Novel 6-substituted benzoyl and non-benzoyl straight chain pyrrolo[2,3-d]pyrimidines as potential antitumor agents with multitargeted inhibition of TS, GARFTase and AICARFTase

Article abstract of DOI:10.1016/j.ejmech.2017.08.032

A novel series of 6-substituted benzoyl and non-benzoyl straight chain pyrrolo[2,3-d]pyrimidines were designed and synthesized as potential antitumor agents targeting both thymidylate and purine nucleotide biosynthesis. Starting from the key intermediate 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidin-6-yl-acetic acid, target compounds 1–6 were successfully obtained through two sequential condensation and saponification reactions in decent yield. The newly synthesized compounds showed antiproliferative potencies against a panel of tumor cell lines including KB, SW620 and MCF7. In particular, most compounds of this series exhibited nanomolar to subnanomolar inhibitory activities toward KB tumor cells, significantly more potent than the positive control methotrexate (MTX) and pemetrexed (PMX). Along with the results of nucleoside protection assays, molecular modeling studies suggested that the antitumor activity of compound 6 could be attributed to multitargeted inhibition of folate-dependent enzymes thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFTase) and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase). Growth inhibition by compound 6 also induced distinct early apoptosis and cell cycle arrest at S-phase, which resulted in cell death.

Full text of DOI:10.1016/j.ejmech.2017.08.032

European Journal of Medicinal Chemistry 139 (2017) 531e541
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel 6-substituted benzoyl and non-benzoyl straight chain pyrrolo
[2,3-d]pyrimidines as potential antitumor agents with multitargeted
inhibition of TS, GARFTase and AICARFTase
,
,
Ruijuan Xing a 1, Hongying Zhang a 1, Jiangsong Yuan a, Kai Zhang a, Lin Li a, Huicai Guo b,
Lijuan Zhao b, Congying Zhang a, Shuolei Li a, Tianfeng Gao a, Yi Liu b *, Lei Wang a
,
, **
a Department of Medicinal Chemistry, School of Pharmacy, Hebei Medical University, Shijiazhuang 050017, China
b Department of Toxicology, School of Public Health, Hebei Medical University, Shijiazhuang 050017, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 6-substituted benzoyl and non-benzoyl straight chain pyrrolo[2,3-d]pyrimidines were
designed and synthesized as potential antitumor agents targeting both thymidylate and purine nucle-
otide biosynthesis. Starting from the key intermediate 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidin-6-yl-
acetic acid, target compounds 1e6 were successfully obtained through two sequential condensation and
saponification reactions in decent yield. The newly synthesized compounds showed antiproliferative
potencies against a panel of tumor cell lines including KB, SW620 and MCF7. In particular, most com-
pounds of this series exhibited nanomolar to subnanomolar inhibitory activities toward KB tumor cells,
significantly more potent than the positive control methotrexate (MTX) and pemetrexed (PMX). Along
with the results of nucleoside protection assays, molecular modeling studies suggested that the anti-
tumor activity of compound 6 could be attributed to multitargeted inhibition of folate-dependent en-
zymes thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFTase) and 5-
aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase). Growth inhibition by
compound 6 also induced distinct early apoptosis and cell cycle arrest at S-phase, which resulted in cell
death.
Received 15 December 2016
Received in revised form
25 March 2017
Accepted 11 August 2017
Available online 14 August 2017
Keywords:
pyrrolo[2,3-d]pyrimidines
Antiproliferation
Multitargeted inhibition
Cell cycle arrest
Apoptosis
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
of thymidine monophosphate (dTMP) from deoxyuridine mono-
phosphate (dUMP) [9]. Inhibition of DHFR results in partial deple-
Classical antifolates, analogs of folic acid, were among the first
effective chemotherapeutic agents and were clinically used as
antimicrobial, antifungal, antiinflammatory and antitumor agents
[1e3]. Dihydrofolate reductase (DHFR) and thymidylate synthase
(TS) are two main intracellular targets of antifolates [4e7], albeit
several other important enzymes are involved in the folate cycle.
DHFR catalyzes the reduction of folic acid to dihyrodrofolate which
was further reduced to active folate, tetrahydrofolate (THF), as one
carbon carrier that donates methyl groups to end target molecules
[8]. TS, however, functions as a catalyst for the de novo biosynthesis
tion of intracellular reduced folates, causing cells incapable of
undergoing accurate DNA replication, ultimately leading to cell
death [8]. TS inhibition leads to so called “thymineless state”, in
which cells experience starvation of thymidine triphosphate (dTTP)
and undergo irreversible cell death [9]. The most commonly used
DHFR inhibitor is methotrexate (MTX, Fig. 1) [10], which binds to
DHFR reversibly and impedes its function by decreasing supplies of
thymidine and purine bases for DNA and RNA synthesis. Another
representative DHFR inhibitor is pralatrexate (PDX, Fig. 1) [11,12],
which was approved in 2009 as a chemotherapy of relapsed or
refractory peripheral T-cell lymphoma. Structurally,
a 2,4-
diaminopyrimidine moiety is required for binding to the active
sites of DHFR. TS inhibitors, however, have a 2-amino-4-oxo-py-
rimidine part in their scaffolds as in pemetrexed (PMX, Fig. 1) [13],
or a 2-methyl-4-oxo-pyrimidine moiety as in raltitrexed (RTX,
Fig. 1) [14].
* Corresponding author.
** Corresponding author.
E-mail addresses: liuyi78@hebmu.edu.cn (Y. Liu), hmuwanglei@hebmu.edu.cn
(L. Wang).
1
Equally contributed to this work.
http://dx.doi.org/10.1016/j.ejmech.2017.08.032
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

