Synthesis, characterization and in vitro anti-tumor activities of novel 9-ethyl-9H-purine derivatives

Article abstract of DOI:10.1007/s10637-009-9309-6

Newer series of 9-ethyl-9H-purine derivatives (EPD) were synthesized and screened for their efficacy in inhibiting the proliferation of various tumor cells in vitro. We evaluated the effects of EPD against HeLa, SiHa, CaSki (human cervical cancer cells),

Full text of DOI:10.1007/s10637-009-9309-6

Invest New Drugs (2010) 28:754–765
DOI 10.1007/s10637-009-9309-6
PRECLINICAL STUDIES
Synthesis, characterization and in vitro anti-tumor activities
of novel 9-ethyl-9H-purine derivatives
Raghu Ningegowda & Amit Grover & Basappa & S. Ranjith &
Kanchugarakoppal S. Rangappa & B. S. Priya & S. Nanjunda Swamy
Received: 4 July 2009 /Accepted: 13 August 2009 /Published online: 16 September 2009
#
Springer Science + Business Media, LLC 2009
Summary Newer series of 9-ethyl-9H-purine derivatives
(EPD) were synthesized and screened for their efficacy in
inhibiting the proliferation of various tumor cells in vitro.
We evaluated the effects of EPD against HeLa, SiHa,
CaSki (human cervical cancer cells), LM8, LM8G7
(murine osteosarcoma cells), OVSAHO and SKOV-3
(human ovarian cancer cells). The chemical structures of
the EPD were confirmed by 1H NMR and LCMS analyses.
The inhibitory effects of EPD were studied by using
trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) and TetraColor One
reagents. Furthermore, SAR studies revealed that the
presence of trifluoromethoxy and trifluromethyl group in
4b and 4g, respectively are responsible for the significant
activity of the EPD against cervical cancer cells and the
presence of isopropoxy group in 4f has influence in
inhibiting the proliferation of osteosarcoma and ovarian
cancer cell types.
.
.
.
Keywords Purines Cytotoxicity Cervical cancer
1
.
.
.
.
Murine osteosarcoma Ovarian cancer H NMR LCMS
TetraColor One reagents
Raghu Ningegowda and Amit Grover contributed equally to the work.
:
R. Ningegowda S. Ranjith
Syngene International Ltd,
Biocon Park,
Introduction
Bangalore 560 099, Karnataka, India
Cervical cancer is a disease of significant morbidity and
mortality. It is the second most common cancer in
women worldwide and is particularly common in
developing countries, where 80% of the world’s cervical
cancer occur. Current evidence indicates that the main
cause of cervical cancer is cervical infection with certain
types of human papilloma virus (HPV) that are transmit-
ted sexually. In addition, osteosarcoma is the most
common type of malignant bone cancer, accounting for
35% of primary bone malignancies. Also, ovarian cancer
has been called "the silent killer" because it frequently
causes non-specific symptoms, which contribute to
diagnostic in late stage and poor prognosis [1]. From an
epidemiologic and cancer prevention perspective, great
strides have been made to dramatically reduce the
incidence and mortality from these diseases. Several
approaches including surgical, radiotherapeutic, and more
recently, chemoradiotherapy comprise the successful treat-
ment modalities for different types of cancer. Recently, a
A. Grover
National Centre for Biological Sciences,
Tata Institute of Fundamental Research GKVK,
Bellary Road,
Bangalore 560065, India
Basappa
Department of Chemistry, Bangalore University,
Bangalore 570006, Karnataka, India
S. Nanjunda Swamy (*)
Department of Biotechnology,
Sri Jayachamarajendra College of Engineering,
Mysore 570006, India
e-mail: nanju_chem@yahoo.com
:
K. S. Rangappa (*) B. S. Priya (*)
Department of Studies in Chemistry, University of Mysore,
Manasagangotri,
Mysore 570006, India
e-mail: rangappaks@yahoo.com
e-mail: priyabs_chem@yahoo.com

