Synthesis and antiproliferative activity of a series of novel 6-substituted pyrido[3,2-d]pyrimidines as potential nonclassical lipophilic antifolates targeting dihydrofolate reductase

Article abstract of DOI:10.1016/j.ejmech.2017.01.033

Dihydrofolate reductase (DHFR) has been a well-recognized target for the treatment of many diseases. Based on 8,10-dideazaminopterins, which are classical antifolates that potently inhibit DHFR, we have designed a series of novel 2,4-diamino-6-substituted pyrido[3,2-d]pyrimidines. By removing the glutamate moiety and introducing lipophilic groups, we hoped to improve passive diffuse through the cell membranes. The target compounds were efficiently synthesized using one-pot procedure and evaluated in?vitro for DHFR inhibition and antitumor activity. Compounds 5e, 5h, 5i and 5k were the most potent inhibitors of recombinant human DHFR (rhDHFR) with IC₅₀values in the range 0.2–1.0?μM. Analysis using flow cytometric indicated that the effect of compound 5k on cell cycle progression was linked to induction of S phase arrest. Compounds 5g, 5h, 5i and 5k showed broad spectrum antitumor activity against four different tumor cell lines, with IC₅₀values in the range 0.07–23?μM. Molecular docking investigations showed that the trimethoyphenyl ring of compound 5k occupied a position near the cofactor-binding site in the rhDHFR-inhibitor complex, with close intermolecular contacts with Asp21, Phe31, Ser59, Ile60 and Pro61.

Full text of DOI:10.1016/j.ejmech.2017.01.033

Accepted Manuscript
Synthesis and antiproliferative activity of a series of novel 6-substituted
pyrido[3,2-d]pyrimidines as potential nonclassical lipophilic antifolates targeting
dihydrofolate reductase
Meng Wang, Jiajia Yang, Mengmeng Yuan, Liangmin Xue, Hao Li, Chao Tian,
Xiaowei Wang, Junyi Liu, Zhili Zhang
PII:
S0223-5234(17)30043-0
10.1016/j.ejmech.2017.01.033
EJMECH 9184
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 November 2016
Revised Date: 20 January 2017
Accepted Date: 21 January 2017
Please cite this article as: M. Wang, J. Yang, M. Yuan, L. Xue, H. Li, C. Tian, X. Wang, J. Liu, Z. Zhang,
Synthesis and antiproliferative activity of a series of novel 6-substituted pyrido[3,2-d]pyrimidines as
potential nonclassical lipophilic antifolates targeting dihydrofolate reductase, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.01.033.
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ACCEPTED MANUSCRIPT
OCH3
OCH3
OCH3
OCH3
NH2
N
NH2
N
N
N
OCH3
N
OCH3
H N
H N
N
2
2
IC =4.00±0.24 µM
IC50=0.29±0.02 µM
5
0
ILE7
GLU30
GLU30
1

ACCEPTED MANUSCRIPT
Synthesis and antiproliferative activity of a series of novel 6-substituted
pyrido[3,2-d]pyrimidines as potential nonclassical lipophilic antifolates
targeting dihydrofolate reductase
a
a
a
a
a
a
Meng Wang , Jiajia Yang , Mengmeng Yuan , Liangmin Xue , Hao Li , Chao Tian ,
a
a,b,**
a*
Xiaowei Wang , Junyi Liu
, Zhili Zhang
a
Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
b
Abstract
Dihydrofolate reductase (DHFR) has been a well-recognized target for the treatment of many
diseases. Based on 8,10-dideazaminopterins, which are classical antifolates that potently inhibit DHFR,
we have designed a series of novel 2,4-diamino-6-substituted pyrido[3,2-d]pyrimidines. By removing
the glutamate moiety and introducing lipophilic groups, we hoped to improve passive diffuse through
the cell membranes. The target compounds were efficiently synthesized using one-pot procedure and
evaluated in vitro for DHFR inhibition and antitumor activity. Compounds 5e, 5h, 5i and 5k were the
most potent inhibitors of recombinant human DHFR (rhDHFR) with IC values in the range 0.2
–
1.0
M. Analysis using flow cytometric indicated that the effect of compound 5k on cell cycle progression
was linked to induction of S phase arrest. Compounds 5g, 5h, 5i and 5k showed broad spectrum
antitumor activity against four different tumor cell lines, with IC values in the range 0.07 23 ꢀM.
5
0
ꢀ
–
5
0
Molecular docking investigations showed that the trimethoyphenyl ring of compound 5k occupied a
position near the cofactor-binding site in the rhDHFR-inhibitor complex, with close intermolecular
contacts with Asp21, Phe31, Ser59, Ile60 and Pro61.
Keywords: pyrido[3,2-d]pyrimidines, antifolate, dihydrofolate reductase inhibitor, anticancer
1
. Introduction
Dihydrofolate reductase (DHFR) catalyzes the reduction of 7,8-dihydrofolate to
,6,7,8-tetrahydrofolate, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as
5
cofactor. DHFR plays a major role in the biosynthesis of nucleic acids and has become an important
target for the treatment of malignant, microbial, parasitic and chronic inflammatory diseases [1–8].
1

ACCEPTED MANUSCRIPT
Classical antifolates, such as methotrexate (MTX) [9], raltitrexed (RTX) [10], pemetrexed (PMX)
[
11] and pralatrexate [12] (Fig.1), are analogs of folate containing a pterin ring, an aromatic ring and a
glutamate tail. Because of the charged glutamate tail, these inhibitors do not passively diffuse across
cell membranes and must be actively transported using the reduced folate carrier system, which entails
folate receptors (FR) [13], the reduced folate carrier [14], and/or the proton-coupled folate transporters
[
15]. Impaired active transport into cells and polyglutamylation of classic antifolate lead to drug
resistance [16 18]. To overcome this, nonclassical antifolates, which are liphphilic molecules that
–
passively diffuse into cells and without the need for folate transport systems, have been developed.
Lipophilic nonclassical antifolates, such as piritrexim [19], trimetrexate [20] and nolatrexed [21] (Fig.
1
), have demonstrated significant growth inhibition of tumor cell.
Fig. 1 Representative examples of classical and nonclassical antifolates.
Derivatives of 8,10-dideazaminopterin (Fig. 2) are very potent inhibitors of DHFR, but these
compounds share the shortcomings of classical antifolates because of their folate-like structures [22].
There has been a continuing effort in our laboratory to develop novel nonclassical antifolates based on
8
,10-dideazaminopterins. The aim of the present study was to design and synthesize more lipophilic
derivatives that lack a glutamate tail and to explore structure-activity relationships within a series of
-substituted pyrido[3,2-d]pyrimidines. Compounds that inhibit recombinant human DHFR (rhDHFR)
may be suitable for treating cancer.
We have now developed
6
an
efficient
synthesis
of
6-phenethyl
and
6
-phenylethenylpyrido[3,2-d]pyrimidines. In addition to removal of the glutamate moiety, different
2

ACCEPTED MANUSCRIPT
lipophilic substituents were introduced into the benzene ring to improve membrane permeability. To
identify more effective anticancer agents, and to investigate the structure activity relationships of
6
-phenylethenyl and phenethyl derivatives of 2,4-diaminopyrido[3,2-d]pyrimidine, both
electron-donating substituents (methyl, ethyl, isopropyl, tert-butyl, methoxy) and electron-withdrawing
halogen) groups were chosen as the substituent R. Different substituents were attached at the
(
same position on the benzene ring and same substituent was attached at different positions on the
benzene ring to examine both electronic and spatial effects of substitution on antiproliferative activity.
The activities of compounds containing carbon-carbon double bonds and single bonds were compared
to explore the effect of conformational flexibility on activity against rhDHFR. The antiproliferative
activity of the compounds was evaluated in four different cancer cell lines. Molecular docking studies
were conducted to elucidate the structure-activity relationships.
Fig. 2 Design of 6-substituted pyrido[3,2-d]pyrimidines 4a-4k and 5a-5k.
2
. Chemistry
The key to the synthesis of the target compounds is carbon-carbon bond formation, an important
and challenging procedure in organic synthesis. Previously reported syntheses of the
carbon-carbon bond of 8,10-dideazaaminopterins involve reaction of
,4-diamino-6-bromomethylpyrido[3,2-d]pyrimidine with methyl phenylacetate or benzaldehyde
2
derivatives [22-24], but activation of 6-methyl group of pteridine to 6-bromomethyl is a multi-step
procedure [24,25]. We have previously reported we an efficient one-pot reaction for construction of
3
carbon-carbon
bonds
through
C(sp )-H
bond
functionalization
of
2,4-dihydroxy-6-methylpyrido[3,2-d]pyrimidine [26]. The synthetic strategy to obtain targets of general
formulae 4 and 5 (Fig. 2) is depicted in Scheme 1. Commercially available 5-aminouracil (6) and
crotonaldehyde were cyclized in 20% HCl using the Skraup reaction to give
3

