Design and synthesis of pyrimidine molecules endowed with thiazolidin-4-one as new anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2014.06.033

Design and synthesis of new pyrimidine derivatives clubbed with thiazolidin-4-one from 4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2- amine and their in vitro anticancer activities were screened at National Cancer Institute (NCI), USA against full

Full text of DOI:10.1016/j.ejmech.2014.06.033

Accepted Manuscript
Design and Synthesis of Pyrimidine Molecules Endowed with Thiazolidin-4-one as
New Anticancer agents
Mohd Rashid, Asif Husain, Mohammad Shaharyar, Ravinesh Mishra, Afzal Hussain,
Obaid Afzal
PII:
S0223-5234(14)00552-2
10.1016/j.ejmech.2014.06.033
EJMECH 7076
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 December 2013
Revised Date: 10 June 2014
Accepted Date: 16 June 2014
Please cite this article as: M. Rashid, A. Husain, M. Shaharyar, R. Mishra, A. Hussain, O. Afzal, Design
and Synthesis of Pyrimidine Molecules Endowed with Thiazolidin-4-one as New Anticancer agents,
European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.06.033.
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Graphical abstract:
Design and Synthesis of Pyrimidine Molecules Endowed with Thiazolidin-4-one as New
Anticancer agents
*
Mohd Rashid, Asif Husain , Mohammad Shaharyar, Ravinesh Mishra, Afzal Hussain, Obaid
Afzal
A series of new pyrimidine
molecules bearing thiazolidin-
4
-one were synthesized under
microwave irradiation from 4-
2-chlorophenyl)-6-(2,4dichloro
(
phenyl)pyrimidin-2-amine for
getting promising anticancer
agents at NCI against full NCI 60 cell lines panel. The present study may result in the
development of new anticancer agents in the near future.

ACCEPTED MANUSCRIPT
Design and Synthesis of Pyrimidine Molecules Endowed with Thiazolidin-4-one as New
Anticancer agents
*
Mohd Rashid, Asif Husain , Mohammad Shaharyar, Ravinesh Mishra, Afzal Hussain, Obaid
Afzal
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard
University), Hamdard Nagar, New Delhi-110062, India
*
Corresponding author. Address: Tel.: +91-11-26059681, 26059688, ext. 5647; Mobile: +91-9891116086; Fax:
+
91-11-16988874. Email address: drasifhusain@yahoo.com, ahusain@jamiahamdard.ac.in (Asif Husain),
rashidpharm2008@gmail.com (Mohd Rashid).
1

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Abstract
Design and synthesis of new pyrimidine derivatives clubbed with thiazolidin-4-one from 4-(2-
chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-amine and their in vitro anticancer activities
were screened at National Cancer Institute (NCI), USA against full NCI 60 cell lines. Compound
2
(NSC: 765735) exhibited remarkable growth inhibition at single dose (10 µM) and encourage
chosen for broadcast at 10-fold dilutions of five different concentrations (0.01, 0.1, 1, 10 and 100
µM). The compound 2 was found better quality for Lung cancer cell line (HOP-92) by viewing
growth inhibition (GI 0.52) and no cytotoxicity seen (LC >100). Molecular docking study was
5
0
50
performed using Maestro 9.0 (Schrodinger Inc. USA) to provide binding mode into binding sites
of CDK2. Compound 2 could be used as a lead compound for developing new potential
anticancer agents.
Keywords: In-vitro anticancer activity, NCI 60 cell line, CDK 2 enzyme, Thiazolidin-4-one.
2

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1
. Introduction
The cell cycle is a highly proscribed and prearranged mechanism which is fundamental in each
cell of an organism. A term of the cell cycle malady in which cells carve up mitosis without
manage called as cancer and it’s not only affect the appropriateness but also financial system of
the patient. Therefore, it is buildup a monetary shock and amplified financially viable load for
family as evenly for patient to be exact with a lot of panic and hopelessness everywhere [1-3].
The value of many accessible anticancer molecules is restricted by their toxicity to usual quickly
growing cells in the intestinal and bone marrow areas. The solemn difficulty of on hand
anticancer agents is that the cancerous cells which are primarily covered up by a precise medicine
may build up resistance for that exact medicine. While foremost altered have been ended in the
accessible chemotherapeutic through molecular biology, empirical screening, rationale drug
discovery and development and up till now nonstop painstaking duty of medicinal chemistry for
pointed new anticancer agents [4-6].
At the moment more than 60% of the medicines, which are in practice, are synthetic molecules
and step by step the scale of synthetic medicinal chemistry is widened. In the middle of the
extensive series of heterocyclic and fused heterocyclic compounds were evaluated as prospective
anticancer agents, the derivatives of pyrimidine heterocyclic compounds are of immense
significant in their biological plus synthetic loom of medicinal chemistry essentially subsequent
to the finding of Fluorouracil (1) [7], Tegafur (2) [8], NU6027 (3) [9], Uramustine (4) [10],
Cytarabine (5) [11], Thiarabine (6) [12], Ara-C (7) [13], Gemcitabine (8) [14], CNDAC (9) [15],
Capecitabine (10) [16], CGP60474 (11) [17], Nimustine (12) [18], Trimetrexate (13) [19],
ClNK4 (14) [20] and some other related heterocyclic moieties for example; Troxacitabine (15)
[
21], Decitabine (16) [22], Forodesine (17) [23], Mercaptopurine (18) [24] and Olomoucine (R
1
3

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=
H, R
2
= Me), N-9-Isopropylolomoucine (R1 = H, R2 = iPr) (19) [25] as revealed in Figure 1.
The chemically pinched of recently geared up compounds as lucid drawing with Fluorouracil and
additional interrelated heterocyclic compounds structurally shown in Figure 2.
In excess of the pasted few years, pyrimidine has been given a great deal curiosity and reported to
have prospective antitumor/antiproliferative/anticancer activity [26-32] along with antimicrobial
[
[
33, 34], antiviral including anti-HIV [35], anti-inflammatory including analgesic and ulcerogenic
36, 37], antidiabetic [38], antiparkinsonism [39], antipsychotic [40], anticonvulsant [41],
antihistaminic [42], antinociceptive [43], molluscicidal [44], herbicidal [45], steroidal like
compound [46] and anthelmintic activity [47]. In persistence of our earlier and ongoing research
works on the heterocyclic and fused heterocyclic compounds as new anticancer agents was found
to be reasonable choosy towards Leukemia cancer with mean growth inhibition (GI) as GI50 6.59,
GI50 12.62 and GI50 1.04 [48-51]. In sight of these points, it was consideration important to
design and synthesized new molecules that to have substituted pyrimidine. The pyrimidine
molecules were also clubbed with other heterocyclic ring like thiazolidinone, thiazolo-pyrimidine
and thiazolo-isoxazoline with a glance forward to produce capable anticancer agents. For that
reason, a number of new compounds were synthesized and screened for their in vitro anticancer
activities at Development Therapeutic Program, National Cancer Institute (NCI),
Chemotherapeutic Research division, USA, on nine human systems (Leukemia, Melanoma and
cancers of Lung, Colon, Brain, Breast, Ovary, Kidney and Prostate) against full NCI 60 cell line
panel according their applied protocol.
2
. Result and discussion
2
.1. Chemistry
The title compounds were synthesized in four steps as outlined in the synthetic protocol (Scheme
4