Invest New Drugs (2010) 28:754–765
755
considerable attention has been focused on identification
of synthetic and natural chemotherapeutics capable of
inhibiting and/or retarding the progress of various types of
cancer. For instance Leptomycin B and actinomycin D in
combination can induce p53-dependent apoptotic cell
death in HPV positive human cervical cancer cell lines
[2]. Additionally, a Mullerian inhibiting substance and
different peptide aptamers have also been shown to induce
apoptosis in cervical cancer cell lines [3,4]. The fused
pyrimidine derivatives such as naturally occurring purine
bases and their analogues have been focused of great
interest over many years by reason of their diversified
biological activities [5–9]. Several purine analogues
such as 2-chlorodeoxyadenosine, 2-deoxyadenosine, 3-
deoxyadenosine (cordycepin), adenosine, adenine arabi-
noside, deoxyguanosine and guanine arabinoside have
been used in chemotherapeutic regimens mainly based on
their antiproliferative and cytotoxic effects [10–17]. Some
2,6,9–tri and 2,6,8,9–tetra substituted purines display a
wide range of activities such as kinases [18], tubulin
polymerization [19] and antagonist effects to receptors
[20]. Compounds that contain biaryl group as a key
feature in the sartan family of drugs for high blood
pressure and for important natural products, such as
vancomycin antibiotics. The innocuous nature of boronic
acids which are generally non-toxic, thermally, air and
moisture stable [21,22].
Thus, more modification of biologically active purine
derivatives may lead to achieve the highly expected
effective anticancer drugs, hence we made an effort to
synthesize purine derivatives by introducing different
substitutents in the fourth position of benzene ring of
purine analogues. In the present study, we have
synthesized 9-ethyl-9H-purine derivatives (EPD) deriva-
tives and screened for their efficacy in inhibiting the
proliferation of cancer cell lines in vitro against different
tumor cell types including HeLa, SiHa, CaSki (human
cervical cancer cells), LM8, LM8G7 (murine osteosarco-
ma cells), OVSAHO and SKOV-3 (human ovarian cancer
cells).
Mass and purity were recorded on a LC-MSD-Trap-XCT.
Silica gel column chromatography was performed using
Merck 7734 silica gel (60–120 mesh) and Merck made
TLC plates. The yields are isolated yields and are the
average of two runs.
Preparation of 3-(2-chloro-9H-purin-6-ylamino)-propan-
1-ol (2)
To a stirred solution of 2,6-dichloro-9H-purine 1 (20 g,
0.1058 mol) in n-butanol (200 ml), 3-amino-1-propanol
(9.93 g, 0.1322 mol) and triethylamine (13.37 g,
0.1322 mol) were added. Heated the reaction mixture for
12 h at 120°C under nitrogen atmosphere. The solid
obtained was filtered and triturated with hexane. Yield=
18.9 g, Purity was 99.4%.
1HNMR (400 MHz, DMSO-d6, δ ppm): 1.73(m, 2H)
3.47(t, J=5.24 Hz, 4H)4.51(t, J=5 Hz, 1H) 8.08(S, 2H)
13.03(S, 1H). MS (ESI + ion): m/z =228.6
Preparation of 3-(2-Chloro-9-ethyl-9H-purin-6-ylamino)-
propan-1-ol (3)
To a solution of 3-(2-Chloro-9H-purin-6-ylamino)-
propan-1-ol 2 (15 g, 0.0659 mol) in DMF (200 ml), ethyl
iodide (12.85 g, 0.08238 mol) and anhydrous potassium
carbonate (45.47 g, 0.3295 mol) were added and the
mixture was stirred for 12 h at room temperature under
nitrogen atmosphere. DMF was distilled out and the
residue was diluted with water (250 ml), extracted using
ethyl acetate (2×250 ml). The combined organic layer
was washed with brine, dried the organic layer using
anhydrous Na2SO4, filtered and concentrated to afford a
pure white solid. Yield=14.65 g, , Purity was 99.1%.
1HNMR (400 MHz, DMSO-d6, δ ppm): 1.46(t, J=
7.2 Hz, 3H) 1.82(t, J=6.28 Hz, 2H) 3.52(q, J=5.76 Hz ,
2H) 3.66(bs, 2H) 4.25(q, J=7.12 Hz, 2H) 4.52(s, 1H) 7.78
(S, 1H), MS (ESI + ion): m/z =256.6.
General procedure for the preparation of EPD 4(a-g)
A 100 ml three necked flask equipped with a temper-
ature probe, magnetic stirrer and a septum was charged
with toluene (5 ml), water (1 ml), ethanol (1 ml) under
nitrogen atmosphere. The flask was charged with 3-
(2-chloro-9-ethyl-9H-purin-6-ylamino)-propan-1-ol 3
(250 mg, 0.00097 mol), 4-isopropoxy-phenyl boronic acid
(350 mg, 0.00735 mol), sodium carbonate (0.411 g,
0.00388 mol). Degassed the reaction mixture for about
10 min and palladium catalyst was added (0.055 g,
0.0004 mol). The reaction mixture was then allowed to
warm to 110°C and stirred for 12 h under nitrogen
atmosphere. Reaction was monitored by TLC. Solvent
Experimental
Chemistry
All reactions were performed under nitrogen. N,N-
dimethyl formamide (DMF), n-butanol and toluene were
dried using 4Ao molecular sieves overnight. H1 spectrum
was recorded at 400MHZ and melting points were
determined using SELACO-650 hot stage melting point
apparatus and were uncorrected. Spin multiplets are given
as s (singlet), d (doublet), t (triplet) and m (multiplet).