ACCEPTED MANUSCRIPT
2,4-dihydroxy-6-methylpyrido[3,2-d]pyrimidine (1) as previously described [27]. Compound 1 was
then reacted with substituted benzaldehydes in the presence of p-toluenesulfonamide using
N,N-dimethylacetamide as solvent to form
E)-2,4-dihydroxy-6-phenylethylenylpyrido[3,2-d]pyrimidines (2a-2k). Intermediates (2a-2k) were
treated with an excess of POCl in the presence of catalytic (C H ) N to provide the 2,4-dichloro
(
3
2
5 3
derivatives (3a-3k). Conversion of 3a-3k to the corresponding 2,4-diamino derivatives (4a-4k) was
achieved using a saturated solution of ammonia in dry methanol in a sealed vessel at 150 °C for 8 h.
The products were isolated as light yellow solids. Hydrogenation of the carbon-carbon double bonds in
compounds 4a-4k was carried in ethanol using 10% Pd/C as the catalyst.
o
Scheme 1. Reagents and conditions: (a) 20% HCl, reflux, 4 h (b) p-toluenesulfonamide, N,N-dimethylacetamide, 160 C, 36 h; (c) POCl ,
3
Et N, reflux, 8 h; (d) saturated NH in dry methanol, 150 °C, 8 h; (e) Pd/C, ethanol, rt, 12 h.
3
3
3. Results and discussion
3.1. In vitro antitumor screening
The antiproliferative activities of the compounds were evaluated using four human tumor cell lines:
HL-60, HeLa, A549 and H1299.
Table 1
a
IC values (ꢀM) of 6-substituted pyrido[3,2-d]pyrimidines 4a-4k and 5a-5k against HL-60, HeLa, A549 and H1299
5
0
cell lines.
Compounds
X-Y
R
HL-60
HeLa
A549
H1299
4

ACCEPTED MANUSCRIPT
4
4
a
CH=CH
CH=CH
CH=CH
CH=CH
H
3.85±0.05
4.54±0.50
1.19±0.04
1.04±0.23
13.4±0.9
14.5±3.1
9.86±0.48
10.5±0.1
13.9±0.7
18.9±1.5
9.13±0.24
10.7±0.9
15.2±0.4
ND
’
b
4 -CH
3
’
4
c
4 -C
2
H
5
4.86±0.11
11.2±0.3
’
4
d
4 -CH(CH )
3 2
’
4
e
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
4 -F
2.61±0.04
3.80±0.40
2.51±0.84
1.63±0.13
2.03±0.04
3.60±0.28
2.33±0.59
0.36±0.01
0.31±0.03
0.21±0.02
0.49±0.07
0.58±0.03
0.27±0.09
0.12±0.01
0.0720±0.009
0.42±0.01
0.80±0.01
0.42±0.01
0.0227±0.0004
1.62±0.03
7.98±0.17
7.60±0.85
9.20±0.47
6.50±0.34
18.8±0.9
13.8±0.6
2.10±0.01
8.61±0.15
52.4±1.5
49.4±1.8
14.0±2.4
54.0±2.1
5.30±0.07
4.30±0.81
10.5±0.2
14.3±0.2
10.4±0.1
2.90±0.20
>100
6.78±0.06
8.17±0.96
11.9±0.9
5.68±0.21
9.10±0.72
7.56±0.24
5.34±0.10
9.78±0.45
34.1±0.9
15.4±0.7
15.9±1.3
4.98±0.46
16.3±1.5
22.6±2.1
18.0±0.3
14.4±0.4
29.9±0.8
11.6±0.5
5.99±0.06
9.39±0.94
6.19±0.02
3.73±0.27
12.2±0.5
8.63±0.13
3.99±0.23
9.16±0.36
1.61±0.09
2.76±0.01
ND
’
4
f
4 -Cl
’
4
4
g
4 -Br
’
h
4 -OCH
3
’
4
i
3 -OCH
3
’
4
j
2 -OCH
3
’
‘
’
4k
5a
5b
3 ,4 ,5 -(OCH )
3 3
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
H
2
2
2
2
2
2
2
2
2
2
2
’
4 -CH
3
’
5
c
4 -C
2
H
5
’
5
d
4 -CH(CH )
3 2
’
5
e
4 -F
14.8±2.0
1.06±0.04
1.49±0.03
2.15±0.13
7.03±0.05
4.73±0.21
2.17±0.04
’
5
f
4 -Cl
’
5
5
g
4 -Br
’
h
4 -OCH
3
’
5
i
3 -OCH
3
’
5
j
2 -OCH
3
’
‘
’
5
k
3 ,4 ,5 -(OCH )
3 3
MTX
-FU
0.0135±0.0020 0.0720±0.0023
ND 8.91±0.11
5
62.2±1.2
a
Calculated from three replicates
ND (not detected)
Both the position and nature of the substituent influenced the antiproliferative activity (Table 1).
,4-Diamino-6-phenethylpyrido[3,2-d]pyrimidine 5a had greater antitumor activity than
2
(E)-2,4-diamino-6-styrylpyrido[3,2-d]pyrimidine 4a against all four cancer cell lines. With the
’
exception of compound 4b, alkyl groups (-CH , -C H and -CH(CH ) ) at the 4 -position of the benzene
3
2
5
3 2
5

ACCEPTED MANUSCRIPT
ring in the 6-styryl derivatives were found to enhanced antitumor activity. Introduction of
’
electron-withdrawing substituents at the 4 -position of the benzene ring (F, Cl, Br, 4e-4g) or
’
’
’
electron-donating substituents (4 -OCH , 3 -OCH , 2 -OCH , 4h-4j) also increased antiproliferative
3
3
3
activity in all cell lines. Compound 4k with three methoxy groups on the benzene ring had the best
overall activity with IC50 values of 2.33, 2.10, 5.34 and 3.99 ꢀM against HL-60, HeLa, A549 and
H1299 cells, respectively.
With the exception of compound 5e, 6-phenethyl derivatives (5a-5k) showed better antitumor
activity than the corresponding 6-phenylethenyl derivatives (4a-4k) against HL-60 and H1299 cells.
For example, compounds 5f-5h inhibited growth of HL-60 with IC values of 0.27, 0.12 and 0.072 ꢀM,
5
0
respectively. These 6-phenethyl derivatives are thus approximately 14-, 21- and 23-fold more active
than the corresponding 6-phenylethenyl derivatives 4f-4h. Similarly, compounds 5f and 5g, with IC50
values against H1299 cells of 1.49 and 2.15 ꢀM respectively are approximately 9 and 4 times more
’
potent than compounds 4f and 4g in this cell line. Incorporation of alkyl groups at the 4 -position of the
benzene ring in the 6-phenethyl derivatives (5b-5d) decreased antitumor activity against HeLa and
A549 cells but improved activity against H1299 cells. In comparison with compound 5a, compounds 5f
’
’
(
4 -Cl) and 5g (4 -Br) exhibited higher antiproliferative activity against all except A549 cells with IC
50
’
’
’
values in the range 0.12-5.30 ꢀM. The presence of a methoxy group at the 4 , 3 or 2 -position of the
benzene ring in the 6-phenethyl derivatives decreased antiproliferative activity against HeLa and A549
cells, but increased activity against H1299 cells. Compound 5h inhibited the growth of HL-60 cells
with an IC value of 0.072 ꢀM and was thus approximately 6-fold more potent than compound 5k (Fig.
5
0
3
).
1
00
100
80
a)
b)
8
6
4
2
0
0
0
0
0
60
40
20
0
0
.01
0.1
1
10
100
0.01
0.1
1
10
100
compound 5h
compound 5k
Fig. 3 Inhibition growth of HL-60 cells by compounds 5h (a) and 5k (b). Cell proliferation inhibition was measured over a range of
6

