Mechanism-based design, synthesis and biological studies of N 5-substituted tetrahydrofolate analogs as inhibitors of cobalamin-dependent methionine synthase and potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.09.027

A number of 8-deazatetrahydrofolates bearing electrophilic groups on N ⁵ were designed and synthesized based on the action mechanism of methionine synthase, and their biological activities were investigated as well. Compounds (11b, 12b and 16)

Full text of DOI:10.1016/j.ejmech.2012.09.027

European Journal of Medicinal Chemistry 58 (2012) 228e236
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Mechanism-based design, synthesis and biological studies of N5-substituted
tetrahydrofolate analogs as inhibitors of cobalamin-dependent methionine
synthase and potential anticancer agents
Zhili Zhang b, Chao Tian b, Shouxin Zhou b, Wei Wang b, Ying Guo b, Jie Xia a, Zhenming Liu a, Biao Wang b,
,b,
Xiaowei Wang b, Bernard T. Golding c, Roger J. Griff c, Yansheng Du d, Junyi Liu a
*
a State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
b Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
c Department of Chemistry, University of Newcastle, England
d Department of Neurology, Indiana University School of Medicine, USA
a r t i c l e i n f o
a b s t r a c t
A number of 8-deazatetrahydrofolates bearing electrophilic groups on N5 were designed and synthesized
based on the action mechanism of methionine synthase, and their biological activities were investigated
Article history:
Received 27 April 2012
Received in revised form
17 August 2012
Accepted 23 September 2012
Available online 9 October 2012
as well. Compounds (11b, 12b and 16) showed the most active against methionine synthase (IC50
8.11 M, 1.73 M, 1.43 M). In addition, the cytotoxicity to human tumor cell lines and dihydrofolate
reductase (DHFR) inhibition by target compounds were evaluated.
:
m
m
m
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
8-Deazatetrahydrofolates
Antitumor
Methionine synthase
Folate inhibitor
1. Introduction
synthase was found in plants and the cobalamin-dependent
methionine synthase was found in mammalian tissues, and either
one or both could be found in microorganisms [2].
Methionine synthase (EC.2.1.1.13) plays a crucial role in folate
metabolism due to its catalytic activity of the conversion of
homocysteine to methionine. The conversion of homocysteine to
methionine can be catalyzed by two distinct enzymes, which are
the cobalamin-independent and cobalamin-dependent methionine
synthase. The cobalamin-independent enzyme catalyzes the direct
transfer of a methyl group from 5-methyltetrahydrofolate (CH3e
H4folate) to homocysteine, whereas the cobalamin-dependent
methionine synthase (MetS), as an intermediary methyl carrier,
promotes the transfer of the methyl group in two steps. Firstly, the
methyl group is transferred from CH3eH4folate to enzyme-bound
cob(I)alamin (Cbl(I)) to generate methyl-cob(III)alamin (CH3eCbl)
and H4folate, secondly it is transferred from the generated CH3e
Cbl to homocysteine to produce methionine and regenerate the
Cbl(I) (Fig. 1) [1]. The cobalamineindependent methionine
Since free cob(I)alamin is a strong nucleophile [3], and can easily
react with alkyl halides following SN2 mechanism [4], the postu-
lated double-transfer mechanism has stereochemical and kinetic
constraints on the reaction. Zydowsky [2] observed that, using
isotope labeled N5 (HDT) CH3eH4folate, steric effect supported the
proposed two-step reaction mechanism in which the methyl group
was transferred sequentially from CH3eH4folate to Cb1(I) and then
from the generated enzyme-bound CH3eCbl to homocysteine.
The methionine and H4folate will be further metabolized
through the one-carbon methionine transmethylation and folate
cycles. Therefore, MetS is involved in many important biochemical
pathways. According to the proposed mechanism, inhibition of
MetS will result in the decline of the intracellular methionine and
S-adenosylmethionine (AdoMet), and accumulation of CH3e
H4folate in the folate circulation. Besides the redistribution of
cellular folate derivatives, inhibition of methionine synthase also
leads to a decline in the total intracellular folate level. The major
circulation form of folate is CH3eH4folate, and MetS activity is
required to convert this derivative to forms that can be used for
* Corresponding author. Department of Chemical Biology, School of Pharma-
ceutical Sciences, Peking University, Beijing 100191, China.
E-mail address: lilybmu@bjmu.edu.cn (J. Liu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.027

Z. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 228e236
229
Fig. 1. The cobalamin-dependent methionine synthase catalyzed methyl group transfer. Cbl(I): Cob(I)alamin, CH3eCbl: methylcob(III)alamin, Glu: glutamate.
nucleotide biosynthesis [5]. Thus, the deleterious effect of MetS
inhibition on cellular nucleotide biosynthesis and overall folate
distribution renders this enzyme a particularly interesting target
for chemotherapeutic intervention.
Based on the action mechanism and the model with an active
site for docking the substrate CH3eH4folate, we designed and
synthesized a series of N8-deazaH4folates bearing electrophilic
substituents at N5-position in which carbons have partial positive
charge due to the polarity of the carboneoxygen
p bond or carbone
halogen bond (Fig. 3) as novel inhibitors of MetS. These compounds
should bind to MetS competitively to replace CH3eH4folate, and
inhibit MetS activity by formation of covalent bonds with the
nucleophilic cob(I)alamin cofactor of the enzyme, as shown in
Fig. 4.
2. Design of inhibitors
Although the biochemical role of MetS is well understood and it
can be an excellent target for rational drug design, no anticancer
agents directed toward this enzyme is available to date. The
enzyme is efficiently and specifically inhibited by the cell-signaling
molecule nitrous oxide (N2O) [6,7] through the oxidation of the
cobalamin cofactor [8]. There are also other inhibitors such as
methylmercury through binding to homocysteine [9], ethanol and
Literature survey revealed that numerous derivatives of folate
have been synthesized to date as potential antagonists. There were
structural modifications in either the pteridine ring, the p-amino-
benzoyl region, or the glutamate side-chain [14]. So far, very few
studies on the effect of N5-substituent of H4folate have been reported
[15]. One obstacle in the synthesis of such compounds is the
autoxidation tendency of H4folates under relatively mild conditions
[15]. In contrast, their 5-or 8-deaza analogs are significantly more
stable. Therefore, we designed 8-deaza and N5-substituted H4folate
analogs, which should have higher reactivity and stability than CH3e
H4folate. The N5-electron-deficient substituent of N8-deazatetrahy-
drofolates could easily form covalent bonds with the nucleophilic
cob(I)alamin of MetS, thus inhibiting the activity of MetS.
acetaldehyde through acetaldehyde-induced inhibition (IC50
:
2 mM) [10], S-AdoMet derivatives [11] and various cobalamin
analogs through decrease of the coenzyme cobalamin (activity
decreased to 20%) [12] reported in literature. However, it appears
that the design of folate analogs as inhibitors of MetS has been
overlooked. Consequently, no prototype folate analog inhibitor is
available for synthetic studies.