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R. Xing et al. / European Journal of Medicinal Chemistry 139 (2017) 531e541
Fig. 1. Structures of classical antifolates.
Fig. 2. Design of 6-substituted pyrrolo[2,3-d]pyrimidines 1e6.
The two folate-dependent enzymes glycinamide ribonucleotide
formyltransferase (GARFTase) and 5-aminoimidazole-4-
multitargeted agent against TS, DHFR, GARFTase and AICARFTase
[21,22]. Thus, multitargeted antifolates would seem to have sub-
stantial advantages over mono-targeted antifolates when tumors
become resistant due to alterations in one or the other targeted
enzyme. In our previous studies [23,24], we designed and synthe-
sized a series of multitargeted antifolates with a pyrrolo[2,3-d]py-
rimidine core structure (1a, Fig. 2). These compounds showed
potent antiproliferative activities against several tumor cell lines
including KB, A549 and HepG2, while the intracellular targets were
identified as TS, GARFTase and AICARFTase. Our interest is to
develop novel multitargeted antifolates as antitumor agents and to
optimize the structures for better inhibition of folate metabolizing
enzymes than PMX. In this study, the side chain pyridyl-3-
methylamino group of the lead compound 1a (Fig. 2) was
replaced by a glutamate to provide target compound 6 (Fig. 2) as a
classical analog. This isosteric replacement would enable 6 to un-
carboxamide ribonucleotide formyltransferase (AICARFTase) were
also considered as intracellular targets of antifolates. GARFTase
catalyzes the incorporation of C-8 into inosine monophosphate
(IMP) by transferring the formyl group from N10-formyl-tetrahy-
drofolate (N10-formyl-THF) to the side-chain amino group of gly-
cinamide ribonucleotide (GAR) to produce formylglycinamide
ribonucleotide (FGAR) and THF [15,16]. AICARFTase, however, cat-
alyzes the formation of C-2 of IMP through transferring the formyl
group from N10-formyl-THF to 5-amino group of 5-
aminoimidazole-4-carboxamide ribonucleotide (AICAR) to yield
5-formylaminoimidazole-4-carboxamide ribonucleotide (FAICAR)
[16,17].
Compared with the number of DHFR or TS inhibitors used in
clinics, specific inhibitors of GARFTase or AICARFTase have been
scarce. Lometrexol (LMTX, Fig. 1) [18,19], the first GARFTase inhib-
itor to enter clinical trials, was abandoned eventually due to severe
and cumulative myelosuppression and mucositis in phase I clinical
trials. BW1540 (Fig. 1) [20], a sulfamido-bridged 5,8-dideazafolate
analog with potent inhibition of AICARFTase (Ki ¼ 8 nM), was not
taken into clinical trials yet.
dergo polyglutamylation at the
g-carboxyl moiety by the enzyme
folypoly- -glutamate synthetase (FPGS) [25], resulting in increased
g
binding to target enzymes at low concentrations and intracellular
retention through metabolic trapping. The non-benzoyl straight
chain analogs 1e5 (Fig. 2) were also designed and synthesized to
explore the importance of the side chain phenyl ring for antitumor
activity, as well as the optimal distance of the alkyl amide linker
Drug resistance is easily developed for antifolates with specific
inhibition of only one folate-dependent enzyme. As an exception,
PMX, originally considered as a specific TS inhibitor, is actually a
between the core structure of pyrrolo[2,3-d]pyrimidine and the L-
glutamate moiety.