756
Invest New Drugs (2010) 28:754–765
was evaporated in vacuo. The residue was taken up in
ethyl acetate (50 ml), organic layer was washed with water
(20 ml), brine (20 ml), dried over anhydrous Na2SO4,
filtered and concentrated. The crude residue was
adsorbed on to silica gel, purified by column chroma-
tography using 9:1 chloroform: methanol as eluent.
(dd, J=2.08 Hz, 1H), 7.35 (t, J=7.88 Hz, 1H), 7.71 (bs,
1H), 7.96 (t, J=9.04 Hz, 2H), 8.16 (s, 1H). MS (ESI + ion):
m/z =341.40.
Preparation of 3-[9-ethyl-2-(4-methoxy-phenyl)-9H-purine-
6-ylamino]-propan-1-ol (4d)
Preparation of 3-[2-(4-chloro-phenyl)-9-ethyl-9H-purine-6-
ylamino]-propan-1-ol (4a)
The general experimental procedure described afforded 4d
from 3-(2-chloro-9-ethyl-9H-purin-6-ylamino)-propan-1-ol
3 (0.250 g, 0.00097 mol) in toluene (5 ml), 4-methoxy
phenyl boronic acid (0.296 g, 0.00195 mol), sodium
carbonate (0.411 g, 0.00388 mol), absolute ethanol (1 ml)
and water (1 ml) were added. Yield=0.222 g , Purity was
98.5%.
The general experimental procedure described afforded
4a from 3-(2-chloro-9-ethyl-9H-purin-6-ylamino)-propan-
1-ol 3 (0.25 g , 0.00097 mol) in toluene (5 ml), 4-chloro
phenyl boronic acid (0.304 g, 0.00195 mol), sodium
carbonate (0.411 g, 0.00388 mol), absolute ethanol
(1 ml) and water (1 ml) were added. Yield=0.165 g,
Purity 99.2%.
1HNMR (400 MHz, DMSO-d6, δ ppm): 1.46 (t, J=
7.24 Hz, 3H), 1.81 (t, J=6.52 Hz, 2H), 3.52 (t, J=6.08 Hz,
2H), 3.64 (bs, 2H), 3.82 (s, 3H), 4.23 (q, J=7.24 Hz, 2H),
4.53 (bs, 1H), 7.0 (dd, J1=1.96 Hz, J2=8.1 Hz, 1H), 7.37 (t,
J=7.92 Hz, 2H), 7.72 (bs, 1H), 7.98 (t, J=7.32 Hz, 2H),
8.16 (s, 1H). MS (ESI + ion): m/z =327.38.
1HNMR (400 MHz, DMSO-d6, δ ppm): 1.45(t, J=
7.24 Hz, 3H), 1.81 (t, J=6 Hz, 2H), 3.51 (q, J=5.52 Hz,
2H), 3.64 (bs, 2H), 4.23 (q, J=7.12 Hz, 2H), 4.52 (bs, 1H),
7.52 (d, J=8.36 Hz, 2H), 7.75 (bs, 1H), 8.17 (S, 1H), 8.39
(d, J=7.72 Hz, 2H). MS (ESI + ion): m/z =331.8
Preparation of 3-[9-ethyl-2-(4-fluoro-phenyl)-9H-purine-6-
ylamino]-propan-1-ol (4e)
Preparation of 3-[9-ethyl-2-(4-trifluoromethoxy-phenyl)-
9H-purine-6-ylamino] propan-1-ol (4b)
The general experimental procedure described afforded
4e from 3-(2-chloro-9-ethyl-9H-purin-6-ylamino)-propan-
1-ol 3 (0.250 g, 0.00097 mol) in toluene (5 ml), 4-fluoro
phenyl boronic acid (0.272 g, 0.00195 mol), sodium
carbonate (0.411 g, 0.00388 mol), absolute ethanol
(1 ml) and water (1 ml) were added. Yield=0.147 g,
Purity was 99.1%.
The general experimental procedure described afforded 4b
from 3-(2-chloro-9-ethyl-9H-purin-6-ylamino)-propan-1-ol 3
(0.250 mg, 0.00097 mol) in toluene (5 ml), 4-
trifluoromethoxy phenyl boronic acid (401 mg,
0.00195 mol), sodium carbonate (0.411 g, 0.00388 mol),
absolute ethanol (1 ml) and water (1 ml) were added. Yield=
0.197 g , Purity was 99.2%.
1HNMR (400 MHz, DMSO-d6, δ ppm): 1.45 (t, J=
7.16 Hz, 3H), 1.81 (t, J=6.36 Hz, 2H), 3.51 (q, J=5.72 Hz,
2H), 3.64 (bs, 2H), 4.23 (q, J=7.08 Hz, 2H), 4.52 (bs, 1H),
7.28 (t, J=8.56 Hz, 2H), 7.72 (bs, 1H), 8.15 (s, 1H), 8.43
(bs, 2H). MS (ESI + ion): m/z =315.34.
1HNMR (400 MHz, DMSO-d6, δ ppm): 1.45 (t, J=
7.2 Hz, 3H), 1.81 (t, J=6.4 Hz, 2H), 3.51 (q, J=5.76 Hz,
2H),3.64 (bs, 2H), 4.23 (q, J=7.16 Hz, 2H), 4.52 (bs, 1H),
7.45 (d, J=8.28 Hz, 2H), 7.7 (bs, 1H), 8.18 (S, 1H), 8.49
(d, J=8 Hz, 2H). MS (ESI + ion): m/z =381.35.
Preparation of 3-[9-ethyl-2-(4-isopropoxy-phenyl)-9H-
purine-6-ylamino]-propan-1-ol (4f)
Preparation of 3-[2-(4-ethoxy-phenyl)-9-ethyl-9H-purine-6-
ylamino]-propan-1-ol (4c)
The general experimental procedure described afforded 4f
from 3-(2-chloro-9-ethyl-9H-purin-6-ylamino)-propan-1-ol
3 (0.250 g, 0.00097 mol) in toluene (5 ml), 4-isopropoxy
phenyl boronic acid (0.350 g, 0.00195 mol), sodium
carbonate (0.411 g, 0.00388 mol), absolute ethanol (1 ml)
and water (1 ml) were added. Yield=0.170 g, Purity was
98.5%.
The general experimental procedure described afforded 4c
from 3-(2-chloro-9-ethyl-9H-purin-6-ylamino)-propan-1-ol
3 (0.250 g, 0.00097 mol) in toluene (5 ml), 4-ethoxy
phenyl boronic acid (0.323 g, 0.00195 mol), sodium
carbonate (0.411 g, 0.00388 mol), absolute ethanol (1 ml)
and water (1 ml) were added. Yield=0.212 g, Purity was
98.6%.
1HNMR (400 MHz, DMSO-d6, δ ppm): 1.29 (d, J=
5.96 Hz, 6H), 1.45 (t, J=7.20 Hz, 3H), 1.81 (t, J=6.44 Hz,
2H), 3.51 (t, J=6.12 Hz, 2H), 3.64 (bs, 2H), 4.22 (q, J=
7.20 Hz, 2H), 4.52 (bs, 1H), 4.67 (m, 1H), 6.97 (d, J=8.64 Hz,
2H), 7.61 (bs, 1H), 8.11 (s, 1H), 8.31 (d, J=8.16 Hz, 2H). MS
(ESI + ion): m/z =355.43.
1HNMR (400 MHz, DMSO-d6, δ ppm): 1.36 (t, J=
6.96 Hz, 3H), 1.45 (t, J=7.28 Hz, 3H), 1.81 (m, 2H), 3.52
(q, J=6.04 Hz, 2H), 3.64 (bs, 2H), 4.091 (q, J=6.96 Hz,
2H), 4.23 (q, J=7.24 Hz, 2H), 4.53 (t, J=4.48 Hz, 1H), 6.98