Recently, benzimidazole and benzothiadiazole derivatives have
been designed and evaluated in a cell free system as specific MetS
inhibitors by Banks et al. (Fig. 2) [13]. The activity of the inhibitors
and the action mechanism have been investigated by using purified
rat liver MetS. It was found that some benzimidazoles and nitro-
3. Chemistry
The synthesis of N5-substituted 8-deazatetrahydrofolate deriv-
atives 8be11b was accomplished via the reactions outlined in
Scheme 1. Intermediate 6-bromomethyl-2,4-diaminopyrido[3,2-d]
pyrimidine (1) was prepared by a procedure initially reported by
Srinivasan and Broom [16] and then modified by DeGraw et al. [17]
Chlorination of 6-acetoxymethyl-2,4-dioxopyrido[3,2-d]pyrimi-
dine with POCl3 in the presence of a catalytic amount of triethyl-
amine under reflux to afford 6-acetoxymethyl-2,4-dichloro
benzothiadiazoles gave IC50 values close to or below 100
mM.
According to CH3eH4folate-binding model with MetS, some
structural requirements for the design of inhibitors should be met.
For example, N1 and 2-amino group of CH3eH4folate forming
hydrogen bonds with amino acid residues of MetS should be kept. A
planar region like pyrimidine ring that can be positioned in the
center of the active site, with two or three possible hydrogen-
bonding groups, is required.
Y
N
N
N
S
N
R
X
Z
X = H, OCH3; Y = H, NO2, NH2, OCH3; R = H, CH3; Z = H, NO2, NH2
R1 = -CHO, -TS, -CH2CHBrCH2Br; R2=H, -COCH3, -CH3; R3=H, Br
Fig. 2. Structures of benzimidazole and benzothiadiazole derivatives synthesized by
Banks et al.
Fig. 3. N8-deazatetrahydrofolates bearing N5 electrophilic substituents.

230
Z. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 228e236
O
COOH
MetS
R1
N
NH2
N
N
H
COOH
N
N
+
H2N
Co(I)balamin
R3
R2
MetS=Methionine Synthase
R1=-CHO,-TS, -CH2CHBrCH2Br, -CH2CH=CH2
R2=-CH3, -COCH3, H
R3=H, Br
MetS
Co(III)balamin
O
COOH
R1'
N
NH2
N
N
H
COOH
N
N
R2
R3
H2N
Fig. 4. Reactions of designed inhibitors with MetS.
pyrido[3,2-d]pyrimidine. Subsequent hydrolytic amination with
ammoniaemethanol in a sealed vessel at 150 ꢀC furnished 2,4-
diamino-6-hydroxymethylpyrido[3,2-d]pyrimidine, which was
further transformed to crude product 1 by bromination with PBr3.
Intermediate 1 [16] was condensed with diethyl N-(p-amino-
benzoyl)- -glutamate and diethyl N-[p-(methylamino)benzoyl]-L-
glutamate in dry dimethylacetamide (DMAc) to afford the
L
compounds 2a and 3a, respectively. The reaction partner N-[p-
Scheme 1. Reagents and conditions: (a) DMAc, diethyl N-(p-aminobenzoyl) -L-glutamate or diethyl N-[p-(methylamino)benzoyl]-L-glutamate, rt, 48 h; (b) acetic anhydride, acetic
acid, rt, 2 h; (c) PtO2, 0.3e0.4 MPa, AcOH or trifluoroactyl acid, ethanol, rt, 2e3 d; (d) 8a: paraformaldehyde, THF, H2O, sonication, rt, 4 h; 9a: allyl bromide, Cs2CO3, DMF, rt, 15 h; 10a:
Tos-Cl, (CH3CH2)3N, CH3CN, rt, 10 h; 11a: formic acid, ethyl formate, rt, 1 h; 12a: formic acid, 60 ꢀC, 2 h; (e) 1 mol/L NaOH solution, THF, rt.

Z. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 228e236
231
spectrum and signals at
d
65.8 in the 13C NMR (DMSO-d6) spectrum
were assigned to NeCH2eN. In addition, correlation signals
between the long-range J-coupled H of methylene groups and C-6
(
d
56.3), C-4a (
at cross points (Fig. 5). Also the coupling signals between the
carbon of methylene and H-6 ( 3.65), H-9 ( 3.33) could be found.
d 113.7), C-9 (d 51.2), C-11 (d 148.2) could be observed
d
d
Additional gHMBC results confirmed the assigned structure of 8a.
The N5-allyl N10-methyl derivative 9a was synthesized in 40%
yield from 6a and allyl bromide (1.1 eq) in DMF with K2CO3 as the
base, and the yield was improved to 76% when using Cs2CO3 instead
of K2CO3. Treatment of 6a with TsCl and (C2H5)3N gave the N5-tosyl
Fig. 5. Long-range coupled correlation of the methylene groups of 8a./1H of methy-
lene group and 13C in three-bond distance, ꢁꢁꢁꢁ/13C of methylene group and 1H in
three-bond distance.
N
10-methyl derivative 10a. Treatment of 6a and 7a with a mixture
of ethyl formate and formic acid gave the N5-formyl derivatives 11a
and 12a in 75 and 86%, respectively.
(methylamino)benzoyl]-
ercially available diethyl N-(p-aminobenzoyl)-
a modified method reported Yang et al. [18] Reductive benzylation
and methylation of N-(p-aminobenzoyl)- -glutamate by benzalde-
L-glutamate was synthesized from comm-
L-glutamate by using
Finally saponification of the diesters of 2ae12a with 0.3 mol/L
NaOH in ethanol or THF gave the target compounds 2be11b.
L
hyde, sodium cyanoborohydride, acetic acid, and formaldehyde in
one pot afforded diethyl N-[p-(N-benzoyl-N-methylamino)
4. Results and discussion
benzoyl]-
ylation with Pd/C in 1 N HCl at 0.3 MP furnished diethyl N-[(p-
methylamino)benzoyl]- -glutamate.
L
-glutamate
[19].
Then
hydrogenolytic
debenz
4.1. Methionine synthase and dihydrofolate reductase (DHFR)
inhibition
L
With the condensed products in hand, treatment of diethyl 8-
deaza aminopterin 2a with a mixture of acetic anhydride and
acetic acid gave the N10-acetyl derivative 4a directly. Reduction of
2a and 3a with PtO2 and HOAc in ethanol at 40 ꢀC gave compounds
5a and 6a in 45% yield along with over-reduced by-products [14].
By treatment with trifluoroacetic acid, 4a could be transformed to
7a in a high yield (74%), possibly because of the electron-
withdrawing acetyl group. Compounds 5a, 6a and 7a were diaste-
reomeric mixtures due to the appearance of another chiral center
C6, and the ratio determined by chiral high performance liquid
chromatography is 1:1.
The target compounds and intermediates were evaluated
against MetS of HL-60 and recombinant human DHFR, respectively,
and the half-inhibitory concentrations (IC50) are presented in
Table 1. Compounds 13, 14, 15 and 16 previously reported [14] as
inhibitors of DHFR have the same substitution on N5-position
[Fig. 6]. So they were evaluated at the same time to get more
information of structure activity relationship.