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533
2. Chemistry
continuously exposed to a wide range of drug concentrations in
folate-free RPMI/10% dFBS, supplemented with 2 nM leucovorin
[(6R,S)5-formyl tetrahydrofolate (LCV)]. Relative cell numbers were
assessed after 72 h. Table 1 was a summary of growth inhibitory
effects of compounds 1e6 in comparison with those for the classical
antifolates MTX and PMX (Fig. 1).
Target compounds 1e6 were synthesized as shown in Scheme 1
and Scheme 2. The key intermediate 2-amino-4-oxo-pyrrolo[2,3-d]
pyrimidin-6-yl-acetic acid, 10, was obtained by a condensation re-
action of 2,4-diamino-6-hydroxypyrimidine, 7, and ethyl-4-
chloroacetoacetate, 8, followed by hydrolysis, through a proced-
ure reported by our group [23,24]. Compound 10 was then reacted
with various amino acid methyl esters or 4-aminobenzoic acid
methyl ester followed by saponification to produce carboxylic acids
12a-f. Final condensation with glutamic acid diethyl ester hydro-
chloride followed by hydrolysis afforded target compounds 1e6. As
an example, compound 6 was characterized to elucidate the
structures of our target compounds. The elemental composition of
6 was determined to C20H20N6O7 from its HRMS (ESI) at m/z
As shown in Table 1, compounds 1e6 were all potent inhibitors
against KB, SW620 and MCF7 tumor cells. In particular, all com-
pounds demonstrated nanomolar to subnanomolar antiprolierative
activities toward KB cells. Compared with the standard MTX (IC50 of
10 nM against KB tumor cells) and PMX (IC50 of 70 nM against KB
tumor cells), compound 1 (Fig. 3), the most potent compound of
this series, had an IC50 of 0.078 nM against KB cells in culture,
which was about 140-fold and 1000-fold more potent than that of
MTX and PMX, respectively. The only benzoyl side chain compound
of this series, 6 (Fig. 3), was also a nanomolar inhibitor against KB
cells with an IC50 of 7.5 nM. Compounds 1e6 inhibited proliferation
of KB cells in the following order of potency (numbers of bridge
carbons are noted in parentheses): 1 (1C) > 5 (5C) > 6 (benzene) > 4
(4C) > 2 (2C) ~ 3 (3C), indicating that the side chain phenyl ring of
this series was not necessary for inhibitory activity and could be
replaced by a straight chain with even enhanced antitumor activ-
ities. A optimal distance of the alkyl amide linker between the core
455.1330 [M-H]-, which was further verified by the 1H NMR and 13
C
NMR data (experimental section) suggesting the presence of 20
protons and 20 carbons in 6. On 1H NMR spectrum, one singlet at dH
6.04 (5-CH) and one broad singlet at dH 6.30 (-NH2), along with the
protons at dH 11.08 (7-NH) and 10.80 (3-NH), showed pyrrolo[2,3-d]
pyrimidine core structure. The glutamic acid moiety was indicated
as four protons at dH 1.92e2.37 (-CH2-), one proton at dH 4.35e4.41
(-CH-), one doublet at dH 8.52 (-CONH-) and one broad singlet at dH
12.39(-COOH). The double doublet at dH 7.75e7.87 (benzene) was
attributed to the benzene ring. The singlet present at dH 3.66
belonged to methylene group, whereas the singlet at dH 10.42 was
amide proton. Consequently, 6 was characterized as (S)-2-{4-[2-(2-
amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-ace-
tylamino]-benzoylamino}-pentanedioic acid.
structure of pyrrolo[2,3-d]pyrimidine and the L-glutamate moiety is
essential. In contrast to the results for KB cells, compounds 1e6
only showed micromolar to submicromolar inhibitory activities
toward SW620 and MCF7 cells, which were less potent that of MTX
and PMX.
In attempt to confirm that the inhibitory effect of compounds
1e6 leads to cytotoxicity, colony-forming assays were performed
towards KB cells and the results were shown in Fig. 4. In these
experiments, cells were plated at limiting dilutions, and colonies
were counted after 10e14 days. Results were compared with those
for KB cells treated with MTX and PMX, and for untreated cells as
well. Consistent to the results of inhibition assays (Table 1), the
antiproliferative effects of compounds 1 and 6 were irreversible at
the highest concentration, reaching greater than 90% and 80% in-
hibition of colony formation, respectively (Fig. 4), thereby estab-
lishing these compounds as cytotoxic.
3. Biological evaluation and discussion
3.1. 6-Substituted pyrrolo [2,3-d]pyrimidines as inhibitors of tumor
cell proliferation
The newly synthesized compounds 1e6 were initially evaluated
for their antiprolierative effects toward a panel of tumor cells
including KB (nasopharyngeal), SW620 (colon) and MCF7 (breast)
cells
through
the
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. Cells were
Scheme 1. Reagents and conditions: a) NaOAc, H2O, reflux, 18 h; b) i) 1 N NaOH, RT, 1.5 h; ii) 3 N HCl; c) EDCI, HOBt, Et3N, DMF, RT, 5 h; d) NMM, CDMT, DMF, RT, 5 h.

