Design, synthesis and biological evaluation of novel 6-substituted pyrrolo [3,2-d] pyrimidine analogues as antifolate antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2017.07.002

A series of novel 6-substituted pyrrolo[3,2-d]pyrimidine analogues (10a, 11a-13a, 15a, 17a, 18a, 27a and 28a) have been designed and synthesized as antifolate antitumor agents. The anti-proliferative activities of these compounds against HL60, A549, H1299, Hela, HCT116 and HT29 tumor cells were evaluated. Most of the compounds exhibited micromolar anti-proliferative potencies. Compound 15a, the most potent one, has GI₅₀ value of 0.73, 1.72, and 8.92 μM against A549, H1299 and HL60 cells, respectively. The cell cycle distribution assay displayed that 15a could increase the accumulation of G2/M-phase cells. 15a showed low potency in induction of apoptosis. However, the inhibition of A549 cell colony formation was observed. These indicated that the tumor cell death relied on the irreversible effect of 15a on clonogenicity and cell proliferation. The identification of targeted pathway of 15a implied that the anti-proliferative potencies of 15a probably act through dual inhibition of thymidylate synthase (TS) and dihydrofolate reductase (DHFR).

Full text of DOI:10.1016/j.ejmech.2017.07.002

Accepted Manuscript
Design, synthesis and biological evaluation of novel 6-substituted pyrrolo [3,2-d]
pyrimidine analogues as antifolate antitumor agents
Chao Tian, Meng Wang, Zifei Han, Fang Fang, Zhili Zhang, Xiaowei Wang, Junyi Liu
PII:
S0223-5234(17)30524-X
10.1016/j.ejmech.2017.07.002
EJMECH 9565
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 April 2017
Revised Date: 30 June 2017
Accepted Date: 2 July 2017
Please cite this article as: C. Tian, M. Wang, Z. Han, F. Fang, Z. Zhang, X. Wang, J. Liu, Design,
synthesis and biological evaluation of novel 6-substituted pyrrolo [3,2-d] pyrimidine analogues
as antifolate antitumor agents, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.07.002.
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ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Novel 6-Substituted Pyrrolo [3,2-d]
Pyrimidine Analogues as Antifolate Antitumor Agents
Chao Tian, Meng Wang, Zifei Han, Fang fang, Zhili Zhang, Xiaowei Wang, Junyi Liu

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Novel 6-Substituted Pyrrolo [3,2-d]
Pyrimidine Analogues as Antifolate Antitumor Agents
Chao Tiana, Meng Wanga, Zifei Hana, Fang fanga, Zhili Zhanga, Xiaowei Wanga, Junyi
Liua,b,
a Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
b State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
Abstract
A series of novel 6-substituted pyrrolo[3,2-d]pyrimidine analogues (10a, 11a-13a, 15a, 17a, 18a,
27a and 28a) have been designed and synthesized as antifolate antitumor agents. The
anti-proliferative activities of these compounds against HL60, A549, H1299, Hela, HCT116 and
HT29 tumor cells were evaluated. Most of the compounds exhibited micromolar anti-proliferative
potencies. Compound 15a, the most potent one, has GI50 value of 0.73, 1.72, and 8.92 µM against
A549, H1299 and HL60 cells, respectively. The cell cycle distribution assay displayed that 15a
could increase the accumulation of G2/M-phase cells. 15a showed low potency in induction of
apoptosis. However, the inhibition of A549 cell colony formation was observed. These indicated
that the tumor cell death relied on the irreversible effect of 15a on clonogenicity and cell
proliferation. The identification of targeted pathway of 15a implied that the anti-proliferative
potencies of 15a probably act through dual inhibition of thymidylate synthase (TS) and
dihydrofolate reductase (DHFR).
Keywords:
6-substituted pyrrolo[3,2-d]pyrimidines;
Anti-proliferation;
Structure–activity relationship;
Dihydrofolate reductase;
Thymidylate synthase;
G2/M-phase increased;
Colony formation inhibition;
Corresponding author. Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University,
Beijing 100191, China.
E-mail address: jyliu@bjmu.edu.cn.

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Introduction
Folate-dependent metabolic pathway, which is required in plenty of interrelated enzymatic
reactions, has long been recognized as an attractive target of cancer chemotherapy[1, 2]. Among
the enzymes involved in the folate-dependent metabolic pathway, dihydrofolate synthase (DHFR),
thymidylate synthase (TS), glycinamide ribonucleotide formhyltransferase (GARFTase),
5-aminoimidazole-4-carboxamide and ribonucleotide formyltransferase (AICARFTase) are critical
targets in the development of antifolate agents. DHFR reduces 7,8-dihydrofolic acid to
tetrahydrofolic acid [3, 4], while TS catalyze the de novo synthesis of
2’-deoxythymidine-5’-monophosphate (dTMP) from 2’-deoxyuridine5’-monophosphate (dUMP)
by transferring a methyl group from 5,10-methylenetetrahydrofolate to afford 7,8-dihydrofolate
[5]. GARFTase or AICARFTase catalyze
a
formyl group transferring from
10-formyltetrahydrofolate to glycinamide ribonucleotide monophosphate (GARMP) or
5-aminoimidazole-4-carboxamide ribonucleotide monophosphate (AICARMP), which are
involved in the de novo purine nucleotide biosynthesis [6]. The DHFR inhibitors (e.g.,
methotrexate (MTX) [7] and pralatrexate, (PDX) [8]) and TS inhibitors (e.g., pemetrexed (PMX)
[9] and raltiterxed (RTX) [10]) play important roles in the clinical treatment of solid tumor
currently. Furthermore, GARFTase inhibitors such as lometrexol (LMTX) [11], LY309887 [12]
and AG2034 [13] are undergoing clinical trials. Furthermore, there are three major membrane
transport systems, including the reduced folate carrier (RFC) [14], the proton-coupled folate
transporters (PCFT) [15], and folate receptors (FRs) α and β [16], involving in the cellular uptake
of (anti)folate. The latter two are highly expressed in human tumors [17]. Folate-based compounds
with transport selectivity for FRs or PCFT over RFC are reported to possess improved tumor
targeting properties these years [18-20].
Since the introduction of MTX in 1950s [21], plenty of compounds were synthesized and
evaluated as antifolate anticancer candidates. However, most of them were failed due to
dose-limiting toxicities and tumor resistance [18]. In the development of antifolate agents,
discovery of novel antifolate agents with low dose-limiting toxicities and low tumor resistance
remain to be an attractive research area [22, 23].
Fig. 1. Representative examples of classical antifolates and the structure of folic acid
The structure of folate acid could be divided in to four regions (Fig. 1), including the pteridine
region, the bridge region [24], the benzoyl region and the L-glutamate acid region. The structure
activity relationship (SAR) studies of the classical antifolates usually vary in these four regions.
Our previous studies mainly focus on the varying in the pteridine and the bridge region [25-27].
Although the pteridine regions of the approved antifolate anticancer drugs are mostly 6-6 fused
ring system, more and more antifolates with 6-5 fused scaffold, such as pyrrolo[2,3-d]pyrimidines,
thieno[2,3-d]pyrimidine and furo[2,3-d]pyrimidine, are reported in recent years [18-20, 28-31].

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However, there are limited reports on the antifolates with pyrrolo[3,2-d]pyrimidines scaffold. As a
continued effort to discovery novel antifolates with multi-targeted antifolate potencies and
improved dose-limiting toxicities, in this article, we report a series of 6-substituted
2-amino-4-oxo-pyrrolo[3,2-d]pyrimidine analogues varied at position 5, 7 and 9 as novel
antifolate antitumor agents (Fig. 2).
Fig. 2. The structures of target compounds
2. Chemistry
A key intermediate 9 was synthesized as shown in Scheme 1. The synthesis started from benzyl
alcohol, which was condensed with ethyl bromoacetate to obtain compound 1. Then 1 was reacted
with acetonitrile lithium, which was prepared by adding acetonitrile to 2.5 M n-butyl lithium in
THF dropwise, to afford 2. Compound 2 was condensed with diethyl aminomalonate in the
present of acetic acid to achieve 3, which was further cyclized under basic condition to produce
compound 4. Thus, the pyrrole
4
was then subjected to condensation with
1,3-bis(methoxycarbonyl)-2-methylthiopseudourea [32] with acetic acid as catalyst in MeOH to
afford 5. The self-condensation reaction of compound 5 could be processed in the present of
sodium methoxide in MeOH to obtain 6. Hydrolysis of the carbamate group with aqueous sodium
hydroxide at 60 ºC afforded compound 7. Then, the benzyl group was removed by hydrolysis in 6
N aqueous hydrochloric solution at 60 ºC to obtain 8. Subsequently, the crucial intermediate
compound 9 was achieved through bromination of 8 by phosphorus tribromide.
Scheme 1. Synthetic route to compounds 9. Reagents and conditions: a) NaH, BrCH2COOEt, rt; b) n-BuLi,
CH3CN, -78 ºC to rt; c) diethyl aminomalonate, AcOH, rt; d) NaOEt, EtOH, rt; e)
1,3-bis(methoxycarbonyl)-2-methylthiopseudourea, AcOH, N2, rt; f) NaOMe, MeOH, rt; g) i)1 N NaOH, 50 ºC; ii)
AcOH, pH=6; h) 6 N HCl, 60 ºC; i) PBr3, THF, reflux.
With compound 9 in hands, condensation reaction with diethyl (4-aminobenzoyl)-L-glutamate was
first processed to afford 10. The later one could be alkylated by several alkyl halides (methyl
iodide, bromoethane or propargyl bromide) at N9 position to obtain compounds 11-13. Meanwhile,
trifluoroacetylation of 10 could achieve compound 16, which would be halogenated by NBS or I2
at C7 position to produce the 7-halogenated target compounds 17 and 18. To obtain C-C bridge
analogue 15, phosphorus ylide (14) was prepared with compound 9. Without further purification,