IC50 values of N5-substituted compounds 8be11b and 13e16 on
MetS were below 66.38
mM except that of 9b. Comparing the
activity results, it was apparent that the alkyl and acyl groups at N5-
position were associated with stronger inhibition (5b compared
with 13, 6b compared with 11b, 7b compared with 12b) whereas
the N5-unsubstituted analogs (2b and 5e7b) showed no inhibition
of MetS.
The N5,N10-methylenated compound 8a was synthesized from
5a and paraformaldehyde at room temperature by nucleophilic
addition and dehydration reactions. It was found that the reaction
rate could be increased under sonication. And no products of
hydroxymethylation on N5 or N10 of 5a was observed in the reac-
tion. The structure assignment of 8a was achieved based on
detailed MS and NMR spectroscopic studies. The ESI-TOF-MS
showed the molecular ion peak [M þ H]þ was at m/z 512. Signals
5-Dibromosubstituted compound 16, the most potent inhibitor
of MetS in this series, showed a greater activity, about 15-fold with
IC50 of 1.43
mM, than the corresponding 5-alky compound 15
(IC50 ¼ 21.00
mM). Similarly, compounds 11b and 12b with formyl
3.78, 4.92 (d, each, J ¼ 4.8 Hz) in the 1H NMR (DMSO-d6)
groups on N5 were stronger MetS inhibitors than 6b and 7b. The
at
d
Table 1
In vitro inhibitory effects against MetS and DHFR.
R1
R2
R3
MetS (IC50
,
m
M)
DHFR (IC50, mM)
2b
5b
6b
7b
13
14
8b
9b
10b
11b
12b
15
e
e
e
H
H
H
H
H
H
H
H
H
H
Br
Br
>100
0.018 ꢂ 0.00098
0.56 ꢂ 0.060a
0.033 ꢂ 0.0027
0.033 ꢂ 0.0051
1.41 ꢂ 0.12a
2.15 ꢂ 0.16a
0.80 ꢂ 0.00005
0.42 ꢂ 0.063
1.07 ꢂ 0.32
H
H
H
H
87.91 ꢂ 5.81
eCH3
eCOCH3
H
eCH2CH]CH2
eCH2e
eCH3
eCH3
eCH3
eCOCH3
H
>100
>100
eCH2CH]CH2
eCH2CH]CH2
eCH2e
eCH2CH]CH2
eTS
eCHO
eCHO
eCH2CH]CH2
eCH2CHBrCH2Br
27.83 ꢂ 5.19
15.87 ꢂ 1.43
66.38 ꢂ 10.99
>100
55.00 ꢂ 3.74
8.11 ꢂ 1.43
1.73 ꢂ 0.67
21.00 ꢂ 1.31
1.43 ꢂ 0.40
0.032 ꢂ 0.0017
>100
2.37 ꢂ 0.11a
16
H
9.34 ꢂ 0.56a
a
Date derived from ref. [14].

232
Z. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 228e236
Fig. 6. Structures of compounds 13, 14, 15 and 16.
results indicated that the partial positive charge on the N5-
substituents was necessary as inhibitors of nucleophilic MetS.
Moreover, the results that 11b, 12b and 16 had higher inhibitory
activity were consistent with our prediction. However, as shown in
Table 1, weaker inhibition was found for 11b compared to 8b. The
later docking poses of them revealed that the specific tricyclic
geometry of 8b didn’t fit well into the enzyme pocket.
aminobromobenzoic acid side chain and Asn-323 as well as Arg-516,
similar to the interaction with CH3eH4folate. No matter whether the
N5eBr was in S or R configuration, the skeleton was in the same
position and formed the same hydrogen bonds. The dibromopropyl
group on N5 in both configurations would be in suitable cave which
was near the “opening” site of the protein [Fig. 8].
Compounds 2b, 6e8b, 11e12b were evaluated against
recombinant human DHFR also because the bicyclic pyrimidine
analogs of folic acid containing 2,4-diamino substitution pattern
are usually inhibitors of dihydrofolate reductase [20]. As shown in
Table 2, compound 2b, a 8-deaza derivative of aminopterin which is
a classical antifolate and antitumor agent, was the strongest
4.3. Evaluation of human tumor cell lines
Compounds 7be12b and 13e16 were evaluated for their cyto-
toxicity toward tumor cell lines. For comparison, the intermediates
2b and 5b were evaluated as well. The potency of inhibiting the
growth of tumor cells was measured as IC50 compared to MTX, and
the results are listed in Table 2. To discuss adequately, the previ-
ously reported values of compounds 5b, 13e16 against BGC-823,
Hela, Bel-7402 were indicated [14]
inhibitor of DHFR (IC50 ¼ 0.018
were almost equipotent against the DHFR (IC50 ¼ 0.033
0.033 M, and 0.032 m
m
M). Compounds 6b, 7b and 11b
M,
M).
m
m
m
M) to the standard MTX (IC50 ¼ 0.0312
Compared to 6b, compounds 9b and 10 bearing bulkier substitu-
Compound 2b, an 8-deaza counterpart of aminopterin, dis-
played a much stronger cytotoxicity against all five cell lines than
MTX. Besides 2b, only 10b among the compounds showed good
activity against KB and Bei-7402 cell lines. IC50 values of other
compounds were more than 1 M and even more than 50 M. That
means the 8-deazafolate derivatives might be insensitive against
these two cell lines and might be tissue-specific chemotherapeutic
agents for cancers.
ents on N5 position showed weaker inhibition.
4.2. Molecular modeling
To understand the possible binding mode of 8-deazate-
trahydrofolate derivatives with electrophilic groups on MetS,
compound 16 was docked into the CH3eH4folate-binding pocket of
cobalamin-dependent methionine synthase (1e566) from ther-
motoga maritima (cd2þ, hcy, methyltetrahydrofolate complex) (PDB
ID: 1Q8J) by using the ligand minimization of Discovery Studio (DS)
2.5. Default parameters were used as described in the manual unless
otherwise specified. It was found that compound 16 bound to MetS
in the pocket of CH3eH4folate in an extended mode, and formed
almost the same key hydrogen bonds with the protein [Fig. 7].
The pterin ring, similar to that of CH3eH4folate, was positioned
by hydrogen bonds with the same three residues Asn-411, Asp-473,
and Asn-508 except Asp-390 which initially formed a hydrogen
bond with N8 of CH3eH4folate. There was an interaction between p-
It’s interesting to note that the inhibition values of N5,N10
-
methylenated tricyclic compound 8b against BGC-823 and Hela
tumor cells was better than those of 9b and 11b, almost close to that
of MTX. These data suggest that 8b maybe inhibit other enzyme of
folic acid metabolism except MetH.
Analog 16, with the dibromopropyl group on N5, inhibited the
growth of SKOV3 cells by 50% at concentrations of 0.042 mM, which
clearly indicated that 16 was a considerably stronger anti-tumor
agent than MTX by the fact that it was 1.4 times more effective
for SKOV3. Another two compounds 11b and 12b, which inhibited
MetS better too, have poor activity. Maybe we need more evidence
Table 2
Inhibition concentrations (IC50, in
mM) against selected tumor cell lines.