Invest New Drugs (2010) 28:754–765
757
HeLa
4a
HeLa
Control
DMSO
10µM
Control
4b
4d
4f
DMSO
10µM
50
40
30
20
10
0
50
40
30
20
10
0
50µM
50µM
100µM
250µM
100µM
250µM
0
1
2
3
4
5
0
1
2
3
4
5
Days
Days
HeLa
HeLa
Control
DMSO
10µM
Control
DMSO
10µM
4c
50
40
30
20
10
0
50
40
30
20
10
0
50µM
50µM
100µM
250µM
100µM
250µM
0
1
2
3
4
5
0
1
2
3
4
5
Days
Days
HeLa
Control
DMSO
10µM
HeLa
Control
DMSO
10µM
4e
50
40
30
20
10
0
50
40
30
20
10
0
50µM
50µM
100µM
250µM
100µM
250µM
0
1
2
3
4
5
0
1
2
3
4
5
Days
Days
HeLa
Control
DMSO
10µM
4g
45
40
50µM
35
30
25
20
100µM
250µM
15
10
5
0
0
1
2
3
4
5
Days
Fig. 1 Dose and time-dependent effect of EPD 4(a–g) on the viability of
HeLa cells by Trypan Blue assay. Approximately 1×105cells/ml were
cultured in a six well sterilized tissue culture plate in DMEM
supplemented with 10% FBS and incubated at 37°C. Compounds 4(a–
g) were added after 24 h of plating at a concentration of 10, 50, 100 and
250µM. Control treatment was performed with 0.5% final concentration
of DMSO. After every 24 h from the time of addition of compounds, cell
viability was determined by trypan exclusion assay and data was
represented as graph. Each experiment was repeated a minimum of three
times and the values obtained were plotted as a function of time
Preparation of 3-[9-ethyl-2-(4-trifluoromethyl-phenyl)-9H-
purine-6-ylamino]-propan-1-ol (4g)
1-ol 3 (0.250 g, 0.00097 mol) in toluene (5 ml), 4-trifluoro
phenyl boronic acid (0.370 g, 0.00195 mol), sodium
carbonate (0.411 g, 0.00388 mol), absolute ethanol
(1 ml) and water (1 ml) were added. Yield=0.202 g,
Purity was 98.9%.
The general experimental procedure described afforded
4g from 3-(2-chloro-9-ethyl-9H-purin-6-ylamino)-propan-

758
Invest New Drugs (2010) 28:754–765
1HNMR (400 MHz, DMSO-d6, δ ppm): 1.46 (t, J=
HeLa cells. Approximately 1×105cells/ml were seeded in a
six well tissue culture plate. HeLa cells growing in log
phase were treated with 10µM, 50µM, 100µM and 250µM
concentrations of EPD 4(a-g) after every 24 h. For the
determination of growth rate, smaller aliquots were collect-
ed in 0.5 ml tubes, trypan blue (0.4%) was added to the cell
suspension, number of cells (viable-unstained and non
viable-blue) were counted using a haemocytometer. IC50
values (concentration of compound causing 50% inhibition
of cell growth) were estimated after 72 h of treatment with
the compounds 4(a-g) Table 2. Since the compounds were
dissolved in DMSO, it was used as negative control. The
amount of DMSO used was corresponding to the DMSO in
highest concentration of compound tested, which did not
have effect on the cell lines tested, as observed in Fig. 1.
Each experiment was repeated a minimum of 3 times and
the values obtained were plotted as a function of time, as
shown in Fig. 1.
7.2 Hz, 3H), 1.82 (t, J=6.28 Hz, 2H), 3.52 (q, J=3.76 Hz,
2H), 3.66 (bs, 2H), 4.25 (q, J=7.12 Hz, 2H), 4.52 (s, 1H),
7.83 (d, J=7.96 Hz, 3H), 8.21 (bs, 1H), 8.58 (d, J=7.48 Hz,
2H). MS (ESI + ion): m/z =365.35.
Biology
Cell lines and cultures
Cervical cancer cell lines transformed with HPV-16 (CaSki
and SiHa) and HPV-18 (HeLa) were used in experiments.
All cell lines were maintained in Dulbecco's Modified
Eagle's Medium (DMEM) containing 10% FBS (GIBCO
BRL,USA), 100 U of Penicillin G/ml and 100 μg of
streptomycin/ml (Sigma–Aldrich, USA) at 37°C in a
humidified atmosphere containing 5% CO2. The murine
osteosarcoma cell line LM8G7, which has a high metastatic
potential to the liver and LM8 cells, which has a metastatic
potential to lung were maintained in DMEM supplemented
with 10% FBS, streptomycin (100 μg/ml), penicillin
(100 units/ml), 100X non-essential amino acids,
β-mercaptoethanol (50 μM), 100X sodiumpyruvate, and
L-glutamine (2 mM) at 37°C in a humidified 5% CO2
atmosphere [24]. Human ovarian cancer cells (OVSAHO)
were cultured in RPMI media supplemented with 10%
FBS, L-glutamine (2 mM), and NaHCO3 (10%). The
SKOV-3 cells were cultured in McCoy’s 5A medium
supplemented with 10% FBS and L-glutamine (2 mM).
The cells were grown to 80% confluency before passage,
and experiments were restricted to passages 5–20.
In vitro cell proliferation assay (MTT assay)
Cell survival was assessed by 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) dye reduc-
tion assay [25], which is based on the ability of active
mitochondria living cells to reduce a yellow methylthia-
zoletetrazolium salt to violet formazan salt that can be
detected spectrophotometrically. Plating of cells was
preceded by five passages to ensure log phase growth
and active transit through the cell cycle. CaSki, SiHa and
HeLa cells were harvested at 80% confluency and plated
on 96-well plates at 5,000 cells per well. After 24 h, cells
were treated with escalating doses of 4(a–g) and incubated
in 5% CO2 atmosphere with high humidity. After 72 h of
exposure with compounds, cells were washed with 1X
PBS before adding 100µl of 1: 4 diluted MTT solution
(5 mg/ml) in PBS and incubated for 4 h at 37°C. The blue
In vitro cell viability assay—Trypan blue exclusion assay
Trypan blue exclusion assay was performed to assess the
effect of newly synthesized EPD 4(a-g) on viability of
Scheme 1 Synthesis of EPD 4
(a–g)
H3C
N
N
N
N
N
N
N
(i)
N
(ii)
N
Cl
N
Cl
Cl
N
N
OH
Cl
N
N
OH
3
1
2
(iii)
Where R = -Cl, -OCF3, -OCH2CH3, OCH3, -F, -OCH(CH3)2, -CF3
H3C
N
Reagents and conditions
N
N
(i) Triethylamine, n-butanol, 3-Amino-1-propanol at 120 0C for 24 h
(ii) Ethyl Iodide, K2CO3 , DMF at room temperature
N
N
OH
(iii) Boronic acid, Na2CO3, Pd[P(Ph3)]4, H2O, EtOH, Toluene, Reflux
H
R
4(a-g)

