Synthesis and cytotoxicity of 8-cyano-3-substitutedalkyl-5-methyl-4- methylene-7-methoxy-3,4-dihydropyrido[4,3-d]pyrimidines

Article abstract of DOI:10.1016/j.bmcl.2011.07.067

Synthesis and cytotoxicity of 11 4-methylene pyrido[4,3-d]pyrimidines 5a-k were described. Cytotoxicity assay results showed that some compounds had much stronger antitumor activity than Fluorouracil against KB cell lines. The most active compound 5i exhi

Full text of DOI:10.1016/j.bmcl.2011.07.067

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5975–5977
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and cytotoxicity of 8-cyano-3-substitutedalkyl-5-methyl-4-methy-
lene-7-methoxy-3,4-dihydropyrido[4,3-d]pyrimidines
Wen-Yan Mo a, Yong-Ju Liang b, Yu-Cheng Gu a,c, Li-Wu Fu b, , Hong-Wu He a,
⇑
⇑
a Key Lab of Pesticide and Chemical Biology, Ministry of Education, School of Chemistry, Central China Normal University, Wuhan 430079, PR China
b State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou 510060, PR China
c Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and cytotoxicity of 11 4-methylene pyrido[4,3-d]pyrimidines 5a–k were described. Cytotoxic-
ity assay results showed that some compounds had much stronger antitumor activity than Fluorouracil
against KB cell lines. The most active compound 5i exhibited high potency against KB, CNE2, MGC-803
cell lines with IC50 values of 0.48, 0.15, 0.59 lM, respectively. The preliminary structure–activity relation-
ships indicated that the introduction of benzyl groups bearing electron-donating function groups is
Received 1 June 2011
Revised 2 July 2011
Accepted 14 July 2011
Available online 28 July 2011
favorable for the activity.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Pyrido[4,3-d]pyrimidine
Synthesis
Antitumor activity
MTT
Pyrido[4,3-d]pyrimidines display various remarkable biological
activities, such as fungicidal, antiviral, antiinflammatory, and anti-
microbial properties.1–6 And some compounds have also been dis-
covered as EGFr-TK inhibitors7–9 or DHFR inhibitors,10 which
stimulated our interest intensely.
As known so far, the reported synthesis of pyrido[4,3-d]pyrim-
idine derivatives usually involved in the formation of a pyrimidine
ring from a suitable substituted pyridine ring.11–13 However, the
preparation of the substituted pyridine or the formation of the
pyrimidine ring required relatively strict reaction condition and
long reaction time.
In our previous work, a protocol via the aza-Wittig reaction
have been developed for the construction of pyrido[4,3-d]pyrimi-
din-4-ones in order to find compounds with some particular bio-
logical functions.14 And recently, a highly efficient regioselective
intramolecular ring closure reaction was also developed and
utilized successfully for the synthesis of bioactive 4-methylene
pyrido[4,3-d]pyrimidines.15–17
Based on our previous research work, pyrido[4,3-d]pyrimidine
derivatives exhibited some interesting antitumor activity,18 which
attracted our attention. As literatures described,9,11 this class of
pyrido[4,3-d]pyrimidines are most likely to be the EGFr inhibitors.
And reports ever pointed out that electron-donating groups in
ortho-position of pyridine ring would contribute to the potency.8
So here we designed and synthesized a series of 4-methylene
pyrido[4,3-d]pyrimidines, in which the electron-donating methoxy
group was introduced to the ortho-position of pyridine and a vari-
ety of the substituents were introduced to the pyrimidine ring.
As shown in the Scheme 1, the intermediate 1 was prepared
according to the literature.16 The intermediate 1 was readily oxi-
dated by hydrogen peroxide to give intermediate 2, and then con-
verted into 7-methoxy-4-amino-pyridine
3 by nucleophilic
substitution reaction. Followed by the reaction of compound 3
with triethyl orthoformate, formamidate 4 was obtained in good
yield. Then the formamidate 4 was cyclized smoothly with alkyl
amines or ammonia at mild condition to afford title compounds
5 in moderate to good yields (Table 1).
All the structures of title compounds 5a–k were elucidated by
comprehensive 1H NMR, IR and mass spectra, together with
elemental analysis. In the 1H NMR spectra, two protons of 4-meth-
ylene group in pyrimidine are nuclear isoequivalent, appear at d
4.50–4.70 as two doublets. 2-H appears at d 7.50 as singlet,
7-OCH3 group at d 4.03 and 5-CH3 group at d 2.60. For the 13C
NMR spectra, the carbon atoms in the pyridine and pyrimidine ring
show representative bands. For example, C(5) appears at d 30 ppm,
C(7) at d 55 ppm and C(4) at d 82 ppm, and the carbon atom in
cyano group appears at d 85 ppm. The IR spectra shows typical
absorption bands at 2220 (C„N) and 1600, 1550, 1520 cmꢀ1
(C@N, C@C).
The cytotoxicity of all the title compounds were evaluated
against KB cells by MTT assay,19 and two representative com-
pounds 5i and 5j were further screened against CNE2, MGC-803
cell lines. All the IC50 values derived from the experimental data
are summarized in Tables 1 and 2. As the results listed, some
⇑
Corresponding authors. Tel.: +86 20 87343163; fax: +86 20 87343170 (L.F.);
tel./fax: +86 27 67867960 (H.H.).
E-mail address: he1208@mail.ccnu.edu.cn (H.-W. He).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.067