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R. Xing et al. / European Journal of Medicinal Chemistry 139 (2017) 531e541
Scheme 2. Reagents and conditions: a) EDCI, HOBt, Et3N, DMF, RT, 5 h; b) i) 1 N NaOH, RT, 1.5 h; ii) 3 N HCl; c) NMM, CDMT, DMF, RT, 5 h.
m
M)a of 6-substituted pyrrolo[2,3-d]pyrimidines 1e6 and classical antifolates against KB, SW620 and MCF7 cell lines.
Table 1
IC50 values (in
antifolate
X
KB
SW620
MCF7
1
2
3
4
5
6
-CH2-
7.8 ꢀ 10ꢁ5 (1 ꢀ 10ꢁ6
0.092 (0.006)
0.107 (0.007)
)
0.786 (0.029)
1.102 (0.211)
0.948 (0.072)
1.137 (0.142)
0.563 (0.031)
0.977 (0.105)
0.913 (0.211)
2.375 (0.543)
3.643 (0.821)
3.808 (0.704)
1.084 (0.137)
1.128 (0.150)
-(CH2)2-
-(CH2)3-
-(CH2)4-
-(CH2)5-
0.043 (0.005)
1.4 ꢀ 10ꢁ4 (3 ꢀ 10ꢁ6
)
)
7.5 ꢀ 10ꢁ3 (1 ꢀ 10ꢁ5
MTX
PMX
e
e
0.01 (0.001)
0.07 (0.012)
0.022 (0.003)
0.09 (0.017)
0.097 (0.006)
0.65 (0.028)
a
Calculated from three replicates (plus/minus SEM in parentheses).
Fig. 3. Growth inhibition of KB cells by compounds 1 and 6 in the presence of physiologic concentrations of reduced folate. a) Growth inhibition of KB cells by compound 1. b)
Growth inhibition of KB cells by compound 6. Cell proliferation inhibition was measured over a range of concentrations of compounds 1 and 6 in complete folate-free RPMI1640 in
the presence of LCV at 2 nM. Cell densities were measured with a MTT assay. Results were normalized to cell density in the absence of drug. Results shown are representative data of
experiments performed in triplicate.
3.2. Identification of TS, GARFTase and AICARFTase as the targeted
pathway of compound 6
circumvent the biosynthetic requirements for reduced folates. If TS
and/or purine biosynthesis are targeted, the inhibitory effects of the
new series of compounds against KB cells would be abolished by
excessive thymidine or adenosine. In addition, KB cells were also
Nucleoside protection experiments with thymidine (10
adenosine (60 M) were conducted to identify the targeted
pathway for compound 6, as that nucleoside salvage pathways
mM) and
m
treated with 5-aminoimidazole-4-carboxamide (AICA, 320
mM) in
the presence of compound 6 to confirm the likely targets in purine

R. Xing et al. / European Journal of Medicinal Chemistry 139 (2017) 531e541
535
Fig. 4. Colony-forming inhibition assay. a) KB cells were inoculated into 60 mm dishes with a density of 300 cells per dish, in the presence of MTX (10 nM), PMX (100 nM) or
compounds 1e6 (100 nM). b) The percentage of colony-forming in the presence of compounds 1 and 6 (1 nM and 10 nM). Colonies were enumerated after 10e14 days after staining
with methylene blue. Data were calculated as percent of controls treated identically but without drugs. Results are presented as mean values from three experiments (plus/minus
SEM).
Fig. 5. Cell proliferation assays with protection by nucleosides including thymidine and adenosine and by 5-aminoimidazole-4-carboxamide (AICA) to identify intracellular targets
of PMX and compound 6. To identify the targeted pathways and the folate-dependent intracellular enzymes in KB cells treated with compound 6 (1 nM-10
assays were performed in the presence of 10 M thymidine, 60 M adenosine, or 320 M AICA. a) The results for 6, normalized to those for untreated cells (no drug). b) The results
for PMX. Details are provided in the Experimental section. The results shown are representative of triplicate experiments.
mM), cell proliferation
m
m
m

