Synthesis and biological evaluation of novel 2,4,5-substituted pyrimidine derivatives for anticancer activity

Article abstract of DOI:10.1016/j.bmcl.2008.09.067

A series of novel 2,4,5-substituted pyrimidine derivatives were synthesized and evaluated for inhibition against the human hepatocellular carcinoma BEL-7402 cancer cell line. Several compounds showed potent inhibition with an IC₅₀ value less than 0.10 μM. Structure-activity relationships for this class of compounds at the 2- and 5-position of the pyrimidine scaffold have been elucidated. The most active compound 7gc showed good inhibition of several different human cancer cell lines with IC₅₀ values from 0.024 to 0.55 μM.

Full text of DOI:10.1016/j.bmcl.2008.09.067

Bioorganic & Medicinal Chemistry Letters 19 (2009) 275–278
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel 2,4,5-substituted pyrimidine
derivatives for anticancer activity
*
*
Fuchun Xie , Hongbing Zhao , Lizhi Zhao, Liguang Lou , Youhong Hu
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Medicinal Chemistry, 555 Zu Chong Zhi Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 2,4,5-substituted pyrimidine derivatives were synthesized and evaluated for inhibition
against the human hepatocellular carcinoma BEL-7402 cancer cell line. Several compounds showed
potent inhibition with an IC50 value less than 0.10 lM. Structure–activity relationships for this class of
compounds at the 2- and 5-position of the pyrimidine scaffold have been elucidated. The most active
compound 7gc showed good inhibition of several different human cancer cell lines with IC50 values from
Received 8 August 2008
Revised 14 September 2008
Accepted 16 September 2008
Available online 21 September 2008
0.024 to 0.55 lM.
Keywords:
Pyrimidine
Anticancer
BEL-7402
Ó 2008 Elsevier Ltd. All rights reserved.
Pyrimidine is found as a core structure in a large variety of com-
pounds that exhibit important biological activity.1–9
O
B
Specifically, 2,4-disubstituted and 2,4,6-trisubstituted pyrimi-
N
A
N
dines have shown potent anticancer activity as CDK inhibitors,10
a
inhibitors,11 Abl tyrosine protein kinase inhibitors,12 PI-3
C
TNF-
kinase inhibitors,13 Akt kinase inhibitors,14 and cytokines
HO
inhibitors.15
1
The use of combinatorial approaches toward the synthesis of
drug-like scaffolds is a powerful tool in helping to speed up drug
discovery. Recently, we have developed an efficient method to gen-
erate 2,4,5-substituted pyrimidine libraries using a three-compo-
nent one-pot reaction.16 Through biological activity screening,
compound 1 (Fig. 1) was found to exhibit potent inhibition against
the human hepatocellular carcinoma cell line BEL-7402
Figure 1. Molecular structure of active compound 1.
tion of intermediate 2 with commercially available amidines, com-
pounds 1a–c were prepared. The subsequent protection of 1 with a
Bn group and then oxidation with SeO2 gave the carboxylic acid 3.
Compound 3 was refluxed with SOCl2 in methanol to afford methyl
ester 4. Both 3 and 4 were hydrogenated in the presence of 10% Pd–
C to obtain the desired products 1d and 1e. In addition, methyl es-
ter 4 was exchanged with methyl amine and dimethyl amine, fol-
lowed by deprotection of the Bn group to give pyrimidines 1f and
1g.
(IC50 = 1.02 lM) and lung cancer cell line A549 (IC50 = 2.40 lM).
Based on the activity of compound 1, a series of novel 2,4,5-pyrim-
idine derivatives were synthesized using modifications at the 2-po-
sition and ring B of the pyrimidine moiety. These compounds were
evaluated for inhibition against the human hepatocellular carci-
noma BEL-7402 cancer cell line. The structure–activity relation-
ships (SARs) for this class of compounds have been clarified.