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1
and 2). In first step, 3-(2-chlorophenyl)-1-(2,4-dichlorophenyl)prop-2-en-1-one (1) was
synthesized by reacting 2,4-dichloroacetophenone with 2-chlorobenzaldehyde in the presence of
rectified spirit. In second step, 4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-amine (2)
was prepared by reacting compound 1 with guanidine HCl and DMF used as a solvent. The
compound 2 showed different R value and melting point while comparing with starting
f
compound 1. The IR spectra of compound 2 showed characteristic stretching band at 3420,
-
1
3
022, 1652 and 1383 cm due to N-H, C-H, C=N and C-N groups. The band appeared in IR
-1
-1
spectra at 1515 cm confirmed the presence of C=C and at 818 cm indicative the presence of C-
1
Cl. In H-NMR spectra the singlet appeared at δ 8.9 could be accounted for the presence of C-H
1
of pyrimidine ring and another singlet appeared at around δ 4.9 indicated the NH
2
group. In H
NMR spectra there appeared signals in the form of singlet, two doublet and multiple due to H-3,
1
3
6
, 5 and H-4, protons at around δ 8.7, 8.3 (J = 6.3 Hz), 8.1(J = 6.9 Hz), 7.5-7.2 and C NMR
spectra showed the chemical shift at around δ 132.9, 129.6, 128.7, 128.3, 128.2, 126.9, 126.0,
1
3
1
25.8, 117.4, 116.6, 109.8 and 89.7 indicated the aromatic carbon. The C NMR spectra showed
the signals at around δ 153.8, 151.9, 143.8 and 138.6 due to the presence of pyrimidine carbon.
The mass spectrum of compound 2 exhibited molecular ion peak at m/z 350 according to their
molecular formula C H Cl N . In third step, compound 2 was treated with KSCN in the
1
6
10
3
3
presence of dry acetone to form 2-(4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidinylimino)
-1
thiazolidin-4-one (3). The characteristic peaks appeared in IR spectra at around 751 and 701 cm
1
due to C-Cl and C-S-C of compound 3. In H NMR spectra, appeared a singlet at around δ 13.7
2
indicative of N-H and disappeared by addition of D O as confirmation for this group. The
1
characteristic singlet appeared in H NMR at δ 4.5 indicative the presence of CH of
2
1
3
thiazolidinone and chemical shift in C NMR at around δ 140.5 and 50.1 indicated
5

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thiazolidinone carbon. In fourth step, compounds, 2-(4-(2-chlorophenyl)-6-(2,4-dichloro phenyl)
pyrimidin-2-yl-imino)-5-(substituted) thiazolidin-4-one (4a-i) were synthesized by reacting
compound 3 with a variety of aromatic aldehydes in presence of acetic acid. In general IR spectra
-
1
of the compounds showed characteristic band at around 3323 and 3103 cm for N-H and C-H
1
(
=CH) group. In H NMR spectra, one singlet appeared at around δ 12.7 indicated N-H of
thiazolidinone and disappeared by addition of D O as confirmation for this group. The presence
2
-
1
of C=O and C=N confirmed with the band appeared at around 1728 and 1668 cm in IR spectra.
1
3
1
The C NMR spectra showed the peaks at around δ 170.2 and 132.8 due to C=O and =CH. In H
NMR spectra, two singlet appeared at around δ 9.1 and 7.14 could be accounted for C-H
1
3
(
pyrimidine ring) and C-H (=C-H), respectively. The signals were appeared in C NMR spectra
at around δ 135.4 and 107.9 due to thiazolidinone carbon. The pyrimidine carbons were observed
1
3
in C NMR spectra, at around δ 148.65, 144.20, 136.98 and 94.39. The aromatic protons
appeared in the form of doublet, singlet for 2,4-dichlorophenyl and multiplet for 2-chlorophenyl
at around δ 7.9 (J = 6.9 Hz), 7.0 and 7.0-6.7.
2
.2. Anticancer screening
Results at a single dose for every one tested compounds were reported as a mean graph of the
percent growth of the treated cells and deliberated by comparing with untreated control cells as
percentages of growth inhibitions over all the tested cell lines [52, 53]. Compound 2 exhibited
0
5
.07% growth against MDA-MB-435 cell line (Melanoma cancer), compound 3 exhibited
8.00% growth against UO-31 cell line (Renal cancer), compound 4g exhibited 32.13% growth
against MDA-MB-435 cell line (Melanoma cancer). The compounds which reduced the growth
of the cell lines to 32% or less (negative number indicate kills) were considered in vitro active
[
54] (Table 1). Compound 2 (NSC: 765735/1) fulfilled prearranged doorstep growth inhibition
6

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criteria and further selected for NCI full panel five dose assay at 10-fold dilutions of five
different concentrations (0.01, 0.1, 1, 10 and 100 µM). The result of tested compound was given
by three response parameters (GI , TGI and LC ) for each cell line from log concentration vs %
5
0
50
growth inhibition curves on nine cancer disease. Compound 2 (NSC: 765735/1) under research
exhibited marvelous anticancer activity against all the tested cell lines with GI values between
5
0
0
.52 to 81.1 µM beneath responsive range an outstanding activity (Table 2). With regard to the
sensitivity against some individual cell lines the compound showed high activity against non-
small cell Lung cancer cell line like HOP-92 (GI 0.52, TGI 7.62, LC >100 and log GI -6.28,
5
0
50
10
50
log TGI -5.12, log LC >-4.00). Obtained data revealed an noticeable sensitivity profile
1
0
10
50
towards Melanoma cancer subpanel (GI50 value ranging from 1.97-20.9 µM), least for MDA-
MB-435 and maximum for SK-MEL-28 cell line. The compound proved to be sensitive towards
all the tested Leukemia cancer, Colon cancer, Renal cancer, Prostate cancer and Breast cancer
cell lines with not more than 13.8 µM concentrations. All the tested Leukemia cancer cell lines
were sensitive with not more than GI 4.82 µM concentrations of the tested compound. The
5
0
highest growth inhibitory activity was observed against the non-small cell Lung cancer cell lines
like HOP-92 cell line with GI value 0.53 µM and minimum growth inhibitory activity against
5
0
NCI-H226 cell line of non-small cell Lung cancer with GI value 81.1µM. The all remaining
5
0
subpanel cell line showed maximum sensitive towards tested compound with not more than GI50
value 13.8 µM, except NCI-H322M (31.2 µM), SK-MEL-28 (20.9 µM), 786-0 (20.2 µM),
MALME-3M (17.7 µM), SNB-19 (16.1µM) and NCI/ADR-RES (14.8 µM). Over all the GI50
values of the screening process resulted in a sensitive range inferior of 4.17 µM denoting an
outstanding activity and values of LC in most of the cell lines >100 µM except COLO 205
5
0
(53.0 µM) and SK-MEL-5 (71.1 µM) (Table 2). A mean graph midpoint (MG-MID) for all cell
7