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compound 14 was condensed with diethyl-N-[4-(formyl)benzoyl]-L-glutamate and hydrogenated
subsequently to achieve compound 15.
Scheme 2. Synthetic route to compounds 10-18. Reagents and conditions: a) DMAC, 80 ºC; b) CH3I, CH3CH2Br
or propargyl bromide, K2CO3, anhydrous DMF, rt; c) (CF3CO)2O, DCM, rt; d) NBS or I2, DCM, rt; e) (C6H5)3P,
anhydrous DMF, argon, 60 ºC, 2h, then NaOMe, rt, 1h; f) i) anhydrous DMF, 60 ºC, 24h; ii) H2, Pd/C; g) 1 mol/L
NaOH, r.t.
The N5-substituted analogues were obtained through the synthetic route outlined in scheme 3.
Acylation of compound 7 with pivaloyl chloride afforded 2-NH2-protected compound 19, which
was proceeded chlorination with POCl3. Alkylation of compound 20 by alkyl halides (methyl
iodide or bromopropylene) afforded compounds 21 and 22. Hydrolysis of 21 and 22 could obtain
compounds 23 and 24. The same method as preparation of compound 8 was used to remove the
benzyl group to produce 25 and 26. Bromonation of 25 and 26 and subsequently condensation
with diethyl (4-aminobenzoyl)-L-glutamate respectively afforded compounds 27 and 28.
Scheme 3. Synthetic route to compounds 19-27. Reagents and conditions: a) PivCl, DCM, reflux, 5h; b) POCl3,
reflux, 10h; c) MeI or Allyl bromide, NaH; d) 2 mol/L NaOH, 1,4-dioxane, reflux; 6h; e) 6 mol/L HCl, reflux, 6h; f)
i) PBr3, THF; ii) diethyl (4-aminobenzoyl)-L-glutamate, DMAC, 80 ºC; g) 1 mol/L NaOH, r.t.
Hydrolysis of compounds 10, 11-13, 15, 17, 18, 27, and 28 could finally obtain our target
compounds 10a, 11a-13a, 15a, 17a, 18a, 27a, and 28a as carboxylic acid form [27].
3. Biological evaluation and discussion
3.1. Anti-proliferative effects of target compounds

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The anti-proliferative effects of 10a, 11a-13a, 15a, 17a, 18a, 27a, and 28a were evaluated
toward HL60, A549, H1299, Hela, HCT116 and HT29 cell lines using MTS method. The growth
inhibitory effects (GI50) results are illustrated in Table 1 and compared with MTX.
The biological data in Table 1 established that compounds15a, 27a and 28a showed moderate to
good inhibitory effect against most of the cell lines. 15a was the best, obviously. Although most of
the compounds exhibited micromolar anti-proliferative potencies toward cancer cell lines, some
structure activity relationships about this kind of compounds could be summarized as follow.
Firstly, the inhibitory activities of 10a-13a indicated that introducing substitution to the bridge
region could significantly diminish the inhibitory effects against the cancer cell lines. Secondly,
introduction of bromine or iodine at position 7 of 10a (afforded 17a and 18a) would also decrease
their anti-proliferative effects. Thirdly, the inhibitory activities of 27a and 28a showed that
substituents at position 5 of 10a would not reduce the inhibitory effects. In turn, they could
moderately increase the inhibitory activities against HL60, A549 and H1299 cell lines. Finally,
15a established much better anti-proliferative effects than 10a against these tumor cell lines,
especially for HL60, A549 and H1299 cell lines, although their only difference is position 9. In
brief, we could conclude that position 7 and 9 of this kind of compounds play important roles in
the anti-proliferative effects and introduction of certain substituents on position 5 was conducive
to the inhibitory effects.
Table 1. The anti-proliferative effects of the target compounds
Cpd
10a
11a
X
H
H
Y
R
H
H
HL60
A549
H1299
Hela
HCT116
>100
HT29
>100
>100
NH
47.42±6.94
>100
79.03±7.30
>100
73.07±4.01
>100
85.14±3.21
>100
NCH3
NCH2C
H3
>100
H
H
H
H
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
12a
13a
NCH2C
≡CH
CH2
NH
H
Br
I
H
8.92±1.60
40.06±9.38
54.20±3.64
13.51±5.68
0.73±0.16
83.03±8.46
>100
1.72±0.67
>100
73.08±3.46
>100
54.61±8.66
>100
99.14±4.90
>100
15a
17a
18a
27a
H
NH
H
>100
>100
>100
>100
H
NH
CH3
CH2CH
=CH2
46.01±5.99
51.33±5.71
94.37±5.28
>100
>100
H
NH
15.45±1.93
57.27±17.70
0.014±0.002
68.39±16.86
0.072±0.002
82.72±4.24
>100
>100
98.55±10.84
>100
28a
MTX
0.038±0.001
0.13±0.02
3.2. Inhibitory activities toward DHFR
To identify the targeted enzyme of these compounds, first of all, compounds 10a, 11a-13a, 15a,
17a, 18a, 27a and 28a were evaluated as inhibitors of human DHFR. The inhibitory ratio at 1 µM,

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10 µM and 100 µM are showed in Figure 3. The DHFR inhibition results established that most of
the compounds showed inhibitory potencies in some degree toward DHFR. 15a, which has a
inhibitory ratio of 66.7% at 100 µM, was the best one among these compounds. As 15a displayed
poor inhibitory potencies at low concentration, considering of its high anti-proliferative potency
against tumor cells, it seemed probably that DHFR was not the main targeted enzyme of 15a.
Fig. 3. The inhibitory activities toward DHFR
3.3. Identification of the targeted enzyme of 6-Substituted Pyrrolo [3,2-d] Pyrimidines
antifolates
As the substrates for folate-dependent enzymes, including DHFR, TS, GARFTase and
AICARFTase, share a similar scaffold, antifolate anticancer drugs always exhibited multi-targeted
effects currently.
To determine the targeted pathway of compound 15a, the nucleoside protection experiments
were conducted to distinguish de novo purine nucleotide from thymidylate biosynthesis
employing adenosine (60 µM) and thymidine (10 µM), respectively [33, 34]. As there are two
folate-dependent enzymes (GARFTase and AICARFTase) involving in de novo purine nucleotide
biosynthesis [20], in order to further identify the targets, HL60 cells were treated in presence of
AICA (320 µM), which is the substrate for AICARFTase, thus bypassing the step catalyzed by
GARFTase.
The nucleoside/AICA protection results for compound 15a were showed in Fig. 4. For 15a,
single addition of excess adenosine of AICA was slightly protective, while single addition of
excess thymidine was protective to some extent. These results implied that compound 15a was
probably an inhibitor of thymidylate synthase (TS). Based on the above results and the inhibitory
activities toward DHFR, the anti-proliferative potency of 15a probably act through dual inhibition
of TS and DHFR.

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Fig. 4. Cell proliferation assays with protection by nucleosides including thymidine and
adenosine and by 5-aminoimidazole-4-carboxamide (AICA) to identify intracellular targets of
compound 15a. To identify the targeted pathways and the folate-dependent intracellular enzymes
in HL60 cells treated with compound 15a (0.1-100 µM), cell proliferation assays were performed
in the presence of 10 µM thymidine, 60 µM adenosine, or 320 µM AICA. The results were
normalized to those for untreated cells (no drug).
3.4. Effect of compound 15a on cell cycle distribution in A549 cells
In order to verify the effect of compound 15a on the cell cycle distribution, A549 cells were
treated with compound 15a (1µM and 10µM) and MTX (1µM) for 72 h, along with an untreated
control. Cells were washed with PBS, fixed with ice-cold 70% ethanol overnight, stained with
propidium iodide (PI), and analyzed for cell cycle distribution by flow cytometry. As shown in Fig.
5, MTX induced a significant G1-phase arrest relative to the untreated control. Beyond our
expectation, 15a displayed a different effect on the cell cycle distribution. The percent of
G2/M-phase cells increased to 15.0% and 19.4% for 15a (1 µM) and 15a (10 µM) from 7.8%
(untreated control). These results indicated that 15a could effectively induce G2/M-phase arrest.

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Cell Cycle Distribution
(e)
100
80
60
40
20
0
Blank MTX 1 µM 15a 1 µM 15a 10 µM
G1
S
40.9
51.3
7.8
62.0
38.0
0.0
33.7
51.3
15.0
35.0
45.6
19.4
G2
Fig. 5. Cell cycle assay. A549 cells were treated with 1 µM MTX, 1 µM 15a or 10µM 15a for 72
h, washed, fixed, and stained with PI. Cell cycle distribution was detected by flow cytometry. The
results are compared with those treated with DMSO instead of drug. a) DMSO; b) MTX (1µM); c)
15a (1 µM); d) 15a (10 µM); e) The percentage of cells in different phase of cell cycle (G1, S,
G2/M).
3.5. Apoptosis Analysis
An additional experiment were performed with A549 cells treated with compound 15a to assess its
effect on induction of apoptosis. As shown in Fig. 6, the fraction of cells in early apoptosis were

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0.39%, 4.40% and 0.32% for 15a (1 µM), 15a (10 µM) and untreated control. The fraction of cells
in late apoptosis were 1.07%, 3.21% and 1.24% for 15a (1 µM), 15a (10 µM) and untreated
control. These results indicated that compound 15a could induce cell apoptosis to some extent at
higher concentration. However, the low ratio of apoptosis implied that the anti-proliferative effect
of compound 15a was largely independence of apoptosis.
Fig. 6. Analysis of apoptosis by Annexin V-FITC/PI staining and flow cytometry is shown for
A549 cells treated with 15a 1 µM or 10 µM for 72 h along with untreated control. UL: dead cells;
UR: late apoptosis cells; LL: viable cells; LR: early apoptosis cells.
3.6 Colony Formation Inhibition Assay
In the colony formation inhibition assay, A549 tumor cells were treated with compound 15a in
various concentrations (1-20 µM) for 48 h, then washed and incubated in absence of drug.
Colonies were enumerated after 8 days (Fig 7). These results exhibited that 15a could totally
inhibit colony formation at highest drug concentration and the effect of 15a on clonogenicity were
irreversible, which indicated that compound 15a was cytotoxic, but not cytostatic.
15a
100
50
0
NA
1
5
10
20
Concentration ( µM)
Fig. 7. Colony formation assay. A549 cells were plated in six-well plate (100 cells per well) and
treated with compound 15a (1-20 µM) for 48h, after which time the drug was removed and
colonies were allowed to grow in drug-free medium over 8 days.

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4. Molecular modeling studies
On the basis of DHFR inhibition and the cellular metabolic data, compound 15a displayed dual
inhibitory potencies toward thymidylate synthase (TS) and DHFR in tumor cells, molecular
modeling studies were performed using Discovery Studio 2.5.
Figure 8 shows the docked pose of compound 15a (cyan) and the overlay of the docked pose of
compound 15a (cyan) with the crystal structure of PMX (purple) in the human TS (PDB ID 1JU6)
active site [35]. The binding site for the folate cofactor moiety consists of three parts: the pteridine
binding site, the benzoylglutamate region, and the bridge region. The docked pose shows the
pyrrolo[3,2-d]pyrimidine scaffold of 15a form a H-bond network with Asp218, Ala312 and
Asn112 in the pteridine binding site. The H-bond between 4-oxo moiety of 15a and Asn112 was
an extra H-bond compared to that of PMX. The glutamate tail of 15a forms two hydrogen bond
with Lys77 and Phe80 in the benzoylglutamate region. The binding pose of PMX indicate that the
pyrrolo[2,3-d]pyrimidine scaffold of PMX and benzoylglutamate tail form a tortuous ‘L’ structure
through the bridge region. 15a established a similar binding pose to that of PMX. The modeling
study imply that 15a should bind and inhibit the thymidylate synthase (TS), which is aligned with
the results of our cellular metabolic assays.
a)

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b)
Fig. 8. a) Docked pose of compound 15a (cyan) in the human TS (PDB ID 1JU6) active site; b)
Overlay of the docked pose of compound 15a (cyan) with the crystal structure of PMX (purple) in
the human TS (PDB ID 1JU6) active site.
Figure 9 shows the docked pose of compound 15a (cyan) and the overlay of the docked pose of
15a (cyan) with the crystal structure of MTX (purple) in the human DHFR (PDB ID 1U72) active
site [36]. The docked pose displays that the pyrrolo[3,2-d]pyrimidine scaffold of 15a form only
two hydrogen bonds with Glu30 residue through 3-NH and 4-oxo. In contrast, there are three
hydrogen bonds for MTX in the corresponding binding site. The benzoylglutamate tail of 15a
form four hydrogen bonds with Gln35, Arg70 and Asn64 residues, which are same as that of MTX.
The overlay of the docked pose of 15a with the crystal structure of MTX indicated that 15a
established a very similar binding mode with that of MTX in the DHFR active site, especially for
the benzoylglutamate tail. The modeling results imply that 15a could be an inhibitor of DHFR, but
should be less potent than MTX.
The above modeling results suggest that 15a should be a dual inhibitor of TS and DHFR, which is
consistent with the results of cellular metabolic assays and DHFR inhibition assay.

