Novel Synthesis of 8-deaza-5,6,7,8-tetrahydroaminopterin analogues via an aziridine intermediate

Article abstract of DOI:10.3390/molecules17055604

An efficient method for the construction of the tetrahydrofolate skeleton is described. Starting from pterin analogues and aromatic amines, 8-deaza-5,6,7,8- tetrahydroaminopterin derivatives and the heterocyclic benzoyl isosteres were synthesized via a novel aziridine intermediate. Following this method, the byproducts of carbonnitrogen bond hydrogenolysis in traditional synthetic strategy can be completely avoided.

Full text of DOI:10.3390/molecules17055604

Molecules 2012, 17, 5604-5614; doi:10.3390/molecules17055604
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Novel Synthesis of 8-Deaza-5,6,7,8-tetrahydroaminopterin
Analogues via an Aziridine Intermediate
Shouxin Zhou 1, Chao Tian 1, Chao Li 1, Ying Guo 1, Xiaowei Wang 1, Junyi Liu 1,2 and
Zhili Zhang 1,*
1
Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University,
Beijing 100191, China
2
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
* Author to whom correspondence should be addressed; E-Mail: lilybmu@bjmu.edu.cn;
Tel.: +86-10-8280-5203; Fax: +86-10-8280-1706.
Received: 10 April 2012; in revised form: 3 May 2012 / Accepted: 5 May 2012 /
Published: 10 May 2012
Abstract: An efficient method for the construction of the tetrahydrofolate skeleton is
described. Starting from pterin analogues and aromatic amines, 8-deaza-5,6,7,8-
tetrahydroaminopterin derivatives and the heterocyclic benzoyl isosteres were synthesized
via a novel aziridine intermediate. Following this method, the byproducts of carbon-
nitrogen bond hydrogenolysis in traditional synthetic strategy can be completely avoided.
Keywords: tetrahydrofolate; aziridine; catalytic hydrogenation; solvent-free
1. Introduction
Tetrahydrofolate plays a central role in one-carbon metabolism involving DNA biosynthesis, and
the biologically active cofactor forms are substrates for at least 15 relevant enzymes [1–4]. Hence,
tetrahydrofolate analogues are widely used as inhibitors of folate-dependent enzymes for
chemotherapy. Early in 1960s, tetrahydrohomofolate (Figure 1) was found to be a potent inhibitor
against TS (thymidine synthase) [5]. After then, more tetrahydrofolate analogues were developed
as antitumor drugs, including lometrexol [6] as a specific inhibitor of GARFT (glycinamide
ribonucleotide formyl transferase), AG2034 [7] and LY309887 [8] as next-generation GARFT
inhibitors, side chain modified 5-deazatetrahydrofolate analogues [9] as potential dual inhibitors of

Molecules 2012, 17
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FPGS (folylpolyglutamate synthetase) and GARFT as therapeutic agents, compound 1 and its
analogues as MS (methionine synthase) and DHFR (dihydrofolate reductase) inhibitors [10].
Figure 1. Tetrahydrofolate and tetrahydroaminopterin analogues.
In our research on new antifolate inhibitors, we have identified N-[4-{(2-[2,4-diamino-5-(2,3-
dibromopropane)-5,6,7,8-tetrahydropyrido(3,2-d) pyrimidin-6-yl]methyl)amino}-3-bromobenzoyl]-L-
glutamic acid (ZL033) [10,11], which inhibits methionine synthase (IC50: 1.4 ± 0.4 µmol/L) and
proliferation of HL-60 cells (IC50: 2.0 ± 1.0 µmol/L). Despite the remarkable biological importance of
tetrahydrofolate analogues, not many synthetic approaches have been reported. The common synthesis
strategy [12–15] is based on a critical step which is catalytic hydrogenation of folate derivatives, and
was the strategy we used in synthesis of ZL033 and a series of novel 8-deazatetrahydrofolate
derivatives as DHFR and MS inhibitors (Scheme 1). The reductive hydrogenation is a critical step in
the entire synthetic scheme due to the benzylic hydrogenolysis of the C9-N10 bond as a side reaction.
Careful adjustment of reaction solution acidity and protection of N10 with an electron-withdrawing
substituent before reduction didn’t work well either. Against this background, we now report a
practical and novel method for synthesizing 8-deazatetrahydrofolate 1 by ring-opening of an aziridine
intermediate. Following this method, ZL033 and its heterocyclic benzoyl isosteres can be synthesized.
And the modification in the side chain of 8-deazatetrahydrofolate can lead to more optional
tetrahydrofolate analogues.
Scheme 1. Reported route for compound 1.