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a
lines were calculated from response parameter GI50, represented as MG-MID and it was found
9
.16. The mean graph midpoint for all subpanel cell lines were also considered, accessible as
b
MG-MID and found values shown in Table 2. The log molar concentration of the resulted
1
0
screening as log GI ranged from -6.28 to -4.51, minimum concentration for HOP-92 cell line
1
0
50
(-6.28) of non-small cell Lung cancer subpanel and maximum for NCI-H322M cell line (-4.51)
of non-small cell Lung cancer subpanel, log TGI value for most cell lines showed more than > -
1
0
4
.00 and mostly cell lines of subpanel showed log LC >-4.00 except COLO 205 (-4.28) and
10 50
SK-MEL-5 (-4.15) cell line. A mean graph midpoint (MG-MID) calculated for each of response
parameters in terms of log molar concentration and given as log GI (-5.19), log TGI (-4.24)
1
0
10
50
10
and log10LC50 (-4.01) (Table 3).
The Dose response curves for each cell line were converted into endpoint patterns, which
represent a snapshot of the activity of tested compounds. Three endpoints are routinely calculated
as GI50 (growth inhibitory activity) the concentration at which growth is 50% of the no drug
control, TGI (total growth inhibition) the concentration where the number of cells is equal to
those at time zero, when drug is added and LC (cytotoxic activity), the concentration at which
5
0
the number of viable cells is 50% of those present at time zero. Dose response curves for all cell
lines in the NCI 60 panel exposed to synthetic compound 2 (NSC: 765735/1) were color coded
by tissue of origin: red, leukemia cell line; blue, lung cancer; green, colon cancer; gray, central
nervous system cancer; coral, melanoma; purple, ovarian cancer; gold, renal cancer; turquoise,
prostate cancer; pink, breast cancer (Figure 3). The dose response curve was created by plotting
the PGs against log10 of corresponding sample concentration (molar) for each cell line.
Horizontal lines provided at the PG values of +50, 0 and -50. The concentrations of tested
8

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compound corresponding to points where the curves cross these lines are GI50, TGI and LC50
respectively. [55, 56].
,
Dose response curve of compound 2 (NSC: 765735) explained in colon cancer subpanel among
nine human cancer disease, showing end point calculations after treating at five dose level (1 log
-4
-8
dilutions from 10 mol/L to 10 mol/L). Growth percent of 100 corresponds to growth seen in
untreated cells. Growth percent of 0 indicates no net growth over the course of the assay (i.e.
equal to the number of cells at time zero). Growth percent of -100 results, when all cell lines
were killed. GI , log mol/L concentration yielding a growth percent of 50; TGI, log mol/L
5
0
concentration producing a growth of 0 and LC , log mol/L concentration obtaining growth of -
5
0
5
0 or lethality in 50% of the starting cells. These three end points were illustrated for COLO 205
cell line (red open circle) of colon cancer subpanel in Figure 4 and calculated values as GI50
.52, TGI = 15.5 and LC50 = 53.0. No other cell lines of colon cancer subpanel were show
=
3
lethality. Other cell lines of same subpanel were displayed as HCC-2998 (red open diamond),
HCT-116 (red open triangle), HCT-15 (red open square), HT29 (blue solid circle), KM12 (blue
solid diamond) and SW-620 (blue solid triangle) (Figure 4). The obtained results from
Developmental Therapeutics Program, National Cancer Institute were comparing with rationale
design drug (Fluorouracil) in terms of potency by three response parameter (ꢀmol/L) (Table 4).
2
.3. Structural activity relationship
From reported anticancer activity data of all the tested compounds obtained from NCI, an attempt
was made to establish relationship between chemical structure of the compounds and its activity.
2
The electron donating group like -NH in pyrimidine ring have high influence to anticancer
activity as observed by compound 2 (pyrimidin-2-aminesubstituted). The anticancer activity was
also influenced by the presence of electron withdrawing group like chloro (-Cl) and carbonyl
9

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(C=O) group at ortho or para position of aromatic ring. It indicated that substitution on aromatic
ring is essential for anticancer activity as shown by compound 4g and 3. On the other hand the
electron releasing groups like methoxy (-OCH3), hydroxyl (-OH) attached to phenyl ring and
electron donating group like -S- and -O- in thiazolidine ring did not show any appreciable
anticancer activity. So free electron donating group (s) were significantly improved anticancer
activity against the human cancer cell lines of subpanels in comparison to electron withdrawing
groups. Compound 2 may serve as novel template for development of potential and selective
agents in the field of cancer chemotherapy.
2
.4. Molecular docking studies
Cell cycle progression (the traverse through G1, S, G2 and M phases) is tightly controlled by
CDK holoenzyme complexes consisting of catalytic subunits (CDKs) and regulatory subunits
(cyclins). 13 CDKs and 25 cyclin box containing proteins have been identified through human
genome sequencing. Among these, CDK2 and cyclins-A are directly involved in regulating
sequence through cell cycle, it controls G1/S transition phase of cell cycle which is a turning
phase. The activity of CDK is negatively regulated by direct interactions with proteins referred to
as CDK inhibitors (INK4). To gain further evidence for obtaining a good cell cycle inhibitor a
molecular docking study was carried out on the 3D structure of cyclin dependent kinases-2
(CDK2) enzyme using Maestro 9.0 program (Schrodinger Inc. USA) by 64 bit operating system
under window 7 with an HCl computer [Intel (R) Core (TM) i5-2400 CPU @ 3.10 GHz, 8GB
memory]. The enzyme used in the study was obtained from the Protein Data Bank structure
(
PDB ID: 3S2P) as structure checked for missing atoms, bonds and contacts. Water molecules
and all residues other than ligands were manually deleted and the structure was refined as follows
57]. The ligands molecule were constructed using the builder molecule and were energy
[
10

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minimized. The active site was generated using the grid box. The lowest energy conformation
was selected and subjected to an energy minimization. The best binding modes of the compound
2
in the active site of 3S2P was shown in Figure 5 and 6.
Docking of compound 2 into CDK 2 active site revealed that several molecular interactions
hydrogen bond, π interaction and hydrophobic interactions) were considered to be responsible
(
for the observed affinity of the compound. In contrast, compound 2 lacks zwitter ion but
hydrogen bond form to the enzyme through its amino group with the carboxyl group of Asp 145
residue (binding distance 1.69 Å, binding energy -12.42 kcal/mol and docking energy -11.89
kcal/mol) that is at the same residue where the natural inhibitors bind (INK4) [57]. Hydrogen
bond interaction between the N-H of the amino group (NH
hydrogen bond donor with the carboxyl group (C=O) of the side chain residue of Asp 145
distance 1.69 Å) as it act as hydrogen bond acceptor (Figure 5). Pi interaction was also appears
2
) of the compound 2 as it acts as a
(
between dichlorophenyl ring of compound 2 and phenyl ring of Lys 33 residue (distance 4.39 Å)
to play a crucial role in inhibitory activity as shown in Lig plot (Figure 6). In addition to many
hydrophobic interactions between the phenyl ring, pyrimidine ring and chloro atom with the
following amino acids residues; Lys 129, Asp 132, Phe 80, Ala 144, Glu 81, Ala 31, Ile 10, Glu
1
2, Glu 11, Glu 13, Thr 14, Gln 131, Val 18, Leu 134, Phe 82, Leu 83 and Hie 84 as shown in
Figure 5 and 6. The docking results revealed that compound 2 exhibited better binding
interaction to CDK2 (PDB ID: 3S2P) than the known CDK2 inhibitors (INK4). The glide score
value of compound 2 was found to be -7.12 as it indicated that molecule interacts better with the
enzyme.
3
. Conclusion
11

