Novel 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-one derivatives as potential anti-cancer agents

Article abstract of DOI:10.1016/j.bmcl.2010.01.029

A novel series of 3,5,6-trisubstituted pyrazolo[4,3-d]pyrimidin-7-one derivatives, especially 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-ones were synthesized and evaluated for their in vitro anticancer activities against various human cancer cell line

Full text of DOI:10.1016/j.bmcl.2010.01.029

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1630–1633
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-one derivatives
as potential anti-cancer agents
Vani N. Devegowda a,b, Jung Hyun Kim a, Ki-Cheol Han a, Eun Gyeong Yang a,b, Hyunah Choo a,
a,b,
Ae Nim Pae a,b, Ghilsoo Nam a, , Kyung Il Choi
*
*
a Life Sciences Research Division, Korea Institute of Science and Technology, 39-1 Hawolgok-Dong, Seongbuk-Gu, Seoul 136-791, South Korea
b University of Science and Technology, 113 Gwahangno, Yuseong-Gu, Daejeon 305-333, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 3,5,6-trisubstituted pyrazolo[4,3-d]pyrimidin-7-one derivatives, especially 6-N-arylcar-
boxamidopyrazolo[4,3-d]pyrimidin-7-ones were synthesized and evaluated for their in vitro anticancer
activities against various human cancer cell lines. The inhibitory activities for several kinases have also
been tested. The prepared compounds library exhibited significant anticancer activity towards HT-29
colon and DU-145 prostate cancer cell lines. The structure–activity relationships of the 6-N-arylcarbox-
amidopyrazolo[4,3-d]pyrimidin-7-one scaffold at R1, R2 and R3 have been elucidated. Among the synthe-
sized compounds, 12b was the most active compound with GI50 value of 0.44 lM and 1.07 lM against
HT-29 and DU-145 cell lines, respectively, and 13a was the most selective compound towards colon can-
cer cell line.
Received 7 September 2009
Revised 8 December 2009
Accepted 13 January 2010
Available online 20 January 2010
Keywords:
6-N-Arylcarboxamidopyrazolo[4,3-d]-
pyrimidin-7-one
Anticancer activity
Structure–activity relationship
Ó 2010 Published by Elsevier Ltd.
Cancer, a diverse group of diseases characterized by uncon-
trolled growth of abnormal cells, is a major worldwide problem.
It is a fatal disease standing next to the cardiovascular disease.
Although the cancer research has led to a number of new and effec-
tive solutions, the medicines used as treatments have clear limita-
tions and unfortunately cancer is projected as the primary cause of
death in the future.1,2 Currently there is a huge scientific and com-
mercial interest in the discovery of new anticancer drugs. There-
fore the search for potent, safe and selective anticancer
compounds is a crucial aspect of modern cancer research.
Pyrazolopyrimidines have extremely rich biological activities
because of their structural similarities with purines.3 The research
by other groups have confirmed that pyrazolo[4,3-d]pyrimdin-7-
one derivatives are potent and selective inhibitors of type 5 cyclic
guanosine-30,50-monophospahate phosphodiesterase (cGMP) PDE-
54 and, as such, have utility in the treatment of erectile dysfunc-
tion.5 The other pharmacological applications such as memory
improvement,6 CNS stimulation,7 anti-inflammation,8 and treat-
ment of heart diseases9 have been reported. Substituted pyrazo-
lopyridmidinones are also useful as cardiotonic,10 herbicidal,11
anticancer12 and antiviral13 agents. Although much attention has
been paid to the complexes and the biological activities thereof,
our interests have been focused onto the relationships between
structure of pyrazolo[4,3-d]pyrimidinone and their anticancer
activities. Herein, we report the synthesis and structure–activity
relationship (SAR) of a series of 6-N-arylcarboxamidopyrazol-
o[4,3-d]pyrimidin-7-one derivatives as anti-cancer agents.
The general procedure to obtain 6-N-arylcarboxamidopyrazol-
o[4,3-d]pyrimidin-7-one derivatives is shown in Schemes 1 and 2.
Based on the consideration of simple processes and ample diver-
sity, we derived a concise strategy for synthesizing 3,5,6-trisubsti-
tuted pyrazolo[4,3-d]pyrimidin-7-one derivatives.