ACCEPTED MANUSCRIPT
concentrations of compounds 5h and 5k in RPMI1640 medium. Cell densities were measured using an MTS assay. Results were
normalized to cell density in the absence of drug. Results shown are representative data of experiments performed in triplicate.
3.2. DHFR inhibition
Compounds 4a-4k and 5a-5k with structural modifications of the benzene ring were evaluated as
inhibitors of rhDHFR as previously described [28-30]. Results are reported as IC values (Table 2).
5
0
6-Phenethyl derivatives with a flexible saturated carbon-carbon linker showed better rhDHFR inhibition
than 6-phenylethenyl derivatives. For example, compounds 5f, 5g, 5h, 5i and 5k showed rhDHFR
inhibitory potency with IC values of 2.84, 1.26, 0.71, 0.45 and 0.29 ꢀM, respectively, and were thus
50
approximately 6-, 12-, 6-, 9- and 14-fold more potent than the corresponding compounds 4f, 4g, 4h, 4i
and 4k with a less flexible unsaturated linker. The inhibitory activity of other 6-phenethyl derivatives
was also improved to varying degrees, relative to the corresponding 6-phenylethenyl derivatives.
6-Phenethyl derivatives with a methoxyl group at the 4’- or 3’-position of the benzene ring (compounds
5h and 5i) were more potent inhibitors of rhDHFR than compound 5a.
Table 2
IC values (ꢀM) of 6-substituted pyrido[3,2-d]pyrimidines 4a-4k and 5a-5k against rhDHFR.
a
5
0
Compounds
X-Y
R
H
DHFR
Compounds
X-Y
R
H
DHFR
inhibition
5.33±0.41
inhibition
2.25±0.20
4
4
a
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
5a
5b
5c
5d
5e
5f
CH
2
2
2
2
2
2
2
CH
2
2
2
2
2
2
2
’
’
b
4 -CH
3
3.19±0.76
5.28±1.12
20.1±1.1
4.78±0.98
16.9±3.2
14.8±3.5
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
4 -CH
3
2.55±0.45
2.48±0.10
19.0±0.6
’
’
4
c
4 -C
2
H
5
4 -C
2
H
5
’
’
4
d
4 -CH(CH
3
)
2
4 -CH(CH )
3 2
’
’
4
e
4 -F
4 -F
0.95±0.21
2.84±0.52
1.26±0.08
’
’
4
f
4 -Cl
4 -Cl
’
’
4
g
4 -Br
5g
4 -Br
7

ACCEPTED MANUSCRIPT
’
’
4
h
CH=CH
CH=CH
CH=CH
CH=CH
4 -OCH
3
4.34±0.71
4.13±0.34
5h
5i
CH
CH
CH
CH
2
CH
CH
CH
CH
2
4 -OCH
3
0.71±0.10
0.45±0.06
1.67±0.18
0.29±0.02
’
’
4
i
3 -OCH
3
3
2
2
2
2
3 -OCH
3
3
’
’
4
j
2 -OCH
3.61±0.92
5j
2 -OCH
’
‘
’
’
‘
’
4
k
3 ,4 ,5 -(OCH
3
)
3
4.00±0.24
5k
2
2
3 ,4 ,5 -(OCH )
3 3
MTX
0.0309±0.0010
a
Calculated from three replicates
3.3. Docking studies of compounds 4k and 5k
To explore possible structural reasons for the different rhDHFR inhibitory activities of
6-phenylethenylpyrido[3,2-d]pyrimidine (4k) and 6-phenethylpyrido[3,2-d]pyrimidine (5k), the two
compounds were docked into the binding site of the known structure of rhDHFR. Compounds 4k and
5k have the same trimethoxy substitution pattern on the benzene ring and differ only in the nature of the
9-10 carbon-carbon bond (double bond in 4k and single bond in 5k). The docked poses of compound
5k (green) in human DHFR (PDB 4QJC) (Fig. 4 A) shows the 6-phenethylpyrido[3,2-d]pyrimidine
6
6
scaffold buried deep in the active site and occupying the same location as N -methyl-N -(3,4,5-
trifluorophenyl)-2,4,6-triamine pyrido[2,3-d]pyrimidine [31], (Supplementary material, Fig. 1). This
orientation of the scaffold permits the N1 nitrogen and 2-NH moiety to form hydrogen bonds to the
2
backbone Glu30. The 4-NH group also forms a hydrogen bond with the side chain of Ile7. The
2
trimethoxyphenyl ring of compound 5k occupies a position near the cofactor-binding site, with close
intermolecular contacts with Asp21, Phe31, Ser59, Ile60 and Pro61 (Supplementary material, Fig. 2).
The docked pose of compound 4k (Fig. 4 B) shows that the 6-phenylethenylpyrido[3,2-d]pyrimidine
group is less well accommodated in the active site, compared with compound 5k which contains a
flexible carbon-carbon linker. This could partially explain the greater potency of compound 5k against
DHFR compared with compound 4k.
8

ACCEPTED MANUSCRIPT
A
B
Fig. 4 Docking of compound 5k (A) and compound 4k (B) into the active site of rhDHFR (PDB code 4QJC).
3.4. Effects of compound 5k on cell cycle progression in HL-60 cells
To elucidate the mechanism by which compound 5k inhibits cell proliferation, its effect of on cell
cycle progression and apoptosis were evaluated using flow cytometry. Treatment of HL-60 cells with
compound 5k significantly increased the population of cells in S phase (Fig. 5). The proportion of cells
in S phase increased by 4.3%, 5.6%, 14.5%, 18.9% and 29.8% with concentrations of 0.05, 0.1, 0.2, 0.4
and 0.8 ꢀM of compound 5k, respectively. The percentage of cells in G /G phase was also markedly
1
2
reduced suggesting that inhibition of proliferation by compound 5k may be associated with induction of
S phase arrest and induction of apoptosis.
a)
b)
9

ACCEPTED MANUSCRIPT
c)
d)
e)
f)
1
0

ACCEPTED MANUSCRIPT
G /M
g)
2
1
00
2
%
4
%
S
G1
7
%
11%
63%
10%
1
2%
9%
8
6
4
2
0
0
0
0
0
5
64%
7
3%
0%
78%
88%
29%
26%
26%
22/0.1
2
18%
10%
DMSO
22/0.05
22/0.2
22/0.4
22/0.8
Compound Concentration
ꢀM)
Cell cycle distribution (%)
G1 G /M
(
S
2
DMSO
29.1±0.5 58.7±0.8 12.2±1.1
26.3±1.0 63.0±2.3 10.7±1.4
25.7±0.7 64.3±0.70 10.0±0.3
5k
5k
5k
5k
5k
0.05
0.1
0.2
0.4
0.8
20.2±0.5 73.2±0.3
18.0±1.4 77.6±1.5
10.0±0.5 88.5±0.4
6.6±0.8
4.4±0.4
1.5±0.3
Fig. 5 Cell cycle assay. HL-60 cells were untreated (DMSO) or treated with compound 5k (0.05, 0.1, 0.2, 0.4 or 0.8 ꢀM) for 24 h, washed,
fixed, and stained with propidium iodide. Cell cycle distributions were analyzed by flow cytometry. Cell cycle distributions: a) DMSO, b)
0
.05 ꢀM compound 5k, c) 0.1 ꢀM compound 5k, d) 0.2 ꢀM compound 5k, e) 0.4 ꢀM compound 5k, f) 0.8 ꢀM compound5k, g)
Percentage of cells in each phase of the cell cycle (G , S, and G ).
1
2
4. Conclusion
A series of novel 2,4-diamino-6-substituted pyrido[3,2-d]pyrimidines were designed and
synthesized using an efficient one-pot reaction. Compounds 5e, 5h, 5i and 5k were the most potent
rhDHFR inhibitors, with IC values in the range 0.29–1.0 ꢀM. Compounds 5g-5i and 5k showed broad
5
0
spectrum antitumor activity, with IC values in the range 0.07-23 ꢀM. Docking studies indicated that a
5
0
flexible carbon-carbon linker (single bond rather than double bond) was more important for good
rhDHFR inhibition than the nature or position of substituents in the benzene ring. Compound 5k led to
G /M checkpoint activation and S arrest of HL-60 cells, followed by DNA damage.
1
1
1