Invest New Drugs (2010) 28:754–765
759
Table 1 Chemical structure, yield and melting point of the EPD 4(a–g)
Compound
Product
M.p
(oC)
Yield
(%)
Purity
(%)
H3C
N
N
N
134.1-135.6
66
99.2
4a
N
N
OH
Cl
C16H18ClN5O
H3C
N
4b
4c
N
N
N
N
OH
138-139.2
79
85
98.9
98.6
O
F
F
F
C17H18F3N5O2
H3C
N
N
N
104.8-106.2
N
N
OH
O
CH3
C17H23N5O2
H3C
N
4d
4e
N
N
108.1-109.2
157.6-159.4
89
59
98.5
99.1
N
N
OH
H3C
O
C17H21N5O2
H3C
N
N
N
N
N
OH
F
C
16H18F N5O
H3C
N
4f
N
N
N
N
OH
144.2-145.8
148.2-150.1
68
81
98.5
98.9
O
H3C
CH3
C19H25N5O2
H3C
N
4g
N
N
N
N
OH
F
F
F
C17H18F3 N5O
MTT formazan precipitate was then solubilized by adding
100µl of acidic isopropyl alcohol. Absorbance was
measured at 570 nm on a multiwell ELISA plate reader.
The mean absorbance of medium control served as blank
and was subtracted. IC50 values were estimated after 24 h,
48 h and 72 h exposure of compound. The experiment was
repeated a minimum of three times. The absorbance of
control cells was taken as 100% viability and the values of
treated cells were calculated as a percentage of control. All
experiments were performed in triplicates. Wherever
appropriate, the data was expressed as the mean SEM
of the number of determinations. Statistical significance of
differences between control (0μM) and compound 4(a-g)
treated cells were determined by two tailed Student’s t test.

760
Invest New Drugs (2010) 28:754–765
HeLa
120
80
40
0
4a 4b 4c 4d 4e 4f 4g
4a 4b 4c 4d 4e 4f 4g
4a 4b 4c 4d 4e 4f 4g
4a 4b 4c 4d 4e 4f 4g
0 µM
25 µM
50 µM
100 µM
Fig. 2 Effect of compounds on cell proliferation of HeLa cells by
MTT assay. After 72 h of exposure of cells with 4(a–g) at 10, 50 and
100 μM concentrations, cells were incubated with MTT (5 mg/ml) in
duplicate for 4 h in 5% CO2 at 37°C. Resulting blue formazan
precipitate was dissolved in acidic isopropanol solution and incubated
for additional 2 h. Absorbance was measured at 570 nm. Results are
presented as percentage of viable cells (the cell viability of vehicle
cells were regarded as 100%). Each experiment was repeated a
minimum of three times. The percentage cell proliferation was
calculated as a ratio of OD value of sample to the OD value of
control. The data was expressed as the mean SEM of the number of
determinations. Statistical significance of differences between control
(0μM) and compound 4(a–g) treated cells were determined by two
tailed Student’s t test. Statistical significance was defined as P<0.05
for all the tests
Statistical significance was defined as P<0.05 for all the
tests.
and followed by dilution with DMEM to yield the indicated
final concentration in 150 μl volume (final concentration of
DMSO was <0.1%). After the indicated time, 5µl of
TetraColor One was added to each well and the mixture
was incubated for an additional 2 h. The viability of cells
treated with DMSO was measured using TetraColor One.
Absorbance at 450 nm was monitored and the IC50 values of
the cells were calculated.
In vitro cell proliferation assay (TetraColor One
Reagent assay): LM8, LM8G7, OVSAHO and SKOV-3
cells were seeded at a density of 10,000 cells/well in a 96-well
plate and incubated overnight at 37°C. The cells were treated
with indicated concentrations of the compounds 4(a-g), for
additional 48 h. EPD were added by dissolving in DMSO
SiHa
120
80
40
0
4g
4a 4b 4c 4d 4e 4f 4g
4a 4b 4c 4d 4e 4f 4g
4a 4b 4c 4d 4e 4f
4a4b 4c 4d 4e 4f 4g
0 µM
25 µM
50 µM
100 µM
Fig. 3 Effect of compounds on cell proliferation of SiHa cells by
MTT assay. After 72 h of exposure of cells with 4(a–g) at 10, 50 and
100 μM concentrations, cells were incubated with MTT (5 mg/ml) in
duplicate for 4 h in 5% CO2 at 37°C. Resulting blue formazan
precipitate was dissolved in acidic isopropanol solution and incubated
for additional 2 h. Absorbance was measured at 570 nm. Results are
presented as percentage of viable cells (the cell viability of vehicle
cells were regarded as 100%). Each experiment was repeated a
minimum of three times. The percentage cell proliferation was
calculated as a ratio of OD value of sample to the OD value of
control. The data was expressed as the mean SEM of the number of
determinations. Statistical significance of differences between control
(0μM) and compound 4(a–g) treated cells were determined by two
tailed Student’s t test. Statistical significance was defined as P<0.05
for all the tests

