Synthesis, antiproliferative and apoptosis-inducing activity of thiazolo[5,4-d]pyrimidines

Article abstract of DOI:10.1016/j.ejmech.2013.10.039

Thiazolo[5,4-d]pyrimidines are important class of heterocyclic compounds possessing diverse range of biological activities. Herein, we report an efficient synthesis of thiazolo[5,4-d]pyrimidines using recyclable KF/alumina catalyst. The reaction of 4,6-di

Full text of DOI:10.1016/j.ejmech.2013.10.039

European Journal of Medicinal Chemistry 70 (2013) 864e874
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiproliferative and apoptosis-inducing activity of thiazolo
[5,4-d]pyrimidinesq
,
,
,
,b
Baljinder Singh a b, Santosh K. Guru c, Smit Kour b c, Shreyans K. Jain a b, Rajni Sharma a
,
Parduman R. Sharma c, Shashank K. Singh b c, Shashi Bhushan b c, Sandip B. Bharate b
,
,
,
,d,
*
Ram A. Vishwakarma a
,b,d,
*
a Natural Products Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, India
b Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi Marg, New Delhi 110001, India
c Cancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, India
d Medicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Thiazolo[5,4-d]pyrimidines are important class of heterocyclic compounds possessing diverse range of
biological activities. Herein, we report an efficient synthesis of thiazolo[5,4-d]pyrimidines using recy-
clable KF/alumina catalyst. The reaction of 4,6-dichloro-5-aminopyrimidine with isothiocyanates in
presence of 20 mol% KF/alumina produced thiazolo[5,4-d]pyrimidines in excellent yields without any
chromatographic purifications. The method is operationally simple, fast and the catalyst can be reused
without any significant loss of activity. These compounds were tested for antiproliferative activity in a
panel of 8 cancer cell lines, including lung (NCI-H322 and A549), epidermal (A431), glioblastoma (T98G),
pancreatic (MIAPaCa-2), prostate (PC-3), human leukemia (HL-60) and breast (T47D) cells. The N,N0-
diethylamino-substituted analog, 2-(4-chlorophenylamino)-7-diethylamino-thiazolo[5,4-d]pyrimidine
4k showed antiproliferative activity in lung (NCI-H322 and A549), epidermal (A431) and glioblastoma
Received 7 June 2013
Received in revised form
18 September 2013
Accepted 15 October 2013
Available online 22 October 2013
Keywords:
Thiazolo[5,4-d]pyrimidines
Anticancer
Apoptosis
(T98G) cancer cell lines with IC50 values of 7.1, 1.4, 3.1 and 3.4
substituted analog 4a displayed activity in HL-60 cells with IC50 value of 8
induction of apoptosis in A549 cells at 10 M, as indicated by the increase in the sub-G1 population. The
nuclear morphology of A549 cells after treatment with 4k was also investigated. Similarly, the mor-
pholine substituted analog 4a induced apoptosis in HL-60 cells at 20 M. The effect of compound 4a on
m
M, respectively. The morpholine
KF/alumina
HL-60
A549
m
M. The compound 4k showed
m
m
mitochondrial potential loss in HL-60 cells was also studied. Further, western blotting of 4a and 4k
showed cleavage of PARP-1 and procaspase-3 inhibition which confirms their apoptosis-inducing
activity.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
biological importance, numerous methods have been published for
synthesis of this bicyclic system [2,13,17e23]. Among various re-
Thiazolo[5,4-d]pyrimidines is an important class of heterocyclic
compounds displaying diverse range of biological activities
including anticancer [1e7], anti-inflammatory [8], antidiabetic [9],
human cytomegalovirus inhibitory [10,11], TRPV1 inhibitory [12],
antimicrobial [13], neuroprotective [14], anti-angiogenic [15],
CDC25B phosphatase inhibitory activity [16], etc. Due to its
ported methods, Liu et al.’s [17] method involving treatment of 4,6-
dichloro-5-aminopyrimidine with isothiocyanates in presence of a
base is one of the shortest and most direct approach. Thus, the
development of an efficient and economical synthesis using this
approach will be of great value. With the advent of green protocols,
heterogeneous catalysts [24e27] are becoming overwhelmingly
important. Among various easily accessible heterogeneous cata-
lysts, the potassium fluoride impregnated over aluminum oxide
(KF/alumina) has been recognized as a remarkably useful hetero-
geneous surface to promote many base-catalyzed organic trans-
formations such as O-alkylation of alcohols and phenols [28],
Michael addition [29], Aldol condensation [30], Darzen’s conden-
sation [31], etc. In the present paper, we report an efficient
q
Note: IIIM communication number: IIIM/1588/2013.
* Corresponding authors. Medicinal Chemistry Division, Indian Institute of Inte-
grative Medicine (CSIR), Canal Road, Jammu 180001, India. Tel.: þ91 191 2569111;
fax: þ91 191 2569333.
E-mail addresses: sbharate@iiim.ac.in (S.B. Bharate), ram@iiim.ac.in (R.
A. Vishwakarma).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.039

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874
865
Table 1
synthesis of thiazolo[5,4-d]pyrimidines
3 starting from 4,6-
Optimization of reaction conditions for synthesis of thiazolo[5,4-d]pyrimidine 3a.a
dichloro-5-aminopyrimidine 1 and isothiocyanates 2 using KF/
alumina catalyst (Fig. 1). Furthermore, the thiazolo[5,4-d]pyrimi-
dines 3 were reacted with secondary amines to prepare biologically
important disubstituted thiazolo[5,4-d]pyrimidines 4.
Cl
Cl
+
Ph N C
S
NH2
Cl
N
S
2a
N
N
NH
Ph
Catalyst
All synthesized thiazolo[5,4-d]pyrimidines
3 and 4 were
N
N
screened for antiproliferative activity in a panel of cancer cell lines
including lung (NCI-H322 and A549), epidermal (A431), glioblas-
toma (T98G), pancreatic (MIAPaCa-2), prostate (PC-3), human
leukemia (HL-60) and breast (T47D) cells. The apoptotic potential of
promising antiproliferative compounds 4a and 4k was investigated
by cell cycle and western-blot analysis.
3a
1
Entry
Catalyst
Mol%
Solvent
Time (h)
Temp (ꢀC)
Yield (%)
1.
2.
3.
4.
5.
6.
7.
8.