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R. Xing et al. / European Journal of Medicinal Chemistry 139 (2017) 531e541
nucleotide biosynthesis (GARFTase versus AICARFTase). In cells,
AICA is metabolized to AICAR, the substrate for AICARFTase,
therefore it would bypass the step catalyzed by GARFTase.
3.3. Effect of compounds 1 and 6 on cell cycle progression and
apoptosis in KB cells
Fig. 5 shows the nucleoside/AICA protection results for com-
pound 6 compared to those for PMX. As reported in literature, PMX
is a multitargeted antifolate against TS, DHFR, GARFTase and
AICARFTase. The experiment data clearly showed that the growth
inhibitory effect of the PMX with KB cells was partially protected
by excess thymidine and adenosine, but was completely reversed
in the presence of combined thymidine and adenosine. Similar
results were obtained for compound 6 in that single addition of
excess thymidine or adenosine was partially protective in different
extent, while combined thymidine and adenosine completely
protected KB cells from the growth inhibitory effects of 6, indi-
cating that both thymidylate and de novo purine nucleotide syn-
thesis was being targeted. For PMX and compound 6, AICA alone or
combined with thymidine was only partially protective, suggesting
that inhibition of purine nucleotide biosynthesis by these two
Studies of cell cycle with apoptosis are of key importance in
understanding intracellular mechanism of action of antitumor
chemotherapies. In KB cells, etoposide, a topoisomerase II inhibitor
[26], induced S-phase inhibition and G1-phase accumulation [23],
whereas, PMX, a multitargeted antifolate, resulted in cell cycle ar-
rest at G1-phase with G2-phase suppression [23]. To explore the
impact of folate-dependent enzyme inhibition of the synthesized
compounds on cell-cycle progression, KB cells were treated with
compound 1 (1
mM) and compound 6 (1
mM), along with etoposide
(5 mM), PMX (10
mM) and a vehicle control for 96 h. Then, cells were
washed, fixed, stained with propidium iodide (PI), and analyzed for
cell cycle distribution by flow cytometry. Similar to the result of
PMX, treatment of compound 1 or 6 caused G2-phase inhibition
such that the G2-fraction decreased from 9.59% in the absence of
drugs to 2.43% with compound 1, even disappeared with PMX or
compound 6 (Fig. 6). For the S-population, treatment of compound
1 or 6 significantly increased cell distribution from 35.66% to
44.96% and 68.62%, respectively (Fig. 6). The G1-fraction decreased
upon treatment with compound 1 or 6 from 54.75% to 52.6% and
31.38%, respectively, yet slightly increased to 61.95% with PMX
(Fig. 6). The results suggested that compound 1 and 6 induced cell
cycle arrest at S-phase with G2-phase suppression.
compounds is likely
a composite effect of dual inhibitions
involving both GARFTase and AICARFTase. Therefore, the results
strongly imply that compound 6 is a multitargeted antifolate tar-
geting TS, GARFTase and AICARFTase. Nucleoside protection ex-
periments of the non-benzoyl straight chain compounds 1 and 5
were also performed to identify their molecular targets. Similar to
the results of 6, the antiproliferative activity of compounds 1 and 5
was attributable to multitargeted inhibition of folate-dependent
enzymes TS, GARFTase and AICARFTase (Fig. S1 and Fig. S2, Sup-
plementary Material).
KB cells were stained with annexin V-FITC/PI and analyzed by
flow cytometry to monitor the induction of apoptosis by our newly
synthesized compounds. It has been reported that PMX induces
Fig. 6. Cell cycle assay. KB were treated with etoposide (5 mM), PMX (10 mM), compound 1 (1 mM), compound 6 (1 mM) or left untreated (DMSO) for 96 h, washed, fixed, and stained
with PI. Cell cycle distributions were analyzed by flow cytometry. Cell cycle distributions: a) DMSO, b) etoposide, c) PMX, d) compound 1, e) compound 6, f) The percentages of cells
in each phase of the cell cycle (G1, S, and G2).