The synthesis of the key intermediate ethyl 4-amino-3-substi-
tuted-pyrazol-5-carboxylate 5 is depicted in Scheme 1. The diketo
ester 2 was prepared from the commercially available correspond-
ing ketone 1 and diethyl oxalate using sodium ethoxide as a base in
90–95% yield. Addition of sodium nitrite to the ester 2 in glacial
acetic acid gave the oxime 3 in 50–60% yield.14 Treatment of diketo
O
O
O
O
i
ii
OEt
R1
CO2Et
R1
EtO
O
2
1
O
X
O
O
H
N
X = NO
X = NH2
4
5
EtO
iii
R1
HO
CO2Et
iv
N
N
R1
3
Scheme 1. Synthesis of ethyl 4-amino-3-substituted-pyrazol-5-carboxylate inter-
mediate. Reagents and conditions: (i) NaOEt, EtOH, rt, overnight; (ii) Aq NaNO2,
AcOH, 1 h; (iii) hydrazine hydrate, EtOH, 3 h; (iv) In, HCl, aq THF, 4 h.
* Corresponding authors. Tel.: +82 2 958 5166; fax: +82 2 958 5189.
E-mail addresses: gsnam@kist.re.kr (G. Nam), kichoi@kist.re.kr (K.I. Choi).
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.01.029

V. N. Devegowda et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1630–1633
1631
O
The coupling reaction between acid 7 and various arylhydrazine
were carried out by activating the carboxyl group with diisopro-
pylcarbodiimide (DIC) in ethyl acetate. After completion of the
reaction, pure solid 8 was collected with a good yield of 60–
70%.19 The polyphosphoric acid (PPA) induced cyclo-condensation
of 8 in toluene resulted in the final cyclized 6-N-arylcarboxamido-
pyrazolo[4,3-d]pyrimidin-7-one derivatives 9–13 in 50–70%
yield.20 The structures of the synthesized compounds were deter-
mined by analyses with 1H NMR and 13C NMR spectra.21
All the isolated 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-
7-one derivatives were evaluated for anticancer activity against
various human cancer cell lines using doxorubicin as internal stan-
dard by SRB assay.22 The cell lines used for this study were the hu-
man lung cancer (A-549), human prostate cancer (DU-145), human
colon adenocarcinoma (HT-29), human malignant melanoma (SK-
MEL-2) and human ovarian carcinoma (SK-OV-3).
O
O
H
H
H
N
N
EtO
O
N
v
HO
O
vi
EtO
H2N
N
N
N
NH
NH
R1
R1
R1
R2
R2
6
5
7
O
O
N
H
H
N
H
N
R3
N
H
R3
viii
N
vii
N
N
N
H
N
O
O
O
R2
NH
R1
R1
R2
9, R3 = Pyr
8
10, R3 = Ph
11, R3 = Bn
12, R3 = Tolyl
13, R3 = t-Bu-Ph
Scheme 2. Synthesis of 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-one
derivatives. Reagents and conditions: (v) R2COCl, pyridine, DMAP, CH2Cl2, rt, 2 h;
(vi) 0.5 N NaOH, EtOH, 70 °C, 2.5 h; (vii) R3CONHNH2, DIC, EtOAc, rt, 3 h; (viii) PPA,
Benzene, 80 °C, overnight.
As preliminarily evaluation for anticancer activity, the inhibi-
tion percentage of selected compounds at 100 lM concentration
was evaluated against five human cancer cell lines. The results of
the assay are summarized in Table 1.
The structure–activity relationships of 6-N-arylcarboxamidopy-
razolo[4,3-d]pyrimidin-7-one scaffold have been studied based on
the in vitro cytotoxicity (Table 1). The data were divided into two
groups. The first group of compounds from 9a to 9k is comprised of
one-nitrogen atom in Y or Z (i.e., a pyridine on R3) and the second
group from 10a to 10j has carbon atoms on both Y and Z making R3
a phenyl group. All the compounds in the first group showed low
inhibition percentage within 50% on all cancer cell lines. The best
compound in the first group was 9e (R1 = 4-ClPh) with the inhibi-
tion percentages of 45.97% (HT-29), 54.41% (DU-145), 46.02%
(A-549), 39.57% (SK-MEL-2) and 42.48% (SK-OV-3). Compounds
9a, 9b and 9h having alkyl and heterocyclic functionality on R1
showed poor inhibition indicating the importance of p-chloro-
phenyl group on R1 position. The second group consisting phenyl
oxime 3 with hydrazine hydrate in EtOH yielded nitroso pyrazole 4
in 95–100%,15 which was then reduced via indium mediated reduc-
tion in the presence of HCl at room temperature in aqueous THF,16
and purified by neutral alumina column chromatography to give
55–60% yield of desired intermediate ethyl 4-amino-3-substi-
tuted-pyrazol-5-carboxylate 5. By introducing various R1 groups
at position 3 of pyrazole ring, several ethyl 4-amino-3-substi-
tuted-pyrazol-5-carboxylate 5 were synthesized and further reac-
tions were carried out.