ACCEPTED MANUSCRIPT
5. Experimental section
5.1. Chemistry
o
All evaporations were carried out in vacuo with a rotary evaporator. Melting points ( C) were
1
13
determined on SGW® X-4 melting point apparatus and are uncorrected. H, C NMR spectra were
recorded on a Bruker Avance III 400 MHz NMR spectrometer; chemical shifts are expressed in δ ppm
with reference to TMS; s =singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad singlet.
High-resolution electrospray ionization (HR-ESI) mass spectra were recorded on a Waters Xevo G2
Q-TOF mass spectrometer. Thin-layer chromatography (TLC) was carried out on Silica Gel GF254
plates, and the spots were visualized under 254 and 365 nm illumination. Silica gel (200-300 mesh) was
employed for routine column chromatography separations.
5.1.1. General procedure for the synthesis of intermediates 3a-3k
A mixture of compounds 2a-2k (1 g), phosphorous oxychloride (20 mL) and triethylamine (1 mL)
was heated at reflux for 8 h then evaporated in vacuo. The residue was poured onto ice and extracted
with dichloromethane. The organic phase was dried over magnesium sulphate and concentrated in
vacuo. Purification by column chromatography (petroleum ether/ethyl acetate, 3/1) afforded the title
compound as a yellow solid.
5.1.1.1 (E)-2,4-Dichloro-6-styrylpyrido[3,2-d]pyrimidine (3a)
1
Yield 70%, mp: 168-169 °C; H NMR (400 MHz, CDCl ) δ: 8.24 (d, J=8.0 Hz, 1H, 8-CH), 8.08 (d,
3
’
’
J=8.0 Hz, 1H, 7-CH), 7.87 (d, J=16.0 Hz, 1H, 10-CH), 7.68 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.48-7.39
2
’
’
’
13
(
m, 4H, 9-CH, 3 ,5 -(CH) & 4 -CH); C NMR (100 MHz, CDCl ) δ: 165.07, 158.95, 154.54, 148.15,
2
3
138.39, 136.89, 135.99, 135.52, 129.75, 128.98, 128.66, 127.78, 126.69; LRMS (EI): m/z calcd for
+
C H Cl N [M ], 301.0; found, 301.0.
15
9
2
3
5.1.1.2 (E)-2,4-Dichloro-6-(4-methylstyryl)pyrido[3,2-d]pyrimidine (3b)
1
Yield 65%, mp: 215-217 °C. H NMR (400 MHz, CDCl ) δ: 8.22 (d, J=8.0 Hz, 1H, 8-CH), 8.08 (d,
3
’
’
J=8.0 Hz, 1H, 7-CH), 7.84 (d, J=16.0 Hz, 1H, 10-CH), 7.58 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.38 (d,
2
’
’
13
J=16.0 Hz, 1H, 9-CH), 7.26 (d, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 2.42 (s, 3H, CH ); C NMR (100 MHz,
2
3
CDCl ) δ: 164.92, 159.20, 154.37, 148.14, 140.18, 138.45, 136.89, 135.90, 132.75, 129.73, 128.66,
3
+
1
27.79, 125.73, 21.50; LRMS (EI): m/z calcd for C H Cl N [M ], 315.0; found, 315.0.
16
11
2
3
5.1.1.3 (E)-2,4-Dichloro-6-(4-ethylstyryl)pyrido[3,2-d]pyrimidine (3c)
1
Yield 70%, mp: 174-175 °C. H NMR (400 MHz, CDCl ) δ: 8.21 (d, J=8.0 Hz, 1H, 8-CH), 8.07 (d,
3
1
2

ACCEPTED MANUSCRIPT
’
’
J=8.0 Hz, 1H, 7-CH), 7.84 (d, J=16.0 Hz, 1H, 10-CH), 7.59 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.37 (d,
2
’
’
J=16.0 Hz, 1H, 9-CH), 7.28 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 2.72 (q, J=8.0 Hz, 2H, CH ), 1.29 (t, J=8.0
2
2
13
Hz, 3H, CH ); C NMR (100 MHz, CDCl ) δ: 164.92, 159.20, 154.37, 148.13, 146.47, 138.43, 136.92,
3
3
1
35.86, 133.01, 128.60, 128.52, 127.86, 125.75, 28.80, 15.32; LRMS (EI): m/z calcd for C H Cl N
17 13 2 3
+
[
M ], 329.0; found, 329.0.
5.1.1.4 (E)-2,4-Dichloro-6-(4-isopropylstyryl)pyrido[3,2-d]pyrimidine (3d)
1
Yield 72%, mp: 169-170 °C. H NMR (400 MHz, CDCl ) δ: 8.22 (d, J=8.0 Hz, 1H, 8-CH), 8.08 (d,
3
’
’
J=8.0 Hz, 1H, 7-CH), 7.84 (d, J=16.0 Hz, 1H, 10-CH), 7.61 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.38 (d,
2
’
’
J=16.0 Hz, 1H, 9-CH), 7.31 (d, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 3.01-2.94 (m, 1H, CH), 1.31 (d, J=8.0 Hz,
2
13
6
H, (CH )) ; C NMR (100 MHz, CDCl ) δ: 164.90, 159.22, 154.36, 151.10, 148.14, 138.43, 136.88,
3 2 3
1
35.88, 133.13, 128.63, 127.90, 127.12, 125.77, 34.09, 23.82; LRMS (EI): m/z calcd for C H Cl N
3
18
15
2
+
[
M ], 343.1; found, 343.1.
5.1.1.5 (E)-2,4-Dichloro-6-(4-fluorostyryl)pyrido[3,2-d]pyrimidine (3e)
1
Yield 63%, mp: 248-249 °C. H NMR (400 MHz, CDCl ) δ: 8.25 (d, J=8.0 Hz, 1H, 8-CH), 8.06 (d,
3
’
’
J=8.0 Hz, 1H, 7-CH), 7.85 (d, J=16.0 Hz, 1H, 10-CH), 7.67 (t, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.34 (d,
2
’
’
13
J=16.0 Hz, 1H, 9-CH), 7.15 (d, J=8.0 Hz, 2H, 3 ,5 -(CH) ); C NMR (100 MHz, CDCl ) δ: 165.08,
2
3
1
1
5
64.83, 162.33, 158.76, 154.52, 148.07, 137.09, 136.89, 136.01, 131.79, 129.60, 129.51, 128.70,
+
26.39, 116.24, 116.02; LRMS (EI): m/z calcd for C H Cl FN [M ], 319.0; found, 319.0.
1
5
8
2
3
.1.1.6 (E)-2,4-Dichloro-6-(4-chlorostyryl)pyrido[3,2-d]pyrimidine (3f)
1
Yield 76%, mp: 260-262 °C. H NMR (400 MHz, CDCl ) δ: 8.21 (d, J=8.0 Hz, 1H, 8-CH), 8.07 (d,
3
’
’
J=8.0 Hz, 1H, 7-CH), 7.84 (d, J=16.0 Hz, 1H, 10-CH), 7.59 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.37 (d,
2
’
’
13
J=16.0 Hz, 1H, 9-CH), 7.31 (d, J=8.0 Hz, 2H, 3 ,5 -(CH) ); C NMR (100 MHz, CDCl ) δ: 164.15,
2
3
161.11, 158.54, 164.75, 148,14, 136.92, 136.13, 135.58, 134.04, 129.24, 128.90, 128.73, 127.13, 111.00;
+
LRMS (EI): m/z calcd for C H Cl N [M ], 335.0; found, 335.0.
1
5
8
3
3
5.1.1.7 (E)-2,4-Dichloro-6-(4-bromostyryl)pyrido[3,2-d]pyrimidine (3g)
1
Yield 75%, mp: 244-246 °C. H NMR (400 MHz, CDCl ) δ: 8.25 (d, J=8.0 Hz, 1H, 8-CH), 8.06 (d,
3
’
’
J=8.0 Hz, 1H, 7-CH), 7.82 (d, J=16.0 Hz, 1H, 10-CH), 7.59 (d, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 7.54 (d,
2
’
’
13
J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.40 (d, J=16.0 Hz, 1H, 9-CH); C NMR (100 MHz, CDCl ) δ: 165.16,
2
3
158.52, 154.71, 148.14, 136.98, 136.90, 136.15, 134.47, 132.20, 129.14, 128.74, 127.22, 123.88;
+
LRMS (EI): m/z calcd for C H BrCl N [M ], 378.9; found, 378.9.
1
5
8
2
3
1
3