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a)
b)
Fig. 9. a) Docked pose of compound 15a (cyan) in the human DHFR (PDB ID 1U72) active site; b)
Overlay of the docked pose of compound 15a (cyan) with the crystal structure of MTX (purple) in
the human DHFR (PDB ID 1U72) active site.

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5. Conclusion
In a continued effort to discovery of novel antifolate anticancer candidates, in this study, a series
of novel 6-substituted pyrrolo [3,2-d] pyrimidine analogues 10a, 11a-13a, 15a, 17a, 18a, 27a, and
28a were designed and synthesized as potential antifolate agents. The biological activities of these
compounds toward HL60, A549, H1299, Hela, HCT116, and HT29 cell lines were evaluated.
Most of the compounds exhibited micromolar anti-proliferative potencies against most of these
tumor cell lines. The structure activity relationships about this kind of compounds were
summarized. Compound 15a, which has GI50 of 0.73, 1.72 and 8.92 µM against A549, H1299 and
HL60 tumor cells respectively, was the most potent one of this series. 15a could significantly
affect the cell cycle distribution of tumor cells and induce G2/M-phase arrest to some extent. The
apoptosis assay and colony formation inhibition assay demonstrated that the tumor cell death
relied on the irreversible effect of 15a on clonogenicity. The identification of targeted enzymes of
15a implied that the anti-proliferative activities of 15a probably act through dual inhibition of TS
and DHFR. This conclusion was supported by the molecular modeling studies. These results make
15a as a good lead compound as multi-targeted antifolate agents for further study.

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6. Experimental Section
6.1. Chemistry
6.1.1. General information
Reagents and solvents were purchased from common commercial suppliers and were used without
further purification. Organic solvents were dried according to standard procedures. Melting points
were determined with a SGW® X4 apparatus. Mass spectra were recorded on MDS SCIEX
QSTAR system. 1H-and 13C NMR spectra were recorded with a Varian INOVA-400 or
INOVA-600 with CDCl3 or DMSO-d6 as solvent. Tetramethylsilane was used as an internal
standard to express the chemical shift in ppm (parts per million): s, singlet; d, doublet; t, triplet; q,
quartet; quin, quintet; m, multiplet; and bs, broad singlet. Thin-layer chromatography (TLC) with
a fluorescent indicator were utilized to monitor the reaction progress, and the spots were
visualized under 254 and 365 nm illumination.
6.1.2. Synthesis of Ethyl 2-(benzyloxy)acetate (1)
To a 250 mL round-bottomed flask was added PhMe (100 mL) and NaH (4.0 g, purity 60%, 0.1
mol). The mixture was cooled in an ice bath. Phemethylol (10 mL, 0.097 mol) was then added
dropwise. Once the reaction reached completion, a solution of ethyl bromoacetate (10.7 mL, 0.097
mmol) in PhMe was added and the mixture was stirred at room temperature for 24 h. The reaction
mixture was poured into ice water (200 mL) and neutralized with dilute hydrochloric acid.
Then, the mixture was extracted by ethyl acetate (3 × 100 mL), and the organic phase was washed
with brine (200 mL), dried with Na2SO4 and concentrated. The crude product was purified by
silica gel chromatography using n-hexane-ethyl acetate (30:1, v/v) to yield compound 1 (12.3 g,
65.6%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 1.31 (t, J = 6.8 Hz, 3H, CH3), 4.12 (s, 2H,
CH2CO), 4.26 (q, J = 6.8 Hz, 2H, CH2CH3), 4.67 (s, 2H, PhCH2), 7.36-7.42 (m, 5 H, Ph); 13C
NMR (100 MHz, CDCl3) δ: 14.2, 60.9, 67.3, 73.3, 128.0, 128.1, 128.5, 137.2, 170.4.
ESI-TOF-MS: [M+H]+ m/z: 195.2, [M+Na]+ m/z: 217.2.
6.1.3. Synthesis of 4-(Benzyloxy)-3-oxobutanenitrile (2)
To a 150 mL round-bottomed flask was added 50 mL THF. The flask was cooled to -78 ºC, and 2.2
N n-BuLi (4.7 mL, 10.3 mmol) and acetonitrile (0.55 mL, 10.6 mmol) were added sequentially.
After stirring at -78 ºC for 1 h, 50 mL ethyl 2-(benzyloxy)acetate (compound 1, 2.0 g, 10.3 mmol)
in THF was added. The solution was stirred at -78 ºC for 0.5 h and then warmed to room
temperature. The mixture was poured into ice water (200 mL) and neutralized with dilute
hydrochloric acid. Then, the mixture was extracted with ethyl acetate (3×100 mL), and the organic
phase was washed with brine (200 mL), dried with Na2SO4 and concentrated. The crude product
was then purified by silica gel chromatography using n-hexane–ethyl acetate (3:1, v/v) to give
1
compound 2 (1.61 g, 82.6%) as colorless oil. H NMR (400 MHz, CDCl3) δ: 3.69 (s, 2H,
13
=CH2CN), 4.15 (s, 2H, CH2), 4.62 (s, 2H, PhCH2), 7.35-7.42 (m, 5H, Ph); C NMR (100 MHz,
CDCl3) δ: 29.7, 73.9, 74.1, 112.4, 128.1, 128.5, 128.8, 136.3, 196.8.
6.1.4. Diethyl (Z)-2-((3-(benzyloxy)-1-cyanoprop-1-en-2-yl)amino)malonate (3)

ACCEPTED MANUSCRIPT
Compound 2 (22.0 g, 116 mmol) was dissolved in a mixture of MeOH and acetic acid (1:1, 50
mL), and diethyl aminomalonate hydrochloride (25 g, 118 mmol) were added sequentially. After
stirring at room temperature overnight, the reaction mixture was poured into water (100 mL).
Then the mixture was neutralized with 1 N NaOH solution and extracted by ethyl acetate (3×50
mL), washed with brine (100 mL), dried with Na2SO4. The solvent was evaporated under vacuum
1
to obtain compound 3 (36g, 89.4%)as viscous oil. H NMR (400 MHz,CDCl3)δ：1.30 (t, J = 7.2
Hz, 6H, 2 × CH3), 4.28~4.30 (q, J = 7.2 Hz, 4H, 2 × CH2), 4.43 (d, J = 8.0 Hz, 1H, CHNH), 4.57
(s, 1H, =CHCN), 4.67 (s, 4H, CH2OCH2), 7.36~7.37 (m, 5H, ph); 13C NMR (100 MHz, CDCl3) δ:
14.0, 59.1, 63.1, 65.3, 67.2, 73.2, 118.7, 127.0, 127.6, 128.5, 140.9, 158.2, 165.5; ESI-TOF-MS:
[M+H]+ m/z: 347.18, [M+Na]+ m/z: 369.16.
6.1.5. Ethyl 3-amino-5-(benzyloxymethyl)-1H-pyrrole-2-carboxylate (4)
173 mg (7.52 mmol) Na was added to anhydrous EtOH (20 mL) to form a solution of NaOEt in
ethanol. Then a solution of 2.06 g (7.51 mmol) compound 3 in ethanol (10 mL) was dropwise
added to the NaOEt solution. The reaction mixture was stirred for 6 h at room temperature. The
solvent was evaporated under reduced pressure. The crude product was purified by column
chromatography on silica gel with n-hexane–ethyl acetate (3:1, v/v) as the eluent to yield 4 (1.18 g,
57.3%) as an off-white solid. mp 86-87 °C; 1H NMR (400 MHz, CDCl3) δ: 1.33 (t, J = 6.8 Hz, 3H,
CH3), 4.26-4.31 (m, 2H, OCH2CH3), 4.40 (s, 2H, CH2OBn), 4.49 (s, 2H, PhCH2), 5.63 (s, 1H,
4-CH), 7.29-7.34 (m, 5H, Ph); 13C NMR (100 MHz,CDCl3) δ: 14.7, 59.4, 64.6, 72.1, 98.9, 110.0,
127.9, 127.9, 128.5, 130.9, 133.6, 137.6; ESI-TOF-MS: [M+H]+m/z: 275.15.
6.1.6. Ethyl
(Z)-5-(benzyloxymethyl)-3-(2,3-bis(methoxycarbonyl)guanidino)-1H-pyrrole-2-carboxylate (5)
The pyrrole 4 (1.03 g, 3.76 mmol) was dissolved in anhydrous MeOH (20 mL), and
1,3-bis(methoxycarbonyl)-2-methyl-2-thiopseudourea (0.81 g, 3.93 mmol) and AcOH (10 mL)
were added sequentially. The mixture was stirred at room temperature overnight and became a
thick paste. Then, the mixture was filtered to give compound 5 (1.24, 76.4%) as a white solid. mp
155-157 °C. 1H NMR (400 MHz, CDCl3) δ: 1.38 (t, J = 6.8 Hz, 3H, CH3), 3.76 (s, 3H, OCH3),
3.86 (s, 3H, OCH3), 4.42 (q, J = 6.8 Hz, 2H, OCH2), 4.53-4.54 (d, 4H, CH2OCH2), 7.14 (s, 1H,
4-CH), 7.26~7.36 (m, 5H, ph), 8.84 (bs, 1H, CONH), 11.50 (bs, 1H, 1-NH), 11.82 (s, 1H, N1H);
13C NMR (100 MHz, CDCl3) δ: 14.6, 52.7, 53.5, 60.5, 64.7, 72.6, 104.1, 128.0, 128.0, 128.5,
132.5, 137.4, 152.6, 153.8, 160.7, 164.3; ESI-TOF-MS: [M+H]+ m/z：433.2476, [M+Na]+ m/z：
455.2319.
6.1.7. Methyl
(6-benzyloxymethyl-4-oxo-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-2-yl)carbamate (6)
1.27 g compound 5 was dissolved in anhydrous MeOH, then 0.925g (17.1 mmol) NaOMe was
added in batches. The mixture was stirred at room temperature for 2 h. The mixture was
neutralized with AcOH, and the solid was collected by filtration and washed thoroughly with
1
water. After drying, 6 (0.81 g, 83.9%) was obtained as an off-white solid. mp 220–221 °C; H
NMR (400 MHz, DMSO-d6) δ: 3.73 (s, 3H, CH3), 4.53 (s, 2H, 8-CH2), 4.55 (s, 2H, OCH2), 6.23
(s, 1H, 7-CH), 7.27-7.36 (m, 5H, Ph), 11.14 (bs, 2H, N3H, CONH), 12.11 (s, 1H, N5H); 13C NMR