The 4-amine group of pyrazole was acylated by using substi-
tuted carbonyl chlorides (Scheme 2). It was carried out according
to the previously reported procedure to obtain 6 in 90–95% yield.17
Hydrolysis of the ester released the free acid 7 in 95–100% yield.18
Table 1
Cytotoxicity of selected 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-one derivatives against various human cancer cell lines
Y
Z
O
N
H
N
H
N
N
N
O
R2
R1
Compounds
Substituents
R2
Cytotoxicity (% inhibition, 100 lM)
R1
Y
Z
HT-29
DU-145
A-549
SK-MEL-2
SK-OV-3
9a
9b
9c
9d
9e
9f
9g
9h
n-Pr
n-Pr
i-Bu
3-NO2Ph
3-OMePh
3-NO2Ph
3-NO2Ph
3-OMePh
3-NO2Ph
3-OMePh
3-NO2Ph
3-OMePh
3-NO2Ph
3-NO2Ph
3-NO2Ph
3-OMePh
3-NO2Ph
3-OMePh
3-NO2Ph
3-OMePh
3-NO2Ph
3-NO2Ph
3-OMePh
3-NO2Ph
—
CH
CH
CH
N
CH
N
CH
N
N
N
N
N
CH
N
ꢀ0.83
0.07
ND
15.12
45.97
24.73
28.09
11.73
12.8
7.51
9.13
9.95
11.18
ND
19.66
46.02
28.84
32.4
14.21
11.41
38.73
16.28
39.57
24.50
23.38
19.18
34.14
3.26
28.49
22.85
16.70
78.23
81.87
88.95
84.94
88.47
23.01
43.16
91.96
ND
15.11
4.04
55.29
30.98
54.41
24.86
36.99
1.66
30.08
32.77
22.46
27.76
23.56
93.82
93.54
94.25
94.10
90.81
16.05
50.54
92.51
62.79
35.77
30.19
42.48
19.28
18.22
-0.61
24.99
ND
30.15
28.82
9.96
ND
ND
86.81
81.65
77.67
17.88
35.19
ND
4-ClPh
4-ClPh
4-FPh
(4-Piperidine)Ph
2-Furan
2-Furan
2-Thiophen
2-Phenethyl
n-Pr
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
—
1.51
9i
9j
9k
14.87
10.22
18.98
33.23
20.73
ND
N
N
3.9
20.12
18.66
6.27
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
Doxorubicin
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
—
n-Pr
3-ClPh
3-ClPh
4-ClPh
4-ClPh
4-FPh
2-Furan
2-Furan
2-Phenethyl
—
89.77
90.97
87.81
87.28
92.03
21.18
49.83
89.68
60.89
ND
88.64
81.79
80.68
18.74
53.78
73.88
ND
ND
ND, not determined.

1632
V. N. Devegowda et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1630–1633
Table 2
GI50 values of 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-one derivatives
O
N
H
N
H
R3
N
N
N
O
R2
Cl
Compounds
Substituents
HT-29
DU-145
R2
R3
%Inhibition,
(100 M)
GI50
(lM)
% Inhibition,
(100 M)
GI50 (lM)
a
a
l
l
10e
10f
10k
10l
10m
10n
10o
10p
10q
11a
11b
12a
12b
13a
13b
Doxorubicin
3-NO2Ph
3-OMePh
Ph
3-OMeBn
2-Phenethyl
n-Pr
n-Bu
n-Pent
2-Cyclopentylethyl
n-Pr
cyclohexylmethyl
n-Pent
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Bn
Bn
3-Tolyl
3-Tolyl
4-t-BuPh
4-t-BuPh
—
87.81
87.28
92.03
89.26
86.49
91.09
89.48
90.14
87.29
91.38
85.53
90.54
88.67
72.35
76.77
60.89
18.45 1.5
20.36 1.7
20.56 1.6
22.08 4.0
23.44 7.58
9.26 3.27
0.71 0.29
2.26 0.94
1.80 0.59
15.23 1.44
6.39 0.80
5.37 2.40
0.44 0.31
1.06 0.51
20.57 1.89
0.85 0.05
94.25
94.10
90.21
93.19
85.59
89.37
93.05
88.68
89.61
89.68
79.78
88.76
85.86
65.17
64.16
62.79
17.09 2.3
17.90 0.9
20.10 1.3
44.92 2.719
86.72 10.08
29.24 12.15
29.13 5.97
22.10 5.71
10.17 1.98
15.96 2.54
15.09 3.71
12.86 1.51
1.07 0.67
>100
Cyclohexylmethyl
Cyclohexylmethyl
2-Cyclopentylethyl
—
21.48 0.74
0.35 0.02
ND, not determined.