BGC-823
Hela
SKOV3
KB
Bel-7402
2b
5b
7b
13
8b
9b
10b
11b
12b
14
0.010 ꢂ 0.002
0.44 ꢂ 0.10a
>100
0.003 ꢂ 0.0007
0.96 ꢂ 0.10a
>100
0.010 ꢂ 0.0025
0.24 ꢂ 0.063
>100
0.87 ꢂ 0.21
37.41 ꢂ 7.77
0.573 ꢂ 0.0565
0.450 ꢂ 0.0711
>100
2.70 ꢂ 0.64
>100
18.30 ꢂ 3.12
87.9 ꢂ 1.97a
>100
31.05 ꢂ 2.327
5.20 ꢂ 0.92a
0.18 ꢂ 0.04
2.09 ꢂ 0.41
0.289 ꢂ 0.0538
6.90 ꢂ 1.43
27.01 ꢂ 0.619
2.43 ꢂ 0.41a
6.59 ꢂ 1.5a
22.56 ꢂ 5.05a
0.11 ꢂ 0.01a
7.85 ꢂ 1.73a
0.13 ꢂ 0.023
0.2273 ꢂ 0.0382
0.434 ꢂ 0.0873
6.0 ꢂ 1.30
>100
42.2 ꢂ 8.6a
64.7 ꢂ 13.8
61.5 ꢂ 14.9
0.325 ꢂ 0.0640
65.2 ꢂ 12.5
12.35 ꢂ 0.762
7.1 ꢂ 1.8a
>100
>100
0.336 ꢂ 0.0623
>130
41.59 ꢂ 0.992
27.1 ꢂ 5.60a
0.90 ꢂ 0.21a
19.4 ꢂ 6.12a
0.10 ꢂ 0.023a
34.62 ꢂ 0.531
50.24 ꢂ 1.195
>100
20.03 ꢂ 0.556
6.722 ꢂ 0.449
>100
>100
20.9 ꢂ 3.70
15
16
MTX
1.2 ꢂ 0.23a
>100a
0.042 ꢂ 0.01
0.06 ꢂ 0.012
82.3 ꢂ 10.2a
a
Date derived from ref [14].

Z. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 228e236
233
cinoma (BCG-823), SKOV3, KB and cervical cancer (Hela) were
grown and maintained in RPMI-1640 medium supplemented with
10% fetal bovine serum, penicillin (100 units mLꢁ1), streptomycin
concentration at 37 ꢀC in a humidified incubators in an atmosphere
of 5% CO2. All of the experiments were performed on exponentially
growing cancer cells. Inhibition of cell growth was analyzed by
using MTT and SRB assays [21,22] after 48 h treatment of different
dosages of compounds. The data was presented in Table 1 as the
means of three independent experiments.
6.1.2. Methionine synthase activity assay
Cells of HL60 at the density of 1 ꢃ 106 cells/well in 24-well
plates were serum-starved before treating with various concen-
trations of test compounds for 3 h. Then the cells were harvested
and lysed with lysis buffer (20 mmol/L HEPES pH 7.5, 3 mmol/L
MgCl2, 14 mmol/L NaCl, 5% glycerol, 0.5% Igepal CA-630, 1 mmol/L
DTT, 1 mg/mL aprotonin, 1 mg/mL eupeptin, and 1 mmol/L phe-
nylmethylsulfonyl fluoride) [23]. After centrifugation (10,000 rpm,
10 min, at 4 ꢀC), the supernatants were assayed in 96-well micro-
plates for MetS activity, following the method of Drummond et al.
[24] and Jarret et al. [25] with modification. The assay solution
contained 1 mol/L potassium phosphate buffer (pH 7.2) 20
1 mol/L DTT 5 L, 4.2 mmol/L 5-methyltetrahydrofolate 12
0.76 mmol/L SAM 5 mL, 97 L double distilled water, and 40
m
m
L,
L,
m
m
m
L cell
lysate. After adding 0.5 mmol/L hydroxocobalamin 20
mL into the
mixture, it was immediately preincubated at 37 ꢀC for 5 min
immediately. Then the reaction was initiated by mixing with
100 mmol/L
The reaction was terminated by the addition of 5 mmol/L HCl/60%
formic acid 50
L and incubated at 80 ꢀC for 10 min. The total
volume in the well of a 96-well microplate was 250 L. The plate
L-homocysteine 1 m
L and incubated for 10 min at 37 ꢀC.
Fig. 7. Superimposition of the 16’s best docking pose into 1Q8J considering the
hydrogen bonds with water and amino acid residues. The viewpoint is approximately
along the expected approach of the corrin ring. On the piperidine ring of the pterin, the
C6 side chain substituent is axial and the N5-dibromo propyl group is equatorial,
pointing toward the reader.
m
m
was read at 350 nm by using FLUOstar OPTIMA microplate multi-
detection reader (BMG Offenburg, Germany).
to support the association of MetH inhibition with cancer cell
death. Further studies against more cancer cell lines and on the
transport mechanism and accumulation will be required.
6.1.3. Dihydrofolate reductase (DHFR) assay
All recombinant human (rh) DHFR enzymes were assayed
spectrophotometrically in a solution containing 0.1 mM dihy-
drofolate, 0.3 mM NADPH, and 100 mM KCl at pH 7.5 and 32 ꢀC. The
5. Conclusion
Based on the action mechanism of methionine synthase, 8-
deaza-5,6,7,8-tetrahydrofolate bearing electrophilic substituents
on N5 were designed and synthesized to explore their MetS inhi-
bition activity as well as antitumor activity. Compounds 11b, 12b
and 16 showed high activities against MetS. And the function was
confirmed by docking study into the CH3eH4folate-binding pocket
of MetS. Compounds 11b, 12b didn’t show good activity against
cancer cell lines. Further studies will be done on this class of
compounds.
6. Experimental section
6.1. Biological evaluation
Human dihydrofolate reductase and MTX were purchased from
Sigma Chemical Co. RPMI-1640 medium was produced from
GIBCO.; Sulforhodamine B (SRB), 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT), tetrazolium salt and propi-
dium iodide were purchased from Sigma Chemical co.; Microplate
reader (FLUOsta OPTIMA, Germany); BECScan flow cytometer
(Becton Sickinson FACScan, American).
6.1.1. Cell lines and culture conditions
Human promyelocytic leukemic cell line (HL-60), human
hepatocellular carcinoma cell line (Bel-7402), stomach adenocar-
Fig. 8. Superposition of MetS structure in complex with 16 (orange) and CH3eH4folate
(magenta). (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)

234
Z. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 228e236
change in absorbance was measured at 340 nm by microplate
spectrophotometer [26,27]
7.53e7.66 (m, 3H), 7.81e7.84 (d, 2H, J ¼ 8.1 Hz). 13C NMR (75 MHz,
CDCl3)
d 14.1, 22.6, 26.8, 29.6, 52.5, 54.2, 60.9, 61.8, 126.7, 127.8,
128.7, 131.2, 133.2,145.9,151.6, 159.1, 162.2, 166.2, 170.4, 172.0, 173.3,
6.2. Chemistry
177.5. MS(ESI) m/z 538.2 [M þ H]þ.
Melting points (uncorrected) were determined with an X4-type
apparatus. All evaporations were carried out in vacuo with a rotary
evaporator.1H-and 13C NMR spectra were recorded with Jeol-AL-
300 and Varian INOVA-500 with DMSO-d6 as solvent and Me4Si
as an internal standard. J-values are expressed in Hz. The ESI-TOF
spectra were taken on a QSTAR (ABI. SUA) mass spectrometer by
using methanol as the solvent. HPLC purity determinations were
carried out using a DIKMA AS-H 4.6 ꢃ 250 nm column. Mobile
phase was isopropanol/n-hexane (1:1e1:3.5). All test compounds
were confirmed to be ꢄ95% by HPLC. Thin-layer chromatography
(TLC) was performed on POLYGRAM Sil G/UV254 silica gel plates
with fluorescent indicator, and the spots were visualized under
254 nm illumination.