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2. Results and Discussion
2.1. Retrosynthetic Analysis of 1
Our synthetic strategy is displayed in Scheme 2. Compound 1 is a key intermediate which can be
converted into ZL033 easily by the same method in Scheme 1, so we focused on the convenient
synthesis of 1. We envisaged that target compound 1 and its derivatives could be constructed by
ring-opening of aziridine 5 with aromatic amines. The aziridine 5 would be synthesized via cyclization
of compound 6, which could be obtained by catalytic hydrogenation of compound 3. Accordingly, we
started our synthetic studies by seeking a general and practical method for preparing the reduced
compound 6.
Scheme 2. Synthetic strategy.
2.2. Catalytic Hydrogenation of 3
Compound 3 was prepared in the same way as before [16,17], and was catalytically hydrogenated
directly before linking with the side-chain amine to avoid C9-N10 hydrogenolysis (Scheme 3). As there
is no fragile C9-N10 bond in 3, stronger acidic conditions could be used compared to the similar
hydrogenation in Scheme 1. An initial screening of the influence of different acidic conditions (Table 1)
revealed that moderate acidity gave better results. The reduction reaction didn’t happen without acid
added (Table 1, entry 1). Weak acids such as acetic acid gave product in 50~60% yield, even when
excess acid was used as solvent (Table 1, entries 2~4), while the yield increased obviously with
hydrochloric acid (Table 1, entries 5~7). Treatment of 3 with 8 equivalents of HCl for 24 h gave
compound 6 in 93% yield, but with 16 equivalents of HCl, the yield was only 78%. The reason is that
the hyroxymethyl group at 6-position of compound 3 could be reduced to 6-methyl group over PtO2
under strong acidic conditions. We actually got the 6-methyl isoster of compound 6 in 12% yield under
the conditions of entry 7.
Scheme 3. Catalytic hydrogenation of 3.