ACCEPTED MANUSCRIPT
(100 MHz, DMSO-d6) δ: 53.2, 64.9, 71.9, 102.0, 114.9, 127.9, 128.1, 128.7, 138.6, 139.4, 144.4,
152.9, 156.0. ESI-TOF-MS: [M+H]+ m/z: 328.17, [M+Na]+ m/z: 351.16.
6.1.8. 2-Amino-6-benzyloxymethyl-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (7)
To a 50 mL round-bottomed flask was added 6 (0.84 g, 2.54 mmol) suspended in 1 N NaOH (10
mL). The reaction mixture was heated at 50 °C for 6 h. The resulting solution was cooled in an ice
bath and neutralized with AcOH. The precipitated solid was collected by filtration washed with
1
water, and dried in vacuo to afford 7 (0.68 g, 99.0%) as a white solid. mp 231-233 °C; H NMR
(400 MHz, DMSO-d6) δ: 4.49 (s, 4H, CH2OCH2), 5.97 (s, 1H, 7-CH), 6.17 (s, 2H, 2-NH2),
7.28-7.35 (m, 5H, Ph), 10.94 (bs, 1H, N3H), 11.58 (s, 1H, N5H); 13C NMR (100 MHz, DMSO-d6)
δ: 65.0, 71.7, 100.7, 113.1, 127.9, 128.0, 128.7, 138.2, 138.7, 151.4, 155.1. ESI-TOF-MS: [M+H]+
m/z: 271.17, [M+Na]+ m/z: 293.14.
6.1.9. 2-Amino-6-hydroxymethyl-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (8)
Compound 7 (80 mg, 0.296 mmol) was suspended in 5 N HCl (10 mL). The mixture was stirred at
60 °C for 6 h under an atmosphere of argon. After TLC showed the disappearance of starting
material, the reaction mixture was filtered and neutralized with aqueous NaOH. Further
purification by the silica gel chromatography using dichloromethane–methanol (5:1, v/v) yielded 8
1
(45 mg, 84.4%) as a white solid. mp > 300 °C; H NMR (400 MHz, DMSO-d6) δ: 4.42 (s, 2H,
8-CH2), 5.18 (s, 1H, OH), 5.86 (s, 1H, 7-CH), 6.09 (s, 2H, 2-NH2), 10.82 (bs, 1H, N3H), 11.30 (s,
1H, N5H); 13C NMR (100 MHz, DMSO-d6) δ: 57.0, 98.8, 112.3, 142.7, 151.2, 151.3, 154.7.
ESI-TOF-MS: [M+H]+ m/z: 181.07.
6.1.10. 2-Amino-6-(bromomethyl)-1,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (9)
0.20 g (1.11 mmol) 8 was suspended in THF (5 mL), and then 0.03 mL PBr3 was added. The
mixture was refluxed for 4 h at an atmosphere of argon. After cooled to room temperature, 100 mL
diethyl ether was added to the mixture. The precipitate was collected by filtration and washed with
diethyl ether (3×100 mL) to afford 9 (0.28 g) as a crude yellow solid which was used without
further purification.
6.1.11. Diethyl 4-((2-amino-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl) methylamino)
benzoyl-L-glutamate (10)
0.32 g (1.32 mmol) 9 and 0.40 g (1.24 mmol) diethyl (4-aminobenzoyl)-L-glutamate was
dissolved in anhydrous DMAC (5 mL). The mixture was heated at 60 °C for 6 h under an
atmosphere of argon. TLC showed the disappearance of 9 and formation of one major spot. The
reaction mixture was added CH2Cl2 (50 mL), extracted with H2O (10 mL × 3) and dried over
Na2SO4. After evaporation of solvent, the residue was loaded on a silica gel column and
chromatographed with CH2Cl2/MeOH (9:1, v/v), and the desired fraction were pooled. After
evaporation of the solvent, the residue was dried to afford 369 mg (57.9%) of 10 as a light-yellow
solid; mp > 300 °C. 1H NMR (400 MHz, DMSO-d6) δ: 1.14-1.19 (m, 6H, 2 × CH3), 1.97-2.08 (m,
2H, CH2CH2CO), 2.42 (t, 2H, CH2O, J = 6.8 Hz), 4.01-4.09 (m, 4H, 2 × OCH2), 4.35~4.36 (m, 3H,
8-CH2, NCHCO), 5.97 (s, 1H, 7-CH), 6.67 (d, J = 8.4 Hz, 2H, ph), 6.95 (m, 1H, N9H), 7.11 (bs,
2H, 2-NH2), 7.69 (d, J = 8.4 Hz, 2H, ph), 8.42 (d, J = 7.2 Hz, 1H, CONH), 11.89 (s, 1H, N5H); 13C
NMR (100 MHz, DMSO-d6) δ: 14.5, 26.2, 30.7, 48.9, 52.3, 60.3, 60.8, 98.0, 111.7, 121.3, 129.4,

ACCEPTED MANUSCRIPT
141.1, 151.5, 151.6, 153.5, 167.0, 172.5, 172.7; ESI-TOF-MS: [M+H]+ m/z: 485.22; HRMS:
[M+H]+ m/z: 485.21435, C23H29N6O6 Exact Mass: 485.21431.
6.1.12. General procedure for preparation of substituted diethyl
4-((2-amino-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl) methyl) amino)
benzoyl-L-glutamate (11-13)
To a solution of 10 (100 mg, 0.21 mmol) in anhydrous DMF (5 mL) was added an 1.1 equivalent
(0.23 mmol) of required halohydrocarbon (methyl iodide, ethyl bromide or propargyl bromide)
and K2CO3 (30 mg, 0.21 mmol). The reaction mixture was stirred under argon at room
temperature for different times as reported below. TLC showed the disappearance of 10 and
formation of one major spot. The reaction mixture was added CH2Cl2 (50 mL), extracted with
H2O (10 mL × 3) and dried over Na2SO4. After evaporation of solvent, the residue was loaded on a
silica gel column and chromatographed with CH2Cl2/MeOH (9:1, v/v), and the desired fraction
were pooled. After evaporation of the solvent, the residue was dried to afford 11-13.
6.1.13. Diethyl 4-(((2-amino-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)
methyl)(methyl)amino) benzoyl-L-glutamate (11)
This compound was obtained at 85.0 % yield starting from 10 (100 mg, 0.21 mmol) and methyl
iodide (32.4 mg, 0.23 mmol) after 24 h; mp 136-137 ºC; 1H NMR (600 MHz, CD3OD) δ: 1.21 (t, J
= 7.1 Hz, 3H, CH3), 1.26 (t, J = 7.1 Hz, 3H, CH3), 2.05-2.12 (m, 1H, CHaHbCH2CO), 2.21-2.29
(m, 1H, CHaHbCH2CO), 2.46 (t, J = 7.3 Hz, 2H, CH2CO), 3,12 (s, 3H, NCH3), 4.10 (q, J = 7.1 Hz,
2H, OCH2), 4.19 (q, J = 7.1 Hz, 2H, OCH2), 4.60-4.63 (m, 1H, NCHCO), 4.67 (s, 2H, 8-CH2),
5.89 (s, 1H, 7-CH), 6.83 (d, J = 9.0 Hz, 2H, ph), 7.74 (d, J = 9.0 Hz, 2H, ph); 13C NMR (150 MHz,
CD3OD) δ: 13.2, 14.5, 18.4, 27.5, 31.6, 39.1, 43.8, 50.6, 53.8, 55.9, 58.3, 61.7, 62.4, 99.5, 112.7,
122.3, 130.1, 141.5, 153.4, 170.3, 173.6, 174.6. ESI-TOF-MS: [M-H]+ m/z: 497.21; HRMS:
[M-H]+ m/z: 497.2143, C24H29N6O6 Exact Mass: 497.2149.
6.1.14. Diethyl 4-(((2-amino-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)
methyl)(ethyl)amino) benzoyl-L-glutamate (12)
This compound was obtained at 78.0 % yield starting from 10 (100 mg, 0.21 mmol) and ethyl
bromide (25.0 mg, 0.23 mmol) after 48 h; mp 148-149 ºC; 1H NMR (600 MHz, DMSO-d6) δ: 1.11
(t, J = 7.0 Hz, 3H, N9-CH2CH3), 1.16 (t, J = 7.1 Hz, 3H, CH3), 1.18 (t, J = 7.1 Hz, 3H, CH3),
1.93-2.02(m, 1H, CHaHbCH2CO), 2.07-2.11 (m, 1H, CHaHbCH2CO), 2.41 (t, J = 7.6 Hz, 2H,
CH2CO), 3.53 (q, J = 7.0 Hz, 2H, N9-CH2), 4.02-4.07 (m, 2H, OCH2), 4.07-4.13 (m, 2H, OCH2),
4.35–4.44 (m, 1H, NCHCO), 4.53 (s, 2H, 8-CH2), 5.73 (s, 1H, 7-CH), 5.83 (s, 2H, 2-NH2), 6.74 (d,
J = 9.0 Hz, 2H, ph), 7.71 (d, J = 9.0 Hz, 2H, ph), 8.28 (d, J = 7.5 Hz, 1H, CONH), 11.48 (s, 1H,
N5H); 13C NMR (150 MHz, DMSO-d6) δ: 12.0, 14.1, 25.8, 30. 2, 44.7, 46.8, 51.8, 59.9, 60.4, 98.8,
110.8, 111.9, 120.3, 129.0, 138.9, 149.9, 150.8, 154.0, 166.5, 172.1, 172.3; ESI-TOF-MS: [M+H]+
m/z: 513.34; HRMS: [M+H]+ m/z: 513.2455, C25H33N6O6 Exact Mass: 513.2462.
6.1.15. Diethyl 4-(((2-amino-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)
methyl)(propargyl)amino) benzoyl-L-glutamate (13)
This compound was obtained at 78.0 % yield starting from 10 (100 mg, 0.21 mmol) and propargyl
1
bromide (27.0 mg, 0.23 mmol) after 48 h; mp 148-149 ºC; H NMR (400 MHz, DMSO-d6) δ:

ACCEPTED MANUSCRIPT
1.13-1.19 (m, 6H, 2 × CH3), 1.92-2.01(m, 1H, CHaHbCH2CO), 2.06 (dd, J = 13.5, 5.4 Hz, 1H,
CHaHbCH2CO), 2.40 (t, J = 7.5 Hz, 2H, CH2CO), 3.24 (s, 1H, ≡CH), 4.01-4.05 (m, 2H, OCH2),
4.06-4.10 (m, 2H, OCH2), 4.32 and 4.33 (2 × S, 2H, N9-CH2), 4.35-4.38 (m, 1H, NCHCO), 4.83 (s,
2H, 8-CH2), 5.90 (s, 1H, 7-CH), 6.63 (d, J = 8.7 Hz, 2H, ph), 7.64 (d, J = 8.7 Hz, 2H, ph), 8.23 (d,
J = 7.5 Hz, 1H, CONH), 9.75 (s, 1H, N3H), 11.17 (bs, 1H, N5H); 13C NMR (150 MHz, DMSO-d6)
δ: 14.0, 22.0, 25.7, 28.9, 30.1, 31.2, 33.6, 59.8, 60.3, 74.0, 111.2, 112.3, 120.7, 128.8, 129.0, 150.0,
150.9, 166.4, 172.0, 172.1, 182.6; ESI-TOF-MS: [M+H]+ m/z: 523.33.
6.1.16. Diethyl
(4-(N-((2-amino-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)methyl)-2,2,2-trifluoroacet
amido)benzoyl)-L-glutamate (16)
To a solution of 10 (110 mg, 0.23 mmol) in anhydrous CH2Cl2 (5 mL) was added an excess (2 mL)
of trifluoroacetic anhydride (TFAA). The reaction mixture was stirred under argon at room
temperature overnight. TLC showed the disappearance of 10 and formation of one major spot. The
reaction solution was washed with 5% NaHCO3 solution, extracted with H2O (10 mL × 3), and
dried over Na2SO4. After evaporation of solvent, the residue was loaded onto a silica gel column
and chromatographed with CH2Cl2/MeOH (15:1, v/v), and the desired fraction were pooled. After
evaporation of the solvent, the residue was dried to afford 123 mg (93.3%) of 16 as light-yellow
1
solid; mp 129-130 °C. H NMR (400 MHz, DMSO-d6) δ: 1.13-1.20 (m, 6H, 2 × CH3), 1.98-2.12
(m, 2H, CH2CH2CO), 2.42-2.45 (t, J = 7.4 Hz, 2H, CH2CO), 4.00-4.11 (m, 4H, 2 × OCH2),
4.38-4.45 (m, H, NCHCO), 4.95 (s, 2H, 8-CH2), 5.62-5.87 (m, 3H, 2-NH2, 7-CH), 7.36-7.38 (d, J
= 8.3 Hz, 2H, ph), 7.85-7.87 (d, J = 8.3 Hz, 2H, ph), 8.82-8.84 (d, 1H, CONH), 10.51 (bs, 1H,
N3H), 11.58 (s, 1H, N5H); 13C NMR (150 MHz, DMSO-d6) δ: 14.6, 26.0, 31.2, 49.5, 53.2, 59.6,
60.5, 98.4, 111.9, 116.0, 121.8, 128.8, 141.7, 152.1, 152.6, 154.3, 155.8, 167.4, 172.1, 172.4;
HRMS: [M+H]+ m/z: 581.19773; C25H28F3N6O7 Exact Mass: 581.19661.
6.1.17. General procedure for preparation of 7-halogenated diethyl (4-(N-((2-amino
-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzoyl)-
L-glutamates (17, 18)
To a solution of 16 (100 mg, 0.17 mmol) in anhydrous CH2Cl2 (10 mL) was added 1.1 equivalent
of required halogenating reagent (NBS or I2). The reaction mixture was stirred under argon at
room temperature overnight. After the disappearance of 16 as indicated by TLC, the reaction
solution was washed with 5% NaHSO3 solution to remove the excess halogen. The resulting
mixture was extracted with H2O (10 mL × 3) and dried over Na2SO4. After evaporation of solvent,
the residue was loaded onto a silica gel column and chromatographed with CH2Cl2/MeOH (15:1,
v/v), and the desired fraction were pooled. After evaporation of the solvent, the residue was dried
to afford 17 or 18.
6.1.17. Diethyl
(4-(N-((2-amino-7-bromo-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)methyl)-2,2,2-trifl
uoroacetamido)benzoyl)-L-glutamates (17)
This compound was obtained at 89.0 % yield starting from 16 and N-bromosuccinimide (NBS);
mp 149-150 °C. 1H NMR (600 MHz, DMSO-d6) δ: 1.15 (t, J=7.1 Hz, 3H, CH3), 1.19 (t, J =7.1 Hz,
3H, CH3), 1.97-2.02 (m, 1H, CHaHbCH2CO), 2.07-2.11 (m, 1H, CHaHbCH2CO), 2.43-2.45 (m, 2H,

ACCEPTED MANUSCRIPT
CH2CO), 4.03 (q, J = 7.1 Hz, 2H, OCH2), 4.09-4.12 (m, 2H, OCH2), 4.41 (dt, J = 9.5, 7.3 Hz, 1H,
NCHCO), 6.12 (s, 2H, 8-CH2) 7.35 (d, J = 8.2 Hz, 2H, ph), 7.84 (d, J = 8.4 Hz, 2H, ph), 8.81 (d, J
= 7.3 Hz, 1H, CONH), 10.63 (bs, 1H, N3H), 12.10 (s, 1H, N5H); 13C NMR (150 MHz, DMSO-d6)
δ: 14.0, 25.5, 30.1, 45.4, 52.0, 59.8, 60.5, 112.2, 128.2, 128.4, 130.9, 134.1, 139.9, 151.3, 153.5,
165.7, 171.5, 172.1; ESI-TOF-MS: [M-H]- m/z: 657.31 and 659.30; HRMS: [M-H]- m/z: 657.0907
and 659.0894, C25H25BrF3N6O7 Exact Mass: 657.0920.
6.1.18. Diethyl
(4-(N-((2-amino-7-bromo-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)methyl)-2,2,2-trifl
uoroacetamido)benzoyl)-L-glutamates (18)
1
This compound was obtained at 85.0 % yield starting from 16 and I2; mp 140-141 °C. H NMR
(600 MHz, DMSO-d6) δ: 1.14 (t, J = 7.1 Hz, 3H, CH3), 1.17 (t, J = 7.1 Hz, 3H, CH3), 1.91-2.03
(m, 1H, CHaHbCH2CO), 2.05-2.11 (m, 1H, CHaHbCH2CO), 2.41-2.44 (m, 2H, CH2CO), 4.02 (q, J
= 7.1 Hz, 2H, OCH2), 4.10 (q, J = 7.1 Hz, 2H, OCH2), 4.37-4.41 (m, NCHCO, 1H), 6.02 (s, 2H,
8-CH2), 7.33 (d, J = 8.3 Hz, 2H, ph), 7.81 (d, J = 8.6 Hz, 2H, ph), 8.78 (d, J = 7.4 Hz, 1H, CONH),
10.51 (s, 1H, N3H), 12.09 (s, 1H, N5H); 13C NMR (150 MHz, DMSO-d6) δ: 13.9, 25.5, 30.0, 46.8,
52.0, 59.8, 60.5, 113.0, 115.1, 117.0, 128.1, 128.5, 133.8, 134.1, 139.8, 147.6, 151.1, 153.6, 165.6,
171.5, 172.1; ESI-TOF-MS: [M-H]- m/z: 705.23; HRMS: [M-H]- m/z: 705.0765 C25H25F3IN6O7
Exact Mass: 705.0781.
6.1.19. Diethyl
(4-(2-(2-amino-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-6-yl)ethyl)benzoyl)glutamate (15)
To a solution of 9 (90 mg, 0.37 mmol) in 15 mL anhydrous DMF was added 118 mg (0.45 mmol)
dry triphenylphosphine and heated at 60 ºC for 2 h under argon. TLC in CHCl3-CH3OH (9:1)
indicated the disappearance of 9. The reaction mixture was cooled to room temperature and was
added 60 mg (1.11 mmol) NaOMe and 127 mg (0.38 mmol) diethyl
N-[4-(formyl)benzoyl]-L-glutamate in sequence, the solution was heated at 60 ºC for another 60 h
under argon. After evaporation of solvent, water (30 mL) was added to the gummy residue. The
pH of the suspension was adjusted to 7 by 2 M acetic acid and the resulting solution was extracted
with dichloromethane (50 mL × 3). The organic layer was concentrated to a small volume, and
was chromatographed on silica gel and eluted with CH2Cl2/CH3OH (9:1, v/v), and the desired
fraction were pooled. After evaporation of the solvent, 50 mg of critical intermediate was afford as
a yellow solid and was dissolved in 10 mL of anhydrous ethanol. After addition of 5 mg Pd/C, the
resulting suspension was hydrogenated (0.4 MPa) for 12 h. The result mixture was filtered through
celite and the filtrate was evaporated to dryness under reduced pressure. The residue was loaded
onto a silica gel column and chromatographed with CH2Cl2/MeOH (15:1, v/v), and the desired
fraction were pooled. After evaporation of the solvent, the residue was dried to afford 20 mg (11.1%
1
of three steps) of 15 as a yellow solid. mp 156-157 °C. H NMR (400 MHz, DMSO-d6) δ:
1.14-1.20 (m, 6H, 2 × CH3), 1.98-2.02 (m, 1H, CHaHbCH2CO), 2.08-2.11 (m, 1H, CHaHbCH2CO),
2.41-2.45 (t, J = 7.4 Hz, 2H, CH2CO), 2.87-2.90 (t, J = 7.8 Hz, 2H, 8-CH2), 2.98-3.01 (t, J = 7.8
Hz, 2H, 9-CH2), 4.02-4.13 (m, 4H, 2 × OCH2), 4.39-4.45 (m, 1H, NCHCO), 5.71-5.73 (m, 3H：
2-NH2, 7-CH), 7.30-7.32 (d, J = 8.1 Hz, 2H, ph), 7.78-7.80 (d, J=8.1 Hz, 2H, ph), 8.63-8.65 (d, J
= 7.4 Hz, 1H, CONH), 10.31 (bs, 1H, N3H), 11.28 (s, 1H, N5H); 13C NMR (150 MHz, DMSO-d6)
δ: 14.5, 26.2, 29.6, 30.6, 34.9, 52.4, 60.4, 61.0, 99.2, 112.0, 127.9, 128.6, 131.9, 134.5, 141.7,