KF/alumina
K2CO3
NaOH
KF
Alumina
20
20
20
20
20
20
20
20
20
20
20
20
20
10
20
20
20
20
ACN
ACN
ACN
ACN
ACN
DMSO
DMSO
Acetone
DCM
DMF
Water
Water
ACN
24
24
24
24
24
24
24
24
24
24
24
24
24
24
12
5
50
50
50
50
50
50
70
50
50
50
50
90
60
60
60
60
rt
88
34
24
0
2. Results and discussion
0
KF/alumina
KF/alumina
KF/alumina
KF/alumina
KF/alumina
KF/alumina
KF/alumina
KF/alumina
KF/alumina
KF/alumina
KF/alumina
KF/alumina
KF/alumina
40
98
35
30
90
0
2.1. Chemistry
9.
In order to develop an efficient synthetic protocol for prepara-
10.
11.
12.
13.
14.
15.
16.
17.
18.
tion
of
thiazolo[5,4-d]pyrimidines
from
4,6-dichloro-5-
0
aminopyrimidines and phenylisothiocyanates, we investigated
different heterogeneous catalysts under varying reaction condi-
tions. The reaction of 4,6-dichloro-5-aminopyrimidine (1) with
phenylisothiocyanate (2a) was chosen as a model reaction to syn-
thesize thiazolo[5,4-d]pyrimidine 3a. Amongst various catalysts
investigated, the KF/alumina [28,32] catalyst was found to be highly
efficient producing desired product 3a in 4e5 h in excellent yields.
The control experiments with only KF or alumina have not pro-
duced desired product 3a. Amongst various solvents tested (DMSO,
DMF, MeCN, acetone, DCM), only DMSO and MeCN produced
desired product 3a in excellent yield. However, MeCN was selected
for further optimization studies due to its ease of handling and
lower boiling point. Mole % analysis indicated 20 mol% as optimum
catalyst loading required for completion of the reaction (Table 1).
With optimal conditions in our hand, the scope of this reaction
was explored for variety of substrates in order to establish the
generality of the methodology. The substrate scope for different
substituted 4-chloro-5-aminopyrimidines and isothiocyanates is
shown in Table 2. Different phenylisothiocyanates substituted with
both electron-donating (entries 3, 5) as well as electron-
withdrawing (entry 6) groups participated well in this reaction.
Interestingly, when benzyl isothiocyanate was reacted with 6-
dichloro-5-aminopyrimidine (1), two products 3g1 and 3g2 were
formed in 30% and 42%, respectively. The additional product 3g2
was formed via thioamidation of NH of the product 3g1 [17]. The
formation of products 3g1 and 3g2 is depicted in Fig. 2. The catalyst
was reused repeatedly to prove its heterogeneous nature and its
recyclability. Reaction of 4,6-dichloro-5-aminopyrimidine (1) with
98
70
98
96
0
ACN
ACN
ACN
ACN
24
24
ACN
80
76
a
Reagents and conditions: 1 (1 mmol), phenylisothiocyanate (1 mmol).
phenylisothiocyanate in presence of 20 mol% of KF/alumina for 5 h
7-chloro-N-phenylthiazolo[5,4-d]pyrimidin-2-amine
(3a) in 96, 92, 84, and 82% over four cycles, respectively.
In order to demonstrate the utility of synthesized compounds,
we have synthesized compounds 4aen by substituting the 7-chloro
with various secondary amines. Treatment of secondary amines
(1 mmol) with methanolic solution of 3a or 3c (0.25 mmol) at room
temperature produced 4aen in 85e98% yield (Table 3).
produced
2.2. In vitro antiproliferative assay
All synthesized compounds were tested for antiproliferative
activity in eight different cancer cell lines including lung (NCI-H322
and A549), epidermal (A431), glioblastoma (T98G), pancreatic
(MIAPaCa-2), prostate (PC-3), human leukemia (HL-60) and breast
(T47D) cancer cell lines. The antiproliferative screening data of
compounds at 10 mM concentration is provided in Table 4. The re-
sults indicate that the diethylamino-substituted thiazolo[5,4-d]
pyrimidine 4k showed significant activity in four cell lines viz. NCI-
H322, A549, A431 and T98G with 57, 81, 82 and 74% growth inhi-
bition, respectively at 10 mM. The morpholine-substituted analog
Cl
N
Cl
4a showed 38, 45 and 59% growth inhibition in A549, PC-3 and HL-
60 cells. The piperidine-substituted analog 4b (41 and 54%) and 3c
(47 and 46%) showed significant growth inhibition in NCI-H322 and
A549 cells. Another diethylamino-substituted analog 4c also dis-
played significant growth inhibition in A549 (48%) and T98G (49%)
NH2
Cl
N
S
N
N
KF/alumina (20 mol%)
ACN, 60 oC, 5 h
NH
R1
N
1
3
cells at 10 mM. The compound 4j was found to be active in NCI-
MeOH,
rt, 4-5 h
2o amine
+
R1 N C
S
H322, A549 and T98G cells with 63, 57 and 45% growth inhibi-
tion, respectively. The diethylamino-substituted thiazolo[5,4-d]
pyrimidine 4k was the most potent among the series and thus was
selected for further calculation of IC50 values. The IC50 value for 4a
was also determined in selected cell lines as depicted in Table 5.
2
R2
N
N
NH
R1
Analog 4k showed IC50 values of 7.0, 1.4, 3.1 and 3.4
H322, A549, A431 and T98G cells, respectively. The analog 4a
showed IC50 value of 8 M in HL-60 cells. These two compounds
were chosen for further studies.
mM in NCI-
S
N
4
m
Fig. 1. KF/alumina catalyzed synthesis of thiazolo[5,4-d]pyrimidines 3 and 4.

866
B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874
Table 2
inhibited the G2 cell cycle phase in A549 cells. The sub-G1 (G0)
apoptotic population was increased up to 24% in 4a-treated HL-60
cells as shown in Fig. 4.
Scope of the reaction for various isothiocyanates.
Cl
Cl
N
NH2
N
S
R1
N C S
N
N
NH
R1
2.4. Nuclear morphology of cells by fluorescence microscopy
KF/Alumina (20%)
ACN, 60 oC, 5 h
N
1
Cl
3a-f
Flow cytometry alone cannot conclusively identify apoptotic
cells; thus the results were further corroborated by studying nu-
clear morphological changes of cells by fluorescence microscopy. To
evaluate the drug-induced apoptosis, DNA specific fluorescent
nuclear dye 4,6-diamidine-2-phenylindoledihydro-chloride (DAPI)
stained A549 cells were analyzed under fluorescent microscope.