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Variation of solvent also affected the yield strongly (Table 1, entries 6 and 8~9). Ethanol was a
better solvent under these reaction conditions than methanol and acetone. The best conditions we
found featured eight equivalents of hydrochloric acid in ethanol (Table 1, entry 6), resulting in a higher
yield (93%) and the workup is easier than with the reduction reaction in Scheme 1.
Table 1. Optimization of the catalytic reduction conditions a.
Entry
Acid
-
Equiv.
Solvent
Ethanol
Ethanol
Ethanol
AcOH
Ethanol
Ethanol
Ethanol
Methanol
Acetone
Time (h)
Yield (%) b
1
2
3
4
5
6
7
8
9
-
4
8
120
60
60
60
24
24
24
8
-
AcOH
AcOH
AcOH
HCl c
HCl c
HCl c
HCl c
HCl c
54
47
59
82
93
78
21
46
excess
4
8
16
8
8
48
a
Conditions: Compound 3 (1 mmol), H2 (0.3 MPa), PtO2 (0.1 mmol), acid (4~16 mmol or as
solvent), solvent (15 mL), H2O (0~8 mL, from HCl solution). b Yield of isolated product. c 2 mol/L
aqueous HCl solution was used.
2.3. Optimized Synthesis of 5
Before trying this synthetic strategy, we attempted to directly synthesize 1 from 6 in several ways,
but none of them succeeded. The hypothetical Mitsunobu reaction between 6 and diethyl
N-(p-aminobenzoyl)-L-glutamate 4 wouldn’t proceed, and methods of transferring the hydroxymethyl
group in 6 to aldehyde or halidemethyl group couldn’t improve this condensation reaction.
Inspired by the work of Singh [18], which described silica gel induced condensation of aziridines
and aromatic amines, we designed aziridine 5 as the key intermediate [19–21]. Compound 6 was
converted to the 6-chloromethyl derivative 7 in refluxing POCl3 (yield 40%), which was then
treated with KOH, K2CO3 or 1,8-diazabicycloundec-7-ene (DBU) to give 5 in 86% yield after
chromatographic purification, as shown in Scheme 4.
Scheme 4. Two-step synthesis of compound 5.
1
1
Compounds 7 and 5 were both identified by H-NMR, 13C-NMR and ESI-MS. The H-NMR
(DMSO-d6) spectrum of 7 revealed a multiplet signal at δ 4.06~3.92 ppm assignable to CH2Cl, which
13
obviously differed from the CH2OH group signal (δ 4.97~4.90 ppm) of compound 6. The C-NMR
(DMSO-d6) spectrum of 7 also displayed a different signal at δ 47.35 ppm assignable to CH2Cl with
the CH2OH group signal at δ 51.79 ppm for 6. The ESI-MS result of 7 gave the intuitive proof of a

Molecules 2012, 17
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1
chloride atom by a pair of isotopic peaks (214.1 and 216.1, 3:1). The H-NMR (DMSO-d6) spectrum
of 5 displayed two doublet signals at δ 2.14 ppm (d, J = 5.2 Hz, 1H) and 1.56 ppm (d, J = 4.0 Hz, 1H)
which were readily assigned to the two hydrogen atoms attached at methylene position of the aziridine
ring and the 13C-NMR (DMSO-d6) spectrum of 5 displayed a new signal at δ 33.33 ppm assignable to
NCH2 in aziridine ring and a signal at δ 35.23 ppm assignable to NCH which gave both a relatively
high-field shift compared to compounds 7 or 6. High resolution MS provided further confirmation for
the two products.
The reaction above was somewhat low-yielding and its work-up procedure was complicated. After
further investigation, we found an alternative method to obtain 5 in one step by treatment of 6 with
p-toluenesulfonyl chloride, aqueous NaOH and tetrabutylammonium iodide in a two-phase system
(Scheme 5). The yield increased to about 50%, and the work-up was simplified.
Scheme 5. One-step synthesis of compound 5.
NH2
N
NH2
N
H
N
N
TsCl, NaOH, TBAI
THF, H2O, 2 h
N
N
OH
H2N
H2N
6
5
2.4. Synthesis of 1
Compounds 5, 4 and actived silica gel were mixed and shaken thoroughly under solvent-free
conditions (Scheme 6). The desired product 1 was obtained in 90% yield, and the small amount of
impurities could be removed easily by column chromatography. Compared with usual ring-opening
reactions of aziridines using Lewis acid catalysis [22–25], the silica gel-induced method was
1
convenient, green and cheap. Product 1 was verified by H-NMR and ESI-MS, and it was compared
with the same compound prepared by Scheme 1 to prove the structural consistency.
Scheme 6. Synthesis of 1 under solvent-free conditions.
2.5. Synthesis of ZL033
Compound 1 can be converted to ZL033 in the same reaction sequence of allyl substitution,
bromination and hydrolysis as before [11] (Scheme 7).