Invest New Drugs (2010) 28:754–765
761
CaSki
120
80
40
0
4g
4a 4b 4c4d 4e 4f 4g
4a 4b 4c 4d 4e 4f 4g
4a 4b 4c 4d 4e 4f
4a 4b 4c 4d 4e 4f 4g
0 µM
25 µM
50 µM
100 µM
Fig. 4 Effect of compounds on cell proliferation of CaSki cells by
MTT assay. After 72 h of exposure of cells with 4(a–g) at 10, 50 and
100 μM concentrations, cells were incubated with MTT (5 mg/ml) in
duplicate for 4 h in 5% CO2 at 37°C. Resulting blue formazan
precipitate was dissolved in acidic isopropanol solution and incubated
for additional 2 h. Absorbance was measured at 570 nm. Results are
presented as percentage of viable cells (the cell viability of vehicle
cells were regarded as 100%). Each experiment was repeated a
minimum of three times. The percentage cell proliferation was
calculated as a ratio of OD value of sample to the OD value of
control. The data was expressed as the mean SEM of the number of
determinations. Statistical significance of differences between control
(0μM) and compound 4(a–g) treated cells were determined by two
tailed Student’s t test. Statistical significance was defined as P<0.05
for all the tests
Results and discussion
under nitrogen atmosphere to obtain the compound 3-(2-
chloro-9H-purin-6-ylamino)-propan-1-ol (2). The com-
pound 3 was obtained by reported procedure [23],
compound 2 was ethylated using ethyl iodide and N,N-
dimethylformamide as solvent in presence of potassium
carbonate. Finally, the target EPD 4(a-g) were synthesized
via Suzuki coupling, by treating the ethylated compound 3
with different substituted boronic acids, sodium carbonate,
Chemistry
The procedure for the preparation of EPD 4(a-g) involved
three steps as shown in Scheme 1. Initially, the compound
2,6-dichloro-9H-purine (1) was condensed with the amino
alcohol in the presence of triethylamine at 120°C for 24 h
LM8
0 µM
25 µM
50 µM
100 µM
Fig. 5 Effect of compounds on cell proliferation of LM8 cells by
TetraColor One reagent. Cells were seeded at a density of 10,000
cells/well in a 96-well plate and incubated overnight at 37°C. The
cells were treated with indicated concentrations of the EPD 4(a–g), for
additional 48 h. EPD were added by dissolving in DMSO and
followed by dilution with DMEM to yield the indicated final
concentration in 150 μl volume (final concentration of DMSO is
<0.1%). After the indicated time, 5µl of TetraColor One was added to
each well and the mixture was incubated for an additional 2 h. The
viability of cells treated with DMSO was measured using TetraColor
One. Absorbance at 450 nm was monitored and the IC50 values of the
cells were calculated. Each experiment was repeated a minimum of
three times. The data was expressed as the mean SEM of the number
of determinations. Statistical significance of differences between
control (0μM) and compound 4(a–g) treated cells were determined
by two tailed Student’s t test. Statistical significance was defined as
P<0.05 for all the tests

762
Invest New Drugs (2010) 28:754–765
LM8G7
0 µM
25 µM
50 µM
100 µM
Fig. 6 Effect of compounds on cell proliferation of LM8G7 cells by
TetraColor One reagent. Cells were seeded at a density of 10,000 cells/
well in a 96-well plate and incubated overnight at 37°C. The cells were
treated with indicated concentrations of the EPD 4(a–g), for additional
48 h. EPD were added by dissolving in DMSO and followed by dilution
with DMEM to yield the indicated final concentration in 150 μl volume
(final concentration of DMSO is <0.1%). After the indicated time, 5µl
of TetraColor One was added to each well and the mixture was
incubated for an additional 2 h. The viability of cells treated with
DMSO was measured using TetraColor One. Absorbance at 450 nm
was monitored and the IC50 values of the cells were calculated. Each
experiment was repeated a minimum of three times. The data was
expressed as the mean SEM of the number of determinations.
Statistical significance of differences between control (0μM) and
compound 4(a–g) treated cells were determined by two tailed Student’s
t test. Statistical significance was defined as P<0.05 for all the tests
absolute ethanol and water. The reaction mixture was
degassed for 10 mins and palladium catalyst was added.
The products obtained were in good yield in the range of 65
to 80% with 98-99% purity. All the synthesized compounds
were characterized by 1H NMR and LC-MS analyses.
Compound 2 showed, triplet for –CH3 with coupling
constant 6.82 Hz in the region of 1.82 and quartet for
–CH2- with coupling constant 7.12 Hz in the region of
OVSAHO
50 µM
0 µM
25 µM
100 µM
Fig. 7 Effect of compounds on cell proliferation of OVSAHO cells
by TetraColor One reagent. Cells were seeded at a density of 10,000
cells/well in a 96-well plate and incubated overnight at 37°C. The
cells were treated with indicated concentrations of the EPD 4(a–g), for
additional 48 h. EPD were added by dissolving in DMSO and
followed by dilution with DMEM to yield the indicated final
concentration in 150 μl volume (final concentration of DMSO is
<0.1%). After the indicated time, 5µl of TetraColor One was added to
each well and the mixture was incubated for an additional 2 h. The
viability of cells treated with DMBO was measured using TetraColor
One. Absorbance at 450 nm was monitored and the IC50 values of the
cells were calculated. Each experiment was repeated a minimum of
three times. The data was expressed as the mean SEM of the number
of determinations. Statistical significance of differences between
control (0μM) and compound 4(a–g) treated cells were determined
by two tailed Student’s t test. Statistical significance was defined as
P<0.05 for all the tests