6.2.4. N-[4-[(2,4-Diaminopyrido[3,2-d]pyrimidin-6-ylmethyl)
acetylamino]benzoyl]- -glutamic acid 4b
L
A procedure similar to the preparation of 3b was followed to
prepare 4b from 4a. The yield of 4b was 56% as a white solid. mp
200 ꢀC (dec). 1H NMR (500 MHz, DMSO-d6)
d 1.93 (s, 3H), 2.04e2.37
(m, 4H), 3.96e4.02 (m, 1H), 5.02 (s, 2H), 6.18 (s, 2H), 6.95 (br, 2H),
7.42e7.43 (d, 2H, J ¼ 8.0 Hz), 7.49e7.54 (m, 2H), 7.78e7.80 (d, 2H,
J ¼ 8.0 Hz), 7.91e7.92 (d 1H, J ¼ 6.5 Hz). MS(ESI) m/z 482.2 [M þ H]þ.
6.2.5. Diethyl N-[4-[2-(2,4-diamino-5,6,7,8-tetrahydropyrido[3,2-d]
pyrimidin-6-ylmethyl) methylamino]benzoyl]- -glutamate 6a
L
A solution of 3a (397 mg, 0.78 mmol) in 80 mL of ethanol and
acetic acid (v/v ¼ 1/70) was added to platinum oxide catalyst
(40 mg), and the suspension was hydrogenated (0.4 MPa) for
48 h. The reaction mixture was filtered through celite and the
filtrate was adjusted to pH7e8 with NaHCO3 (5%), and evaporated
to dryness under reduced pressure. The residue was added to
CHCl3 (100 mL ꢃ 2) and filtered. The filtrate was concentrated to
a small volume, and was purified by column chromatography
(silica gel). Elution with CH2Cl2eCH3OH (12:1) gave pure 6a
(173 mg, 43%) as a light-yellow solid; mp 119e121 ꢀC 1H NMR
6.2.1. Diethyl N-[4-[(2,4-diaminopyrido[3,2-d]pyrimidin-6-
ylmethyl)methylamino]benzoyl]e
A solution of dried 1 (2.15 g, 7.96 mmol) in dimethylacetamide
(180 mL) was added to diethyl N-(p-methylaminobenzoyl)-
L
-glutamate 3a
L-
glutamate (2.85 g, 8.43 mmol) and the solution was stirred for 2
days at room temperature. The solvent was removed under reduced
pressure and water (100 mL) was added to the gummy residue. The
pH of the suspension was adjusted to 8 by adding NaHCO3 (5%) and
the resulting solution was extracted with chloroform (3 ꢃ 100 mL).
The organic layer was concentrated to a small volume and was
purified by column chromatography (silica gel). Elution with
CH2Cl2eCH3OH (15:1) gave pure 3a (2.9 g, 72%) as a yellow solid.
(500 MHz, DMSO-d6)
d 1.15e1.20 (m, 6H), 1.65e1.71 (m, 1H),
1.91e1.95 (m, 1H), 1.97e2.12 (m, 2H), 2.40e2.43 (t, 2H, J ¼ 7.5 Hz),
2.56e2.58 (m, 2H), 3.04 (s, 3H), 3.37e3.44 (m, 2H), 3.57e3.61 (m,
1H), 4.03e4.12 (m, 4H), 4.37e4.41 (m, 1H), 4.79 (s, 1H), 6.73e6.75
(m, 4H), 7.58 (bs, 2H), 7.75e7.76 (d, 2H, J ¼ 8.5 Hz), 8.32e8.33 (d,
mp 78e80 ꢀC; 1H NMR (300 MHz, CDCl3)
d
1.18e1.31 (m, 6H), 2.09e
1H, J ¼ 7.5 Hz). 13C NMR (500 MHz, DMSO-d6)
d 14.1, 21.0, 22.6,
2.15 (m, 1H), 2.26e2.29 (m, 1H), 2.41e2.90 (m, 2H), 3.18 (s, 3H),
4.05e4.25 (m, 4H), 4.72 (s, 2H), 4.75e4.80 (m, 1H), 5.17 (bs, 2H),
6.71e6.74 (d, 2H, J ¼ 9.0 Hz), 6.84e6.87 (d,1H, J ¼ 9.0 Hz), 7.32e7.35
(d, 1H, J ¼ 9.0 Hz), 7.62e7.65 (d, 1H, J ¼ 9.0 Hz), 7.69e7.72 (d, 2H,
J ¼ 9.0 Hz). MS (ESI) m/z 510.3 [M þ H]þ.
23.8, 25.8, 30.2, 48.8, 51.8, 55.5, 59.9, 60.4, 110.4, 115.8, 120.1,
128.9, 132, 151.2, 151.4, 157.3, 166.4, 172.2, 172.2. MS (ESI) m/z
514.1 [M þ H]þ.
6.2.6. N-[4-[2-(2,4-Diamino-5,6,7,8-tetrahydropyrido[3,2-d]
pyrimidin-6-ylmethyl)methylamino] benzoyl]- -glutamic acid 6b
L
6.2.2. N-[4-[(2,4-Diaminopyrido[3,2-d]pyrimidin-6-ylmethyl)
A procedure similar to the preparation of 3b was followed to
prepare 6b from 6a. The yield of 6b was 46% as a light-yellow solid.
methylamino]benzoyl]- -glutamic acid 3b
L
A solution of 3a (110 mg, 0.216 mmol) in THF (2 mL) was added
to 1 N NaOH (1 mL), and the mixture was stirred at room
temperature about 2 h indicted by the TLC. The organic solvent was
removed by evaporation under reduced pressure, the remaining
aqueous solution was acidified carefully with 0.5 N HCL, and the
solid was collected by filtration, washed with water, and dried in
vacuo to give 3b (70 mg, 71%) as a white solid. mp 200e202 ꢀC 1H
mp 204e206 ꢀC 1H NMR (500 MHz, DMSO-d6)
d 1.92e1.93 (m, 1H),
2.03e2.05 (m, 1H), 2.31e2.32 (m, 4H), 2.40e2.45 (m, 2H), 2.88e
3.01 (m, 2H), 3.07 and 3.18 (s, 3H), 3.44e3.45 (m, 1H), 4.33 (m,
1H), 4.73e4.76 (s, 1H), 6.54 (bs, 2H), 6.76e6.79 (m, 2H), 7.71e7.74
(m, 2H), 8.11e8.14 (m, 1H). MS (ESI) m/z 458.2 [M þ H]þ. HRMS
calcd for C21H28N7O5 (M þ Hþ), 458.21464; found, 458.21509.