Entry
R1
Product
Product yield (%)a
1
2
3
4
3a
3b
3c
3d
96
91
94
81
After the treatment at 1, 3, 5 and 10 mM of compound 4k, charac-
teristic changes of apoptosis such as nuclear condensation, mem-
brane blebbing and formation of apoptotic bodies were observed in
the morphology of treated cells in a concentration-dependent
manner, whereas untreated cells were found to be of normal
intact cell morphology. The results suggest that compound 4k was
able to induce apoptotic cell morphology in A549 cells (Fig. 5).
Similarly, the compound 4a was able to induce apoptotic cell
morphology in HL-60 cells as shown in Fig. 6.
H3CO
Cl
O
5
3e
3f
90
90
2.5. Effect on mitochondrial membrane potential loss
6
The effect of compound 4a on mitochondrial membrane po-
tential loss in human leukemia HL-60 cells was investigated. The
mitochondrial membrane potential (MMP) loss in treated and un-
treated cells is measured by rhodamine-123 dye (Rh-123) which is
reduced by healthy mitochondria into fluorescent probe whose
fluorescence is measured by Flow cytometer in FL-1 channel. The
untreated control cells showed 2% MMP loss, while compound 4a
O2N
a
Isolated yield.
2.3. Cell cycle analysis by flow cytometry
Apoptosis is characterized as programmed cell death which
leads to characteristic changes including blebbing, cell shrinkage,
nuclear fragmentation, chromatin condensation, and chromosomal
DNA fragmentation. It has been observed that many cytotoxic drugs
shows anticancer activity by inducing apoptosis [33]. Thus, in order
to address the cell death caused by compounds 4a and 4k, the
extent of apoptotic death in HL-60 and A549 cell lines was
assessed using flow cytometry through determination of sub-G1
cell population by propidium iodide (PI) staining. Due to DNA
degradation by the apoptosis-associated endonucleases, the DNA
content measurement by staining with DNA specific fluorochrome,
propidium iodide discriminates apoptotic cells [34,35]. As depicted
in Fig. 3, the region marked with different colors represents %
population at different phases of the cell cycle. The percentage of
treated HL-60 cells showed 9%, 16% and 26% at 5, 20 and 30
concentrations, respectively (Fig. 7).
mM
2.6. Apoptosis induction by western-blot analysis with PARP-1 and
procaspase-3
In order to confirm the induction of apoptosis by compounds 4a
and 4k, western blot analysis was performed with poly(ADP-
ribose) polymerase-1 (PARP-1) and procaspase-3. PARP-1 is an
enzyme responsible for the synthesis of poly(ADP-ribose) which is
essential for many cell processes including DNA repair and
apoptosis. It is believed that cleavage of PARP-1 promotes apoptosis
by preventing DNA repair-induced survival and by blocking energy
depletion-induced necrosis [36]. Procaspase-3 has important role
in the development of cancer and it is found that the levels of
procaspase-3 are often elevated in the cancer cells [37]. Treatment
of compound 4a and 4k showed clear cleavage of PARP-1 and in-
hibition of procaspase-3 in a dose-dependent manner (Fig. 8).
These results along with cycle analysis and fluorescence micro-
scopy suggest that both compounds 4a and 4k induces apoptosis.
apoptotic cells exposed to compound 4k increased in
a
concentration-dependent manner after 24 h of incubation. The
sub-G1 (G0) apoptotic population was found to be 22, 44 and 53%
following 1, 3 and 10
mM of 4k treatment compared to control
(untreated cells e 7%). As depicted in Fig. 3, the compound 4k
Cl
3. Experimental section
Cl
N
N
NH2
Cl
NH
N
3.1. General
S
N
N
1
1H and 13C NMR spectra were recorded on Brucker-Avance DPX
FT-NMR 500 and 400 MHz instruments. NMR experiments were
carried out in the indicated solvent. Chemical data for protons are
reported in parts per million (ppm) downfield from tetrame-
thylsilane and are referenced to the residual proton in the NMR
solvent [(CD3)2SO, 2.50; CD3OD, 3.31; CDCl3, 7.26 ppm]. Carbon
nuclear magnetic resonance spectra (13C NMR) were recorded at
125 MHz or 100 MHz: chemical data for carbons are reported in
3g1
KF/Alumina (20%)
ACN, 60 oC, 5 h
Cl
N
S
SCN
NH
N
S
N
N
2g
3g2
Fig. 2. Reaction of 6-dichloro-5-aminopyrimidine (1) with benzyl isothiocyanate (2g).
parts per million (ppm, d scale) downfield from tetramethylsilane

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874
867
Table 3
Table 4
Reaction of 7-chlorothiazolo[5,4-d]pyrimidines 3a or 3c with different secondary
amines.
Antiproliferative profile of thiazolo[5,4-d]pyrimidines 3aed and 4aem in a panel of
cell lines.a
Cl
N
R2
N
Entry % Growth inhibition at 10
mM
2o amine
N
S
N
S
NCI-H322 A549 A431 T98G MIAPaCa-2 PC-3 HL-60 T47D
N
N
NH
R1
NH
R1
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
0
25
47
37
7
41
39
18
39
35
27
30
42
63
57
6
3
0
1
9
0
0
4
22
12
10
19
18
0
10
0
22
82
0
1
4
0
4
0
20
26
18
21
45
2
19
36
29
35
19
34
39
2
21
31
18
21
59
22
31
28
12
39
24
39
33
21
22
39
20
41
22
19
9
MeOH, rt, 4-5 h
3
4
46
29
38
54
48
28
19
19
25
19
24
57
81
23
18
26
10
15
12
17
9
23
26
42
49
45
36
12
19
13
22
45
74
23
20
6
32
12
31
27
31
13
21
29
21
40
8
Entry
R1
R2
Product
Product yield (%)
9
10
30
9
23
21
1
14
28
5
1
2
-Ph
-Ph
4a
4b
90
91
O
N
N
22
28
18
31
18
21
21
38
3
4
-Ph
-Ph
4c
98
96
N
4m
4n
10
27
21
17
37
4d
N
a
NCI-H322, pulmonary bronchioalveolar-carcinoma cell line; A549, human lung
adenocarnoma epithelial cell line; A431, epidermoid carcinoma cell line; T98G,
human caucasian glioblastoma cell line; MIAPaCa-2, human pancreatic carcinoma
cell line; PC-3, human prostate cancer cell line; HL-60, human promyelocytic leu-
kemia cell line; T47D, human breast cancer cell line.