ACCEPTED MANUSCRIPT
145.5, 149.5, 151.0, 167.0, 172.2, 172.6. ESI-TOF-MS: [M+H]+ m/z: 484.36; HRMS : [M+H]- m/z:
484.2197 C24H30N5O6 Exact Mass: 484.2196.
6.1.20. N-(6-((benzyloxy)methyl)-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin-2-yl)pivalamide
(19)
To a solution of 7 (200 mg, 0.74 mmol) in 20 mL CH2Cl2 was added 182 µl (1.48 mmol) pivaloyl
chloride, 9 mg (0.07 mmol) DMAP and 216 µl (1.55 mmol) triethylamine. The resulting mixture
was reflux for 5h. TLC showed the disappearance of 7 and formation of one major spot. The
reaction mixture was extracted with H2O (10 mL × 3) and dried over Na2SO4. After evaporation of
solvent, the residue was loaded onto a silica gel column and chromatographed with PE/EtOAc
(1:1, v/v), and the desired fraction were pooled. After evaporation of the solvent, the residue was
dried to afford 257 mg (98.0%) of 19 as light yellow solid. mp 170-171 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 1.25 (s, 9H, 3 × CH3), 4.54-4.57 (s, 4H, CH2OCH2), 6.26 (s, 1H, 7-CH), 7.27-7.37
(m, 5H, ph), 10.80 (s, 1H, N3H), 12.01 (s, 1H, N5H), 12.16 (s, 1H, CONH); 13C NMR (100 MHz,
DMSO-d6) δ: 26.9, 64.9, 72.0, 102.1, 115.2, 127.9, 128.1, 128.7, 138.6, 139.5, 144.7 145.4, 152.5,
181.1.
6.1.21. N-(6-((benzyloxy)methyl)-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-2-yl)pivalamide (20)
A suspension of 19 (257 mg, 0.73 mmol) in 10 mL POCl3 was reflux for 10 h. After evaporation
of POCl3, the residue was added 10 mL of cold water to remove the excess POCl3. The pH was
adjusted to 7 by aqueous ammonia. The resulting solution was extracted with dichloromethane (50
mL × 3) and dried over Na2SO4. The organic layer was concentrated to a small volume, and was
chromatographed on silica gel and eluted with PE/EtOAc (1:1, v/v), and the desired fraction were
pooled. After evaporation of the solvent, 243 mg (89.9%) of 20 was obtained as a white solid. mp
178-179 °C. 1H NMR (400 MHz, CDCl3) δ: 1.33 (s, 9H, 3 × CH3), 4.60 (s, 2H, 8-CH2), 4.75 (s,
2H, phCH2), 6.45 (s, 1H, 7-CH), 7.31-7.37 (m, 5H, ph), 8.13 (s, 1H, CONH), 9.60 (s, 1H, N5H);
13C NMR (100 MHz, CDCl3) δ: 27.5, 40.1, 64.9, 73.1, 101.2, 122.2, 128.1, 128.2, 128.6, 137.0,
142.1, 145.5, 150.3, 152.3, 175.8.
6.1.22.
N-(6-((benzyloxy)methyl)-4-chloro-5-methyl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)pivalamide (21)
To as solution of 20 (75 mg, 0.20 mmol) in 2 mL of anhydrous DMF was added 9 mg (0.38 mmol)
NaH in batches. The suspension was stirred for 10 min and was dropwise added 13 µl (0.26 mmol)
of methyl iodide in 1 mL of anhydrous DMF. The resulting solution was continued to stir for 6 h.
TLC indicated the disappearance of 20. The reaction mixture was added 10 mL of H2O and
extracted with dichloromethane (20 mL × 3) and dried over Na2SO4. The organic layer was
concentrated to a small volume, and was chromatographed on silica gel and eluted with PE/EtOAc
(2:1, v/v), and the desired fraction were pooled. After evaporation of the solvent, 70 mg (89.9%)
of 21 was obtained as a white solid. 1H NMR (400 MHz, CDCl3) δ: 1.34 (s, 9H, 3 × CH3), 4.05 (s,
3H, N5-CH3), 4.55 (s, 2H, 8-CH2), 4.67 (s, 2H, phCH2), 6.62 (s, 1H, 7-CH), 7.31-7.36 (m, 5H, ph),
8.08 (s, 1H, CONH); 13C NMR (100 MHz, CDCl3) δ: 27.5, 32.9, 40.1, 63.4, 72.3, 103.9, 123.0,
128.0, 128.1, 128.6, 137.0, 142.2,145.6, 149.9, 152.1, 175.6.

ACCEPTED MANUSCRIPT
6.1.23. N-(5-Allyl-6-(benzyloxymethyl)-4-chloro-5H-pyrrolo[3,2-d]pyrimidin-2-yl)pivalamide
(22)
86 mg (97.1%) of 22 was obtained as a white solid through a similar procedure as 21 using 80 mg
(0.22 mmol) of 20 and 20 µl (0.24 mmol) allyl bromide as starting material. 1H NMR (400 MHz,
CDCl3) δ: 1.34 (s, 9H, 3 × CH3), 4.54 (s, 2H, 8-CH2), 4.63-4.64 (m, 3H, phCH2, =CHaHb),
5.09-5.14 (m, 3H, N5-CH2, =CHaHb), 5.92-6.01 (m, 1H, CH=CH2), 6.69 (s, 1H, 7-CH), 7.32-7.39
(m, 5H, ph), 8.09 (s, 1H, CONH); 13C NMR (100 MHz, CDCl3) δ: 27.5, 40.1, 47.2, 67.3, 72.3,
104.4, 116.1, 122.5, 128.1, 128.1, 128.6, 133.8, 137.0, 142.1, 145.7, 150.1, 152.4, 175.6.
6.1.24. 5-Methyl-2-amino-6-(hydroxymethyl)-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one (23)
To a solution of 21 (48 mg, 0.124 mmol) in 1,4-Dioxane (5 mL) was added 10 mL of 2 N NaOH.
The resulting solution was reflux for 6 h. TLC indicated the disappearance of 21. After cooled to
room temperature, the reaction mixture was neutralized by acetic acid and extracted with
dichloromethane (20 mL × 3) and dried over Na2SO4. The organic layer was concentrated to a
small volume, and was chromatographed on silica gel and eluted with CH2Cl2/MeOH (12:1, v/v),
and the desired fraction were pooled. After evaporation of the solvent, 30 mg (84.9%) of 23 was
obtained as a white solid. mp 249-250 °C. 1H NMR (400 MHz, DMSO-d6) δ: 3.87 (s, 3H, CH3),
4.50 (s, 2H, 8-CH2), 4.54 (s, 2H, phCH2), 5.81 (bs, 2H, 2-NH2), 5.97 (s, 1H, 7-CH), 7.28-7.38 (m,
13
5H, ph), 10.53 (bs, 1H, N3H); C NMR (100 MHz, DMSO-d6) δ: 32.1, 63.4, 71.7, 101.7, 113.7,
125.3, 128.0, 128.1, 128.7, 138.5, 138.7, 151.1, 155.4; HRMS: [M+H]+ m/z: 285.13414,
C15H17N4O2 Exact Mass: 285.13460.
6.1.25. 5-Allyl-2-amino-6-(hydroxymethyl)-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one (24)
80 mg (70.9%) of 24 was obtained as a white solid through a similar procedure as 23 using 150
mg (0.36 mmol) of 22 as starting material. mp 232-233 °C. 1H NMR (400 MHz, DMSO-d6) δ:
4.50 (s, 4H, CH2OCH2), 4.74 (d, J = 17.2 Hz, 1H, =CHaHb), 4.94 (s × 2, 2H, N5-CH2), 5.01 (d, J =
10.4 Hz, 1H, =CHaHb), 5.86 (bs, 2H, 2-NH2), 5.89-5.97 (m, 1H, CH=), 6.02 (s, 1H, 7-CH),
7.30~7.38 (m, 5H, ph), 10.56 (bs, 1H, N3H); 13C NMR (100 MHz, DMSO-d6) δ: 47.1, 63.3, 71.8,
102.2, 113.2, 115.8, 128.0, 128.2, 128.7, 135.9, 138.4, 138.5, 151.3, 155.1; HRMS: [M+H]+m/z:
311.14991, C17H19N4O2 Exact Mass: 311.15025.
6.1.26. 5-Methyl-2-amino-6-(hydroxymethyl)-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one (25)
82 mg (80.0%) of 25 was obtained as a light yellow solid through a similar procedure as 8 using
150 mg (0.528 mmol) of 23 as starting material. mp > 300 °C. 1H NMR (400 MHz, DMSO-d6) δ:
3.86 (s, 3H, N5-CH3), 4.47 (s, 2H, CH2OH), 5.86 (bs, 3H, 2-NH2, 7-CH), 10.69 (s, 1H, N3H); 13C
NMR (100 MHz, DMSO-d6) δ: 32.0, 55.4, 99.6, 113.0, 143.0, 145.6, 151.1, 155.5.
6.1.27. 5-Allyl-2-amino-6-(hydroxymethyl)-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one (26)
80 mg (56.4%) of 26 was obtained as a light yellow solid through a similar procedure as 8 using
200 mg (0.645 mmol) of 24 as starting material. mp > 300 °C. 1H NMR (400 MHz, DMSO-d6) δ:
4.44 (s, 2H, CH2OH), 4.76 (d, J = 17.2 Hz, 1H, =CHaHb), 4.96 (s × 2, 2H, N5-CH2), 5.04 (d, J =
10.0 Hz, 1H, =CHaHb), 5.26 (bs, 1H, OH), 5.89-6.00 (m, 4H, 2-NH2, 7-CH, CH=), 10.56 (bs, 1H,
N3H); 13C NMR (100 MHz, DMSO-d6) δ: 41.0, 55.5, 100.1, 112.5, 115. 7, 136.0, 143.1, 151.3,
155.2.