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We have designed and successfully synthesized a series of new pyrimidine derivatives bearing
thiazolidin-4-one and other heterocyclic moieties. The synthesized compounds were evaluated
for their in vitro anticancer activity at the NCI, USA. The selected compounds were showed good
to remarkable anticancer activity at single dose (10 µM) screening. Among these, the compound
2
satisfied threshold inhibition criteria and emerged as lead compound with remarkable
anticancer activity at single dose level showed 100% sensitivity for MDA-MB-435 cell line
without causing the lethality with a mean value (64.97). Therefore it was further forward for
studied at five dose level with MG-MID value GI (9.16), log GI (-5.19), log TGI (-4.24) and
5
0
10
50
10
log LC (-4.01). Furthermore, molecular docking study was performed using Maestro 9.0
1
0
50
program (Schrodinger Inc. USA) to provide the binding mode of the compound 2 into the
binding sites of CDK2 enzyme. In sight of these points, compound 2 could be a subject of further
investigations for searching potential new anticancer agents.
4
. Experimental
4
.1. Chemistry
Melting points were taken on a liquid paraffin bath in open capillary tubes and are uncorrected.
Progress of the reactions was monitored by using TLC plates (silica gel G), toluene: ethyl acetate:
formic acid (5:4:1, v/v/v) and benzene: acetone (9:1, v/v) used as solvent systems. The spots
1
were located by exposure to iodine vapors or under UV-light. Nuclear magnetic resonance ( H
1
3
NMR and C NMR) spectra were recorded on Bruker spectropsin DPX-300 MHz in DMSO-
d /CDCl ; chemical shift (δ) values reported in parts per million (ppm) and coupling constants (J)
6
3
in Hz using tetramethylsilane as internal reference. The splitting pattern abbreviations are as
follows: s, singlet; bs, broad singlet; d, doublet; dd, double doublet; t, triplet; q, quadruplet; m,
2
multiplet. The exchangeable protons (OH and NH) confirmed by the addition of D O. Mass
12

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spectra recorded on LCMS/MS (Perkin-Elmer and LABINDIA, Applied Biosystem) model no.
API 3000, presented as m/z. IR spectra recorded on FT/IR (Jasco, Japan), model no. 410.
Elemental analyses performed on a Perkin-Elmer 240 analyzer and found in the range of ± 0.4%
for each element analyzed (C, H and N).
3
-(2-chlorophenyl)-1-(2,4-dichlorophenyl)prop-2-en-1-one (1)
To a mixture of 2,4-dichloroacetophenone (0.01 mol, 1.89 gm) and 2-chlorobenzaldehyde (0.01
mol, 1.40 gm) in rectified spirit (15 mL), aqueous solution of sodium hydroxide (2 gm, 10%) was
added. It was stirred until the mixture became thicker and then kept in refrigerator for overnight.
A solid product obtained, which was filtered, washed with cold water and then with n-hexane.
Pale yellow solid; Yield: 3.80 gm; Mp. 187-188°C; R = 0.53 [toluene: ethyl acetate: formic
f
acid (5:4:1)].
4
-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-amine (2)
To a solution of compound 1 (0.01 mol, 3.11 gm) and guanidine HCl (0.015 mol, 1.43 gm) in
DMF (30 mL), aqueous solution of sodium hydroxide (0.03 mol, 1.2 gm) was added. It was
refluxed for 7-8 h and then poured into ice cold water to obtain a solid product. The obtained
solid was washed with water, filtered, dried and recrystallized from ethanol. Creamy solid; Yield:
-
1
3
.73 gm; Mp. 214°C; R = 0.57. IR (KBr, cm ): 3420 (N-H, NH ), 3022 (C-H, Ar-H), 1652
f 2
1
(
C=N), 1515 (C=C), 1383 (C-N), 818 (C-Cl). H NMR (CDCl ): δ 8.98 (s, 1H, pyrimidine H-5),
3
8
6
.78 (s, 1H, dichlorophenyl H-3), 8.33 (d, 1H, J = 6.3 Hz, dichlorophenyl H-6), 8.11 (d, 1H, J =
1
3
.9 Hz, dichlorophenyl H-5), 7.57-7.32 (m, 4H, chlorophenyl), 4.91 (s, 2H, NH2). C NMR
(
DMSO-d ): δ 153.88, 151.90, 143.85, 138.62 (4C, pyrimidine), 132.97, 129.65, 128.70, 128.38,
6
+
1
28.21, 126.91, 126.08, 125.85, 117.41, 116.67, 109.86, 89.71 (Ar-C). ESI MS (m/z): 350 (M ).
3 3
Anal.calcd. for C16H10Cl N : C, 54.81; H, 2.87; N, 11.98. Found: C, 54.87; H, 3.12; N, 12.09.
13

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2
-(4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-ylimino)thiazolidin-4-one (3)
Compound 2 (0.01 mol, 3.51 gm) and KSCN (0.015 mol, 1.46 gm) were suspended in dry
acetone (25 mL), and allowed to reflux for 4 h. After completion of reaction, the mixture was
cooled and poured into cold water. The obtained solid was filtered, washed with water, dried and
recrystallized from ethanol. Brown solid; Yield: 2.98 gm; Mp. 223-224°C; R = 0.55. IR (KBr,
f
-1
cm ): 3299 (N-H), 3103 (C-H, Ar-H), 2971 (C-H, CH ), 1715(C=O), 1666 (C=N), 1531 (C=C),
2
1
1
356 (C-N), 751 (C-Cl), 701 (C-S-C). H NMR (DMSO-d ): δ 13.76 (s, 1H, NH, D O
6
2
exchangeable), 8.86 (s, pyrimidine H-5), 8.68 (s, 1H, dichlorophenyl H-3), 8.23 (d, 2H, J = 9.0
1
3
Hz, dichlorophenyl H-6, 5), 7.76-7.21 (m, 4H, chlorophenyl), 4.53 (s, 2H, CH ). C NMR
2
(
DMSO-d ): δ 168.54 (C=O), 151.32, 148.56, 145.61, 105.28 (4C, pyrimidine), 140.57, 50.16
6
(2C, thiazolidinone), 132.31, 130.64, 129.52, 128.17, 128.20, 126.87, 126.01, 123.25, 120.73,
+
1
5
18.57, 116.68, 110.81 (Ar-C). ESI MS (m/z): 449 (M ). Anal.calcd. for C19
H11Cl
3
N
4
OS: C,
0.74; H, 2.47; N, 12.46. Found: C, 50.91; H, 2.57; N, 12.47.
General procedure for the synthesis of 2-(4-(2-chlorophenyl)-6-(2,4-dichlorophenyl) pyrimidin-
-yl-imino)-5-(substituted)thiazolidin-4-one (4a-i)
2
These compounds were prepared by reacting compound 3 (0.01 mol, 4.49 mg) and different types
of aromatic aldehydes (0.01 mol) in glacial acetic acid (20 mL) in presence of anhydrous sodium
acetate (0.01 mol, 0.82 mg). The reaction mixture was placed into a microwave reaction vessel
and kept into the scientific microwave synthesizer (model No. CATA-R, Catalyst systems, India)
and then irradiated at a power level of 5 (50%, 350 W) for 10-15 min. After completion of
reaction, the mixture was cooled, poured into cold water to obtain a solid precipitate. The
obtained solid was filtered, washed with water to remove inorganic contents, dried and
recrystallized from acetonitrile.
14