5
6
-Ph
-Ph
4e
4f
90
85
N
N
HN
N
3.3. General method for preparation of 2-amino-7 chlorothiazolo
[5,4-d]pyrimidines
7
-Ph
-Ph
4g
85
N
N
To the solution of 4,6-dichloro-5-aminopyrimidine 1 (164 mg,
1 mmol) in ACN (4 mL) was added KF/alumina (20 mol%) followed
by isothiocyanate 2 (1 mmol). The mixture was stirred at 60 ꢀC for
5 h, then cooled to room temperature and concentrated under
reduced pressure. The resulted mixture was dissolved in 10%
methanol in CHCl3 and the catalyst KF/alumina was filtered and
washed with acetone and then dried and reused. The filtrate was
dried and washed with hexane: diethyl ether mixture to get
products 3aef and 3g1eg2 in 80e96% yield.
8
9
4h
4i
90
93
N
N
-p-ClPh
-p-ClPh
O
N
10
4j
92
N
11
12
-p-ClPh
-p-ClPh
4k
4l
92
93
N
3.3.1. 2-Phenylamino-7-chloro-thiazolo[5,4-d]pyrimidine (3a)
Yield, 96%; off-white colored solid; m.p. 268e271 ꢀC; 1H NMR
N
(400 MHz, DMSO-d6, ppm):
d
8.66 (s, 1H), 7.79 (d, 2H, J ¼ 8 Hz), 7.43
(dd, 2H, J ¼ 7.6, 8.4 Hz), 7.14 (t, 2H, J ¼ 7.2 Hz); 13C NMR (125 MHz,
13
14
-p-ClPh
-p-ClPh
4m
4n
89
88
N
N
DMSO-d6, ppm):
d 162.86 (C), 161.17 (C), 150.26 (CH), 145.30 (C),
141.65 (C), 139.19 (C), 129.15 (CH), 129.15 (CH), 123.63 (CH), 118.70
(CH), 118.66 (CH); HRMS: m/z 263.0136 calcd for C11H7ClN4S þ Hþ
(263.0153).
N
3.3.2. 2-(4-Methoxyphenylamino)-7-chloro-thiazolo[5,4-d]
pyrimidine (3b)
and are referenced to the carbon resonance of the solvent
[(CD3)2SO, 39.52; CD3OD, 49.00; CDCl3, 77.16 ppm]. ESI-MS and
HRMS spectra were recorded on Agilent 1100 LC-Q-TOF and HRMS-
6540-UHD machines. Thin layer chromatography (TLC) was per-
formed on precoated silica gel 60 GF254 aluminum sheets (Merck).
All solvents used were of analytical grade and purchased from
Merck.
Yield, 91%; off-white colored solid; m.p. 250e254 ꢀC; 1H NMR
(400 MHz, DMSO-d6, ppm):
d
7.41 (s, 1H), 7.39 (d, 2H, J ¼ 8 Hz), 7.01
(s, 1H), 7.01 (d, 2H, J ¼ 8 Hz), 3.78 (s, 3H); 13C NMR (125 MHz,
DMSO-d6, ppm):
d 162.88 (C), 161.18 (C), 150.27 (CH), 147.77 (C),
145.31 (C), 141.66 (C), 139.19 (C), 129.15 (CH), 129.15 (CH), 118.70
3.2. Preparation of KF/alumina catalyst
Table 5
IC50 values of compounds 4a and 4k in selected cell lines.
KF/alumina catalyst was prepared by impregnating potassium
fluoride on basic alumina using a reported method [28,32]. The
neutral alumina (15 g) was mixed with KF (10 g) in 200 mL of water
for the preparation of KF/Al2O3. Water was then removed at 50e
60 ꢀC in rotary evaporator, and this reagent was further dried in
vacuum oven for 12 h.
Entry IC50
(mM)
NCI-H322 A431 T98G A549 MIAPaCa-2 PC-3 HL-60 T-47D
4a
4k
nd
7.1
nd
3.1
nd
3.4
13
1.4
54
nd
17
nd
8
nd
59
nd
nd, not determined.

868
B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874
Fig. 3. Cell cycle analysis of compound 4k treated A549 cells. A549 cells were treated with different concentrations (1, 3 and 10
fluorescence and cell cycle phase distribution.
mM) of compound 4k for 24 h to determine DNA
(CH), 118.66 (CH); HRMS: m/z calcd 293.0265 for C12H9ClN4OS þ Hþ
3.3.8. 1,3-Dibenzyl-1-(7-chlorothiazolo[5,4-d]pyrimidin-2-yl)
thiourea (3g2) [17]
(293.0258).
Yield, 42%; off white colored solid; m.p. 244e246 ꢀC; 1H NMR
3.3.3. 2-(4-Chlorophenylamino)-7-chloro-thiazolo[5,4-d]
pyrimidine (3c)
(400 MHz, DMSO-d6, ppm): d 8.81 (s, 1H), 7.40e7.18 (m, 10H), 5.90
(s, 2H), 4.82 (s, 2H); HRMS: m/z calcd 426.0612 for
Yield, 94%; off-white colored solid; m.p. 273e276 ꢀC; 1H NMR
C
20H16ClN5S2þHþ (426.0608).
(400 MHz, DMSO-d6, ppm):
d
8.66 (s, 1H), 7.83 (d, 2H, J ¼ 8.0 Hz),
7.50 (d, 2H, J ¼ 8.0 Hz); 13C NMR (125 MHz, DMSO-d6, ppm):
3.4. General method for preparation of compounds 4aen
d
162.84 (C), 161.18 (C), 150.27 (CH), 145.31 (C), 141.66 (C), 139.19 (C),
129.15 (CH), 129.15 (CH), 122.86 (C), 118.70 (CH), 118.70 (CH);
To a solution of 2-amino-7-chlorothiazolo[5,4-d]pyrimidines (3,
0.25 mmol) in MeOH (3 mL) was added secondary amines
(1.0 mmol). The mixture was stirred at room temperature for 12e
24 h and then concentrated under reduced pressure. The reaction
mixture was then dried under vacuum and the final products (4ae
n) were recrystallized with methanol and chloroform mixture
giving 85e98% isolated yield.
HRMS: m/z calcd 294.9601 for C11H6Cl2N4SeHꢁ (294.9612).