ACCEPTED MANUSCRIPT
6.1.28. Diethyl 4-((5-methyl-2-amino-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin
-6-yl)methylamino) benzoyl-L-glutamate (27)
50 mg (0.53 mmol) 25 was suspended in anhydrous THF (10 mL), and then 0.2 mL (2.11 mmol)
PBr3 was added. The mixture was refluxed for 4 h at an atmosphere of argon. After cooled to room
temperature, 100 mL diethyl ether was added to the mixture. The precipitate was collected by
filtration and washed with diethyl ether (3×100 mL) to afford a critical bromide intermediate (60
mg) as a crude yellow solid which was used without further purification. 45 mg (0.18 mmol) of
above bromide and 70 mg (0.22 mmol) diethyl (4-aminobenzoyl)-L-glutamate was dissolved in
anhydrous DMAC (10 mL). The resulting solution was added 45 mg (0.54 mmol) NaHCO3 and
heated at 60 °C under argon for 8 h. TLC showed the disappearance of the bromide and and
formation of a new major spot. After cooled to room temperature, the reaction mixture was added
H2O (30 mL), extracted with CH2Cl2 (50 mL × 3) and dried over Na2SO4. After evaporation of
solvent, the residue was loaded on a silica gel column and chromatographed with CH2Cl2/MeOH
(15:1, v/v), and the desired fraction were pooled. After evaporation of the solvent, the residue was
dried to afford 60 mg (68.5%) of 27 as a light-yellow solid. mp 236-237 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 1.15-1.20 (m, 6H, 2 × CH3), 1.95-2.10 (m, 2H, CH2CH2CO), 2.42 (t, J = 7.4 Hz, 2H,
CH2CO), 3.90 (s, 3H, N5-CH3), 4.02-4.12 (m, 4H, 2 × OCH2), 4.34-4.41 (m, 3H, 8-CH2, NCHCO),
5.79 (bs, 2H, 2-NH2), 5.89 (s, 1H, 7-CH), 6.65-6.70 (m, 3H, ph, N9H), 7.66-7.68 (d, J = 8.5 Hz,
2H, ph), 8.25 (d, J = 7.2 Hz, 1H, CONH), 10.44 (bs, 1H, N3H); 13C NMR (100 MHz, DMSO-d6) δ:
14.5, 26.3, 30.7, 32.0, 39.1, 52.3, 60.3, 60.8, 100.3, 111.6, 113.0, 121.4, 129.4, 140.5, 151.1, 151.5,
167.0, 172.6, 172.7; HRMS: [M+H]+ m/z: 499.23030, C24H31N6O6 Exact Mass: 499.22996.
6.1.29. Diethyl 4-((5-allyl-2-amino-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin
-6-yl)methylamino) benzoyl-L-glutamate (28)
32 mg (78.3%) of 28 was obtained as a light yellow solid through a similar procedure as 27 using
22 mg (0.08 mmol) of 26 as starting material. mp 183-184 °C. 1H NMR (400 MHz, DMSO-d6) δ:
1.14-1.20 (m, 6H, 2 × CH3), 1.94-2.10 (m, 2H, CH2CH2CO), 2.39-2.43 (t, J = 7.4 Hz, 2H,
CH2CO), 4.02-4.11 (m, 4H, 2 × OCH2), 4.30-4.41 (m, 3H, 8-CH2, NCHCO), 4.78 (d, J = 7.4 Hz,
1H, =CHaHb), 5.02 (bs, 2H, N5-CH2), 5.08 (d, J = 10.4 Hz, 1H, =CHaHb), 5.83 (bs, 2H, 2-NH2),
5.90 (s, 1H, 7-CH), 5.94-6.04 (m, 1H, CH=), 6.64 (d, J=8.5 Hz, 2H, ph), 6.70 (t, J = 5.4 Hz, 1H,
N9H), 7.65-7.67 (d, J = 8.5 Hz, 2H, ph),8.25 (d, J = 7.2 Hz, 1H, CONH), 10.46 (bs, 1H, N3H); 13C
NMR (100 MHz, DMSO-d6) δ: 14.5, 26.3, 30.7, 47.0, 50.9, 52.3, 60.3, 60.8, 111.6, 112.6, 115.8,
121.4, 129.4, 135.8, 140.6, 151.3, 151.5, 167.0, 172.6, 172.7; HRMS: [M+H]+ m/z: 525.24555,
C26H33N6O6 Exact Mass: 525.24561.
6.1.30.General procedure for preparation of 10a, 11a-13a, 15a, 17a, 18a, 27a, and 28a
A suspension of 20 mg 10, 11-13, 15, 17, 18, 27 or 28 in THF (2 mL) was added 1 mol/L NaOH (1
mL), and the mixture was stirred at room temperature for 8 h. TLC showed the disappearance of
the starting material. After evaporation of the solvent, the remaining aqueous solution was
acidified carefully with 0.5 mol/L HCl, and the solid was collected by filtration, washed with
water, and dried in vacuo to give 10a, 11a-13a, 15a, 17a, 18a, 27a or 28a as pale yellow solid.
The data are reported as follows.

ACCEPTED MANUSCRIPT
6.1.31. 4-((2-Amino-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)methylamino)
benzoyl-L-glutamate (10a)
10a was obtained as a light yellow solid at 65.3% yield using 90 mg (0.19 mmol) of 10 as starting
1
material. mp 232-233 °C. H NMR (400 MHz, DMSO-d6) δ: 1.92-1.97 (m, 1H, CHaHbCH2CO),
2.04-2.11 (m, 1H, CHaHbCH2CO), 2.31-2.43 (m, 2H, CH2CO), 4.30-4.35 (m, 3H, 8-CH2,
NCHCO), 5.88 (s, 1H, 7-CH), 6.01 (bs, 2H, 2-NH2), 6.56 (m, 1H, N9H), 6.64 (d, J=8.4 Hz, 2H,
ph), 7.66 (d, J = 8.4 Hz, 2H, ph), 8.13 (d, J = 8.4 Hz, 1H, CONH), 11.48 (s, 1H, N5H); 13C NMR
(100 MHz, DMSO-d6) δ: 26.1, 30.5, 51.3, 51.8, 98.8, 111.2, 111.8, 121.2, 128.9, 139.8, 150.8,
151.0, 154.0, 166.5, 173.9, 174.0; HRMS: [M+H]+ m/z: 429.15161, C19H21N6O6 Exact Mass:
429.15171.
6.1.32.
(4-(((2-Amino-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)benzoyl)gluta
mic acid (11a)
11a was obtained as a light yellow solid at 60.5% yield using 20 mg (0.04 mmol) of 11 as starting
1
material. mp>300 °C. H NMR (600 MHz, DMSO-d6) δ: 1.92-1.96 (m,1H, CHaHbCH2CO), 2.27
(dd, J = 14.7, 8.0 Hz, 1H, CHaHbCH2CO), 2.31-2.35 (m, 2H, CH2CO), 3.05 (s, 3H, N9-CH3), 4.32
(dd, J = 14.7, 8.0 Hz, 1H, NCHCO), 4.56 (s, 2H, 8-CH2), 5.74 (s, 2H, 2-NH2), 6.79 (d, J = 9.0 Hz,
2H, ph), 7.70 (d, J = 9.0 H, 2H, ph), 8.04 (s, 1H, CONH), 10.31 (bs, 2H, COOH), 11.43 (s, 1H,
N5H); 13C NMR (150 MHz, DMSO-d6) δ: 22.1, 26.8, 29.0, 31.3, 48.8, 52.0, 99.0, 111.2, 111.9,
121.2, 128.6, 138.0, 150.9, 165.8, 173.7, 174.1; HRMS: [M-H]+ m/z: 441.1520, C20H21N6O6 Exact
Mass: 441.1523.
6.1.33.
(4-(((2-amino-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-6-yl)methyl)(ethyl)amino)benzoyl)glutami
c acid (12a)
12a was obtained as a light yellow solid at 70.5% yield using 40 mg (0.08 mmol) of 12 as starting
material. mp>300 °C. 1H NMR (600 MHz, DMSO-d6) δ: 1.10 (t, J = 7.0 Hz, 3H, CH3), 1.91-1.94
(m, 2H, CH2CH2CO), 2.21-2.34 (m, 2H, CH2CO), 3.51 (q, J = 7.0 Hz, 2H, N5-CH2), 4.29 (dd, J =
14.4, 7.2 Hz, 1H, NCHCO), 4.51 (s, 2H, 8-CH2), 5.71 (s, 1H, 7-CH), 5.82 (s, 2H, 2-NH2), 6.73 (d,
J = 8.8 Hz, 2H, ph), 7.67 (d, J = 8.8 Hz, 2H, ph), 7.93 (d, J = 6.3 Hz, 1H, CONH), 11.44 (s, 1H,
N5H); 13C NMR (150 MHz, DMSO-d6) δ: 11.9, 27.2, 31.6, 44.6, 46.7, 52.1, 98.8, 110.8, 111.9,
120.9, 128.5, 138.7, 149.7, 150.7, 165.5, 173.7, 174.3; ESI: [M+H]+ m/z: 513.34; HRMS: [M-H]+
m/z: 455.1675, C21H23N6O6 Exact Mass: 455.1670.
6.1.34.
(4-(((2-Amino-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-6-yl)methyl)(prop-2-yn-1-yl)amino)benzo
yl)glutamic acid (13a)
13a was obtained as a light yellow solid at 57.5% yield using 20 mg (0.04 mmol) of 13 as starting
1
material. mp>300 °C. H NMR (600 MHz, DMSO-d6) δ: 1.89-1.94 (m, 1H, CHaHbCH2CO),
2.02-2.07 (m, 1H, CHaHbCH2CO), 2.32 (t, J = 7.6 Hz, 2H, CH2CO), 3.29 (m, 1H, ≡CH),
4.30-4.34 (m, 1H, NCHCO), 4.80 (s, 2H, 8-CH2), 5.85 (s, 1H, 7-CH), 6.63 (d, J=8.8 Hz, 2H, ph),
7.64 (d, J = 8.8 Hz, 2H, ph), 8.11 (d, J = 7.8 Hz, 1H, CONH), 11.45 (bs, 1H, N5H); 13C NMR (150