ACCEPTED MANUSCRIPT
5.1.1.8 (E)-2,4-Dichloro-6-(4-methoxylstyryl)pyrido[3,2-d]pyrimidine (3h)
1
Yield 70%, mp: 225-227 °C. H NMR (400 MHz, CDCl ) δ: 8.19 (d, J=8.0 Hz, 1H, 8-CH), 8.04 (d,
3
’
’
J=8.0 Hz, 1H, 7-CH), 7.80 (d, J=16.0 Hz, 1H, 10-CH), 7.61 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.26 (d,
2
’
’
13
J=16.0 Hz, 1H, 9-CH), 6.96 (d, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 3.88 (s, 3H, OCH ); C NMR (100 MHz,
2
3
CDCl ) δ: 164.75, 161.02, 159.34, 154.17, 148.09, 138.07, 136.90, 135.78, 129.36, 128.61, 128.25,
3
+
1
24.41, 114.43, 55.43; LRMS (EI): m/z calcd for C H Cl N O [M ], 331.0; found, 331.0.
16
11
2
3
5.1.1.9 (E)-2,4-Dichloro-6-(3-methoxylstyryl)pyrido[3,2-d]pyrimidine (3i)
1
Yield 73%, mp: 153-154 °C. H NMR (400 MHz, CDCl ) δ: 8.19 (d, J=8.0 Hz, 1H, 8-CH), 8.05 (d,
3
J=8.0 Hz, 1H, 7-CH), 7.76 (d, J=16.0 Hz, 1H, 10-CH), 7.34 (d, J=16.0 Hz, 1H, 9-CH), 7.30 (t, J=8.0
’
’
’
13
Hz, 1H, 5 -CH), 7.20 (d, J=8.0 Hz, 1H, 6 -CH), 7.13 (s, 1H, 2 -CH), 3.87 (s, 1H, OCH ); C NMR (100
3
MHz, CDCl ) δ: 164.97, 159.95, 158.84, 154.48, 148.12, 138.21, 136.81, 136.77, 135.96, 129.92,
3
+
1
28.61, 126.89, 120.53, 115.60, 112.53, 55.37; LRMS (EI): m/z calcd for C H Cl N O [M ], 331.0;
16
11
2
3
found, 331.0.
5.1.1.10 (E)-2,4-Dichloro-6-(2-methoxylstyryl)pyrido[3,2-d]pyrimidine (3j)
1
’
Yield 67%, mp: 180-181 °C. H NMR (400 MHz, CDCl ) δ: 8.22-8.14 (m, 3H, 8-CH, 10-CH, 6 -CH),
3
’
7
.72 (d, J=8.0 Hz, 1H, 7-CH), 7.48 (d, J=16.0 Hz, 1H, 9-CH), 7.38 (t, J=8.0 Hz, 1H, 4 -CH), 7.03 (t,
’
’
13
J=8.0 Hz, 1H, 5 -CH), 6.97 (d, J=8.0 Hz, 1H, 3 -CH), 3.96 (s, 3H, OCH ); C NMR (100 MHz, CDCl )
3
3
δ: 164.88, 159.82, 157.83, 154.30, 148.19, 136.88, 135.70, 133.39, 131.00, 128.44, 127.80, 127.28,
+
1
24.45, 120.91, 111.13, 55.60; LRMS (EI): m/z calcd for C H Cl N O [M ], 331.0; found, 331.0.
1
6
11
2
3
5.1.1.11 (E)-2,4-Dichloro-6-(3,4,5-trimethoxylstyryl)pyrido[3,2-d]pyrimidine (3k)
1
Yield 58%, mp: 201-203 °C. H NMR (400 MHz, CDCl ) δ: 8.24 (d, J=8.0 Hz, 1H, 8-CH), 8.10 (d,
3
J=8.0 Hz, 1H, 7-CH), 7.78 (d, J=16.0 Hz, 1H, 10-CH), 7.34 (d, J=16.0 Hz, 1H, 9-CH), 6.91 (s, 2H,
’
’
13
2
1
,6 -(CH) ), 3.97 (s, 6H, (OCH ) ), 3.93 (s, 3H, OCH ); C NMR (100 MHz, CDCl ) δ: 164.86, 158.97,
2 3 2 3 3
54.49, 153.56, 148.20, 139.78, 138.38, 136.93, 136.72, 136.01, 131.04, 128.48, 126.07, 61.05, 56.27;
+
LRMS (EI): m/z calcd for C H Cl N O [M ], 391.0; found, 391.1
1
8
15
2
3
3
5.1.2. General procedure for the synthesis of intermediates 4a-4k
A mixture of compounds 3a-3k (0.7 g), absolute methanol saturated by ammonia (15 mL) was
o
heated at 150 C for 8 h in sealed vessel. After cooled to room temperature, the light yellow precipitate
was filtered and washed with water to give the title compound as a yellow solid.
5
.1.2.1 (E)-2,4-Diamino-6-styrylpyrido[3,2-d]pyrimidine (4a)
1
4

ACCEPTED MANUSCRIPT
1
Yield 91%, mp: 299-300 °C. H NMR (400 MHz, DMSO-d ) δ: 7.81 (d, J=16.0 Hz, 1H, 10-CH), 7.74
6
’
’
(
d, J=8.0 Hz, 1H, 8-CH), 7.65 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.54 (d, J=8.0 Hz, 1H, 7-CH), 7.48 (s, 2H,
2
’
’
’
4
9
1
-NH ), 7.41 (t, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 7.33 (t, J=8.0 Hz, 1H, 4 -CH), 7.31 (d, J=16.0 Hz, 1H,
2
2
13
-CH), 6.27 (s, 2H, 2-NH ); C NMR (100 MHz, DMSO-d ) δ: 162.53, 161.14, 148.45, 146.85, 137.26,
2
6
+
32.52, 131.85, 129.28, 128.52, 128.25, 128.10, 127.33, 127.26; HRMS (TOF ES ): m/z calcd for
+
C H N [(M+H) ], 264.1249; found, 264.1248.
15
13
5
5.1.2.2 (E)-2,4-Diamino-6-(4-methylstyryl)pyrido[3,2-d]pyrimidine (4b)
1
Yield 95%, mp: >300 °C. H NMR (400 MHz, DMSO-d ) δ: 7.75 (d, J=20.0 Hz, 1H, 10-CH), 7.73 (d,
6
’
’
J=8.0 Hz, 1H, 8-CH), 7.54 (d, J=8.0 Hz, 3H, 2 ,6 -(CH) & 7-CH), 7.42 (s, 2H, 4-NH ), 7.25 (d, J=16.0
2
2
’
’
13
Hz, 1H, 9-CH), 7.23 (d, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 6.21 (s, 2H, 2-NH ), 2.33 (s, 3H, CH ); C NMR
2
2
3
(
100 MHz, DMSO-d ) δ: 162.52, 161.08, 148.72, 146.74, 138.04, 134.50, 132.53, 131.87, 129.89,
6
+
+
1
2
5
28.22, 127.23, 127.17, 21.35; HRMS (TOF ES ): m/z calcd for C H N [(M+H) ], 278.1406; found,
16 16 5
78.1407.
.1.2.3 (E)-2,4-Diamino-6-(4-ethylstyryl)pyrido[3,2-d]pyrimidine (4c)
1
Yield 95%, mp: 314-316 °C. H NMR (400 MHz, DMSO-d ) δ: 7.77 (d, J=16.0 Hz, 1H, 10-CH), 7.73
6
’
’
(
d, J=8.0 Hz, 1H, 8-CH), 7.56 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.53 (d, J=8.0 Hz, 1H, 7-CH), 7.48 (s, 2H,
2
’
’
4
-NH ), 7.26 (d, J=16.0 Hz, 1H, 9-CH), 7.25 (d, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 6.28 (s, 2H, 2-NH ), 2.62
2
2
2
1
3
(
q, J=8.0 Hz, 2H, CH ), 1.19 (q, J=8.0 Hz, 3H, CH ); C NMR (100 MHz, DMSO-d ) δ: 162.51,
2 3 6
1
1
5
61.08, 148.64, 146.75, 144.35, 134.74, 132.50, 131.83, 128.71, 128.20, 127.29, 127.22, 127.16, 28.45,
+
+
5.92; HRMS (TOF ES ): m/z calcd for C H N [(M+H) ], 292.1562; found, 292.1562.
1
7
18
5
.1.2.4 (E)-2,4-Diamino-6-(4-isopropylstyryl)pyrido[3,2-d]pyrimidine (4d)
1
Yield 94%, mp: 292-294 °C. H NMR (400 MHz, DMSO-d ) δ: 7.78 (d, J=16.0 Hz, 1H, 10-CH), 7.74
6
’
’
(
d, J=8.0 Hz, 1H, 8-CH), 7.57 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.54 (d, J=8.0 Hz, 1H, 7-CH), 7.38 (s, 2H,
2
’
’
4
-NH ), 7.28 (d, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 7.25 (d, J=16.0 Hz, 1H, 9-CH), 6.30 (s, 2H, 2-NH ), 2.90
2
2
2
1
3
(
m, 1H, CH(CH ) ), 1.21 (d, J=4.0 Hz, 6H, (CH ) ), ; C NMR (100 MHz, DMSO-d ) δ: 162.52,
3 2 3 2 6
1
2
5
60.89, 148.99, 148.79, 146.38, 134.88, 132.32, 131.89, 128.16, 127.31, 127.25, 127.22, 127.16, 33.71,
+
+
4.22; HRMS (TOF ES ): m/z calcd for C H N [(M+H) ], 306.1719; found, 306.1726.
1
8
20
5
.1.2.5 (E)-2,4-Diamino-6-(4-fluorostyryl)pyrido[3,2-d]pyrimidine (4e)
1
Yield 92%, mp: 288-290 °C. H NMR (400 MHz, DMSO-d ) δ: 7.81 (d, J=16.0 Hz, 1H, 10-CH), 7.72
6
’
’
(
d, J=8.0 Hz, 1H, 8-CH), 7.70 (dd, J=8.0 Hz, J=4.0 Hz, 2H, 2 ,6 -(CH) ), 7.55 (d, J=8.0 Hz, 1H, 7-CH),
2
1
5