ACCEPTED MANUSCRIPT
5
-benzylidene-2-(4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-ylimino) thiazolidin-4-
-1
f
one (4a). Pale brown solid; Yield: 5.09 mg; Mp. 223-224°C; R = 0.61. IR (KBr, cm ): 3356 (N-
H), 3142 (C-H, =C-H), 3071 (C-H, Ar-H), 1720 (C=O), 1668 (C=N), 1557 (C=C), 1384 (C-N),
1
8
1
27 (C-Cl), 756 (C-S-C). H NMR (DMSO-d ): δ 12.32 (s, 1H, NH, D O exchangeable), 8.65 (d,
6
2
H, J = 6.3 Hz, dichlorophenyl H-6), 8.48 (d, 1H, J = 6.3 Hz, dichlorophenyl H-5), 8.23 (s, 1H,
1
3
pyrimidine H-5), 7.64 (s, 1H), 7.51 (s, 1H, dichlorophenyl H-3), 7.41-6.82 (m, 9H,Ar-H). C
NMR (DMSO-d ): δ 172.31(C=O), 148.61, 145.32, 136.71, 105.43 (4C, pyrimidine), 138.41,
6
1
10.52 (2C, thiazolidinone), 134.62 (=CH), 132.23, 130.42, 129.32, 128.56, 128.27, 127.81,
27.68, 127.15, 126.75, 125.82, 123.51, 122.42, 118.43, 117.65, 116.45, 116.12, 115.58, 115.13,
1
+
1
09.26 (Ar-C). ESI MS (m/z): 537 (M ). Anal.calcd. for C H Cl N OS: C, 58.06; H, 2.81; N,
2
6
15
3
4
1
0.42.Found: C, 58.13; H, 2.67; N, 10.53.
-(2-chlorobenzylidene)-2-(4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-ylimino)thiaz
5
-
1
f
olidin -4-one (4b). Dark blue solid; Yield: 5.63 mg; Mp. 219-220°C; R = 0.56. IR (KBr, cm ):
3
317(N-H), 3119 (C-H, =C-H), 3070 (C-H, Ar-H), 1697 (C=O), 1674 (C=N), 1566 (C=C), 1373
1
(
C-N), 739 (C-Cl), 779 (C-S-C). H NMR (DMSO-d
6
): δ 12.17 (s, 1H, NH, D
2
O exchangeable),
8
7
.27 (d, 2H, J = 6.3 Hz, dichlorophenyl H-6, 5), 8.11(s, 1H, pyrimidine H-5), 7.73 (s, 1H, CH),
1
3
.62 (s, 1H, dichlorophenyl H-3), 7.49-6.85 (m, 8H, Ar-H). C NMR (DMSO-d ): δ 168.42
6
(C=O), 150.31, 146.23, 142.56, 107.13 (4C, pyrimidine), 136.34, 109.17 (2C, thiazolidinone),
1
1
35.74 (=CH), 132.36, 130.94, 129.73, 128.95, 128.45, 127.85, 127.31, 126.65, 125.86, 125.09,
+
24.13, 117.31, 116.65, 116.43, 115.81, 115.26, 114.68, 111.37 (Ar-C). ESI MS (m/z): 572 (M ).
Anal.calcd. for C H Cl N OS: C, 54.57; H, 2.47; N, 9.79.Found: C, 54.60; H, 2.56; N, 9.92.
2
6
14
4
4
2
-(4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-ylimino)-5-(4-methoxybenzylidene)
-
thiazolidin-4-one (4c). Dark pink solid; Yield: 5.46 mg; Mp. 211-212°C; R = 0.52. IR (KBr, cm
f
15

ACCEPTED MANUSCRIPT
1
)
: 3313 (N-H), 3111 (C-H, =C-H), 3007 (C-H, Ar-H), 2781 (C-H, OCH
3
), 1728 (C=O), 1604
): δ 12.27 (s, 1H,
1
(C=N), 1485 (C=C), 1342 (C-N), 806 (C-Cl), 759 (C-S-C). H NMR (CDCl
3
NH, D O exchangeable), 8.34 (d, 1H, J = 7.8 Hz, dichlorophenyl H-6), 8.12 (d, 1H, J = 6.9 Hz,
2
dichlorophenyl H-5), 8.16 (s, 1H, pyrimidine H-5), 7.74 (s, 1H, CH), 7.22 (s, 1H, dichlorophenyl
1
3
H-3), 7.29-6.63 (m, 8H, Ar-H), 2.75 (s, 3H, OCH ). C NMR (CDCl ): δ 170.21 (C=O), 148.72,
3
3
1
1
1
47.35, 143.68, 103.46 (4C, pyrimidine), 138.52, 110.62 (2C, thiazolidinone), 136.46 (=CH),
30.81, 130.02, 129.92, 129.23, 128.75, 128.43, 127.53, 126.82, 126.12, 125.77, 125.19, 124.36,
+
17.30, 115.41, 115.01, 114.88, 114.26, 109.37 (Ar-C), 61.38 (OCH ). ESI MS (m/z): 567 (M ).
3
Anal.calcd. for C H Cl N O S: C, 57.11; H, 3.02; N, 9.87. Found: C, 57.13; H, 3.07; N, 9.91.
2
7
17
3
4
2
2
-(4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-ylimino)-5-(furan-2ylmethylene)thiazo
-
1
f
lidin-4-one (4d). Blackish solid; Yield: 4.89 mg; Mp. 244-245°C; R = 0.52. IR (KBr, cm ):
3
323 (N-H), 3103 (C-H, =C-H), 3012 (C-H, Ar-H), 1728 (C=O), 1668 (C=N), 1519 (C=C), 1332
1
(
C-N), 1149 (C-O-C, asymmetric), 1062 (C-O-C, symmetric), 846 (C-Cl), 761 (C-S-C). H NMR
DMSO-d ): δ 12.47 (s, 1H, NH, D O exchangeable), 8.73 (d, 1H, J = 6.3 Hz, dichlorophenyl H-
), 8.59 (d, 1H, J = 8.1 Hz, dichlorophenyl H-5), 8.43 (s, 1H, pyrimidine H-5), 8.10 (s, 1H, CH),
(
6
2
6
7
1
3
.95 (s, 1H, dichlorophenyl H-3), 7.86-6.42 (m, 7H, Ar-H). C NMR (DMSO-d ): δ 167.92
6
(C=O), 150.63, 148.31, 145.56, 109.85 (4C, pyrimidine), 137.41, 111.74 (2C, thiazolidinone),
1
1
34.83 (=CH), 132.21, 130.92, 129.34, 129.11, 128.74, 128.51, 127.93, 127.32, 126.55, 126.07,
+
25.29, 124.32, 120.55, 118.46, 117.63, 115.85 (Ar-C). ESI MS (m/z): 527 (M ). Anal.calcd. for
C H Cl N O S: C, 54.61; H, 2.48; N, 10.61.Found: C, 54.83; H, 2.53; N, 10.72.
2
4
13
3
4
2
2
-(4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-ylimino)-5-(thiophen-2ylmethylene)
-
1
thiazolidin-4-one (4e). Brown solid; Yield: 4.82 mg; Mp. 230-231°C; R = 0.48. IR (KBr, cm ):
f
3
341 (N-H), 3127 (C-H, =C-H), 3051 (C-H, Ar-H), 1721 (C=O), 1662 (C=N), 1546 (C=C), 1375
16