3.3.4. 2-Benzoylamino-7-chloro-thiazolo[5,4-d]pyrimidine (3d)
[38]
Yield, 81%; orange colored solid; m.p. 186e190 ꢀC; 1H NMR
(400 MHz, CDCl3, ppm):
1H), 7.61e7.46 (m, 4H); HRMS: m/z calcd 288.9951 for
12H7ClN4OSeHꢁ (288.9951).
d 8.12 (s, 1H), 8.10 (s, 1H), 7.99e7.87 (m,
C
3.4.1. 2-Phenylamino-7-(morpholin-4-yl)-thiazolo[5,4-d]
pyrimidine (4a)
3.3.5. 2-p-Tolyl-7-chloro-thiazolo[5,4-d]pyrimidine (3e) [17]
Yield, 90%; yellow colored solid; m.p. 243e247 ꢀC; 1H NMR
Yield, 97%; white colored solid; m.p. 241e243 ꢀC; 1H NMR
(500 MHz, CD3OD, ppm):
d
8.09 (s, 1H), 7.48 (d, 2H, J ¼ 8.3 Hz), 7.22
(400 MHz, DMSO-d6, ppm):
d
8.27 (s, 1H), 7.64 (d, 2H, J ¼ 8.0 Hz),
(dd, 2H, J ¼ 8.5, 15.9 Hz), 6.94 (t, 1H, J ¼ 7.3 Hz), 4.17 (d, 4H,
7.45 (d, 2H, J ¼ 8.0 Hz), 2.38 (s, 3H); HRMS: m/z calcd 277.0309 for
J ¼ 4.3 Hz), 3.71 (d, 4H, J ¼ 4.4 Hz); 13C NMR (125 MHz, DMSO-d6,
C
12H9ClN4S þ Hþ (277.0309).
ppm): d 161.17 (C), 156.01 (C), 151.9 (C), 150.85 (CH), 140.01 (C),
129.03 (CH), 129.03 (CH), 128.25 (C), 122.36 (CH), 117.77 (CH), 117.77
(CH), 66.07 (CH2), 66.07 (CH2), 45.95 (CH2), 45.95 (CH2); HRMS: m/z
calcd 314.1060 for C15H15N5OS þ Hþ (314.1070).
3.3.6. 2-(4-Nitrophenylamino)-7-chloro-thiazolo[5,4-d]pyrimidine
(3f)
Yield, 90%; yellow colored solid; m.p. 256e259 ꢀC; 1H NMR
(400 MHz, DMSO-d6, ppm):
d
8.75 (s, 1H), 8.35 (d, 2H, J ¼ 8.0 Hz),
3.4.2. 2-Phenylamino-7-piperidin-1-yl-thiazolo[5,4-d]pyrimidine
(4b)
8.03 (d, 2H, J ¼ 8.0 Hz); 13C NMR (100 MHz, CD3OD þ CDCl3, ppm):
d
161.07 (C),157.71 (C),153.58 (C),151.73 (CH),140.05 (C),130.22 (C),
Yield, 91%; yellow colored solid; m.p. 238e239 ꢀC; 1H NMR
129.78 (CH), 129.78 (CH), 128.37 (C), 120.41 (CH), 120.41 (CH);
(500 MHz, CD3OD, ppm):
d
8.17 (s, 1H), 7.67 (d, 2H, J ¼ 8.3 Hz), 7.35
HRMS: m/z calcd 308.0003 for C11H6ClN5O2S þ Hþ (308.0003).
(dd, 2H, J ¼ 8.5, 15.7 Hz), 7.06 (t, 1H, J ¼ 7.3 Hz), 4.27 (d, 4H,
J ¼ 4.5 Hz), 1.81 (t, 2H, J ¼ 5.6 Hz), 1.75 (d, 4H, J ¼ 4.2 Hz); 13C NMR
3.3.7. 2-Benzylamino-7-chloro-thiazolo[5,4-d]pyrimidine (3g1)
[17]
(125 MHz, DMSO-d6, ppm): d 159.85 (C), 155.31 (C), 151.87 (C),
150.96 (CH), 140.18 (C), 128.89 (CH), 128.89 (CH), 127.87 (C), 122.12
(CH), 117.54 (CH), 117.54 (CH), 46.44 (CH2), 46.44 (CH2), 25.65 (CH2),
25.65 (CH2), 24.21 (CH2); HRMS: m/z calcd 312.1269 for
Yield, 30%; off white colored solid; m.p. 234e237 ꢀC; 1H NMR
(400 MHz, DMSO-d6, ppm):
d 8.81 (s, 1H), 7.40e7.26 (m, 5H), 4.82
(s, 2H); HRMS: m/z calcd 277.0309 for C12H9ClN4S þ Hþ (277.0309).
C
16H17N5S þ Hþ (312.1277).

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874
869
Fig. 4. Cell cycle analysis of compound 4a treated HL-60 cells. HL-60 cells were treated with different concentrations (5, 20 and 30
fluorescence and cell cycle phase distribution.
mM) of compound 4a for 24 h to determine DNA
Fig. 5. Effect of compound 4k on nuclear morphology of A549 cells as observed under fluorescence microscope (40ꢂ). (A) Untreated cells have normal intact nuclei. (B) Positive
control paclitaxel (1 M) treated cells showing typical apoptotic morphology. (CeF) Compound 4k treated A549 cells at concentrations of 1, 3, 5 and 10 M, respectively. White
arrows are indicative of apoptotic nuclei.
m
m

870
B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874
Fig. 6. Effect of compound 4a on cellular and nuclear morphology of HL-60 cells. Cells were treated with indicated concentrations of compound 4a for 24 h time period. Cells were
visualized for cellular morphology. Second row shows nuclear morphology of cells after staining with Hoechst 33258 dye.
3.4.3. 2-Phenylamino-7-diethylamino-thiazolo[5,4-d]pyrimidine
(4c)
3.4.7. 2-Phenylamino-7-(4-ethylpiperazin-1-yl)-thiazolo[5,4-d]
pyrimidine (4g)
Yield, 98%; off white colored solid; m.p. 236e239 ꢀC; 1H NMR
Yield, 85%; off white colored solid; m.p. 233e236 ꢀC; 1H NMR
(500 MHz, CDCl3, ppm):
d
8.29 (s, 1H), 7.53 (d, 2H, J ¼ 7.6 Hz), 7.37
(400 MHz, CD3OD, ppm):
d
8.10 (s, 1H), 7.52 (d, 2H, J ¼ 8.0 Hz), 7.27
(dd, 2H, J ¼ 7.5, 15.8 Hz), 7.15 (s, 1H), 7.10 (t, 1H, J ¼ 7.2 Hz), 3.96e
(t, 2H, J ¼ 8.0, 15.9 Hz), 6.98 (t, 1H, J ¼ 8.0 Hz), 4.26e4.21 (m, 3H),
3.94 (m, 4H), 1.35e1.29 (m, 6H); 13C NMR (125 MHz, CDCl3, ppm):
2.56e2.53 (m, 4H), 2.45e2.39 (m, 3H), 1.19 (t, 3H, J ¼ 8.0 Hz); 13C
d
160.02 (C), 156.02 (C), 152.64 (C), 151.84 (CH), 139.73 (C), 129.28
NMR (125 MHz, CD3OD þ CDCl3, ppm):
d 161.07 (C), 157.71 (C),
(CH), 129.28 (CH), 128.28 (C), 123.36 (CH), 118.55 (CH), 118.55 (CH),
43.77 (CH2), 43.77 (CH2), 13.84 (CH3), 13.84 (CH3); HRMS: m/z calcd
300.1280 for C15H17N5S þ Hþ (300.1277).