ACCEPTED MANUSCRIPT
’
’
7
2
1
.48 (s, 2H, 4-NH ), 7.26 (d, J=16.0 Hz, 1H, 9-CH), 7.25 (t, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 6.29 (s, 2H,
2
2
1
3
-NH ), ; C NMR (100 MHz, DMSO-d ) δ: 163.59, 162.54, 161.13, 148.38, 146.82, 133.89, 133.86,
2
6
+
32.52, 130.69, 129.21, 129.13, 128.23, 127.97, 127.95, 127.33,116.30, 116.09; HRMS (TOF ES ): m/z
+
calcd for C H FN [(M+H) ], 282.1155; found, 282.1156.
1
5
13
5
5.1.2.6 (E)-2,4-Diamino-6-(4-chlorostyryl)pyrido[3,2-d]pyrimidine (4f)
1
Yield 94%, mp: 312-314 °C; H NMR (400 MHz, DMSO-d ) δ: 7.82 (d, J=16.0 Hz, 1H, 10-CH), 7.72
6
’
’
(
d, J=8.0 Hz, 1H, 8-CH), 7.67 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.54 (d, J=8.0 Hz, 1H, 7-CH), 7.48 (d,
2
’
’
13
J=8.0 Hz, 2H, 3 ,5 -(CH) ) & 2H, 4-NH ), 7.34 (d, J=16.0 Hz, 1H, 9-CH), 6.27 (s, 2H, 2-NH ); C
2
2
2
NMR (100 MHz, DMSO-d ) δ: 162.54, 161.19, 148.13, 146.93, 136.26, 132.79, 132.52, 130.48, 129.29,
6
+
+
1
2
5
28.89, 128.30, 127.47; HRMS (TOF ES ): m/z calcd for C H ClN [(M+H) ], 298.0895; found,
15 13 5
98.0865.
.1.2.7 (E)-2,4-Diamino-6-(4-bromostyryl)pyrido[3,2-d]pyrimidine (4g)
1
Yield 88%, mp: 331-333 °C. H NMR (400 MHz, DMSO-d ) δ: 7.80 (d, J=16.0 Hz, 1H, 10-CH), 7.72
6
’
’
(
d, J=8.0 Hz, 1H, 8-CH), 7.61 (s, 2H, 2 ,6 -(CH) & 2H, 4-NH ), 7.54 (d, J=8.0 Hz, 1H, 7-CH), 7.48 (d,
2 2
’
’
13
J=8.0 Hz, 2H, 3 ,5 -(CH) )), 7.35 (d, J=16.0 Hz, 1H, 9-CH), 6.28 (s, 2H, 2-NH ); C NMR (100 MHz,
2
2
DMSO-d ) δ: 162.53, 161.20, 148.11, 146.94, 136.61, 132.52, 132.20, 130.54, 129.20, 128.95, 128.32,
6
+
+
1
27.48, 121.40; HRMS (TOF ES ): m/z calcd for C H BrN [(M+H) ], 342.0354; found, 344.0342.
15 13 5
5.1.2.8 (E)-2,4-Diamino-6-(4-methoxylstyryl)pyrido[3,2-d]pyrimidine (4h)
1
Yield 95%, mp: 297-298 °C. H NMR (400 MHz, DMSO-d ) δ: 7.74 (d, J=16.0 Hz, 1H, 10-CH), 7.70
6
’
’
(
d, J=8.0 Hz, 1H, 8-CH), 7.59 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.52 (d, J=8.0 Hz, 1H, 7-CH), 7.43 (s, 2H,
2
’
’
4
-NH ), 7.16 (d, J=16.0 Hz, 1H, 9-CH), 6.98 (d, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 6.22 (s, 2H, 2-NH ), 3.79
2
2
2
1
3
(
s, 3H, OCH ); C NMR (100 MHz, DMSO-d ) δ: 162.48, 161.00, 159.82, 148.93, 146.60, 132.51,
3
6
+
1
31.64, 129.88, 128.66, 128.15, 127.04, 125.84, 114.78, 55.66; HRMS (TOF ES ): m/z calcd for
+
C H N O [(M+H) ], 294.1355; found, 294.1360.
16
16
5
5.1.2.9 (E)-2,4-Diamino-6-(3-methoxylstyryl)pyrido[3,2-d]pyrimidine (4i)
1
Yield 91%, mp: 275-277 °C. H NMR (400 MHz, DMSO-d ) δ: 7.78 (d, J=16.0 Hz, 1H, 10-CH), 7.73
6
(
d, J=8.0 Hz, 1H, 8-CH), 7.55 (d, J=8.0 Hz, 1H, 7-CH), 7.48 (s, 2H, 4-NH ), 7.33 (d, J=16.0 Hz, 1H,
2
’
’
’
9
-CH), 7.32 (d, J=8.0 Hz, 1H, 5 -CH), 7.22 (d, J=8.0 Hz, 1H, 6 -CH), 7.21 (s, 1H, 2 -CH), 6.88 (dd,
’
13
J=8.0 Hz, J=4.0 Hz, 1H, 4 -CH), 6.28 (s, 2H, 2-NH ), 3.81 (s, 3H, OCH ); C NMR (100 MHz,
2
3
DMSO-d ) δ: 162.54, 161.15, 160.11, 148.40, 146.86, 138.72, 132.52, 131.83, 130.26, 128.38, 128.26,
6
1
6

ACCEPTED MANUSCRIPT
+
+
1
2
5
27.36, 119.86, 114.47, 112.21, 55.56; HRMS (TOF ES ): m/z calcd for C H N O [(M+H) ],
1
6
16
5
94.1355; found, 294.1357.
.1.2.10 (E)-2,4-Diamino-6-(2-methoxylstyryl)pyrido[3,2-d]pyrimidine (4j)
1
Yield 90%, mp: 264-266 °C. H NMR (400 MHz, DMSO-d ) δ: 7.84 (d, J=16.0 Hz, 1H, 10-CH), 7.72
6
’
(
d, J=8.0 Hz, 1H, 8-CH), 7.65 (d, J=8.0 Hz, 1H, 6 -CH), 7.54 (d, J=8.0 Hz, 1H, 7-CH), 7.42 (s, 2H,
’
’
4
-NH ), 7.33 (d, J=16.0 Hz, 1H, 9-CH), 7.31 (t, J=8.0 Hz, 1H, 4 -CH), 7.07 (d, J=8.0 Hz, 1H, 3 -CH),
2
’
13
7
.00 (t, J=8.0 Hz, 1H, 5 -CH), 6.29 (s, 2H, 2-NH ), 3.89 (s, 3H, OCH ); C NMR (100 MHz,
2
3
DMSO-d ) δ: 162.48, 161.00, 157.52, 149.10, 146.69, 132.52, 129.85, 129.19, 128.13, 127.91, 127.04,
6
+
+
1
2
5
26.98, 125.66, 121.14, 111.96, 55.96; HRMS (TOF ES ): m/z calcd for C H N O [(M+H) ],
16 16 5
94.1355; found, 294.1351.
.1.2.11 (E)-2,4-Diamino-6-(3,4,5-trimethoxylstyryl)pyrido[3,2-d]pyrimidine (4k)
1
Yield 86%, mp: 248-250 °C. H NMR (400 MHz, DMSO-d ) δ: 7.76 (d, J=16.0 Hz, 1H, 10-CH), 7.72
6
(
d, J=8.0 Hz, 1H, 8-CH), 7.56 (d, J=8.0 Hz, 1H, 7-CH), 7.54 (s, 2H, 4-NH ), 7.29 (d, J=16.0 Hz, 1H,
2
’
’
13
9
-CH), 6.97 (s, 2H, 2 ,6 -(CH) ), 6.37 (s, 2H, 2-NH ), 3.85 (s, 6H, (OCH ) , 3.69 (s, 3H, OCH ); C
2 2 3 2 3
NMR (100 MHz, DMSO-d ) δ: 162.53, 160.78, 153.57, 148.74, 146.17, 138.19, 132.91, 132.26, 132.22,
6
+
+
1
3
5
28.16, 127.37, 127.30, 104.68, 60.57, 56.35; HRMS (TOF ES ): m/z calcd for C H N O [(M+H) ],
18 20 5 3
54.1566; found, 354.1567.
.1.3. General procedure for the synthesis of intermediates 5a-5k
A solution of intermediates 4a-4k (100 mg) and Pd/C (10 %) in ethanol (40 mL) in an autoclave.
The autoclave was evacuated and backfilled with H three times, pressurized to 0.4 Mpa, and stirred at
2
room temperature for 12 h. After the pressure was released, the reaction mixture was filtered through
filter paper and the filtrate was concentrated in vacuo. The resulting residue was purified by using a
silica gel column (eluent: dichloromethane/methanol, 9/1) to obtain the title compound as a white solid.
5.1.3.1 2,4-Diamino-6-phenethylpyrido[3,2-d]pyrimidine (5a)
1
Yield 92%, mp: 206-207 °C. H NMR (400 MHz, DMSO-d ) δ: 7.49 (d, J=8.0 Hz, 1H, 8-CH), 7.41 (d,
6
’
’
’
’
J=8.0 Hz, 1H, 7-CH), 7.35-7.20 (m, 6H, 4-NH , 3 ,5 -(CH ) , 2 ,6 -(CH ) ), 7.19-7.13 (m, 1H, 4’-CH),
2
2 2
2 2
1
3
6
.15 (s, 2H, 2-NH ), 3.09 (s, 4H, CH CH ); C NMR (100 MHz, DMSO-d ) δ: 162.39, 160.71, 154.24,
2 2 2 6
+
1
45.76, 142.13, 132.39, 128.85, 128.68, 128.32, 127.58, 126.23, 39.15, 35.04; HRMS (TOF ES ): m/z
+
calcd for C H N [(M+H) ], 266.1406; found, 266.1402.
1
5
16
5
5
.1.3.2 2,4-Diamino-6-(4-methylstyryl)pyrido[3,2-d]pyrimidine (5b)
1
7