ACCEPTED MANUSCRIPT
1
(
C-N), 830 (C-Cl), 773 (C-S-C). H NMR (DMSO-d
6
): δ 11.93 (s, 1H, NH, D
2
O exchangeable),
8
.61 (d, 1H, J = 6.9 Hz, dichlorophenyl H-6), 8.54 (d, 1H, J = 7.2 Hz, dichlorophenyl H-5), 8.21
(s, 1H, pyrimidine H-5), 7.86 (s, 1H, CH), 7.63 (s, 1H, dichlorophenyl H-3), 7.57-6.85 (m, 7H,
1
3
Ar-H). C NMR (DMSO-d ): δ 169.13 (C=O), 149.31, 146.27, 145.12, 110.75 (4C, pyrimidine),
6
1
1
36.57, 112.80 (2C, thiazolidinone), 135.72 (=CH), 132.70, 130.82, 130.13, 129.45, 128.67,
28.35, 127.96, 127.45, 126.77, 126.25, 125.59, 124.31, 120. 31, 118.92, 117.65, 115.81 (Ar-
+
C).ESI MS (m/z): 543 (M ). Anal.calcd. for C H Cl N OS : C, 53.00; H, 2.41; N, 10.30.
2
4
13
3
4
2
Found: C, 53.04; H, 2.63; N, 10.37.
-(4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-ylimino)-5-methylenethiazolidin-4-one
2
-
1
(
4f). Gray solid; Yield: 4.54 mg; Mp. 210-211°C; R = 0.47. IR (KBr, cm ): 3356 (N-H), 3162
f
(
C-H, =CH
2
), 3071 (C-H, Ar-H), 1706 (C=O), 1654 (C=N), 1533 (C=C), 1362 (C-N), 821 (C-
1
Cl), 765 (C-S-C). H NMR (DMSO-d
6
): δ 12.23 (s, 1H, NH, D
2
O exchangeable), 8.58 (d, 1H, J
=
6.6 Hz, dichlorophenyl H-6), 8.49 (d, 1H, J = 7.2 Hz, dichlorophenyl H-5), 8.32 (s, 1H,
pyrimidine H-5), 7.74 (s, 1H, CH), 7.52 (s, 1H, dichlorophenyl H-3), 7.29-6.89 (m, 4H, Ar-H),
1
3
5
.91 (s, 2H, CH
2 6
). C NMR (DMSO-d ): δ 167.92 (C=O), 150.17, 147.73, 145.35, 112.13 (4C,
pyrimidine), 138.05, 113.74 (2C, thiazolidinone), 135.86 (=CH ), 130.83, 129.25, 128.77,
2
1
28.31, 127.84, 127.35, 126.69, 125.52, 124.78, 123.65, 120.32, 119.81 (Ar-C). ESI MS (m/z):
+
4
61 (M ). Anal. calcd. for C H Cl N OS: C, 52.02; H, 2.40; N, 12.13.Found: C, 52.06; H, 2.43;
2
0
11
3
4
N, 12.09.
5
-(4-chlorobenzylidene)-2-(4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-ylimino)thiaz
-
1
olidin-4-one (4g). Pale brown solid; Yield: 5.77 mg; Mp. 240-241°C; R = 0.60. IR (KBr, cm ):
f
3
317 (N-H), 3151 (C-H, =CH), 3067 (C-H, Ar-H), 1711 (C=O), 1648 (C=N), 1529 (C=C), 1350
1
(
C-N), 807 (C-Cl), 741 (C-S-C). H NMR (DMSO-d ): δ 12.15 (s, 1H, NH, D O exchangeable),
6
2
17

ACCEPTED MANUSCRIPT
8
7
.46 (t, 2H, J = 8.1 Hz, dichlorophenyl H-6, 5), 8.21 (s, 1H, pyrimidine H-5), 7.79 (s, 1H, CH),
1
3
6
.62 (s, 1H, dichlorophenyl H-3), 7.49-6.38 (m, 8H, Ar-H). C NMR (DMSO-d ): δ 170.42
(C=O), 154.21, 148.65, 146.56, 110.74 (4C, pyrimidine), 140.13, 112.82 (2C, thiazolidinone),
1
1
37.26 (=CH ), 132.57, 130.87, 130.65, 130.13, 129.68, 129.29, 128.48, 128.12, 127.54, 127.15,
2
+
26.53, 125.82, 123.43, 123.25, 120.33, 117.51, 115.86, 115.21 (Ar-C). ESI MS (m/z): 572 (M ).
Anal.calcd. for C H Cl N OS: C, 54.57; H, 2.47; N, 9.79.Found: C, 54.71; H, 2.43; N, 9.85.
2
6
14
4
4
2
-(4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-ylimino)-5-(4-hydroxybenzylidene)thia
-
1
zolidin-4-one (4h). Dark blue solid; Yield: 5.68 mg; Mp. 217-218°C; R = 0.64. IR (KBr, cm ):
f
3
506 (O-H), 3335 (N-H), 3120 (C-H, =CH), 3054 (C-H, Ar-H), 1690 (C=O), 1638 (C=N), 1540
1
(
C=C), 1362 (C-N), 795 (C-Cl), 753 (C-S-C). H NMR (DMSO-d ): δ 12.61 (s, 1H, NH, D O
6
2
exchangeable), 9.17 (s, 1H, OH), 8.64 (d, 2H, J = 6.3 Hz, dichlorophenyl H-6,5), 8.37 (s, 1H,
pyrimidine H-5), 7.82 (d, 2H, J = 7.2 Hz, hydroxyphenyl H-2, 6), 7.61 (s, 1H, CH), 7.54 (s, 1H,
CH, dichlorophenyl), 7.44-6.92 (m, 4H, Ar-H), 6.83 (d, 2H, J = 7.2 Hz, hydroxyphenyl H-3,5).
1
3
C NMR (DMSO-d
6
): δ 173.12 (C=O), 152.35, 148.51, 146.27, 111.37 (4C, pyrimidine),
), 132.84, 131.71, 130.87, 130.52, 130.28,
1
1
1
2
2
40.29, 113.67 (2C, thiazolidinone), 135.54 (=CH
2
29.76, 129.48, 128.62, 128.37, 127.69, 127.21, 126.56, 125.13, 118.71, 118.45, 117.64, 115.58,
+
15.34 (Ar-C). ESI MS (m/z): 553 (M ). Anal.calcd. for C H Cl N O S: C, 56.38; H,
2
6
15
3
4
2
.73; N, 10.12. Found: C, 56.43; H, 2.85; N, 10.33.
-(4-(2-chlorophenyl)-6-(2,4-dichlorophenyl)pyrimidin-2-ylimino)-5-(4-hydroxy-3-methoxybenz
-
ylidene)thiazolidin-4-one (4i). Brown solid; Yield: 5.46 mg; Mp. 246°C; R = 0.58. IR (KBr, cm
f
1
): 3508 (O-H), 3323 (N-H), 3103 (C-H, =CH), 3012 (C-H, Ar-H), 1728 (C=O), 1668 (C=N),
1
1
519 (C=C), 1332 (C-N), 846 (C-Cl), 744 (C-S-C). H NMR (DMSO-d ): δ 12.78 (s, 1H, NH,
6
D O exchangeable), 9.59 (s, 1H, OH), 9.16 (s, 1H, pyrimidine H-5), 7.94 (d, 2H, J = 6.9 Hz,
2
18