a
Data are the mean of three or more experiments and are reported as mean standard error of the mean (SEM) by MTT assay.
functionality on R3 have better cytotoxic activities of over 90% inhi-
bition when compared with those of the first group compounds. In
the case of colon cancer cell line HT-29, the maximum inhibition of
92.03% was observed for 10g followed by 10d with 90.97% inhibi-
tion. For cell lines DU-145 and A-549, the maximum inhibition
were observed for 10e (94.25%, 88.64%) and 10f (94.10%, 81.79%),
respectively. In case of SK-MEL-2 cancer cell line, 91.96% inhibitory
potential was observed for 10j. Finally in the case of ovarian cancer
cell line SK-OV-3, the maximum inhibition was 86.81% for 10e. The
second series compounds such as 10c–10g and 10j which have a
substituted phenyl at R1 and a phenyl at R3 showed good inhibitory
potential. The detailed study of SAR helped to point out that the
improvements in growth inhibition of cancer cell lines can be
achieved as follows: (i) The alkyl and heterocyclicgroups at R1 weak-
en the inhibition towards all cell lines; therefore it must be excluded
in order to improve the growth inhibition. (ii) The growth inhibition
can be maximized by changing pyridine ring with phenyl group in
the region R3. (iii) In most of the cell lines, the phenyl group with
chloride at R1 increases the percentage growth inhibition.
effective as R2 in improving the activity. The GI50 values for these
compounds remained in the range of 17–20 M. Furthermore, the
l
compounds 10l and 10m having flexible phenyl ring and their ana-
logs on R2 drastically lost anticancer activity on DU-145 cell line.
In an effort to increase the anticancer activity, we introduced
aliphatic and alicylcic substituents at R2 region. Interestingly, com-
pound 10n (R2 = n-Pr, GI50 = 9.26
pared to 10k (R2 = Ph, GI50 = 20.56
our surprise, elongation of one carbon chain (compound 10o,
R2 = n-Bu, GI50 = 0.71
M) resulted in 13-fold increase in potency
lM) showed better activity com-
lM) against HT-29 cell line. To
l
compared to 10n on HT-29 cell line. However, further increase of
the chain length decreased the activity by threefold (compound
10p, R2 = n-Pen, GI50 = 2.26
lM). The increase in activity was ob-
served when the aliphatic groups on R2 were replaced by alicyclic
groups. The most active compound of the set was 12b, which
showed GI50 values of 0.44 lM and 1.07 lM against HT-29 and
DU-145 cell lines, respectively. For HT-29 cell line, most of the pre-
pared compounds having aliphatic and alicyclic chain on R2
showed potent anticancer activities, while they showed low cyto-
toxic activities against DU-145 cell line except the compound 12b.
Having identified potent anticancer compounds, we next pro-
filed them against a panel of kinases as shown in Table 3. GSK 3b
and Aurora-A kinase in the panel were effectively targeted by all
Based on the structure–activity relationship studies, R1 group
was fixed as p-chlorophenyl group which was the most effective
group for enhancing the cytotoxicity, and further modifications
were made. Selected compounds which showed over 80% inhibi-
tion at 100 lM concentration were evaluated for their GI50 values
against two cell lines, HT-29 and DU-145. GI50 values, which indi-
cate the concentration required to inhibit cancer cell proliferation
by 50% after exposure of cells to test compounds, have been deter-
mined by MTT assay22 using doxorubicin as internal standard. The
results are tabulated in Table 2.