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Scheme 7. Synthesis of ZL033 from 1.
2.6. Synthesis of Other Tetrahydrofolate Analogues
Aside from ZL033, many other tetrahydrofolate analogues can be synthesized from intermediate 1
by simply modifying the substituents on N5 or N10. Furthermore, the strategy above can be used for
synthesis of various compounds, such as heterocyclic benzoyl analogues and modification in the side
chain of tetrahydrofolate. Earlier research proved that substitution of N10-phenyl by aromatic
heterocycles benefited inhibitory activity against several kinases [26–29]. The synthetic route of
heterocyclic benzoyl analogues was similar with that of ZL033, including the ring-opening of aziridine
by aromatic heterocyclic amines (Scheme 8). Compounds 10, 11 and 12 were obtained in acceptable
yields by the solid-phase reaction catalysed by silica gel.
Scheme 8. Synthesis of heterocyclic benzoyl analogues of 1.
NH2
NH2
H
N
O
O
H2N
N
silica gel
48 h
N
N
N
H
COOH
COOH
N
N
COOH
COOH
+
+
HN
S
HN
S
H2N
H2N
N
5
10
NH2
NH2
O
H
N
O
N
silica gel
48 h
COOH
COOH
N
H
COOH
COOH
H2N
S
S
HN
HN
H2N
H2N
N
5
11
NH2
NH2
H
N
O
N
silica gel
36 h
N
N
N
H
OCH3
H2N
+
O
O
OCH3
H2N
N
H2N
O
5
12
Compounds 10, 11 and 12 were transformed into analogues of ZL033 by substitution and
hydrolysis reactions. This novel synthetic route is flexible to a certain extent. With various pteridines
and aniline analogues, more tetrahydroaminopterin and tetrahydrofolate analogues were prepared in
this novel method. The studies of these heterocyclic analogues of 8-deazatetrahydrofolate on the
antifolate activity and the structure-activity relationship will be published in the future.

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3. Experimental
3.1. General
All reactions were monitored by TLC analysis (pre-coated silica gel plates with fluorescent
indicator UV254, 0.2 mm) and visualized with 254 nm UV light. The products were purified by
column chromatography on silica gel (silica gel 230–400 mesh or silica gel H, produced by Qingdao
Ocean-Chem Inc., Qingdao, China), or preparative thin layer chromatography on silica gel (silica gel
GF254, 0.5 mm, also produced by Qingdao Ocean-Chem Inc., Qingdao, China). 1H-NMR spectra were
measured on Bruker avance III 400 spectrometer (400 MHz, Bruker Co., Fällanden, Switzerland) or
JEOL JNM-AL300 spectrometer (300 MHz, Joel, Japan). Data were reported as follows: Chemical
shifts in ppm from tetramethylsilane as an internal standard in CDCl3 or DMSO-d6, multiplicity
(s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constants (Hz),