ACCEPTED MANUSCRIPT
dichlorophenyl H-6, 5), 7.55 (d, 1H, J = 6.3 Hz, OCH
3
and OH-phenyl H-6), 7.36 (t, 1H, J = 8.1
Hz, OCH
3
and OH-phenyl H-5), 7.14 (s, 1H, CH), 7.07 (s, 1H, dichlorophenyl H-3), 7.01-6.75
1
3
(
m, 4H, Ar-H), 6.68 (s, 1H, OCH and OH-phenyl H-2), 3.83 (s, 3H, OCH ). C NMR (DMSO-
3
3
d6): δ 170.23 (C=O), 148.65, 144.20, 136.98, 94.39 (4C, pyrimidine), 135.42, 107.94 (2C,
thiazolidinone), 132.84 (=CH), 130.64, 130.48, 129.47, 128.92, 128.32, 127.92, 127.56, 127.46,
1
26.87, 125.83, 123.55, 117.32, 116.79, 116.49, 116.07, 115.90, 115.76, 109.09 (Ar-C), 68.44
+
(
OCH ). ESI MS (m/z): 583 (M ). Anal.calcd. for C H Cl N O S: C, 55.54; H, 2.93; N, 9.60.
3
27 17
3
4
3
Found: C, 55.63; H, 3.25; N, 9.63.
-(2-chlorophenyl)-N-(4-(2-chlorophenyl)-3a,4-dihydro-1H-pyrido[1,2-a]thiazolo[5,4-e]pyrimid
4
in-2(10aH)-ylidene)-6-(2,4-dichlorophenyl)pyrimidin-2-amine (4bi)
A mixture of compound 4b (0.003 mol, 1.72 mg), 2-aminopyridine (0.003 mol, 0.28 mg) and
DMF (15 mL) was placed into a microwave reaction vessel equipped with a magnetic stir bar.
The vessel was kept into the scientific microwave synthesizer (model No. CATA-R, Catalyst
systems, India) and irradiated at a power level of 5 (50%, 350 W) for 14 min. The reaction
mixture was cooled to room temperature, poured into crushed ice and acidified with glacial acetic
acid to yield a solid precipitate. The solid product was filtered, dried and recrystallized from
-
1
acetonitrile. Buff solid; Yield: 1.52 mg; Mp. 251-252°C; R = 0.64. IR (KBr, cm ): 3306 (N-H),
f
1
3
076 (C-H, Ar-H), 1666 (C=N), 1508 (C=C), 1377 (C-N), 788 (C-Cl), 721 (C-S-C). H NMR
(
DMSO-d ): δ 12.79 (s, 1H, NH, D O exchangeable), 9.16 (s, 1H, pyrimidine H-5), 7.91 (t, 2H, J
6 2
=
6.9 Hz, dichlorophenyl H-6, 5), 7.42-6.61 (m, 12H, Ar-H), 7.14 (s, 1H, dichlorophenyl H-3),
1
3
3
.76, 2.26 and 2.04 (s, 3H, pyrido[1,2-a]thiazolo[5,4-e]pyrimidin-2(10aH)-ylidene H-1, 2, 3). C
NMR (DMSO-d ): δ 154.23, 150.62, 137.68, 101.35 (4C, pyrimidine), 146.29, 143.73, 140.35,
6
1
38.62, 120.56, 110. 71, 82.61, 68.23, 57.10 (9C, pyrido[1,2-a]thiazolo[5,4-e]pyrimidin-2
19

ACCEPTED MANUSCRIPT
(10aH)-ylidene), 135.57, 132.17, 130.72, 130.19, 129.43, 129.27, 129.06, 128.67, 128.24,
1
27.82, 127.53, 127.21, 126.83, 126.54, 123.47, 123.15, 119.37, 117.89 (Ar-C). ESI MS (m/z):
+
6
50 (M ). Anal. calcd. for C H Cl N S: C, 57.25; H, 3.10; N, 12.92.Found: C, 57.41; H, 3.31;
3
1
20
4
6
N, 13.11.
4
6
-(2-chlorophenyl)-N-(3-(2-chlorophenyl)-6,6a-dihydrothiazolo[4,5-c]isoxazol-5-(1H)-ylidene)-
-(2,4-dichlorophenyl)pyrimidin-2-amine (4bii)
A mixture of compound 4b (0.003 mol, 1.72 mg), hydroxylamine (0.003 mol, 0.09 mg) and
DMF (15 mL) was placed into a microwave reaction vessel equipped with a magnetic stir bar.
The reaction vessel was kept into the scientific microwave synthesizer (model No. CATA-R,
Catalyst systems, India) and irradiated at a power level of 6 (60%, 420 W) for 15 min. The
completion of reaction was checked by single-spot TLC by using toluene: ethyl acetate: formic
acid (5:4:1) as solvent system. The mixture was cooled, poured into crushed ice and acidified
with glacial acetic acid to yield a solid product, which was filtered, dried and recrystallized from
-
1
acetonitrile. Dark gray solid; Yield: 1.44 mg; Mp. 263-264°C; R
f
= 0.66. IR (KBr, cm ): 3306
1
(
N-H), 3076 (C-H, Ar-H), 1622 (C=N), 1585 (C=C), 1377 (C-N), 788 (C-Cl), 721 (C-S-C). H
NMR (DMSO-d ): δ 12.75 (s, 1H, NH, D O exchangeable), 9.60 (s, 1H, H-N-O, D O
6
2
2
exchangeable), 9.18 (s, 1H, CH, pyrimidine), 7.38 (d, H, J = 8.4 Hz, dichlorophenyl H-6), 7.23
d, 1H, J = 7.8 Hz, dichlorophenyl H-5), 7.14 (s, 1H, dichlorophenyl H-3), 7.06-6.52 (m, 8H, Ar-
(
1
3
H), 3.83 (s, 1H, CH). C NMR (DMSO-d ): δ 162.73, 145.21, 65.36, 63.56 (4C, thiazolo[4,5-
6
c]isoxazol-5(1H)-ylidene), 155.16, 152.23, 138.74, 103.47 (4C, pyrimidine), 132.25, 130.47,
1
1
30.18, 129.86, 129.23, 128.75, 128.31, 127.69, 127.15, 126.55, 126.13, 124.36, 124.20, 123.87,
+
23.58, 123.12, 120.74, 120.52 (Ar-C). ESI MS (m/z): 587 (M ). Anal.calcd. for
26 4 5
C H15Cl N OS: C, 53.17; H, 2.57; N, 11.92. Found: C, 53.06; H, 2.71; N, 11.95.
20

ACCEPTED MANUSCRIPT
4
.2. Anticancer screening
The human tumor cell lines were grown in RPMI 1640 medium containing 5% fetal bovine
serum and 2mM L-glutamine. After cell inoculation, the microtiter plates incubated at 37°C, 5%
CO , 95% air and 100% relative humidity for 24 h prior to addition of tested compounds. After
2
2
4 h, two plates of each cell line fixed in situ with TCA, to stand for a measurement of the cell
population for each cell line at the time of sample addition (Tz). The sample solubilized in
DMSO at 400-fold the desired final maximum test concentration and stored frozen prior to use.
At the time of compounds addition, an aliquot of frozen concentrate was thawed and diluted to
twice, the desired final maximum test concentration with complete medium containing 50 µg/ml
gentamicin. Additional four, 10-fold or ½ log serial dilutions are made to provide a total of five
drug concentrations plus control. Aliquots of 100 µL of these different sample dilutions added to
the appropriate microtiter wells already containing 100 µL of medium, resulting in the required
final sample concentrations. The tested compounds addition, the plates was incubated for an
2
additional 48 h at 37°C, 5% CO , 95% air and 100% relative humidity. Cells fixed in situ by the
gentle addition of 50 µL of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated
for 60 min at 4°C. The supernatant was discarded and plates washed five times with tap water
and air dried. Sulforhodamine B (SRB) solution (100 µL) at 0.4 % (w/v) in 1% acetic acid added
to each well and plates incubated for 10 min at RT. Bound stain subsequently solubilized with 10
m Mtrizma base and the absorbance was read on an automated plate reader at a wavelength of
5
15 nm [52,58]. By using seven absorbance measurements [time zero, (Tz), control growth (C)
and test growth in the presence of sample at the five concentration levels (Ti)]. Percentage
Growth (PG- measured effect of the compound on a cell line) and calculated as:
21