ACCEPTED MANUSCRIPT
MHz, DMSO-d6) δ: 22.1, 26.0, 29.0, 29.8, 30.4, 31.3, 51.7, 111.2, 121.2, 128.8, 150.2, 151.0,
166.4, 173.7, 173.9, 179.4; HRMS: [M+H]+ m/z: 467.1678, C22H23N6O6 Exact Mass: 467.1679.
6.1.35. (4-(2-(2-Amino-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-6-yl)ethyl)benzoyl)glutamic acid
(15a)
15a was obtained as a light yellow solid at 55.0% yield using 10 mg (0.02 mmol) of 15 as starting
1
material. mp 254-255 °C. H NMR (600 MHz, DMSO-d6) δ: 1.84 (bs, 1H, CHaHbCH2CO), 1.93
(bs, 1H, CHaHbCH2CO), 2.14-2.17 (m, 1H, CHaHbCO), 2.28 (bs, 1H, CHaHbCO), 2.87 (t, J = 7.8
Hz, 2H, 8-CH2), 2.97 (t, J = 7.8 Hz, 2H, 9-CH2), 4.09-4.32 (m, 1H, NCHCO), 5.72 (s, 1H, 7-CH),
5.75 (s, 2H, 2-NH2), 7.29 (d, J = 8.0 Hz, 2H, ph), 7.74 (d, J = 8.0 Hz, 2H, ph), 8.08 (s, 1H,
CONH), 10.33(s, 1H, N3H), 11.25 (s, 1H, N5H); 13C NMR (150 MHz, DMSO-d6) δ: 29.1, 29.9,
34.5, 53.1, 98.8, 111.4, 126.9, 128.1, 131.4, 132.4, 141.2, 144.3, 147.4, 150.5, 164.7, 173.5;
HRMS: [M-H]-m/z: 426.1413, C20H20N5O6 Exact Mass: 426.1414.
6.1.36.
(4-(((2-Amino-7-bromo-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-6-yl)methyl)amino)benzoyl)glut
amic acid (17a)
17a was obtained as a light yellow solid at 67.0% yield using 30 mg (0.05 mmol) of 17 as starting
1
material. mp >300 °C. H NMR (600 MHz, DMSO-d6) δ: 1.91-1.92 (m, 2H, CH2CH2CO),
2.23-2.26 (m, 1H, CHaHbCO), 2.31-2.35 (m, 1H, CHaHbCO), 4.27 (d, J = 5.6 Hz, 1H, NCHCO),
6.08 (s, 2H, 8-CH2), 6.65 (d, J = 8.8 Hz, 2H, ph), 7.62 (d, J = 8.8 Hz, 2H, ph), 7.92 (s, 1H,
CONH), 10.56 (s, 1H, N3H), 11.92 (s, 1H, N5H); 13C NMR (150 MHz, DMSO-d6) δ: 38.6, 51.3,
88.0, 111.1, 111.5, 121.6, 128.5, 135.7, 144.4, 150.4, 151.3, 153.6, 165.6, 173.6, 174.2; HRMS:
[M-H]- m/z: 505.0457, 507.0456, C19H18BrN6O6 Exact Mass: 505.0471.
6.1.37.
(4-(((2-Amino-4-hydroxy-7-iodo-5H-pyrrolo[3,2-d]pyrimidin-6-yl)methyl)amino)benzoyl)glutam
ic acid (18a)
18a was obtained as a light yellow solid at 66.3% yield using 20 mg (0.03 mmol) of 18 as starting
1
material. mp >300 °C. H NMR (600 MHz, DMSO-d6) δ: 1.85-1.92 (m, 2H, CH2CH2CO),
2.24-2.16 (m, 1H, CHaHbCO), 2.26-2.31 (m, 1H, CHaHbCO), 4.22 (q, J = 7.0 Hz, 1H, NCHCO),
4.26 (d, J = 5.6 Hz, 2H), 6.05 (s, 2H, 8-CH2), 6.63 (d, J = 8.7 Hz, 2H, ph), 7.59 (d, J = 8.7 Hz, 2H,
ph), 7.79 (d, J = 6.5 Hz, 1H, CONH), 10.60 (s, 1H, N3H), 11.94 (s, 1H, N5H); 13C NMR (150 MHz,
DMSO-d6) δ: 27.9, 30.0, 32.1, 52.7, 58.5, 111.4, 112.4, 121.9, 128.5, 138.9, 148.0, 150.5, 151.3,
153.7, 165.4, 173.8,174.8; HRMS: [M-H]- m/z: 553.0326, C19H18IN6O6 Exact Mass: 553.0333.
6.1.38.
(4-(((2-Amino-4-hydroxy-5-methyl-5H-pyrrolo[3,2-d]pyrimidin-6-yl)methyl)amino)benzoyl)glut
amic acid (27a)
27a was obtained as a light yellow solid at 78.9% yield using 50 mg (0.10 mmol) of 27 as starting
material. mp 239-240 °C. 1H NMR (400 MHz, DMSO-d6) δ: 1.90-2.04 (m, 2H, CH2CH2CO), 2.31
(t, J = 3.2 Hz, 2H, CH2CO), 3.89 (s, 3H, N5-CH3), 4.33-4.34 (m, 3H, 8-CH2, NCHCO), 5.86 (bs,
2H, 2-NH2), 5.89 (s, 1H, 7-CH), 6.62 (t, J = 5.4 Hz, 1H, N9H), 6.68 (d, J = 8.4 Hz, 2H, ph), 7.76
(d, J = 8.4 Hz, 2H, ph), 8.05 (d, J = 6.8 Hz, 1H, CONH); 13C NMR (100 MHz, DMSO-d6) δ: 25.9,

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30.3, 38.5, 40.0, 46.4, 51.6, 111.0, 111.8, 112.3, 115.3, 121.1, 128.8, 135.1, 150.6, 150.8, 166.3,
173.7, 173.8; HRMS: [M+H]+ m/z: 443.16744, C20H23N6O6 Exact Mass: 443.16736.
6.1.39.
(4-(((5-Allyl-2-amino-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-6-yl)methyl)amino)benzoyl)-L-glut
amic acid (28a)
28a was obtained as a light yellow solid at 78.9% yield using 20 mg (0.04 mmol) of 28 as starting
material. mp 242-243 °C. 1H NMR (400 MHz, DMSO-d6) δ: 1.91 (bs, 2H, CH2CO), 2.19-2.22 (m,
2H, CH2CH2CO), 4.19-4.28 (m, 3H, NCHCO, 8-CH2), 4.78 (d, J = 17.2 Hz, 1H, =CHaHb),
5.01-5.09 (m, 3H, N5-CH2, =CHaHb), 5.89 (s, 1H, 7-CH), 5.94-6.01 (m, 1H, CH=), 6.11 (bs, 2H,
2-NH2), 6.62-6.66 (m, 3H, N9H, ph), 7.60 (d, J = 7.9 Hz, 2H, ph), 7.766 (s, 1H, CONH), 11.153
(bs, 1H, N3H); 13C NMR (100 MHz, DMSO-d6) δ: 22.0, 24.4, 28.4, 28.6, 28.9, 31.2, 31.5, 33.6,
38.6, 51.7, 111.0, 112.5, 120.8, 121.1, 128.5, 128.7, 140.0, 140.4, 150.5, 150.9, 166.2, 173.7,173.9;
HRMS: [M+H]+ m/z: 469.18335, C22H25N6O6 Exact Mass: 469.18301.
6.2. Biological evaluation
6.2.1. In vitro human cancer growth inhibition
Human promyelocytic leukemic cell line (HL-60), human lung adenocarcinoma epithelial cell line
(A549), human lung adenocarcinoma cell line (H1299), human cervix adenocarcinoma cell line
(Hela) and human colon adenocarcinoma cell line (HT29) were grown and maintained in
RPMI-1640 or DMEM medium supplemented with 10% fetal bovine serum, penicillin (100 units
mL-1), streptomycin concentration at 37 ºC in a humidified incubators in an atmosphere of 5%
CO2. Exponentially growing cancer cells were used to perform the experiments. The cell lines
were assessed by trypsinizing and seeding 5 × 103 cells per well into 96-well plates. Cells were
grown for 12 h and then treated with compounds at concentration ranging from 10 nM to 100 µM
and incubated for 48 h in 200 µL media. MTS reagent (20 µL) in serum-free medium was added
to each well and incubated further for 3 h. O. D. value at 490 nm was measured using a
Benchmark Plus Microplate Reader (Bio-Rad). Three individual experiments were performed to
obtain mean cell viability. Cells treated with 0.1% DMSO were used as a control, while MTX
were used as a positive control.
6.2.2. Dihydrofolate Reductase (DHFR) Assay.
The recombinant human (rh) DHFR was purchased from Sigma-Aldrich (Shanghai, China). The
inhibitory activities of the target compounds against rhDHFR enzymes were assayed in a solution
containing 0.1 mM dihydrofolate, 0.3 mM NADPH, and 100 mM KCl at pH 7.5 and 32 ˚C. The
reaction was initiated with an amount of enzyme yielding a change in O.D. value at 340 nm
measured by micro-plate spectrophotometer (BioRad).
6.2.3. Identification of the targeted pathway
To identify the targeted pathways/folate-dependent enzymes (i.e., TS, GARFTase or AICARFTase)
of these pyrrolo[3,2-d]pyrimidine analogues, proliferation assays were tested by co-incubation
with adenosine (60 µM), thymidine (10 µM), or 5-aminoimidazole-4-carboxamide hydrochloride

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(320 µM) and the results compared to those of incubations in parallel without nucleoside/AICA or
drug additions.
6.2.4. Cell Cycle Assay
A549 cells were treated with compound 15a (1 µM and 10 µM) or MTX (1 µM) for 72 h in
RPMI1640 medium/10% dFBS, 5% penicillin-streptomycin, and 2 nM LCV. Cells were washed
twice with ice-cold PBS and fixed in 70% ethanol (4 ºC, overnight), then stained by re-suspension
in 0.5 mL of PBS containing 50 µg/mL PI and 100 µg/mL RNase type I-A (Sigma Aldrich) at least
20 min at room temperature in the dark prior to analysis. The cells were detected by flow
cytometry using the BD FACSCalibur flow cytometer for determining the percentage of cells in
each phase of the cell cycle. In each experiment, 1× 104 cells were assessed for cell cycle
distribution. All data were analyzed with ModFitLT V3.0 software (BD Biosciences).
6.2.5. Apoptosis Analysis
A549 cells were treated with 15a (1 µM and 10 µM) for 72 h in RPMI media complete with 10%
DFBS, 5% penicillin-streptomycin, and 2 nM LCV. Cells were harvested by trypsinization and
washed twice with cold PBS. Cells were assayed for apoptosis by flow cytometry using the BD
FACSCalibur flow cytometer (BD Biosciences). Apoptosis was measured using the annexin
V-FITC/PI kit, as specified by the manufacturer (BD Biosciences), with a minimum of 1× 104
cells analyzed for early apoptosis (annexin-V-FITChigh/PIlow; LR) and late apoptosis/necrosis
(annexin-V-FITChigh/PIhigh; UR); viable cells are shown as LL (annexin-V-FITClow/7-AADlow).
6.2.6. Colony Formation Assay
Colony formation assays were performed with compound 15a to testify a cytotoxic response.
A549 cells (~100 cells) were plated in six-well plate in RPMI media complete with 10% DFBS, 5%
penicillin-streptomycin, and 2 nM LCV for 24 h incubation period at 37 ºC in the presence of 5%
CO2. Concentration from 0 to 20 µM of compound 15a were added for an additional 48 h, After
which the media was aspirated and replaced with new RPMI media complete with 10% DFBS, 5%
penicillin-streptomycin, and 2 nM LCV for 8 days. After that, the cells were washed twice with
room temperature PBS and fixed with methanol for 30 min. Then, the cells were stained by
Giemsa stain for another 30 min. After removing the stain and washing twice with water, the
stained colonies were counted.
6.3. Molecular Modeling and Computational Studies.
The X-ray crystal structures of human TS at 2.89 Å resolution (PDB ID 1JU6) and DHFR at 1.9 Å
resolution (PDB ID 1U72) were downloaded from the Protein Data Bank. Docking studies were
performed using Accelrys Discovery Studio client 2.5 (DS 2.5) software. The protonation state of
the proteins and the ligands were calculated using the default settings. Water molecules in the
active site were removed. The active site was defined by a sphere of 9.0 Å from the native ligand
in the crystal structure. Molecules used for the docking experiments were prepared by DS 2.5. The
CDocker protocol was used to score the docked poses. The docked poses were further visualized
and processed by PyMOL 1.6.x.
Acknowledgments

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The authors thank the Peking University Medical and Health Analysis Center for their help on the
cell cycle assay and apoptosis analysis. This work was supported by the National Natural Science
Foundation of China (21302007).
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