Table 3
Kinase activity of 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-one derivatives
Kinases
Compounds (% of remaining enzyme activity at 10 lM)
10o
10p
11b
12a
12b
As shown in Table 2, the modification of R2 and R3 substituent
led to significant change in anticancer activity. Compound 12b
(R2 = cyclohexylmethyl, R3 = tolyl) was the most active compound
Aurora-A(h)
EGFR(h)a
16
86
11
9
76
8
26
113
11
7
82
6
18
85
11
95
GSK3b(h)b
PDGFR
a
(h)c
105
99
77
92
on HT-29 cell line with GI50 value of 0.44 lM. It was found that
compounds 10e, 10f and 10k with variable R2 have no major differ-
ences in GI50 values. The electron withdrawing or electron donat-
ing group attached to phenyl ring and phenyl ring itself were not
a
b
c
Human epithelial growth factor receptor.
Human glycogen synthase kinase 3-beta.
Human platelet-derived growth factor receptor alpha.

V. N. Devegowda et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1630–1633
1633
16. Lee, J. G.; Choi, K. I.; Koh, H. Y.; Kim, Y.; Kang, Y.; Cho, Y. S. Synthesis 2001, 1, 81.
17. Bartulewicz, D.; Anchim, T.; Dabrowska, M.; Nowaczek, K. M. Il Farmaco 2004,
59, 211.
18. Fischer, D. S.; Allan, G. M.; Bubert, C.; Vicker, N.; Smith, A.; Tutill, H. J.; Purohit,
A.; Wood, L.; Packham, G.; Mahon, M. F.; Reed, M. J.; Potter, B. V. L. J. Med. Chem.
2005, 48, 5749.
the five compounds with 6–11% (89–94% of inhibition) and 7–26%
(74–93% of inhibition) of remaining activity at 10 M concentra-
tion, respectively. The compounds, however, showed negligible
activity on EGFR and PDGFR kinases indicating moderate selectiv-
l
a
ity towards these kinases. Thus, it could be found that 6-N-arylcar-
boxamidopyrazolo[4,3-d]pyrimidin-7-one derivatives selectively
inhibit serine/threonine kinases rather than tyrosine kinases.
In summary, an extensive library of 6-N-arylcarboxamidopyraz-
olo[4,3-d]pyrimidin-7-one derivatives was assembled and exam-
ined for trends with respect to anticancer potency, selectivity
and SARs. The in vitro anticancer activity tests indicated that com-
pound 12b was the most cytotoxic agent against both colon and
prostate cancer cell lines. While 13a was highly selective towards
colon (HT-29) cancer cell line, a few other compounds such as 10o,
10p and 10q exhibited significant anticancer activity against HT-
29. The alicyclic and aliphatic groups on R2 were found to be vital
for potency. Further studies on the structure modification and anti-
cancer activity evaluation are in progress.
19. Hale, K. J.; Lazarides, L. Chem. Commun. 2002, 1832.
20. El-Abadelah, M. M.; Sabri, S. S.; Khanfar, M. A.; Yasin, H. A. J. Heterocycl. Chem.
2002, 39, 1055.
21. Spectral data of selected 6-N-arylcarboxamidopyrazolo[4,3-d]pyrimidin-7-one
derivatives: Compound 10o: White Solid (362 mg, 75.5%); mp 226.0; 1H NMR
(DMSO-d6, 300 MHz): d 9.62 (s, 1H, NH), 8.33 (d, J = 8.4 Hz, 2H), 8.04–7.99 (m,
2H), 7.67–7.58 (m, 5H), 2.86–2.64 (m, 2H), 1.81–1.70 (m, 2H), 1.45–1.35 (m,
2H), 0.89 (t, J = 7.3 Hz, 3H); 13C NMR (DMSO-d6, 75 MHz): d 173.2, 166.5, 157.5,
133.2, 131.7, 129.9, 129.4, 129.3, 129.3, 128.7, 128.2, 128.0, 127.0, 123.6, 33.1,
28.4, 22.0, 14.1; FABMS: m/z 422.138 [M++H]; Compound 12b: White Solid
(328.8 mg, 52.4%); mp 253.7; 1H NMR (DMSO-d6, 300 MHz): d 14.16 (s, 1H,
NH), 9.61 (s, 1H, NH), 7.85 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H), 7.59–7.40
(m, 4H), 2.43 (s, 3H), 2.22 (d, J = 6.6 Hz, 2H), 1.73–1.56 (m, 6H), 1.17–1.10 (m,
3H), 0.98–0.91 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 172.4, 163.9, 139.4,
133.9, 133.2, 129.9, 129.3, 128.8, 127.4, 124.2, 123.5, 117.0, 102.1, 43.7, 34.9,
33.0, 26.2, 25.9, 21.3; FABMS: m/z 476.185 [M++H]; Compound 13a: White Solid
(211.1 mg, 55.8%); mp 296.5; 1H NMR (DMSO-d6, 300 MHz): d 9.61 (s, 1H, NH),
7.97 (d, J = 8.1 Hz, 2H), 7.72 (d, J = 8.2 Hz, 2H), 7.65 (d, J = 8.2 Hz, 2H), 7.57 (d,
J = 8.0 Hz, 2H), 2.21 (d, J = 6.7 Hz, 2H), 1.69–1.53 (m, 6H), 1.32 (s, 9H),
1.10–1.07 (m, 3H), 0.92 (t, J = 10.6 Hz, 2H); 13C NMR (DMSO-d6, 75 MHz): d
172.4, 163.9, 155.6, 133.9, 129.5, 129.3, 128.8, 128.6, 128.2, 126.9, 126.7, 125.7,
120.9, 117.0, 43.7, 35.3, 34.9, 33.1, 31.2, 26.2, 25.9; FABMS: m/z 518.232
[M++H].