Initial SAR data indicated aryl or bulky substitution at the 2-po-
sition of pyrimidine was unfavorable for anticancer activity.16
Therefore, we designed and synthesized compounds with less
bulky groups and different electronic effects at the 2-position to
investigate further SARs (Scheme 1). By the convenient condensa-
The activity of compound 1a is close to the parent compound 1
for against the BEL-7402 cancer cell line. Compound 1c,17a with an
amine group, exhibits good inhibition with an IC50 of 0.67 lM.
Comparing with electron-donating substituted compounds, com-
pounds 1d–g with electron-withdrawing groups at the 2-position
led to poor activity (Table 1).
Based on the preliminary SARs, the focused pyrimidine library
with modifications of ring B was designed and prepared from
iodochromone 5 using various boronic acids and acetamidine or
guanidine by a one-pot reaction (Table 2). Compounds 6a, 7a, 7b,
6c, 6d, and 7d with the different substitutions including methoxy
group at the ortho- or meta-position on the phenyl ring B, did not
* Corresponding author.
E-mail addresses: lglou@mail.shcnc.ac.cn (L. Lou), yhhu@mail.shcnc.ac.cn (Y.
Hu).
These authors contribute equally to this work.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.067

276
F. Xie et al. / Bioorg. Med. Chem. Lett. 19 (2009) 275–278
O
O
O
N
a
1a R1 = H;
R1
N
1b R1 =OMe;
1c R1 = NH2;
O
2
HO
O
d
O
e
O
N
N
N
b, c
HO
MeO
MeO
1
N
N
N
O
O
N
O
BnO
3
BnO
4
HO
1e
g, e
f, e
O
e
O
O
N
N
Me2N
HO
MeHN
N
N
N
O
O
O
HO
HO
1d
HO
1g
1f
Scheme 1. Reactions and conditions: (a) K2CO3, amidine, DMF, 70 °C; (b) BnBr, K2CO3, THF, reflux; (c) SeO2, pyridine, reflux; (d) SOCl2, MeOH, reflux; (e) 10% Pd–C, MeOH,
50 °C; (f) aq MeNH2, MeOH, reflux; (g) aq Me2NH, MeOH, reflux.
Table 1
show inhibitory activity. With Cl, Boc-protected amine, amine, hy-
2-Substitued effect of pyrimidine derivatives for BEL-7402 inhibition
droxyl and trifluromethoxy at the para-position of ring B, the com-
Compound
R1
IC
(
lM)
Compound
R1
IC
(lM)
50
50
pounds also exhibited no activity. Similar to the parent compound
1
Me
H
1.02
1.43
6.00
0.67
1d
1e
1f
CO2H
>10
>10
>10
>10
1, para-methylthio substituted compound 7j17b increased the
1a
1b
1c
CO2Me
CONHMe
CONMe2
activity with an IC50 of 0.09 lM. When bearing a methoxy group
OMe
NH2
at the para-position and a methyl group at the meta-position of
1g
the phenyl ring, compound 6k17c and 7k17d also improved activi-
ties with IC50’s of 0.10 and 0.12 lM, respectively. With methoxy
substitution at either 3,4-position or 3,4,5-position of the phenyl
ring, compounds 7l, 6m, and 7m decrease inhibitory activities.
When the aromatic ring was changed to a pyridine ring, the com-
pounds 6n, 7n, 6o, and 7o did not exhibit inhibition of the BEL-
7402 cancer cell line.