NMR (300 MHz, DMSO-d6)
d
1.93e2.06 (m, 2H), 2.29e2.34 (t, 2H,
6.2.7. Diethyl N-[4-[2-(2,4-diamino-5,6,7,8-tetrahydropyrido[3,2-d]
J ¼ 7.2 Hz), 3.18 (s, 3H), 4.29e4.37 (m, 1H), 4.77 (s, 2H), 6.77e6.80
(d, 2H, J ¼ 8.1 Hz), 7.35e7.38 (d, 1H, J ¼ 8.7 Hz), 7.54e7.57 (d, 1H,
J ¼ 8.4 Hz), 7.71e7.74 (d, 2H, J ¼ 8.4 Hz), 8.10e8.12 (d, 1H, J ¼ 7.5 Hz).
MS (ESI) m/z 454.0 [M þ H]þ.
pyrimidin-6-yl methyl)acetylamino]benzoyl]-L-glutamate 7a
A solution of 4a (520 mg, 0.97 mmol) in ethanol (100 mL) and
trifluoroactyl acid (0.2 mL) was added to platinum oxide catalyst
(55 mg), and the suspension was hydrogenated (0.3 MPa) for 72 h.
The reaction mixture was filtered through celite and the filtrate was
adjusted to pH7e8 with NaHCO3 (5%), and evaporated to dryness
under reduced pressure. The residue was added to CHCl3
(60 mL ꢃ 2) and filtered. The filtrate was concentrated to a small
volume, and was purified by column chromatography (silica gel).
Elution with CH2Cl2eCH3OH (18:1) gave pure 7a (390 mg, 74%) as
a white solid; mp: 104e106 ꢀC 1H NMR (300 MHz, DMSO-d6)
6.2.3. Diethyl N-[4-[(2,4-diaminopyrido[3,2-d]pyrimidin-6-
ylmethyl)acetylamino]benzoyl]- -glutamate 4a
L
A solution of 2a (330 mg, 0.67 mmol) in CH3COOH (20 mL) was
added to acetic anhydride (1 mL), and the solution was stirring at
room temperature for 2 h. Then the mixture was poured into 15 mL
of water, extracted with CHCl3 (3 ꢃ 10 mL) and dried over Na2SO4.
After evaporation of solvent, the residue was purified by column
chromatography (silica gel). Elution with CH2Cl2eCH3OH (13:1)
gave pure 4a (350 mg, 88%) as a white solid. mp 99e100 ꢀC 1H NMR
d
1.14e1.22 (m, 6H), 1.56e1.91 (m, 2H), 1.83 (s, 3H), 1.99e2.14 (m,
2H), 2.43e2.48 (t, 2H, J ¼ 7.5 Hz), 3.23e3.36 (m, 3H), 3.68e3.81 (m,
2H), 4.01e4.15 (m, 4H), 4.41e4.49 (m, 1H), 4.54 (s, 1H), 7.02 (s, 2H),
7.57e7.57 (d, 2H, J ¼ 8.4 Hz), 7.73 (br, 2H), 7.94e7.97 (d, 2H,
J ¼ 8.4 Hz), 8.82e8.85 (d, 1H, J ¼ 7.2 Hz). MS (ESI) m/z 542.3
[M þ H]þ.
(300 MHz, CDCl3):
d 1.19e1.32 (m, 6H), 1.93 (s, 3H), 2.12e2.36 (m,
2H), 2.39e2.55 (m, 2H), 4.07e4.26 (m, 4H), 4.73e4.80 (m, 1H), 5.01
(s, 2H), 6.30 (br, 2H), 6.86 (br, 2H), 7.16e7.18 (d, 2H, J ¼ 7.8 Hz),

Z. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 228e236
235
6.2.8. N-[4-[2-(2,4-Diamino-5,6,7,8-tetrahydropyrido[3,2-d]
pyrimidin-6-ylmethyl)acetylamino] benzoyl]- -glutamic acid 7b
(m, 2H), 2.95e2.96 (d, 3H, J ¼ 3.5 Hz), 3.12e3.12 (d, 1H, J ¼ 4.0 Hz),
3.15 (d, 1H, J ¼ 4.0 Hz), 3.34e3.37 (d, 1H, J ¼ 16.0 Hz), 3,42 (bs, 1H),
3.46e3.49 (d, 1H, J ¼ 13.5 Hz), 4.09e4.25 (m, 4H), 4.76e4.81 (m,
1H), 5.10e5.25 (m, 2H), 5.66e5.73 (m, 1H), 6.11 (bs, 2H), 6.51e6.52
(d, 2H, J ¼ 8.5 Hz), 7.64e7.66 (d, 2H, J ¼ 7.5 Hz). 13C NMR (500 MHz,
L
A procedure similar to the preparation of 3b was followed to
prepare 7b from 7a. The yield of 7b was 76% as a light-yellow solid.
mp 173 ꢀC (dec). 1H NMR (300 MHz, DMSO-d6)
d 1.56e1.91 (m, 2H),
1.83 (s, 3H), 1.99e2.14 (m, 2H), 2.43e2.48 (t, 2H, J ¼ 7.5 Hz), 3.23e
3.68 (m, 3H), 3.73e3.81 (m, 2H), 4.41e4.49 (m, 1H), 4.54 (s, 1H),
7.02 (s, 2H), 7.47 (br, 2H), 7.94e7.97 (d, 2H, J ¼ 7.5 Hz), 7.54e7.57 (d,
2H, J ¼ 7.5 Hz), 8.82e8.85 (d, 1H, J ¼ 7.2 Hz). MS (ESI) m/z 546.5
[M þ H]þ.
CDCl3) d 14.1, 17.6, 21.9, 27.3, 29.6, 30.6, 51.9, 52.3, 52.8, 55.8, 60.8,
60.6, 110.7, 110.8, 116.8, 117.5, 120.5, 128.8, 134.8, 151.5, 151.6, 155.2,
161.1, 167.2, 172.5, 173.4. MS (ESI) m/z 554.2 [M þ H]þ.