Invest New Drugs (2010) 28:754–765
763
SKOV-3
0 µM
25 µM
50 µM
100 µM
Fig. 8 Effect of compounds on cell proliferation of SKOV-3 cells by
TetraColor One reagent. Cells were seeded at a density of 10,000
cells/well in a 96-well plate and incubated overnight at 37°C. The
cells were treated with indicated concentrations of the EPD 4(a–g), for
additional 48 h. EPD were added by dissolving in DMSO and
followed by dilution with DMEM to yield the indicated final
concentration in 150 μl volume (final concentration of DMSO is
<0.1%). After the indicated time, 5µl of TetraColor One was added to
each well and the mixture was incubated for an additional 2 h. The
viability of cells treated with DMBO was measured using TetraColor
One. Absorbance at 450 nm was monitored and the IC50 values of the
cells were calculated. Each experiment was repeated a minimum of
three times. The data was expressed as the mean SEM of the number
of determinations. Statistical significance of differences between
control (0μM) and compound 4(a–g) treated cells were determined
by two tailed Student’s t test. Statistical significance was defined as
P<0.05 for all the tests
4.25 ppm were observed. We have observed peaks in the
region ranging from 6.70 to 8.40 ppm for the EPD 4(a–g).
The chemical structures, physical data and yield of all the
compounds are given in Table 1.
viability assay the cells were counted at intervals of 24 h
till the control attained stationary phase. Results from
trypan blue assay, showed that addition of compounds 4
(a–g) affected the viability of HeLa cells and cytotoxicity
induced by the compounds 4(a–g) was dose and time
dependant Fig. 1, the EPD 4(a–g) showed significant
effect on cell viability at concentrations of 50µM, 100µM
and 250µM. It was found that the effect was maintained
even after prolonged incubation periods. The IC50 values
of the EPD 4(a–g) against the tested tumor cells are
summarized in Table 2. Compounds 4b and 4g exhibited
prominent cytotoxicity on HeLa cell viability which can
be attributed to their trifluoromethoxy and trifluromethyl
groups, respectively.
Biology
Inhibition of cell proliferation is an important property
of chemotherapeutic agents. In the present study, the
efficacy of EPD 4(a–g) against the proliferation of
various cancer cell types such as HeLa, SiHa and CaSki
was using MTT reagent as shown in Figs. 2, 3, and 4.
LM8, LM8G7, OVSAHO and SKOV-3 using TetraColor
One reagent as shown in Figs. 5, 6, 7, and 8. The
inhibitory effects of EPD 4(a–g) against the viability and
proliferation of tumor cells were evaluated. During the cell
Interestingly, some of the derivatives showed selective
cytotoxicity towards cancer cell lines. Compound 4a was
Table 2 The antiproliferative
Compound
IC50 (in µm)
effects of EPD 4(a–g) in HeLa,
SiHa, CasKi, LM8, LM8G7,
OVSAHO and SKOV-3 cells
(IC50 value in µm) based on
MTT and TetraColor One
reagents assay
HeLa
SiHa
Caski
LM8
LM8G7
OVSAHO
SKOV-3
4a
4b
4c
4d
4e
4f
50
30
80
50
45
85
35
45
50
75
20
75
50
50
75
30
41
22
65
34
59
28
84
46
100
50
>100
>100
>100
29
>100
>100
>100
46
>100
>100
>100
35
>100
>100
>100
40
75
100
40
4g
85
76
69
65

764
Invest New Drugs (2010) 28:754–765
potent against HeLa, SiHa and LM8 cells with IC50 values
of 50µM, 45µM and 41µM, respectively. Compound 4b
displayed significant antiproliferative effect on all cancer
cell lines tested with a lower IC50 values Table 2.
Compound 4d showed selective antiproliferative effect
against HeLa, SiHa and CaSki cells with an IC50 value of
50µM, where as compound 4e was effective only to HeLa
and CaSki cells with an IC50 value of 45µM and 50µM,
respectively. On the other hand 4f effectively inhibited the
proliferation of LM8, LM8G7, OVSAHO and SKOV-3
cells with IC50 values of 29µM, 46µM, 35µM and 40µM,
respectively. Compound 4g showed potent selective anti-
proliferative effect towards HeLa, SiHa and CaSki cells
with IC50 value of 35µM, 40µM and 30µM, respectively.
Structure–activity relationship (SAR) for the newly
synthesized EPD 4(a–g) revealed that the presence of
trifluoromethoxy group showed better activity than triflur-
omethyl group, which is evident from high antiproliferative
effect of 4b than 4g. Interestingly, the absence of
trifluoromethoxy group in 4c and 4d, bearing ethoxy and
methoxy groups, respectively showed a reduced anti-
proliferative activity against osteosarcoma and ovarian
cancer cell types. These results enlightened us to obtain a
better molecule, which can inhibit the proliferation of
cervical cancer cells. In addition, compound 4f bearing
isopropoxy group failed to inhibit the proliferation of
cervical cancer cells but showed significant effect on
osteosarcoma and ovarian cancer cell types. When we
explored the potency of 4a and 4e with chloro and fluoro
group, respectively attached directly to the phenyl ring, we
observed compound 4a with chloro group showed better
activity compared to 4e with fluoro group. The SAR study
revealed that the replacement of the fluoro group in 4e to
trifluoro methyl group in 4g, showed a considerable
increase in the activity. Similarly the presence of methoxy
group in 4d decreased the activity compared to the presence
of trifluoro methoxy group in 4b at the same position. Thus
the present study clearly indicated that presence of
trifluoromethoxy and trifluromethyl group respectively in
4b and 4g are responsible for the better activity of the EPD
against the cervical cancer cells and isopropoxy group in 4f
showed significant effect on osteosarcoma and ovarian
cancer cell types.
ative activity on different tumor cell types. SAR studies
revealed that the presence of trifluoromethoxy group of
purine derivatives showed potent anti-proliferative activity
against all the tumor cells tested. Although some of the
purine derivatives presented here showed modest anti-
proliferatve activities, the investigations of these structural
modifications and preliminary SAR would be helpful to
further design and develop more potent compounds.
Acknowledgements This work was supported by World Bank under
Technical Education Quality Improvement Programme (TEQUIP). SNS
thank Prof. Sudhir Krishna, National Center for Biological Sciences
(NCBS) for help related to biological studies. Basappa thanks Prof. K.
Sugahara (Hokkaido University, JAPAN) for providing the facility and
JSPS for the post doctoral fellowship. Amit G acknowledges NCBS
(TIFR) for postdoctoral fellowship. SNS express his sincere
gratitude to JSS Mahavidyapeetha and Dr. B.G. Sangameshwara
for the encouragement towards this research work. We gratefully
acknowledge Dr. Pooja Grover, Dr. Kavitha and Dr. Vinaya for
critically reading the manuscript.
References
1. Goff BA, Mandel L, Muntz HG, Melancon CH (2000) Ovarian
carcinoma diagnosis. Cancer 89:2068–2075
2. Hietanen S, Lain S, Krausz E, Blattner C, Lane DP (2000)
Activation of p53 in cervical carcinoma cells by small molecules.
Proc Natl Acad Sci 97:8501–8506
3. Butz K, Denk C, Ullmann A, Scheffner M, Hoppe-Seyler F
(2000) Induction of apoptosis in human papillomaviruspositive
cancer cells by peptide aptamers targeting the viral E6 oncopro-
tein. Proc Natl Acad Sci 97:6693–6697
4. Thanh U, Barbie David A, Barbie DT, MacLaughlin SM,
Donahoe PK (2003) Mullerian inhibiting substance inhibits
cervical cancer cell growth via a pathway involving p130 and
p107. Proc Natl Acad Sci 100:15601–15606
5. Smith KO, Galloway KS, Kennell WL, Ogilvie KK, Radotus BK
(1982) A new nucleoside analog, 9-[2-hydroxy-1-(hydroxymethyl)
ethoxyl]methyl]guanine, highly active in vitro against herpes
simplex virus types 1 and 2. Antimicrob Agents Chemother 22:55
6. Skiper HE, Montgomery JA, Thomson JR, Schabel FM (1959)
Structure-activity relationships and cross-resistance observed on
evaluation of a series of purine analogs against experimental
neoplasms. Cancer Res 19:287–308
7. Kidder GW, Dewey VC, Parks RE Jr, Woodside GL (1951)
Further evidence on the mode of action of 8-azaguanine
(guanazolo) in tumor inhibition. Cancer Res 11(3):204–211
8. Baker BR, Wood WF (1967) Irreversible enzyme inhibitors. CII.
On the mode of phenyl binding of 9-phenylguanine to guanine
deaminase and xanthine oxidase. J Med Chem 10(6):1101–1105
9. Daly JW, Padgett WL, Shamim MT (1986) Analogues of caffeine
and theophylline: effect of structural alterations on affinity at
adenosine receptors. J Med Chem 29:1305–1308
However, it could be concluded to mention that purine
derivatives with electron-withdrawing and electron-donating
substituents on the phenyl ring have good antiproliferatve
activity against the human cancer cell lines tested.
10. Chen LS, Sheppard TL (2004) Chain termination and inhibition of
Saccharomyces cerevisiae poly(A) polymerase by C-8-modified
ATP analogs. J Biol Chem 279:40405–40411
11. Honma Y, Niitsu N (2000) Vidarabine and 2-deoxycoformycin as
antileukemic agents monocytic leukemia. Leuk Lymphoma 39:57–
66
12. Levenson R, Kernen J, Housman D (1979) Synchronization of
MEL cell commitment with cordycepin. Cell 18:1073–1078
Conclusion
In the present study, we have shown that the com-
pounds 4(a–g) showed selective and strong antiprolifer-