Table 2
Study on the SARs of 5-substituted pyrimidine derivatives for BEL-7402 inhibition
R2
O
NH
NH2
DBU,50-60oC
N
cat. Pd(PPh3)4
I
R2B(OH)2,K2CO3
R1
R1
N
A series of O-alkylated derivatives were prepared from interme-
diate 8 by alkylation with various alkyl halides and then condensa-
O
THF-H2O,reflux
HO
5
6 R1 = Me
7 R1 = NH2
Table 3
Synthesis and activity of 40-alkoxyl pyrimdine derivatives
Compound
R1
R2
Yield (%)
IC50 (lM)
OR2
OH
O
1. R2X, K2CO3
6a
7a
7b
6c
6d
7d
6e
6f
Me
2-FPh
2-FPh
2-OMePh
3-OMePh
3-CF3Ph
3-CF3Ph
4-ClPh
4-NHBocPh
4-NHBocPh
4-NH2Ph
4-NH2Ph
4-OCF3Ph
4-OCF3Ph
4-OHPh
4-SMePh
3-Me-4-OMePh
3-Me-4-OMePh
3,4,-Di-OMePh
3,4,5-Tri-OMePh
3,4,5-Tri-OMePh
3-Pyridinyl
3-Pyridinyl
4-Pyridinyl
4-Pyridinyl
55
50
44
51
58
52
53
45
41
85
80
57
55
40
45
52
49
55
53
47
38
35
39
34
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.09
0.10
0.12
4.10
>10
>10
>10
>10
>10
>10
N
NH2
NH2
Me
Me
NH2
Me
Me
NH2
Me
NH2
Me
NH2
NH2
NH2
Me
NH2
NH2
Me
NH2
Me
NH2
Me
NH2
DM F,40-50oC, 4h
H2N
N
2.guanidine
O
DMF, 60-70oC, 8h
8
HO
7ia-m
Compound
R2
Yield (%)
IC50 (lM)
7f
6ga
7ga
6h
7h
7i
7ia
7ib
7ica
7id
7ie
7if
Et
59
62
89
49
61
62
85
63
92
64
52
43
65
0.24
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
Allyl
Pr
iPr
Bu
7j
2-Methylallyl
7iga
7ih
7iia
7ij
7ik
7il
iBu
6k
7k
7l
6m
7m
6n
7n
6o
7o
3-Methylbut-2-enyl
Isopentyl
CH2CN
CH2CO2Et
2-(Pyrrolidin-1-yl)ethyl
CH2CH2CH2OBn
7im
a
Compounds 7ic, 7ig, and 7ii were prepared from 7ib, 7if, and 7ih by
hydrogenation.

F. Xie et al. / Bioorg. Med. Chem. Lett. 19 (2009) 275–278
277
Boc
NH
Boc
N
H
N
O
Me
O
O
O
Me
NaH, MeI
DMF, rt, 5h
TFA,DCM
rt, 1h
O
O
9
11
10
NH
NH2
K2CO3
DMF, 60-70 oC, 8h
R2
R1
R2
N
N
Me
O
Me
NH
NH2
6ga R1 = Me, R2=H;
R2I or Ac2O
K2CO3
N
7ga R1 = NH2, R2=H;
7gb R1 = NH2, R2=Me;
7gc R1 = NH2, R2=Et;
R1
R1
N
K2CO3
THF,reflux, 6h
O
DMF, 60-70 oC, 8h
HO
7gd
R
1 = NH2, R2=Ac;
12
Scheme 2.
tion with guanidine (Table 3). Only the ethoxy derivative 7ia17e
showed good inhibition with IC50 of 0.24 M. From these results,
man hepatocellular carcinoma cell line BEL-7402 and colon can-
cer line HT29.
l
we suppose a suitable less bulky electron-donating group at the
para-position of aromatic ring B will improve bioactivity.
In summary, a novel series of 2,4,5-substituted pyrimidine
derivatives were synthesized and evaluated in vitro for inhibition
against human hepatocellular carcinoma BEL-7402 cancer cell pro-
liferation. Several compounds showed potent inhibition with an
Encouraged by the further SARs on the ring B, intermediate 9
was methylated by MeI to give compound 10. The Boc group of
10 was removed by TFA to produce 11, which was directly con-
densed with acetamidine and guanidine to afford 6ga and 7ga. Fur-
thermore, compound 11 was alkylated by MeI and EtI and acylated
by Ac2O, followed by condensation with guanidine to obtain com-
pounds 7gb–d in good yields (Scheme 2).