ACCEPTED MANUSCRIPT
1
Yield 98%, mp: 201-202 °C. H NMR (400 MHz, DMSO-d ) δ: 8.17-8.04 (d, 2H, 4-NH ), 7.65 (d,
6
2
’
’
J=8.0 Hz, 1H, 8-CH), 7.55 (d, J=8.0 Hz, 1H, 7-CH), 7.13 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.07 (d, J=8.0
2
’
’
13
Hz, 2H, 3 ,5 -(CH) & s, 2H, 2-NH ), 3.10-3.05 (m, 4H, CH CH ), 2.25 (s, 3H, CH ); C NMR (100
2
2
2
2
3
MHz, DMSO-d ) δ: 162.71, 157.70, 156.47, 138.76, 135.15, 129.29, 129.00, 128.71, 126.82, 39.14,
6
+
+
3
4.44, 21.07; HRMS (TOF ES ): m/z calcd for C H N [(M+H) ],280.1562; found, 280.1563.
16 18 5
5.1.3.3 2,4-Diamino-6-(4-ethylstyryl)pyrido[3,2-d]pyrimidine (5c)
1
Yield 95%, mp: 223-224 °C. H NMR (400 MHz, DMSO-d ) δ: 7.49 (d, J=8.0 Hz, 1H, 8-CH), 7.46 (s,
6
’
’
1
H, 4-NH), 7.41 (d, J=8.0 Hz, 1H, 7-CH), 7.29 (s, 1H, 4-NH), 7.15 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.09
2
’
’
(
d, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 6.23 (s, 2H, 2-NH ), 3.09-3.00 (m, 4H, CH CH ), 2.54 (q, J=8.0 Hz, 2H,
2 2 2 2
1
3
CH ), 1.14 (t, J=8.0 Hz, 3H, CH ); C NMR (100 MHz, DMSO-d ) δ: 162.41, 160.81, 154.25, 145.98,
2
3
6
+
1
41.51, 139.27, 132.51, 128.76, 128.31, 128.06, 127.60, 39.31, 34.70, 28.23, 16.13; HRMS (TOF ES ):
+
m/z calcd for C H N [(M+H) ], 294.1719; found, 294.1725.
1
7
20
5
5.1.3.4 2,4-Diamino-6-(4-isopropylstyryl)pyrido[3,2-d]pyrimidine (5d)
1
Yield 91%, mp: 223-235 °C. H NMR (400 MHz, DMSO-d ) δ: 7.51 (d, J=8.0 Hz, 1H, 8-CH), 7.45 (d,
6
’
’
J=8.0 Hz, 1H, 7-CH), 7.46-7.36 (2H, 4-NH ), 7.17 (d, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 7.13 (d, J=8.0 Hz,
2
2
’
’
2
H, 2 ,6 -(CH) ), 6.29 (s, 2H, 2-NH ), 3.08-3.01 (m, 4H, CH CH ), 2.86-2.80 (m, 1H, CH), 1.17 (d, 6H,
2 2 2 2
1
3
(
CH ) ); C NMR (100 MHz, DMSO-d ) δ: 162.42, 160.34, 154.58, 146.21, 145.07, 139.42, 132.02,
3 2 6
+
1
28.74, 128.42, 127.48, 126.58, 39.26, 34.63, 33.48, 24.43; HRMS (TOF ES ): m/z calcd for C H N
18 22 5
+
[
(M+H) ], 308.1875; found, 308.1875.
5.1.3.5 2,4-Diamino-6-(4-fluorostyryl)pyrido[3,2-d]pyrimidine (5e)
1
Yield 90%, mp: 229-231 °C H NMR (400 MHz, DMSO-d ) δ: 7.76-7.65 (2H, 4-NH ), 7.56 (d, J=8.0
6
2
’
’
Hz, 1H, 8-CH), 7.47 (d, J=8.0 Hz, 1H, 7-CH), 7.27 (t, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 7.08 (t, J=8.0 Hz, 2H,
2
’
’
13
3
1
3
5
,5 -(CH) ), 6.61 (s, 2H, 4-NH ), 3.09 (s, 4H, CH CH ); C NMR (100 MHz, DMSO-d ) δ: 162.52,
2 2 2 2 6
62.26, 159.86, 159.29, 155.01, 142.93, 138.13, 130.81, 130.63, 130.55, 128.80, 127.22, 115.44, 115.23,
+
+
9.10, 33.95; HRMS (TOF ES ): m/z calcd for C H FN [(M+H) ], 284.1311; found, 284.1315.
1
5
15
5
.1.3.6 2,4-Diamino-6-(4-chlorostyryl)pyrido[3,2-d]pyrimidine (5f)
1
Yield 92%, mp: 222-224 °C. H NMR (400 MHz, DMSO-d ) δ: 7.83-7.70 (2H, 4-NH ), 7.57 (d, J=8.0
6
2
’
’
Hz, 1H, 8-CH), 7.47 (d, J=8.0 Hz, 1H, 7-CH), 7.31 (t, J=8.0 Hz, 2H, 3 ,5 -(CH) ), 7.27 (t, J=8.0 Hz, 2H,
2
’
’
13
2
1
,6 -(CH) ), 6.67 (s, 2H, 4-NH ), 3.10 (s, 4H, CH CH ); C NMR (100 MHz, DMSO-d ) δ: 162.54,
2 2 2 2 6
59.11, 155.00, 141.07, 130.85, 130.81, 130.76, 130.63, 128.86, 128.61, 127.18, 38.76, 34.00; HRMS
1
8

