4 (s,1H, H2; m-nitrophenyl), 9.6 (s, lH,
NH-Pyrimidine), 9.9 (s,1H, NH-amide),10.0–10.2 (br, 2H, 2 OH),12.1
(s, 1H, NH 3-aminothiazolidine). Anal. Calcd for C28H22N6O6S2: C,
55.80; H, 3.68; N, 13.95. Found: C, 55.1; H, 4.0; N, 14.3.
(DMSO-d6) (d ppm) 2.3 (s, 3H, CH3-pyrazole), 2.6 (s, 3H, CH3-
Pyrimidine), 4.8 (s, 1H, Pyrimidine-H6), 6.1 (s, 1H, H3, dihydrox-
yphenyl), 6.2–6.5 (d, 2H, j ¼ 8.0 Hz, H5,6; dihydroxyphenyl), 6.8 (s,
1H, ¼ CH methylene), 7.2–7.7 (m, 3H, thiophene ring), 7.8–8.2 (m,
3H, H4,5,6; m-nitrophenyl), 8.6 (s,1H, H2; m-nitrophenyl), 9.8 (s, lH,
NH-Pyrimidine), 10.2 (s, 1H, NH-amide), 10.4–10.6 (br, 2H, 2 OH).
Anal. Calcd for C27H22N6O6S: C, 58.06; H, 3.97; N, 15.05. Found: C,
57.9; H, 3.6; N, 15.5.
5.2. Biology
5.1.25. 4-(2,4-Dihydroxyphenyl)-2-(arylmethylene)-4-oxo-2-
thioxothiazolidin-3-ylamino-6-methyl-N-(3-nitrophenyl)-1,4-
dihydropyrimidine-5-carboxamide 8a–c
5.2.1. Enzymatic activity inhibition assay
The inhibition studies of cell cycle dependent kinase 2 were
performed for the synthesized compounds along with olomoucine,
roscovitine and PYK2104 as reference compounds. Olomoucine was
purchased from Sigma and we synthesized PYK 2104 and roscovi-
tine [21]. CDK2/cyclinA enzyme was purified from infected sf21
insect cells. For baculoviral overexpressions of proteins, we subcl-
oned human CDK2 c-DNA tagged by hexa-histidine on its N-
terminal and human cyclinA c-DNA into pBacPak 8 expression
vector, respectively, and baculovirus which carries each gene was
generated using baculovirus generating kit. CDK2/cyclinA enzyme
was purified using Niþ2-affinity resin from sf21 insect cell culture
into which CDK2 and cyclinA carrying baculoviruses were cotrans-
fected. Enzyme assays were done in 20 mL of 50 mMTris–HCl con-
To a stirred solution of KOH (6.7 mmol, 0.4 g) in absolute ethanol
(30 ml) was added the hydrazinyl derivative 5 (3.33 mmol. 1.33 g)
and carbon disulphide (4 mmol, 0.3 g) and the reaction mixture was
stirred 4h to dissolve carbon disulphide. Chloroacetic acid
(3.33 mmol, 0.31 g) and the appropriate aldehyde (4 mmol) were
added and the stirring was continued for another 2 h then the
reaction mixture was refluxed for 4 h. The reaction mixture was
cooled, acidified to pH 2–3 and the resulting precipitate was filtered
off, dried and recrystallized.
5.1.26. 4-(2,4-Dihydroxyphenyl)-2-(5-(furan-2-ylmethylene)-4-
oxo-2-thioxothiazolidin-3-ylamino)-6-methyl-N-(3-nitrophenyl)-
1,4-dihydropyrimidine-5-carboxamide 8a
taining 10
DTT and 4
m
M ATP, 0.2
m
Ci of gamma-P32 ATP, 10 mM MgCl2, 5 mM
mg of histone H1 was used as a substrate. The reaction was
4-(2,4-Dihydroxyphenyl)-2-(5-(furan-2-ylmethylene)-4-oxo-2-
thioxothiazolidin-3-ylamino)-6-methyl-N-(3-nitrophenyl)-1,4-di-
hydropyrimidine-5-carboxamide 8a was obtained as yellow crys-
tals (56% yield); mp(ꢀC)228–230; MS m/z 594 (Mþ2H)þ; 1H NMR
continued for 10 min in the presence of inhibitors and stopped by
adding 10 mL of 30% phosphoric acid. The stopped mixtures were
spotted onto P81 paper and were washed with 10 mM Tris–HCl (pH
8.0) containing 0.1 M NaCl five times. The radioactivity of each spot

1166
D.A. Ibrahim, A.M. El-Metwally / European Journal of Medicinal Chemistry 45 (2010) 1158–1166
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was quantified with BAS imager. The concentration of inhibitor that
gives 50% inhibition was designated as IC50 value.
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5.2.2. Cell growth inhibition assay
Human cancer cell lines, A431 (cervical cancer cell line) and
HCT116 (colon cancer cell line) were obtained from Cell Bank in
National Cancer Institute. Cells were grown in RPMI 1640 medium
containing 10% fetal bovine serum at 37 ꢀC and 5% CO2. For cell
division inhibition assay, 1000 cells were plated on 96 well plate
and next day, test compounds as well as doxorubicin as reference
compounds were treated to the cells at various concentrations.
Cells were allowed to grow further for two days in the presence of
compounds, then fixed by adding equal volume of 4% formalin for
30 min. Fixed cells were washed with tap water five times and
stained in 0.1% Sulphorhodamine B for 30 min. Subsequently cells
were washed with 1% acetic acid for four times and the dyes
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J.G. Mulheron, R. Batorsky, U. Roongta, A. Kamath, P. Marathe, S.A. Ranadive,
J.S. Sack, J.S. Tokarski, N.P. Pavletich, F.Y.F. Lee, K.R. Webster, S.D.J. Kimball,
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attached to cells were eluted by adding 100 mL of 0.1 M Tris–HCl (pH
8.0) and shaking for 10 min. The absorbance was measured at
520 nm wavelength using microplate reader (Molecular Dynamics).
The absorbance is proportional to cell number in each well.
Measurements were done triplicate and averaged. The absorbance
from cells at the time of compound treatment was designated as 0%
and the absorbance from cells after two days growth with no
compound treatment was assigned as 100%. The GI50 value was
defined as the inhibitor concentration which gives 50% cell growth
inhibition during 2 days period of compound treatment.
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¨
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