Compound 7ga with the NH2 group at the pyrimidine 2-pois-
tion, showed better inhibition than 6ga with a methyl group. Fur-
ther, methylated compound 7gb maintained good inhibition for
the BEL-7402 cancer cell line. The N-methyl ethyl substituted com-
pound 7gc17f exhibited excellent inhibitory activity with an IC50 of
IC50 value less than 0.10 lM. From the current investigation, struc-
ture–activity relationships of those compounds suggest electron-
donating groups at the 2-position of pyrimidine will determine
the anticancer activity and para-substitution of aromatic ring B
with suitable less bulky electron-donating group will increase
the anticancer activity. Additional research on the mechanisms of
these compounds and modification is underway.
Acknowledgments
0.024
l
M for the BEL-7402 cancer cell line. However, N-acetyl
This work was supported by grants from Shanghai Commission
of Science and Technology (06PJ14112) and Chinese Academy of
Sciences (KSCX-2-YW-R-23).
methyl compound 7gd showed no activity, which is similar to
compound 7f (Table 4).
All active compounds were tested by non-cancerous cell line
WI-38 and showed no inhibition (IC50 > 10
lM). Compound 7gc
References and notes
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Table 4
Bioactivity of 40-N-substituted pyrimidine derivatives
Compound
R1
R2
IC50 (lM)
6ga
7ga
7gb
7gc
Me
H
H
Me
Et
Ac
3.40
0.32
0.20
0.024
>10
NH2
NH2
NH2
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A549
Calu-3
H460
SK-BR3
SGC-7901
HT29
7gc
Control
0.55
0.50
0.12
0.30
0.30
0.090
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123.18, 122.14, 118.07, 116.53, 110.06, 55.14, 16.05; MS (EI) m/z 307 (M+).(e)
Compound 7ia: Mp 209–211 °C; 1H NMR (CDCl3, 300 MHz) d 8.32 (s, 1H), 7.18
(dt, J = 7.8, 1.6 Hz, 1H), 7.11 (d, J = 8.5 Hz, 2H), 6.99–6.93 (m, 2H), 6.88 (d,
J = 8.6 Hz, 2H), 6.48 (dt, J = 7.5, 1.3 Hz, 1H), 5.24 (br s, 2H), 4.05 (q, J = 7.0 Hz,
2H), 1.43 (t, J = 7.0 Hz, 3H); 13C NMR (DMSO-d6, 75 MHz) d 162.45, 161.46,
159.61, 157.38, 156.21, 130.36, 130.31, 129.70, 129.55, 123.24, 122.13, 118.12,
116.51, 114.22, 62.90, 14.67; MS (EI) m/z 307 (M+).(f) Compound 7gc: Mp 236–
237 °C; 1H NMR (CDCl3, 300 MHz) d 8.34 (s, 1H), 7.18 (dt, J = 7.7, 1.6 Hz, 1H),
7.09 (dd, J = 8.1, 1.5 Hz, 2H), 7.05 (d, J = 9.0 Hz, 2H), 697 (dd, J = 8.2, 1.1 Hz, 1H),
6.68 (d, J = 8.8 Hz, 2H), 6.51 (dt, J = 7.6, 1.3 Hz, 1H), 5.06 (br s, 2H), 3.42 (q,
J = 7.03 Hz, 2H), 2.93 (s, 3H),1.14 (t, J = 7.15 Hz, 3H); 13C NMR (DMSO-d6,
75 MHz) d 161.95, 160.91, 159.85, 156.58, 147.52, 130.37, 129.31, 124.28,
122.93, 122.44, 118.01, 116.66, 112.00, 45.81, 36.98, 10.83; MS (EI) m/z 320
(M+).