ACCEPTED MANUSCRIPT
+
+
(
TOF ES ): m/z calcd for C H ClN [(M+H) ], 300.1016; found, 300.1019.
15
15
5
5.1.3.7 2,4-Diamino-6-(4-bromostyryl)pyrido[3,2-d]pyrimidine (5g)
1
’
’
Yield 89%, mp: 226-228 °C. H NMR (400 MHz, DMSO-d ) δ: 7.51 (d, J=8.0 Hz, 2H, 3 ,5 -(CH) ),
6
2
’
’
7
.45-7.41 (m, 4H, 8-CH, 7-CH & 4-NH ), 7.21 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 6.33 (s, 2H, 2-NH ), 3.07
2 2 2
1
3
(
s, 4H, CH CH ); C NMR (100 MHz, DMSO-d ) δ: 162.44, 160.16, 154.25, 144.68, 141.56, 131.80,
2 2 6
+
1
31.51, 131.18, 128.52, 127.45, 119.26, 38.71, 34.12; HRMS (TOF ES ): m/z calcd for C H BrN
15 15 5
+
[
(M+H) ], 344.0511; found, 344.0508.
5.1.3.8 2,4-Diamino-6-(4-methoxylstyryl)pyrido[3,2-d]pyrimidine (5h)
1
Yield 95%, mp: 218-219 °C. H NMR (400 MHz, DMSO-d ) δ: 7.48 (d, J=8.0 Hz, 1H, 8-CH), 7.39 (d,
6
’
’
J=8.0 Hz, 1H, 7-CH), 7.23 (s, 2H, 4-NH ), 7.16 (d, J=8.0 Hz, 2H, 2 ,6 -(CH) ), 6.83 (d, J=8.0 Hz, 2H,
2
2
’
’
13
3
1
,5 -(CH) ), 3.71 (s, 3H, OCH ), 3.03 (m, 4H, CH CH ); C NMR (100 MHz, DMSO-d ) δ: 162.38,
2 3 2 2 6
60.83, 157.90, 154.27, 145.99, 133.97, 132.51, 129.76, 128.28, 127.63, 114.14, 55.43, 39.45, 34.20;
+
+
HRMS (TOF ES ): m/z calcd for C H N O [(M+H) ], 296.1511; found, 296.1515.
1
6
18
5
5.1.3.9 2,4-Diamino-6-(3-methoxylstyryl)pyrido[3,2-d]pyrimidine (5i)
1
Yield 89%, mp: 201-202 °C. H NMR (400 MHz, DMSO-d ) δ: 7.48 (d, J=8.0 Hz, 1H, 8-CH), 7.41 (d,
6
’
’
J=8.0 Hz, 1H, 7-CH), 7.30 (s, 2H, 4-NH ), 7.17 (t, J=8.0 Hz, 1H, 5 -CH), 6.83 (s, 1H, 2 -CH), 6.82 (d,
2
’
’
J=8.0 Hz, 1H, 6 -CH), 6.73 (dd, J=8.0 Hz, J=4.0 Hz, 1H, 4 -CH), 6.16 (s, 2H, 2-NH ), 3.71 (s, 3H,
2
13
OCH ), 3.11-3.02 (m, 4H, CH CH ); C NMR (100 MHz, DMSO-d ) δ: 162.38, 160.70, 159.69,
3
2
2
6
154.20, 145.75, 143.75, 132.37, 129.66, 128.35, 127.56, 121.10, 114.54, 111.73, 55.32, 39.01, 34.99;
+
+
HRMS (TOF ES ): m/z calcd for C H N O [(M+H) ], 296.1511; found, 296.1516.
1
6
18
5
5.1.3.10 2,4-Diamino-6-(2-methoxylstyryl)pyrido[3,2-d]pyrimidine (5j)
1
Yield 88%, mp: 299-300 °C; H NMR (400 MHz, DMSO-d ) δ: 7.49 (d, J=8.0 Hz, 1H, 8-CH), 7.38 (d,
6
’
’
J=8.0 Hz, 1H, 6 -CH), 7.25 (s, 2H, 4-NH ), 7.19-7.15 (m, 2H, 5 -CH & 7-CH), 6.95 (d, J=8.0 Hz, 1H,
2
’
’
3
-CH), 6.83 (t, J=8.0 Hz, 1H, 4 -CH), 6.21 (s, 2H, 2-NH ), 3.79 (s, 3H, OCH ), 3.03 (s, 4H, CH CH );
2 3 2 2
1
3
C NMR (100 MHz, DMSO-d ) δ: 162.40, 160.59, 157.62, 154.71, 145.55, 132.27, 130.06, 129.83,
6
+
1
28.28, 127.71, 127.49, 120.64, 111.07, 55.73, 37.62, 29.78; HRMS (TOF ES ): m/z calcd for
+
C H N O [(M+H) ], 296.1511; found, 296.1514.
16
18
5
5.1.3.11 2,4-Diamino-6-(3,4,5-trimethoxylstyryl)pyrido[3,2-d]pyrimidine (5k)
1
Yield 93%, mp: 145-146 °C. H NMR (400 MHz, DMSO-d ) δ: 7.49 (d, J=8.0 Hz, 1H, 8-CH), 7.43 (d,
6
’
’
J=8.0 Hz, 1H, 7-CH), 7.28 (s, 2H, 4-NH ), 6.57 (s, 2H, 2 ,6 -(CH) ), 6.12 (s, 2H, 2-NH ), 3.73 (s, 6H,
2
2
2
1
9

ACCEPTED MANUSCRIPT
13
(
OCH ) ), 3.61 (s, 3H, OCH ), 3.11-3.07 (m, 2H, CH ), 3.03-3.00 (m, 2H, CH ); C NMR (100 MHz,
3 2 3 2 2
DMSO-d ) δ: 162.37, 160.81, 154.20, 153.10, 145.96, 137.85, 136.06, 132.51, 128.34, 127.56, 106.18,
6
+
+
6
3
5
0.41, 56.21, 39.12, 35.31. HRMS (TOF ES ): m/z calcd for C H N O [(M+H) ], 356.1723; found,
18 22 5 3
56.1722.
.2. MTS assay
Human cancer cell lines were obtained from Type Culture Collection of the Chinese Academy of
Sciences (Shanghai, China). HeLa, HL-60, A549 and H1299 cells were routinely cultured in RPMI
o
1
640 medium, supplemented with 10% fetal bovine serum, penicillinestreptomycin solution, at 37 C
with 5% CO . To evaluate antiproliferative properties of the synthesized compounds, the
2
[
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium, inner
salt; (MTS)] assay was used. The cell lines were assessed by trypsinizing each cell line and seeding
000-3000 cells per well into 96-well plates. Cells were grown for 12 h and then treated with
1
compounds at concentrations ranging from 0.03 ꢀM to 100 ꢀM and incubated for 48 h in 200 ꢀL media.
MTS reagent (20 ꢀL) in phosphate buffer solution was added to each well and incubated further for 3 h.
A490 nm was measured using a Thermomax Molecular Device plate reader. The experiments were
performed in triplicate and repeated at least three times for each compound per cell line. MTX and 5-Fu
were used as positive controls.
5.3. Dihydrofolate reductase (DHFR) inhibition assay
The recombinant human (rh) DHFR inhibition assay was performed in a final volume of 100 ꢀL.
The assay systerm contained 0.1 mM dihydrofolate, 0.3 mM NADPH, 100 mM KCl and 0.011 unit rh
DHFR in phosphate buffer, pH 7.5. After addition of the enzyme, the mixture was incubated at room
temperature for 15 min. The absorbance of each well was read in the microplate reader at room
temperature at wavelengths of 340 nm.
5.4. Effects of compound 5k on cell cycle progression in HL-60 cells
6
HL-60 cells at a density of 1.8 × 10 cells per well were cultured in 60 mm petri dish overnight,
then they were harvested and washed twice with ice-cold PBS after treating with compound 5k for 24 h.
o
Next, these cells were fixed in 70% cooled ethanol at 4 C until being washing in PBS just before
analysis by flow cytometry. 0.5 mL PBS within 10 mg/mL RNase A were add to the fixed cells and
o
incubated at 37 C for 30min. After this, 50 mg/mL propidium iodide was added to the cells. The DNA
contents were determined using a CellQuest and analyzed by the Modfit software (Becton Dickinson).
2
0

ACCEPTED MANUSCRIPT
5.5. Docking and molecular modeling study
The three-dimensional structures of compounds 5k and 4k, which presented best and worst
biological profiles, in their neutral forms were constructed using ACCELRYS DISCOVERY STUDIO
client 2.5 (DS 2.5) software. Lowest energy conformer of each new analog ‘global-minima’ was docked
6
6
into the rhDHFR enzyme-binding domain in tertiary complex with NADPH and N -methyl-N -(3,4,5-
trifluorophenyl)-2,4,6-triamine pyrido[2,3-d]pyrimidine [31], code ID 4QJC, was downloaded from the
protein data bank at the Research Collaboratory for Structural Bioinformatics (www.rcsb.org) [32]. All
of hydrogens were added and enzyme structure was subjected to refinement by DS 2.5 software. All
atoms within a 10 Å around the co-crystallized ligand in crystal co-ordinates of DHFR were selected as
binding site. For DS 2.5 docking, the default parameters were used if it was not mentioned.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (21172014,
21302007).
Appendix A. Supplementary data
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[
[
[
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A series of 2,4-diamino-6-substituted pyrido[3,2-d]pyrimidines were efficiently synthesized.
Compounds were evaluated for in vitro DHFR inhibition and antitumor activity.
Compounds 5g, 5h, 5i and 5k showed antitumor activity with IC50 values range of 0.07-23
µM against four cell lines.
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SARs and docking studies were discussed in detail.
Compound 5k arrested in S Phase.