6.2.12. N-[4-[[2-(2,4-Diamino-5-allyl-5,6,7,8-tetrahydropyrido
[3,2-d]pyrimidin-6-yl)methyl] methylamino]benzoyl]-L-glutamic
6.2.9. Diethyl N-[4-[(2-2,4-diamino-5,10-methylene-5,6,7,8-
acid 9b
tetrahydropyrido[3,2-d]pyrimidin-6-yl methyl)amino]benzoyl]-
glutamate 8a
L
-
A procedure similar to the preparation of 3b was followed to
prepare 9b from 9a. The yield of 9b was 69.4% as a light-yellow
A solution of 5a (400 mg, 0.8 mmol) and paraformaldehyde
(120 mg) in THF (1 mL) and water (1.2 mL) was reacted under
sonication for 4 h at room temperature. TLC in CHCl3eCH3OH (8:2)
indicated complete disappearance of compound 8. The organic
solvent was removed by evaporation under reduced pressure and
the remaining aqueous solution was added to 10 mL of water, and
extracted with CHCl3 (5 mL ꢃ 3). The organic layer was dried over
anhydrous sodium sulfate, concentrated to a small volume, and
poured onto a column (silica gel). Elution with CHCl3eCH3OH (16:1)
gave 317 mg (77.4%) of pure 8a as a light yellow solid. mp 95e97 ꢀC
solid. m.p. 208e210 ꢀC 1H NMR (300 MHz, DMSO-d6)
d 1.63e1.67
(m, 1H), 1.90e1.97 (m, 1H), 1.99e2.10 (m, 2H), 2.30e2.35 (t, 2H,
J ¼ 7.2 Hz), 2.44e2.51 (m, 2H), 2.95 (s, 3H), 3.05e3.13 (m, 1H), 3.30
(bs, 4H), 4.30e4.36 (m, 1H), 4.96e5.18 (m, 2H), 5.82e5.84 (m, 1H),
6.58e6.61 (d, 2H, J ¼ 9.0 Hz), 6.79 (bs, 2H), 7.70e7.72 (d, 2H,
J ¼ 7.8 Hz), 8.09e8.11 (d, 1H, J ¼ 6.0 Hz). MS (ESI) m/z 498.3
[M þ H]þ. HRMS calcd for C24H32N7O6 (M þ Hþ), 498.24594; found,
498.24675.
6.2.13. Diethyl N-[4-[2-(2,4-diamino-5-tosyl-5,6,7,8-
tetrahydropyrido[3,2-d]pyrimidin-6-ylmethyl) methylamino]
1H NMR (500 MHz, DMSO-d6)
d
1.15e1.24 (m, 6H), 1.72e1.87 (m,
2H),1.97e2.11 (m, 2H), 2.41e2.46 (m, 2H), 2.50e2.58 (m, 2H), 3.33e
3.34 (m, 1H), 3.63e3.69 (m, 2H), 3.78e3.79 (d,1H, J ¼ 4.5 Hz), 4.03e
4.13 (m, 4H), 4.38e4.43 (m,1H), 4.91e4.92 (d,1H, J ¼ 4.0 Hz), 5.64 (s,
2H), 6.24 (s, 2H), 6.53e6.55 (d, 2H, J ¼ 9.0 Hz), 7.80e7.78 (d, 2H,
J ¼ 9.0 Hz), 8.33e8.35 (d, 1H, J ¼ 7.0 Hz). 13C NMR (125 MHz, DMSO-
benzoyl]-
A solution of 6a (47 mg, 0.092 mmol) in CH3CN (2 mL) was
added to Tos-Cl (22.6 mg, 0.021 mmol) and (CH3CH2)3N (50 L) and
L
-glutamate 10a
m
the mixture was stirred at room temperature for 10 h. Solvent was
removed under reduced pressure. The residue was stirred with
water (30 mL), and the mixture was extracted with CH2Cl2
(2 ꢃ 20 mL). The CH2Cl2 layers were combined and dried over
anhydrous Na2SO4. After evaporation of solvent to a small volume,
the residue was purified by preparative thin layer chromatography
(silica gel). Elution with CHCl3eCH3OH (9:1) gave pure 10a (11 mg,
18%) as a light yellow solid. mp 88e90 ꢀC 1H NMR (300 MHz, CDCl3)
d6) d 14.1, 21.9, 25.8, 28.2, 30.2, 51.2, 51.9, 56.3, 59.9, 60.4, 65.8,110.5,
113.7, 120.6, 129.0, 148.2, 153.7, 158.8, 159.5, 166.5, 172.2, 172.2. MS
(ESI) m/z 512.3 [M þ H]þ, m/z 534.3 [M þ Na]þ.
6.2.10. N-[4-[[2-(2,4-Dimino-5,10-methylene-5,6,7,8-
tetrahydropyrido[3,2-d]pyrimidin-6-yl)methyl] amino]benzoyl]-L-
glutamic acid 8b
d 1.21e1.34 (m, 6H), 1.94e1.99 (m, 2H), 2.09e2.20 (m, 2H), 2.29e
A procedure similar to the preparation of 3b was followed to
prepare 8b from 8a. The yield of 8b was 68% as a white solid. mp
2.33 (m, 2H), 2.40 (s, 3H), 2.44e2.56 (m, 2H), 3.08e3.09 (d, 3H,
J ¼ 1.2 Hz), 3.18e3.24 (m,1H), 3.76e3.85 (m,1H), 4.09e4.16 (m, 2H),
4.21e4.28 (m, 2H), 4.35e4.40 (m, 1H), 4.81e4.82 (m, 1H), 4.93 (bs,
2H), 5.54 (bs, 2H), 6.76e6.79 (d, 2H, J ¼ 8.7 Hz), 7.24e7.27 (d, 2H,
J ¼ 9.0 Hz), 7.50e7.53 (d, 2H, J ¼ 7.8 Hz), 7.75e7.78 (d, 2H,
95 ꢀC (dec). 1H NMR (300 MHz, DMSO-d6)
d 1.76e2.11 (m, 4H),
2.29e2.34 (m, 2H), 2.49e2.73 (m, 2H), 3.33e3.90 (m, 1H), 3.66e
3.69 (m, 2H), 3.79e3.80 (d, 1H, J ¼ 3.9 Hz), 4.29e4.36 (m, 1H),
4.92e4.93 (d, 1H, J ¼ 3.9 Hz), 6.48 (bs, 2H), 6.53e6.56 (d, 2H,
J ¼ 8.7 Hz), 6.77 (bs, 2H), 7.79e7.76 (d, 2H, J ¼ 8.7 Hz), 8.13e8.16 (d,
J ¼ 9.0 Hz). 13C NMR (300 MHz, CDCl3)
d 14.1, 21.6, 27.3, 27.4, 28.1,
29.6, 30.5, 52.1, 54.9, 57.4, 60.7, 61.6, 106.0, 110.9, 120.8, 127.5, 128.9,
129.9,133.4,144.7,150.9,159.9,162.0,167.0,172.4,173.3. MS (ESI) m/
z 668.5 [M þ H]þ.
1H, J ¼ 7.5 Hz). 13C NMR (75 MHz, DMSO-d6)
d 21.5, 26.6, 30.9, 51.2,
52.2, 56.7, 65.8, 110.6, 113.9, 121.2, 128.9, 148.1, 150.1, 157.1, 160.1,
166.1, 174.4. IR (KBr)
y 3330.64, 2928.96, 1607.55, 1505.89, 1392.01,
1275.19, 1201.58. MS (ESI) m/z 456 [M þ H]þ. HRMS calcd for
6.2.14. N-[4-[2-(2,4-Diamino-5-tosyl-5,6,7,8-tetrahydropyrido[3,2-d]
pyrimidin-6-ylmethyl) methylamino]benzoyl]-L-glutamatic acid 10b
C
22H26N6O5 (M þ Hþ), 456.19899; found, 456.19933.