17. Spectral data for representative compounds. (a) Compound 1c: Mp 199–200 °C;
1H NMR (CDCl3, 300 MHz) d 8.32 (s, 1H), 7.19 (td, J = 7.6, 1.2 Hz, 1H), 7.14 (d,
J = 8.7 Hz, 2H), 6.96 (d, J = 7.9 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 6.49 (d, J = 7.8 Hz,
1H), 5.20 (br s, 2H), 3.83 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) d 162.71, 161.55,
159.69, 158.21, 156.07, 130.51, 129.84, 129.77, 123.50, 122.37, 118.38, 116.55,
113.92, 55.10; MS (EI) m/z 293 (M+).(b) Compound 7j: Mp 223–224 °C; 1H NMR
(CDCl3, 300 MHz) d 8.31 (s, 1H), 7.28–7.09 (m, 5H), 7.02–6.91 (m, 2H), 6.50 (t,
J = 7.6 Hz, 1H), 5.21 (br s, 2H), 2.50 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) d
162.72, 161.77, 159.33, 155.77,136.35, 134.05, 130.38, 130.30, 128.95, 125.60,
123.69, 122.03, 118.29, 116.35, 14.48; MS (EI) m/z 309 (M+).(c) Compound 6k:
Mp 83–84 °C; 1H NMR (CDCl3, 300 MHz) d 8.60 (s, 1H), 7.21 (dt, J = 7.7, 1.7 Hz,
1H), 7.08–6.98 (m , 4H), 6.84 (d, J = 9.12 Hz, 1H), 6.50 (dt, J = 7.8, 1.3 Hz, 1H),
3.87 (s, 3H), 2.80 (s, 3H), 2.21 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 163.81,
161.42, 160.25, 159.63, 157.77, 132.21, 131.22, 130.96, 129.38, 128.44, 127.59,
127.46, 118.41, 118.12, 110.33, 55.30, 25.29, 16.18; MS (EI) m/z 306 (M+).(d)
Compound 7k: Mp 204–205 °C; 1H NMR (CDCl3, 300 MHz) d 8.31 (s, 1H), 7.18 (t,
J = 8.2 Hz, 1H), 7.03–6.93 (m, 4H), 6.80 (d, J = 8.0 Hz, 1H), 6.48 (t, J = 7.6 Hz, 1H),
5.16 (br s, 2H), 3.85 (s, 3H), 2.19 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) d 162.34,
161.35, 159.70, 156.31, 156.22, 130.64, 130.36, 130.31, 129.26, 127.17, 125.37,
18. The human hepatocellular carcinoma cell line BEL-7402 and gastric
adenocarcinoma cell line SGC-7901 were obtained from the Cell Bank of the
Shanghai Institute for Biological Sciences, Chinese Academy of Science
(Shanghai, China). Human lung adenocarcinoma cell line A549 and Calu-3,
colon cancer HT29, large cell lung cancer NCI-H460, breast carcinoma SK-BR3
were all purchased from the American Type Culture Collection (Manassas,
USA). The BEL-7402, SGC-7901, NCI-H460, and SK-BR3 cells were cultured in
RPMI 1640 medium. Calu-3 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM). A549 cells were cultured in Ham’s F12K medium. HT29 cells
were cultured in McCoy’s 5a medium (modified). All media were
supplemented with 10% FBS and cells were incubated at 37 °C in an
atmosphere of 5% CO2.Sulforhodamine B cell proliferation assay: Cells were
seeded in 96-well plates and then treated with either vehicle (DMSO) or
different concentrations of compounds. After 72 h of incubation, cells were
fixed with 10% trichloroacetic acid for 1 h at 4 °C, washed five times with tap
water and air-dried. Cells were stained with 0.4% (w/v) sulforhodamine B (SRB)
for 20 min at room temperature and washed five times with 1% acetic acid.
Bound SRB was solubilized with 10 mM Tris and absorbance was measured at
540 nm.