5976
W.-Y. Mo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5975–5977
NH2
O
NH2
O
NH2
O
NC
O
H3CS
O
NC
NC
H2O2
Na2WO4
CH3
CH3
CH3ONa
CH3OH
CH(OEt)3
Ac2O
CH3
CH3
CH3
N
H3CO
N
H3CS
N
1
CH3
3
2
2
1
CHOEt
O
R
R
N
N
N
4
N
N
N
N
N
3
OH
NC
8
NC
NC
H3CO
NC
CH2
-H2O
CH3
CH3
CH3
+
RNH2
CH3
CH3
7
H3CO
N
CH3
5
H3CO
CH3
N
H3CO
N
6
5a
5b~k
4
Scheme 1. Synthesis of pyrido[4,3-d]pyrimidines.
Table 1
Yields and in vitro cytotoxicity of pyrido[4,3-d]pyrimidines 5
benzyl bearing electron-donating groups, contributes to the antitu-
mor activity significantly. For example, the most active compounds
5i and 5j have 4-methyl or 4-methoxy benzyl groups on 3-position
of pyrimidine ring, respectively. Moreover, for the aromatic alkyl
groups, lengthening the methylene chain will decrease the activity.
For instance, compound 5k (4-ClPhCH2CH2–) shows much lower
activity than compound 5g (4-ClPhCH2–).
R
N
N
N
N
NC
NC
CH2
CH3
CH3
H3CO
N
CH3
H3CO
N
5a
5b~k
In conclusion, we synthesized a series of 4-methylene pyr-
ido[4,3-d]pyrimidines 5 and discovered two compounds 5i and 5j
with promising high potency against KB, CNE2, MGC-803 cells.
And cytotoxicity could be further improved by incorporating
appropriate functional groups.
Compd
R
Yielda (%)
IC50 against KBb,c
(lM)
5a
5b
5c
5d
5e
5f
5g
5h
—
76
67
69
62
85
83
71
54
49
65
73
—
>50
>50
>50
>50
>50
>50
20
CH3CH2–
CH3CH2CH2–
CH3(CH2)4CH2–
2-Furfuryl-CH2–
PhCH2–
4-ClPhCH2–
3-CH3PhCH2–
4-CH3PhCH2–
4-OCH3PhCH2–
4-ClPhCH2CH2–
—
Acknowledgments
37
This work was financially supported by the National Basic Re-
search Program of China (No. 2010CB126100), National Natural
Science Foundation of China (No. 20772042, 21002037), the 863
5i
5j
5k
0.48
0.67
>50
12.5
Fluorouracil
Project (No. 2006AA09Z419) and
a fellowship awarded to
a
Isolated yields based on formamidate 3.
IC50 values are presented as mean values of three independent experiments
W.-Y. Mo by Syngenta Ltd, and the research was supported partly
by the PCSIRT (No. IRT0953).
b
done in quadruplicates. Coefficients of variation were <10%.
c
KB cells represent the drug sensitive human oral carcinoma cells.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.07.067.
Table 2
Antitumor activity of representative compounds 5i–j in vitro
a
Compd
R
IC50
(lM)
References and notes
CNE2b
MGC-803c
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5i
5j
4-CH3PhCH2-
4-OCH3PhCH2-
0.15
0.18
0.59
0.67
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a
IC50 values are presented as mean values of three independent experiments
done in quadruplicates. Coefficients of variation were <10%.
b
CNE2 cells represent the human nasopharyngeal carcinoma cells.
MGC803 cells represent the human gastric carcinoma cells.
c
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compounds showed excellent antiproliferative activity against
human tumor cell lines. Among them, two representative
compounds 5i and 5j displayed much higher antitumor activity
against KB cell lines than positive control Fluorouracil. For exam-
ple, compound 5i showed pretty high potency against KB, CNE2
and MGC-803 cells with IC50 values of 0.48, 0.15, 0.59
lM, respec-
tively. And compound 5j also showed IC50 values against KB, CNE2
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and MGC-803 cells low down as 0.67, 0.18 and 0.67
respectively.
As indicated in Table 1, linear alkyl groups on the 3-position of
pyrimidine ring have no good to the cytotoxicity. However, the
introduction of aromatic alkyl groups, especially 4-substituted
lM,
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MTT solution (10
to each well of the culture plate (containing 200
incubation, the formazan crystal formed in the well was solubilized with
100 L DMSO for optical density reading at 540 nm. The inhibition rate was
calculated according to Eq. 1. The assessments of cytotoxic activity were
expressed as the concentration inhibiting 50% of cancer cell growth (IC50).
l
L, 10 mg/mL) in PBS (PBS without MTT as the blank) was fed
lL
medium). After 3 h
l
18. Ren, Q. Y.; Liang, Y. J.; He, H. W.; Fu, L. W.; Gu, Y. C. Bioorg. Med. Chem. Lett.
2009, 19, 6713.
19. Evaluation of cytotoxicity (MTT): The cancer cells harvested during the
exponential growth phase were seeded into 96-well microtitre plates at
5 ꢁ 104–1 ꢁ 105 cells/mL in fresh medium. After overnight growth, the cells
were treated with the compounds (dissolved in DMSO) at selected
concentrations for a period of 3 days. At the end of the treatment period,
IR% ¼ ð1 ꢀ ODaverage; sampleÞ=ODaverage; blank ꢁ 100%
ð1Þ