153.58 (C), 151.73 (CH), 140.05 (C), 129.78 (CH), 129.78 (CH), 121.41
(CH), 120.41 (CH), 120.41 (CH), 54.68 (CH2), 54.68 (CH2), 50.77
(CH2), 43.18 (CH2), 43.18 (CH2), 14.73 (CH3); HRMS: m/z calcd
341.1543 for C17H20N6S þ Hþ (341.1543).
3.4.4. 2-Phenylamino-7-dimethylamino-thiazolo[5,4-d]pyrimidine
(4d)
3.4.8. 2-Phenylamino-7-[4-(piperidin-1-yl)piperidin-1-yl]-thiazolo
[5,4-d]pyrimidine (4h)
Yield, 96%; off white colored solid; m.p. 235e237 ꢀC; 1H NMR
(500 MHz, CD3OD, ppm):
d
8.17 (s, 1H), 7.68 (d, 2H, J ¼ 7.8 Hz), 7.36
Yield, 90%; off white colored solid; m.p. 256e258 ꢀC; 1H NMR
(dd, 2H, J ¼ 7.7, 17.6 Hz), 7.07 (t,1H, J ¼ 7.4 Hz), 3.56 (s, 6H); 13C NMR
(400 MHz, CD3OD, ppm):
d
8.18 (s, 1H), 7.65 (d, 2H, J ¼ 8.0 Hz), 7.35
(125 MHz, CD3OD, ppm):
d
160.20 (C), 157.74 (C), 154.94 (C), 151.89
(dd, 2H, J ¼ 8.0, 16.0 Hz), 7.08 (t, 1H, J ¼ 8.0 Hz), 5.57e5.54 (m, 1H),
3.09e3.03 (m, 2H), 2.71e2.64 (m, 6H), 2.06e2.03 (m, 2H),1.64e1.58
(m, 6H), 1.51e1.49 (m, 2H); 13C NMR (100 MHz, CD3OD þ CDCl3,
(CH), 141.78 (C), 130.09 (CH), 130.09 (CH), 128.28 (C), 123.76 (CH),
119.27 (CH), 119.21 (CH), 39.88 (CH3), 39.88 (CH3); HRMS: m/z calcd
272.0961 for C13H13N5S þ Hþ (272.0964).
ppm):
d 160.95 (C), 158.03 (C), 153.67 (C), 151.91 (CH), 141.69 (C),
130.11 (CH), 130.11 (CH), 123.90 (CH), 119.38 (CH), 119.38 (CH), 64.15
(CH), 51.27 (CH2), 51.27 (CH2), 46.86 (CH2), 46.86 (CH2), 29.23 (CH2),
29.23 (CH2), 26.75 (CH2), 26.75 (CH2), 25.47 (CH2), 25.47 (CH2);
HRMS: m/z calcd 395.2006 for C21H26N6S þ Hþ (395.2012).
3.4.5. 2-Phenylamino-7-(4-methylpiperazin-1-yl)-thiazolo[5,4-d]
pyrimidine (4e)
Yield, 90%; white colored solid; m.p. 231e234 ꢀC; 1H NMR
(400 MHz, CD3OD þ CDCl3, ppm):
d 8.20 (s, 1H), 7.63e7.59 (m, 2H),
7.36e7.32 (m, 2H), 7.08e7.04 (m, 1H), 4.33e4.31 (m, 2H), 2.63e2.61
3.4.9. 2-(4-Chlorophenylamino)-7-(morpholin-4-yl)-thiazolo[5,4-
d]pyrimidine (4i)
(m, 6H), 2.38 (s, 3H); 13C NMR (125 MHz, CD3OD þ CDCl3, ppm):
d
161.27 (C), 157.91 (C), 153.78 (C), 151.93 (CH), 140.23 (C), 129.98
Yield, 93%; white colored solid; m.p. 218e222 ꢀC; 1H NMR
(CH), 129.98 (CH), 128.50 (C), 121.61 (CH), 120.61 (CH), 120.61 (CH),
55.99 (CH2), 55.99 (CH2), 46.55 (CH2), 46.55 (CH2), 46.29 (CH3);
HRMS: m/z calcd 327.1386 for C16H18N6S þ Hþ (327.1386).
(400 MHz, DMSO-d6, ppm):
d
8.27 (s,1H), 7.64 (d, 2H, J ¼ 8.0 Hz), 7.45
(d, 2H, J ¼ 8.0 Hz), 4.17e4.15 (m, 4H), 3.77e3.75 (m, 4H); 13C NMR
(125 MHz, DMSO-d6, ppm): d 161.10 (C), 155.95 (C), 151.91 (C), 150.84
(CH), 140.01 (C), 129.04 (CH), 129.04 (CH), 128.26 (C), 122.37 (CH),
117.77 (CH), 117.77 (CH), 66.08 (CH2), 66.08 (CH2), 45.96 (CH2), 45.96
(CH2); HRMS: m/z calcd 348.0675 for C15H14ClN5OS þ Hþ (348.0680).
3.4.6. 2-Phenylamino-7-(piperazin-1-yl)-thiazolo[5,4-d]pyrimidine
(4f)
Yield, 85%; white colored solid; m.p. 222e226 ꢀC; 1H NMR
(400 MHz, CD3OD þ CDCl3, ppm):
d
8.18 (s, 1H), 7.59 (d, 2H,
3.4.10. 2-(4-Chlorophenylamino)-7-(piperidin-1-yl)-thiazolo[5,4-d]
pyrimidine (4j)
J ¼ 8.0 Hz), 7.33 (dd, 2H, J ¼ 8.0, 15.8 Hz), 7.05 (t, 1H, J ¼ 8.0 Hz),
4.33e4.25 (m, 4H), 3.04e2.96 (m, 4H); 13C NMR (100 MHz,
Yield, 92%; yellow colored solid; m.p. 227e228 ꢀC; 1H NMR
CD3OD þ CDCl3, ppm):
d
160.60 (C), 156.13 (C), 151.87 (C), 151.80
(400 MHz, CD3OD þ CDCl3, ppm):
d 8.14 (s, 1H), 7.63 (d, 2H,
(CH), 140.17 (C), 130.71 (CH), 130.00 (CH), 130.00 (CH), 128.10 (C),
120.52 (CH), 120.52 (CH), 53.23 (CH2), 53.23 (CH2), 45.62 (CH2),
45.62 (CH2); HRMS: m/z calcd 313.1232 for C15H16N6S þ Hþ
(313.1230).