Invest New Drugs (2010) 28:754–765
765
13. Niitsu N, Umeda M, Honma Y (2000) Myeloid and monocytoid
leukemia cells have different sensitivity to differentiation-inducing
activity of deoxyadenosine analogs. Leuk Res 24:1–9
14. Koc Y, Urbano AG, Sweeney EB, McCaVrey R (1996) Induction
of apoptosis by cordycepin in ADA-inhibited TdT-positive
leukemia cells. Leukemia 10:1019–1024
15. Niitsu N, Yamaguchi Y, Umeda M, Honma Y (1998) Human
monocytoid leukemia cells are highly sensitive to apoptosis induced
by 2’-deoxycoformycin and 2’- deoxy adenosine : association with
dATP-dependent activation of caspase-3. Blood 92:3368–3375
16. Wu JY, Zhang QX, Leung PH (2007) Inhibitory effects of ethyl
acetate extract of Cordyceps sinensis mycelium on various cancer
cells in culture and B16 in C57BL/6 mice. Phytomedicine 14:43–
49
17. Wu WC, Hsiao JR, Lian YY, Lin CY, Huang BM (2007) The
apoptotic effect of cordycepin on human OEC-M1 oral cancer cell
line. Cancer Chemother Pharmacol 60:103–111
18. Gray NS, Wodicka L, Thunnissen AM, Nornam TC, Kwon S,
Espinoza FH, Morgan DO, Barnes G, Leclerc S, Meijer L, Kim SH,
Lockhart DJ, Schultz PG (1998) Exploiting chemical libraries,
structure, and genomics in the search for kinase inhibitors. Science
281(5376):533–538
binding molecule with novel cellular effects. Nat Biotechnol
18:304–308
20. Cocuzza AJ, Chidester DR, Culp S, Fitzgerald L, Gilligan P
(1999) Use of the Suzuki reaction for the synthesis of aryl-
substituted heterocycles as corticotropin-releasing hormone
(CRH) antagonists. Bioorg Med Chem Lett 9(7):1063–1066
21. Michal Hocek, Ivan Votruba, Hana Dvoráková (2003) Synthesis
of carba-analogues of myoseverin by regioselective cross-
coupling reactions of 2,6-dichloro-9-isopropyl purine. Tetrahedron
59:607–611
22. Schweitzcr BA, Kool ET (1994) Nonpolar aromatic nucleosides
as hydrophobic isosteres of DNA nucleosides. J Org Chem
59:7238–7242
23. Pathak AK, Pathak V, Seitz LE, Suling WJ, Reynolds RC (2004)
Antimycobacterial Agents. 1. Thio Analogues of Purine. J Med
Chem 47:273–276
24. Basappa MS, Sugahara KN, Lee CM, ten Dam GB, van
Kuppevelt TH, Miyasaka M, Yamada S, Sugahara K (2009)
Involvement of chondroitin sulfate E in the liver tumor focal
formation of murine osteosarcoma cells. Glycobiology 19(7):735–
742
25. Mosmann T (1983) Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65(1–2):55–63
19. Rosania GR, Chang YT, Perez O, Sutherlin D, Dong HL,
Lockhart DJ, Schultz PG (2000) Myoseverin, a microtubule-