22. In vitro cytotoxicity evaluation: Cytotoxic activities of the anticancer drugs
against human cancer cell lines were investigated using the SRB assay or MTT
assay. Human lung cancer (A-549), Human colon adenocarcinoma (HT-29),
Human prostate cancer (DU-145), human ovarian cancer (SK-OV-3) and
human melanoma cancer cell lines (SK-MEL-2) were supplied from the
Korean Cell Line Bank, Seoul National University. All cell lines were grown in
RPMI 1640 (Gibco BRL) supplemented with 10% (V/V) heat inactivated Fetal
Bovine Serum (FBS) and maintained at 37 °C in a humidified atmosphere with
5% CO2. SRB assay; SRB (Sulforhodamine B) were purchased from Sigma. The
cells (3–7 ꢁ 103 cells/well) were seeded into 96-well plate. Various
concentrations of samples were added to each well in duplicate, then
incubated at 37 °C with 5% CO2 for two days such that time cells are in the
exponential phase of growth at the time of drug addition. After incubation, the
Acknowledgements
This research work was supported by the Seoul R&BD Program
(2G07340). V.N.D. thanks the Korean Government MOEHRD for
Korea Research Foundation Grant (KRF-2007-211-C00028). J.H.K.
also thanks the Korean Government for postdoctoral grant (KRF-
2007-355-C00026).
Reference and notes
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100
left for 30 min at room temperature, washed five times with tap water. The
100 of 0.4% SRB solution was added to each well and left at room
temperature for 30 min. SRB was removed and the plates washed five times
with 1% acetic acid before air drying. Bound SRB was solubilized with 200
10 mM unbuffered Tris-base solution (Sigma) and plates were left on a plate
shaker for at least 10 min. The optical density was measured using
lL of formalin solution were gently added to the wells. Microplates were
l
L
l
L
a
microplate reader (Versamax, Molecular Devices) with a 520 nm wavelength
and the anticancer effective concentration was expressed as an GI50. F(x)=
*
*
(T2 ꢀ T0)/T0 100, T2 < T0 (T2 ꢀ T0)/(C ꢀ T0) 100, T2 > T0 or T2 = T0.; MTT
assay; The cells (5 ꢁ 104 cells/mL) were seeded into 96-well plate. Various
concentrations of samples were added to each well in duplicate, then
incubated at 37 °C with 5% CO2 for two days such that time cells are in the
exponential phase of growth at the time of drug addition. Add 15
solution (Promrga, Cell Titer96) to each well. Incubate the plate at 37 °C for up
to 4 h in a humidified, 5% CO2 atmosphere. After incubation, add 100 L of the
solubilization solution/stop mix (Promrga, Cell Titer96) to each well. Allow the
plate to stand overnight in a sealed container with a humidified atmosphere at
room temperature to completely solubilize the formazan crystals. The optical
lL of the Dye
l
density was measured using
Devices) with a 570 nm wavelegth and the anticancer effective concentration
was expressed as a GI50
a microplate raeder (Versamax, Molecular
.
15. Majid, T.; Hopkins, C. R.; Pedgrift, B.; Collar, N. Tetrahedron Lett. 2004, 45, 2137.