J ¼ 8.0 Hz), 7.28 (d, 2H, J ¼ 8.0 Hz), 4.21e4.20 (m, 4H), 1.76e1.72 (m,
6H); 13C NMR (100 MHz, CD3OD þ CDCl3, ppm):
d 160.61 (C), 157.13
(C), 153.78 (C), 151.81 (CH), 140.17 (C), 130.05 (C), 129.71 (CH),
129.71 (CH), 128.12 (C), 120.22 (CH), 120.22 (CH), 45.62 (CH2), 45.62

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874
871
Fig. 7. Compound 4a induced mitochondrial potential loss in human leukemia HL-60 cells. Cells (0.44 ꢂ 106/ml/24 well plate) were treated with these compounds at 5, 20, and
30 mM concentration for 24 h time period. Cells were stained with rhodamine-123 (200 nM) for 30 min and analyzed in FL-1 vs. counts channels of flow cytometer. Data are
representative of one of three similar experiments at different time period.
(CH2), 27.23 (CH2), 27.23 (CH2), 25.72 (CH2); HRMS: m/z calcd
346.0894 for C16H16ClN5S þ Hþ (346.0888).
J ¼ 8.0 Hz), 7.29 (d, 2H, J ¼ 8.0 Hz), 3.62e3.59 (m, 2H), 3.47e3.43 (m,
2H), 2.06e1.99 (m, 4H); 13C NMR (125 MHz, CD3OD þ CDCl3, ppm):
d
159.29 (C), 157.83 (C), 152.96 (C), 152.15 (CH), 140.49 (C), 130.83
3.4.11. 2-(4-Chlorophenylamino)-7-diethylamino-thiazolo[5,4-d]
pyrimidine (4k)
(C), 130.14 (CH), 130.14 (CH), 128.46 (C), 120.32 (CH), 120.32 (CH),
46.68 (CH2), 46.68 (CH2), 25.46 (CH2), 25.46 (CH2); HRMS: m/z calcd
332.0721 for C15H14ClN5S þ Hþ (332.0731).
Yield, 92%; orange colored solid; m.p. 198e202 ꢀC; 1H NMR
(400 MHz, CD3OD þ CDCl3, ppm):
d 8.20 (s, 1H), 7.52 (d, 2H,
J ¼ 8.0 Hz), 7.24 (d, 2H, J ¼ 8.0 Hz), 3.95e3.89 (m, 4H), 1.18 (d, 6H,
3.5. Cell culture, growth conditions and treatment conditions
J ¼ 8.0 Hz); 13C NMR (100 MHz, CDCl3, ppm):
d 159.96 (C), 156.03
(C), 152.67 (C), 151.84 (CH), 139.72 (C), 129.27 (CH), 129.27 (CH),
128.28 (C), 127.34 (C), 118.54 (CH), 118.54 (CH), 43.78 (CH2), 43.78
(CH2), 13.84 (CH3), 13.84 (CH3); HRMS: m/z calcd 334.0886 for
C15H16ClN5S þ Hþ (334.0888).
Human lung carcinoma cell line (A549, NCI-H322), epidermal
(A431), glioblastoma (T98G), pancreatic (MIAPaCa-2), prostate (PC-
3), human leukemia (HL-60) and breast (T47D) were procured from
European Collection of cell cultures (ECACC). The human cancer cell
lines were grown in tissue culture flasks in complete growth me-
dium (RPMI-1640/MEM/DMEM medium supplemented with 10%
3.4.12. 2-(4-Chlorophenylamino)-7-dimethylamino-thiazolo[5,4-d]
pyrimidine (4l)
fetal calf serum, 100 mg/ml streptomycin and 100 units/ml peni-
Yield, 93%; white colored solid; m.p. 212e214 ꢀC; 1H NMR
cillin) in carbon dioxide incubator (New Brunswick, USA) at 37 ꢀC,
5% CO2 and 98% RH.
(400 MHz, CD3OD þ CDCl3, ppm):
d 8.81 (s, 1H), 8.13 (d, 2H,
J ¼ 8.0 Hz), 7.85 (d, 2H, J ¼ 8.0 Hz), 4.16 (s, 6H); 13C NMR (125 MHz,
CD3OD þ CDCl3, ppm):
d 160.18 (C), 157.52 (C), 154.97 (C), 151.85
3.6. In vitro antiproliferative assay
(CH), 140.39 (C), 130.59 (C), 1309.18 (CH), 130.18 (CH), 128.46 (C),
120.53 (CH), 120.53 (CH), 40.68 (CH3), 40.68 (CH3); HRMS: m/z
calcd 306.0565 for C13H12ClN5S þ Hþ (306.0574).
Sulforhodamine B (SRB) assay was performed to determine the
in vitro antiproliferative activity. Panel of human cancer cell lines of
various tissue origin were used to evaluate the antiproliferative
activity of compounds. Cell suspension of optimum cell density
3.4.13. 2-(4-Chlorophenylamino)-7-(4-methylpiperazin-1-yl)-
thiazolo[5,4-d]pyrimidine (4m)
(7500ꢁ15,000 cells/100
plates and incubated for 24 h. Test compounds at different con-
centrations in complete growth medium (100 l) were added after
24 h of incubation along with known cytotoxic agent Erlotinib as
positive control. For preliminary screening, 10 M concentration of
test compound was used whereas for IC50 calculation 0.5, 1, 2.5, 5,
mL) was seeded in 96 well flat bottom
Yield, 89%; off white colored solid; m.p. 234e236 ꢀC; 1H NMR
(400 MHz, CD3OD þ CDCl3, ppm):
d 8.11 (s, 1H), 7.52 (d, 2H,
J ¼ 8.0 Hz), 7.24 (d, 2H, J ¼ 8.0 Hz), 4.23e4.21 (m, 4H), 2.55e2.53 (m,
m
4H), 2.29 (s, 3H); 13C NMR (125 MHz, CD3OD þ CDCl3, ppm):
m
d
161.07 (C),157.71 (C),153.58 (C),151.73 (CH),140.05 (C),130.22 (C),
129.78 (CH), 129.78 (CH), 128.37 (C), 120.41 (CH), 120.41 (CH), 55.79
(CH2), 55.79 (CH2), 46.35 (CH2), 46.35 (CH2), 46.09 (CH3); HRMS: m/
z calcd 361.0988 for C16H17ClN6S þ Hþ (361.0996).