integration. 13C-NMR spectra were recorded on a Bruker Avance III 400 (100 MHz) or JEOL JNM-AL300
spectrometer (75 MHz). Mass spectra were measured with a Quattro micro 2000 (ESI-TOF)
instrument (Bruker Co., Bremen, Germany). HRMS was performed on a Bruker APEX IV FT-MS
(7.0T, Waters, New Castle, DE, USA). Melting points were measured with a SGW X4 microscopic
melting point apparatus. Chemicals were purchased from Aldrich, Acros, AlfaAsar, Strem, OuheChem
or Beijing Chemical Works and were used without further purification, unless otherwise noted.
Hydrogen gas needed in reduction reaction was supplied by a GCD-300A hydrogen generator,
manufactured by the Beijing BCHP Analytical Technology Institute.
(2,4-Diamino-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidin-6-yl)methanol (6): To a suspension of
2,4-diamino-6-hydroxymethylpyrido[3,2-d]pyrimidine (3, 2.01 g, 10.5 mmol) in ethanol (150 mL) was
added aqueous HCl (40 mL, 2 mol/L, 80 mmol) and platinum dioxide monohydrate (250 mg,
1.0 mmol). The mixture was then hydrogenated in a 0.3 MPa hydrogen generator at room temperature
for 24 h. After evaporation of solvents and acid, anhydrous ethanol (300 mL) was added to the residue
and stirred for 1 h. The resulting precipitate was filtered, washed with acetone (100 mL) and dried
1
under vacuum to afford 1.90 g (92.9% yield) of 6 as a white solid; m.p. 241~243 °C. H-NMR
(300 MHz, DMSO-d6): δ = 7.90 (br.s, 2H, 4-NH2), 6.93 (br.s, 2H, 2-NH2), 4.97~4.90 (br.s × 2, 2H,
CH2OH), 3.47~3.41 (m, 1H, 6-CH), 3.16 (m, 1H, 8-CHaHb), 2.68~2.59 (m, 1H, 8-CHaHb), 1.93~1.88
(m, 1H, 7-CHaHb), 1.53 (m, 1H, 7-CHaHb); 13C-NMR (75 MHz, DMSO-d6): δ = 157.47 (C-8a), 150.52
(C-4), 129.18 (C-2), 116.39 (C-4a), 63.92 (CH2OH), 52.74 (C-6), 22.41 (C-7, C-8); MS (ESI): m/z
196.1 (M+H+, 100%); HRMS: calcd for C8H14N5O [M+H]+ 196.11984, found 196.11963.
2,4-Diamino-6-choloromethyl-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine (7): A stirred solution of
compound 6 (502 mg, 5.1 mmol) in POCl3 (10 mL) was refluxed at 110 °C for 2 h. The resulting
solution was evaporated to a minimized volume of about 1 mL under reduced pressure. Then it was
diluted with 50 mL dichloromethane and evaporated to a minimized volume again. This procedure was
repeated twice with diluting solvent changing successively to ethanol and methanol. Acetone (50 mL)
was added to the concentrated sticky liquid, and the resulting white precipitate was filtered, washed
with acetone (20 mL) and dried under vacuum to afford 221 mg (40.2% yield) of 7 as an off-white
1
solid; m.p. 228~232 °C. H-NMR (400 MHz, DMSO-d6): δ = 4.06~3.92 (m, 2H, CH2Cl), 3.70~3.68