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Where: Mean ODtzero = the average of optical density measurements SRB-derived color just
before exposure of cells to the test compound. Mean ODtest = the average of optical density
measurement of SRB-derived color after 48h exposure of cells to the test compound. Mean ODctrl
=
the average of optical density measurements of SRB-derived color after 48 h with no exposure
of cells to the test compound. The first two columns describe the subpanel. The next two
columns list the Mean ODtzero and Mean OCctrl: the next five columns list the Mean ODtest for
each of five different concentrations. Each concentration expressed as the log10 (molar or ug/ml).
The next five columns list the calculated PGs for each concentration [53]. The response
parameters GI50, TGI and LC50 are interpolated values representing the concentrations at which
the PG is +50, 0 and -50 respectively.
The NCI renamed the IC value in to special concentration parameters (GI , TGI and LC ). A
5
0
50
50
plate reader was used to read the optical densities and a microcomputer processed the optical
densities into the special concentration parameters. Percentage growth inhibition calculated as:
Three dose response parameters (GI , TGI and LC ) were calculated for each experimental
5
0
50
agent. Growth inhibition of 50 % (GI ) calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50 (drug
5
0
concentration resulting in a 50% reduction in the net protein increase), Total growth inhibition
TGI) calculated from Ti = Tz (concentration at which the total growth inhibition is 100%) and
LC50 calculated from [(Ti-Tz)/Tz] x 100 = -50 (concentration of drug resulting in a 50%
(
22

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reduction in the measured protein at the end of the drug treatment as compared to that at the
beginning) indicating a net loss of the cells [55, 56].
4
.3. Toxicity Study
The most active found compound 2 was further tested for its toxicity in rats (liver and kidney)
toxicity and fluorouracil used as standard drug for its comparison. Animals were divided into
three groups; Group I was administered with a single dose (0.3mL) of saline solution orally,
which served as control group. Group II was treated orally with a dose (0.12mg/kg body weight,
0
.3mL) of standard drug (fluorouracil). Group III was received a single dose orally (0.12mg/kg
body weight, 0.2mL) of tested compound 2. The animals were observed for a period of 12 days.
They were sacrificed if they lost more than 20% of their body weight or if there were other signs
th
of significant toxicity. On the 12 day the animals were sacrificed, the liver and kidney were
quickly removed and preserved in 40% formalin for pathological processing. Sections were
stained with hematoxyline and eosin before being observed under an Olympus microscope at 100
X and 400 X magnifications [59-62]. No significant changes were observed in liver and kidney of
treated group when compared to standard and control group of animals (Figure 7 and 8). The
results of biochemical parameters as relative to control and data were analyzed by ANOVA
followed by Dunnett’s multiple comparison tests. All data points are presented as the treatment
groups mean ± SEM.
The compound 2 was further studied for its liver toxic effect in comparison with standard drug
(fluorouracil) on biochemical parameters [serum enzymes (SGPT and SGOT)] and
histopathological testing was carried out. Treated animals with compound 2 was showed a
significant decrease (p<0.01) in activity of serum SGPT, SGOT in compare to control group.
Fluorouracil treated group was showed significant (p<0.01) increases in levels of SGOT and
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SGPT as compared to control group (Table 5).
.4. CDK2 Enzymatic activity
4
The cell division cycle is regulated by the activation of cyclin dependent kinase (CDKs), a group
of serine/threonine kinase enzyme activated by the binding to phase specific protein called cyclin.
Several CDK enzymes have been reported and each member of family has a specific function in
cell cycle division but activity of CDK2 restricted to G1-S phase that is required to complete G1
phase and initiate S phase. CDK2 activated by the regulatory subunits of Cyclin A or E (cyclin
A1 and A2 play a role during S phase and cyclin E1 and E2 during G1/S transition stage).
CDK2/Cyclin A appears during late S phase and plays a role in the progression of DNA
replication. The enzymatic activity of CDK regulated at three levels, cyclin association, subunit
phosphorylation and association with CDK inhibitors. When, cyclin initially bind to the CDKs,
the resulting complex is inactive but the phosphorylation of CDKs by Cdk activating kinase leads
to their activation. Two types of main category of CDKs inhibitors were reported in cells (INK
and WAF/Kip). The members of WAF/Kip family like p21Cip1 (CDKN1A), p27Kip1
(CDKN1B) and Kip2 (p57) complexes with the G1/S CDKs. CDK2 phosphorylates multiple
cellular substrates including SMAD3 and FOXO1. Phosphorylation of FOXO1 leads to its
inhibition. The members of the INK family, INK4A (p16), INK4B (p15), INK4C (p18) and
INK4D (p19), bind to Cdk4 and Cdk6 and block their interaction with D type cyclin therefore
inhibiting CDK activity. The major action is reported to be the inhibition of the kinase activity of
Cdk 2/cyclin E complex. The most promising targets involve for the designing inhibitors that
either block CDKs activity or prevent their interaction with cyclin subunit. Most of the currently
available molecules target the ATP-binding site of these enzymes.
ADP-Glo™ kinase and CDK2/Cyclin A2 kinase enzyme titration required dilute enzyme, tested
24

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compound, ATP and inhibitors in kinase buffer. The quantity of enzyme, tested and inhibitor
added to the media as per 2 ꢀl of enzyme, 2 ꢀl of tested/ATP mix and 1 ꢀl of inhibitor or (5%
DMSO), incubate for 10 minutes at room temperature. 5 ꢀl of ADP‐Glo™ reagent was added
into the reaction media and to continue incubate for 40 minutes. 10 ꢀl of kinase detection reagent
was adding and incubate for 30 minutes at room temperature. The luminescence was record at the
integration time of 0.5‐1 second.
ADP‐Glo™ kinase is a luminescent assay that measures ADP which comes from a kinase
reaction; ADP converted into ATP, which is converted into light by ultra‐Glo™ luciferase/
luciferin reaction. The luminescent signal positively correlates with the ADP amount and kinase
activity. The ATP to ADP conversion curve was prepared at 50 ꢀM range and obtained a linearity
curve (Figure 9) and this further used to calculate the amount of ADP formed in kinase reaction.
The data of CDK2/Cyclin A2 enzyme titration are shown as relative light units (RLU) that
directly correlate to the amount of ADP produced. The correlation between percent of ATP
converted to ADP and corresponding signal to background ratio is indicated for each kinase
amount (Table 6). CDK2/Cyclin A2 kinase activity: CDK2/Cyclin A2 enzyme was titrated with
5
0 ꢀM ATP and luminescence signal generated from each of the amounts of enzyme as shown in
Figure 10a. Staurosporine (standard) dose response curve created with 1.6 ng of CDK2/Cyclin
A2 to determine the potency of inhibitor (IC50 = 1.7 nM) (Figure 10b).
Acknowledgments
We are thankful to National Cancer Institute (NCI), Chemotherapeutic Agents Repository and
Fisher Bio Services, USA for in vitro anticancer screening of the compounds. Thanks also due to
University Grant Commission, New Delhi, Government of India, for financial assistance in terms
of Major Research Project [file No. 36-107/2008 (SR)].
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