A procedure similar to the preparation of 3b was followed to
prepare 10b from 10a. The yield of 10b was 59.5% as a light-yellow
6.2.11. Diethyl N-[4-[[2-(2,4-diamino-5-allyl-5,6,7,8-
tetrahydropyrido[3,2-d]pyrimidin-6-yl)methyl]methylamino]
solid. m.p. 191e192 ꢀC 1H NMR (500 MHz, DMSO-d6)
d 1.48 (m,
benzoyl]-
A solution of 6a (80 mg, 0.16 mmol) in anhydrous DMF (1 mL)
was added to allyl bromide (15 L, 0.18 mmol) and Cs2CO3 (60 mg,
L
-glutamate 9a
1H), 1.76 (m, 1H), 1.89e1.97 (m, 2H), 2.08e2.18 (m, 2H), 2.29e2.37
(m, 5H), 2.99 (s, 3H), 3.17e3.21 (m, 1H), 3.74e3.76 (m, 1H), 4.21
(m, 1H), 4.39 (m, 1H), 6.74 (d, 2H, J ¼ 7.0 Hz), 7.39e7.40 (d, 2H,
J ¼ 12.0 Hz), 7.70-7.71 (d, 2H, J ¼ 7.0 Hz), 7.89e7.91 (d, 2H,
J ¼ 9.0 Hz), 8.25e8.26 (d, 1H, J ¼ 5.5 Hz). MS (ESI) m/z 612.3
[M þ H]þ. HRMS calcd for C28H34N7O7S1 (M þ Hþ), 612.22349;
found, 612.22393.
m
0.18 mmol) and the reaction mixture was stirred at room temper-
ature for about 15 h. Solvent was removed under reduced pressure.
The residue was stirred with water (20 mL), and the mixture was
extracted with CH2Cl2 (2 ꢃ 20 mL). The CH2Cl2 layers were
combined and dried over anhydrous Na2SO4. After evaporation of
solvent to a small volume the residue was purified by column
chromatography (silica gel). Elution with CH2Cl2eCH3OH (15:1)
gave pure 9a (66 mg, 77%) as a light yellow solid. mp 88e90 ꢀC 1H
6.2.15. Diethyl N-[4-[2-(2,4-diamino-5-formyl-5,6,7,8-
tetrahydropyrido[3,2-d]pyrimidin-6-yl methyl)methylamino]
benzoyl]- -glutamate 11a
L
NMR (500 MHz, CDCl3)
d:1.21e1.31 (m, 6H), 1.70e1.74 (m, 1H),
A solution of 6a (48 mg, 0.094 mmol) in ethyl formate (10 mL)
2.11e2.19 (m, 2H), 2.28e2.34 (m, 1H), 2.39e2.55 (m, 2H), 2.63e2.77
was added formic acid (0.4 mL, 0.106 mmol) and the mixture was

236
Z. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 228e236
stirred at room temperature for 1 h. Then acetyl anhydride (0.2 mL)
was added and the resulting solution was stirred for 0.5 h again.
NaHCO3 (5%, 10 mL) was added to the reaction mixture and
extracted with CH2Cl2 (10 mL ꢃ 2). The organic layer was dried over
anhydrous sodium sulfate, concentrated to a small volume, and
poured onto a column (silica gel). Elution with CHCl3eCH3OH
(18:1) gave 38 mg (75%) of pure 11a as a white solid. mp 87e
(m, 2H), 6.18 (bs, 2H), 7.53e7.98 (m, 4H), 8.15 (s, 1H), 8.82e8.87 (d,
1H, J ¼ 6.9 Hz). MS (ESI) m/z 514.2 [M þ H]þ.
Acknowledgments
This study was supported by the National Science Foundation of
China (20972011, 21042009, and 21172014) and grants from the
Ministry of Science and Technology of China (2009ZX09301-010)
for financial support.
89 ꢀC. 1H NMR (300 MHz, CDCl3)
d 1.21e1.33 (m, 6H), 1.84 (m,
1H), 2.12e2.17 (m, 1H), 2.28e2.40 (m, 2H), 2.44e2.55 (m, 2H),
2.65e2.73 (m, 2H), 2.99 (s, 3H), 3.12e3.14 (m, 1H), 3.26 (m, 1H),
3.50e3.55 (m, 1H), 4.08e4.27 (m, 4H, J ¼ 7.2 Hz), 4.76e4.83 (m, 1H),
5.06 (bs, 2H), 5.29 (s, 2H), 6.60e6.63 (d, 2H, J ¼ 8.7 Hz), 6.97 (bs,
Appendix A. Supplementary data
1H), 7.73e7.75 (d, 2H, J ¼ 8.7 Hz), 7.87e7.90 (d, 1H, J ¼ 7.2 Hz). 13
C
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2012.09.027.
NMR (300 MHz, CDCl3) d 14.1, 24.0, 26.1, 26.9, 27.3, 30.5, 45.9, 52.2,
54.2, 60.7, 61.9, 110.9, 121.4, 121.4, 129.0, 150.4, 150.4, 158.6, 160.4,
161.1, 166.8, 173.3, 173.3. MS (ESI) m/z 542.3 [M þ H]þ.
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6.2.17. Diethyl N-[4-[2-(2,4-diamino-5-formyl-5,6,7,8-
tetrahydropyrido[3,2-d]pyrimidin-6-yl methyl)acetylamino]
benzoyl]- -glutamate 12a
L
A solution of 7a (50 mg, 0.092 mmol) in formic acid (2 mL) was
stirred at 60 ꢀC for 2 h. After the solution was cooled to room
temperature, the mixture was adjusted to pH 7 with NaHCO3 (5%)
and extracted with CHCl3 (3 mL ꢃ 3). The organic layer was dried
over anhydrous sodium sulfate, concentrated to a small volume,
purified by preparative thin layer chromatography (silica gel).
Elution with CHCl3eCH3OH (9:1) gave pure 12a (45 mg, 86%) as
a light yellow solid. mp 118e120 ꢀC 1H NMR (300 MHz, DMSO-d6)
d
1.15e1.23 (m, 6H), 1.49e1.60 (m, 1H), 1.80 (s, 3H), 1.91e2.14 (m,
3H), 2.32e2.36 (m, 2H), 2.50e2.51 (m, 2H), 3.35e4.01 (m, 3H),
4.02e4.16 (m, 4H), 4.46 (m, 1H), 6.18 (bs, 2H), 7.53e7.98 (m, 4H),
8.15 (s, 1H), 8.82e8.87 (t, 1H, J ¼ 7.2 Hz). MS(ESþ) m/z 570.0.
6.2.18. N-[4-[2-(2,4-Diamino-5-formyl-5,6,7,8-tetrahydropyrido[3,2-d]
pyrimidin-6-ylmethyl)acetylamino]benzoyl]- -glutamic acid 12b
L
A procedure similar to the preparation of 3b was followed to
prepare 12b from 12a. The yield of 12b was 79% as a light-yellow
solid. mp 145e147 ꢀC 1H NMR (300 MHz, DMSO-d6)
d
1.80 (s, 3H),
1.91e2.14 (m, 2H), 2.32e2.51 (m, 4H), 3.35e4.01 (m, 3H), 4.45e4.52