Molecules 2012, 17
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(m, 1H, 6-CH), 2.64~2.44 (m, 2H, 8-CH2), 1.98~1.95 (m, 1H, 7-CHaHb), 1.77~1.70 (m, 1H, 7-CHaHb);
13C-NMR (100 MHz, DMSO-d6): δ = 51.79 (6-CH), 47.35 (CH2Cl), 23.19 (7-CH2), 22.04 (8-CH2);
MS (ESI): m/z 214.1 (M+H+, isotopic peak of 37Cl, 100%), 216.1 (M+H+, isotopic peak of 35Cl,
33.6%); HRMS: calcd for C8H13ClN5 [M+H]+ 214.08595, found 214.08617.
2,4-Diamino-5,6-methylene-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidine (5)
Method A: To a suspension of compound 7 (296 mg, 1.4 mmol) in acetonitrile (25 mL) was added
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.60 mL, 4.1 mmol). The reaction was stirred at room
temperature for 1 h, then concentrated and isolated by preparative TLC with a chloromethane and
methanol mixture (9:1) as eluent. Compound 5 (210 mg) was obtained as a faint yellow solid, the yield
was 85.7%, while the two-step overall yield from compound 6 was 34.5%.
Method B: To a suspension of compound 6 (300 mg, 1.5 mmol) in anhydrous THF (10 mL) was
added 50% aq. sodium hydroxide solution (4.0 mL, 76.3 mmol) and tetrabutylammonium iodide (101 mg,
0.3 mmol). p-Toluenesulfonyl chloride (291 mg, 1.5 mmol) was added after stirring for 10 min. The
reaction mixture was stirred at room temperature for 1 h, and diluted with ethyl acetate (100 mL) and
stirred thoroughly. The resulting solution was then filtered through kieselguhr and washed with
dichloromethane (50 mL). The filtrate and washes were combined, concentrated and subjected to
preparative TLC with a dichloromethane and methanol mixture (9:1) as eluent. Compound 5 (137 mg,
1
50.3%) was obtained as a faint yellow solid; m.p. 223~226 °C. H-NMR (400 MHz, DMSO-d6):
δ = 6.12 (s, 2H, 4-NH2), 5.52 (s, 2H, 2-NH2), 2.48~2.47 (m, 1H, 6-CH), 2.34~2.28 (m, 1H, 8-CHaHb),
2.27~2.19 (m, 1H, 8-CHaHb), 2.14~2.13 (d, J = 5.2 Hz, 1H, NCHaHb), 2.09~2.06 (m, 1H, 7-CHaHb),
1.96~1.91 (m, 1H, 7-CHaHb), 1.56~1.55 (d, J = 4.0 Hz, 1H, NCHaHb); 13C-NMR (100 MHz, DMSO-d6):
δ = 160.50 (2-C), 159.09 (4-C), 154.62 (8a-C), 117.48 (4a-C), 35.23 (6-CH), 33.33 (N-CH2), 25.26
(8-CH2), 21.05 (7-CH2); MS (ESI): m/z 178.1 (M+H+, 100%); HRMS (ESI): calcd for C8H12N5
[M+H]+ 178.10872, found 178.10813.
Diethyl N-{4-[(2,4-diamino-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidin-6-yl)methylamino]benzoyl}-L-
glutamate (1): Silica gel was activated at 120 °C under vacuum for 6 h before use. Compound 5
(139 mg, 0.8 mmol) and diethyl 4-aminobenzoyl-L-glutamate (310 mg, 1.0 mmol) were mixed
uniformly with activated silica gel (1.02 g). The mixture was stirred vigorously for 24 h, and stopped
when TLC showed full transformation of 5. The reactant was purified by column chromatography
(silica gel). Elution with CHCl 3-CH3OH (12:1) gave pure 1 (356 mg, 90.8%) as a white solid; m.p.
1
143 °C (decomp). H-NMR (400 MHz, DMSO-d6): δ = 8.27~8.25 (d, J = 7.3 Hz, 1H, CONH),
7.69~7.67 (d, J = 8.7 Hz, 2H, 2,6-H on benzene ring), 7.29 (s, 2H, 4-NH2), 6.67~6.64 (d, J = 8.7 Hz,
2H, 3,5-H on benzene ring), 6.55~6.53 (d, J = 8.5 Hz, 1H, 5-NH), 6.50~6.47 (t, J = 5.7 Hz, 1H,
CH2NH), 4.40~4.35 (m, 1H, CONHCH), 4.11~4.02 (m, 4H, CH2CH3 × 2), 3.68~3.65 (m, 1H, 6-CH),
3.21~3.17 (m, 2H, CH2NH), 2.43~2.39 (m, 4H, 8-CH2, CHCH2CH2), 2.12~1.97 (m, 4H, 7-CH2,
CHCH2CH2), 1.18~1.16 (m, 6H, CH3 × 2); MS (ESI): m/z 499.3 (M+H+, 100%); HRMS (ESI): calcd
for C24H34N7O5 [M+H]+ 500.26159, found 500.26051.
Diethyl N-{4-[(2,4-diamino-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidin-6-yl)methylamino]thiophene-2-
formyl}-L-glutamate (10): The heterocyclic benzoyl analogue 10 was prepared in the similar way as the