10, 20 and 40 mM were used. Further, after 48 h incubation, cells
were fixed with ice-cold TCA for 1 h at 4 ꢀC. After 1 h, the plates
were washed five times with distilled water and allowed to air dry
followed by the addition of 100 mL of 0.4% SRB dye for 0.5 h at room
3.4.14. 2-(4-Chlorophenylamino)-7-(pyrrolidin-1-yl)-thiazolo[5,4-
d]pyrimidine (4n)
temperature. Plates were then washed with 1% v/v acetic acid to
remove the unbound SRB dye. The bound dye was solubilized by
Yield, 88%; brown colored solid; m.p. 235e237 ꢀC; 1H NMR
adding 100
mL of 10 mM Tris buffer (pH ¼ 10.4) to each well. The
(400 MHz, CD3OD þ CDCl3, ppm):
d 8.13 (s, 1H), 7.66 (d, 2H,
plates were put on the shaker for 5 min to solubilize the dye

872
B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874
Fig. 8. Effect of compounds 4k and 4a on PARP-1 and procaspase-3 expression in A549 and HL-60 cells respectively. Equal amount of protein was loaded on SDS-PAGE gel for
western blot analysis as described in experimental section. -actin was used as an internal control.
b
completely, and finally the reading was taken at 540 nm on
microplate reader (BioTek Synergy HT). IC50 was determined by
plotting OD against concentration [39].
3.9. Effect of compounds on mitochondrial membrane potential
(MMP)
Changes in mitochondrial transmembrane potential (Jmt) as a
result of mitochondrial perturbation were measured after staining
with rhodamine-123. HL-60 cells were seeded in 12 well plates and
incubated with 4a at indicated concentrations for 24 h time period.
Rhodamine-123 (200 nM) was added 30 min before termination of
experiment. Cells were collected at 400ꢂ g, washed once with PBS
and mitochondrial membrane potential was measured in FL-1
channel Vs counts on BD-FACS Calibur flow cytometer.
3.7. Cell cycle analysis by flow cytometry
A549 cells (1.2 ꢂ 105 cells/ml/six-well plate) were exposed to
1, 3 and 10 mM concentrations of compound 4k. For compound
4a, in HL-60 cell cycle analysis 5, 20, and 30 mM concentrations
were used. After 24 h incubation, cells were washed with PBS
and fixed in 70% ethanol at 20 ꢀC, overnight. Cells were thereafter
washed, digested with DNase free RNase (100 m
g/ml) at 37 ꢀC for
3.10. Apoptosis induction by western blot analysis with PARP-1 and
procaspase-3
45 min and stained with propidium iodide to determine the cell
cycle phase distribution. The DNA fluorescence was measured on
a flow cytometer FACS Calibur (Becton Dickinson, USA). Resulting
DNA distributions were analyzed by Modfit (Verity Software
House Inc., Topsham, ME) for the proportions of cells in
apoptosis, G1-phase, S-phase, and G2eM phases of the cell cycle
[40,41].
The HL-60 and A549 cells were treated with compound 4a at 5,
20 and 30
10 M concentrations respectively for 24 h. The cell lysates were
prepared by using RIPA buffer. An equal amount of protein (70 g)
mM concentrations for 24 h and compound 4k at 1, 3 and
m
m
was subjected to SDS-PAGE analysis and transferred to PVDF
membrane. The membrane was blocked with 5% non-fat milk and
probed with respective primary (PARP-1, procaspase-3) and HRP
linked respective secondary antibodies. The signals were detected
by using ECL plus chemiluminescence’s kit [43].
3.8. Fluorescence microscopy
A549 cells (1.5 ꢂ 105 cells/ml/six-well plate) were seeded and
treated with 1, 3, 5 and 10 mM concentration of compound 4k. After
24 h incubation, cells were trypsinized, PBS washed and resus-
3.11. Statistical analysis
pended in PBS. Air dried slides were fixed for 20 min in methanol
at ꢁ20 ꢀC, stained with DAPI (1
m
g/ml) and kept at 37 ꢀC for 20 min.
Data expressed as mean ꢃ SD or representative of one of three
similar experiments unless otherwise indicated. Comparisons were
made between control and treated groups or the entire intra group
using one way ANOVA with post Bonferroni test through GraphPad
Prism 5.00.288 statistical analysis software. p-values *<0.001 were
considered significant.
After PBS washing, glycerol: PBS (90:10) was used for mounting on
cover slip. Slides were then observed under Fluorescence Micro-
scope (Olympus) using UV filter at 40X magnification [42]. Similar
protocol was followed to study the nuclear morphology of HL-60
cells after treatment with compound 4a.

B. Singh et al. / European Journal of Medicinal Chemistry 70 (2013) 864e874
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4. Conclusion
In summary, we have developed
a simple, efficient and
economical protocol for the synthesis of 2-amino-7-chlorothiazolo
[5,4-d]pyrimidines which is important scaffold from medicinal
chemistry point of view. Most importantly, the present protocol is
heterogeneous, involving use of recyclable catalyst. Furthermore,
the KF/alumina catalyst is easy to prepare from bench-top chem-
icals KF and alumina which are generally available in any chemistry
laboratory. The synthesized compounds were tested for anticancer
activity from which the compounds 4a and 4k displayed promising
antiproliferative activity, particularly in leukemia and lung adeno-
carcinoma cells. The cell cycle analysis and western-blot analysis
with PARP-1 and procaspase-3 indicated that both 4a and 4k in-
duces apoptotic cell death in HL-60 and A549 cells, respectively.
The promising antiproliferative and apoptosis-inducing activity of
4a and 4k can be further utilized as medicinal chemistry lead for
future drug discovery.
Acknowledgments
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The authors gratefully acknowledge Dipika Singh and S. Ara-
vinda for analytical support. B.S. is a Senior Research Fellow
receiving financial support from ICMR, New Delhi. This research
was supported in part by a grant from the CSIR 12th FYP project
(BSC-0108).
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.039.
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