Molecules 2012, 17
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synthesis of 1. Compound 5 (144 mg, 0.8 mmol), diethyl N-(4-aminothiophene-2-formyl)-L-glutamate
(600 mg, 1.8 mmol) and activated silica gel (1.6 g) was stirred together for 48 h to complete the
reaction. Product was purified by column chromatography (silica gel). Elution with CHCl3-CH3OH
1
(15:1) gave pure 10 (186 mg, 45.3%) as a faint yellow liquid. H-NMR (400 MHz, CDCl3): δ = 7.16
(s, 1H, 2-H on thiophene ring), 6.10 (s, 1H, 4-H on thiophene ring), 5.21 (br.s, 1H, 5-NH), 4.72 (br.s,
1H, CH2NH), 4.58 (br.s, 1H, CONHCH), 4.18 (m, 2H, CHCOOCH2), 4.10~4.09 (m, 3H,
CH2COOCH2, 6-CH), 3.31 (m, 1H, CHaHbNH), 3.14 (m, 1H, CHaHbNH), 2.64~2.35 (m, 4H, 8-CH2,
CHCH2CH2), 2.27~2.10 (m, 4H, 7-CH2, CHCH2CH2), 1.24~1.22 (m, 6H, CH3 × 2); HRMS (ESI):
calcd for C22H32N7O5S [M+H]+ 506.21801, found 506.21906.
Diethyl N-{5-[(2,4-diamino-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidin-6-yl)methylamino]thiophene-2-
formyl}-L-glutamate (11): The heterocyclic benzoyl analogue 11 was prepared in the same way as the
synthesis of 10. Compound 5 (96 mg, 0.5 mmol), N-(5-aminothiophene-2-formyl)-L-glutamate
(403 mg, 1.2 mmol) and activated silica gel (1.05 g) was stirred together for 48 h to complete the reaction.
Product was purified by column chromatography (silica gel). Elution with CHCl3-CH3OH (15:1) gave
pure 11 (120 mg, 43.8%) as a faint yellow liquid. 1H-NMR (400 MHz, CDCl3): δ = 7.26 (s, 1H, 2-H on
thiophene ring, coincided with CHCl3 peak), 6.42 (s, 1H, 3-H on thiophene ring), 5.30 (s, 1H, 5-NH,
coincided with residual CH2Cl2 peak), 4.63 (m, 2H, CH2NH, CONHCH), 4.18~4.17 (m, 2H,
CHCOOCH2), 4.09~4.08 (m, 2H, CH2COOCH2), 3.60 (m, 1H, 6-CH), 3.41~3.35 (m, 2H, CH2NH),
2.65~2.64 (m, 1H, 8-CHaHb), 2.43 (m, 2H, CHCH2CH2), 2.27~2.26 (m, 1H, 8-CHaHb), 2.22~2.17 (m,
1H, CHCHaHbCH2), 2.08 (m, 2H, CHCHaHbCH2, 7-CHaHb), 1.91 (m, 1H, 7-CHaHb), 1.22~1.19 (m,
6H, CH3 × 2); HRMS (ESI): calcd for C22H32N7O5S [M+H]+ 506.21801, found 506.21947.
Methyl 5-[(2,4-diamino-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidin-6-yl)methylamino]furan-2-carboxylate
(12): The heterocyclic benzoyl analogue 11 was prepared in a similar way as in the synthesis of 1.
Compound 5 (90 mg, 0.5 mmol), methyl 5-aminofuran-2-formate (181 mg, 1.3 mmol) and activated
silica gel (501 mg) was stirred together for 36 h to complete the reaction. Product was purified by
column chromatography (silica gel). Elution with CHCl3-CH3OH (15:1) gave pure 12 (104.5 mg,
40.7%) as a faint yellow liquid. HRMS (ESI): calcd for C14H19N6O3 [M+H]+ 319.15186, found
319.15244.
4. Conclusions
In conclusion, we have developed a novel synthetic route providing the biologically important
compound 1 in good yields via an aziridine intermediate, thus avoiding the benzylic hydrogenolysis of
the carbon-nitrogen bond of earlier methods. It also represents a convenient preparation of
tetrahydroaminopterin or tetrahydrofolate analogues.
Acknowledgments
This study was supported by the National Science Foundation of China (20972011, 21042009,
21172014) and grants from the Ministry of Science and Technology of China (2009ZX09301-010) for
financial support.

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