R. Xing et al. / European Journal of Medicinal Chemistry 139 (2017) 531e541
537
both intrinsic and extrinsic apoptosis through a p53-mediated and
caspase-dependent and -independent signaling pathways [27]. As
shown in Fig. 7, KB cells treated with PMX showed significant in-
creases in both early- and late-apoptotic fractions compared those
for the untreated control (i.e., the fractions of cells in early
apoptosis were 67% and 16.9% for PMX-treated and control cells,
respectively, whereas the fractions in late apoptosis at 48 h were
21.8% and 5.5%, respectively). The results also demonstrated that
both compound 1 and 6 could induce distinct early apoptosis (35.4%
and 36.2% for compound 1 and 6, respectively) but no late apoptosis
(Fig. 7), which is different from the result of PMX.
PMX, the 2-NH2 moiety forms a hydrogen bond with the side chain
of -carboxylic group of Glu948, whereas 3-NH interacts with the
a
backbone of Ala947. The 4-oxo moiety forms two hydrogen bonds
with both Asp951 and H2O1216. Additional hydrogen bond is
formed between 7-NH of the pyrrolo[2,3-d]pyrimidine and the
backbone of Arg897. The side chain amide carbonyl oxygen of 6 has
a hydrogen bond interaction with Asn913. The glutamate side chain
of 6 is oriented similarly to that of PMX, and forms two hydrogen
bonds with the backbone of Met896 and the side chain of Arg897,
respectively.
The docked pose of compound 6 (red) in the human AICARFTase
active site (PDB ID 1P4R) was shown in Fig. 9. Similar to the qui-
nazolinone ring of the native crystal structure ligand (BW1540,
Fig. 1), the pyrrolo[2,3-d]pyrimidine scaffold of 6 resides in a pocket
formed by Phe544, Asn489, Met312, Asp546, Ile452 and Ser450.
This orientation of the scaffold permits the 2-NH2 moiety to form
hydrogen bonds to the side chains of Asp546, whereas the 4-oxo
moiety forms a hydrogen bond with the side chain of Asn547. The
side chain benzoic carbonyl oxygen of 6 interacts with H2O1815 and
H2O1947 to form hydrogen bonds. Similar to BW1540, the gluta-
3.4. Molecular modeling
To provide a molecular explanation for the cellular metabolic
data that compound 6 inhibits TS, GARFTase and AICARFTase in KB
cells, MOE 2009.10 was used to perform molecular modeling
studies, and the docked poses were visualized using CCP4MG [28].
Fig. 8 shows the docked pose of compound 6 (red) in the human
GARFTase active site (PDB ID 4ZZ3). The pyrrolo[2,3-d]pyrimidine
scaffolds of 6 and PMX occupy the same location. Analogous to
mate moiety of 6 is oriented in a manner that aids the a-carbolic
Fig. 7. Apoptosis in KB cells treated with PMX, compound 1 or 6 for 48 h. Apoptosis was analyzed using annexin V-FITC/PI staining and flow cytometry for early apoptosis (Q2-LR),
late apoptosis (Q2-UR) and necrosis (Q2-UL); viable cells are shown in Q2-LL. Results are compared to those for controls treated with DMSO. The percentages of cells in each
quadrant are summarized. a) DMSO, b) PMX, c) compound 1, d) compound 6.

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R. Xing et al. / European Journal of Medicinal Chemistry 139 (2017) 531e541
Fig. 8. Molecular modeling of 6 with GARFTase. Docked pose of 6 (red) with PMX (green) in human GARFTase (PDB ID 4ZZ3). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
Fig. 9. Molecular modeling of 6 with AICARFTase. Docked pose of 6 (red) with BW1540 (green) in human AICARFTase (PDB ID 1P4R). (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
group form two hydrogen bonds with the backbone of Ala566 and
the side chain of Ser565, respectively.
Consistent with the results of our in vitro metabolic assays
(Fig. 5), the docking results clearly demonstrates that compound 6
could bind and inhibit both de novo purine biosynthetic enzymes
(i.e., GARFTase and AICARFTase) and TS.
Fig. 10 shows the overlay of the docked poses of 6 (red) with the
crystal structure of PMX (green) in human TS (PDB ID 1JU6). The
results shows that the pyrrolo[2,3-d]pyrimidine scaffold of 6
occupy the same location as that of PMX and are buried deep in the
active site. For the pyrrolo[2,3-d]pyrimidine core of 6, several
important hydrogen bond interactions are involved. The N1 nitro-
gen forms a hydrogen bond with H2O435, whereas the 2-NH2 in-
teracts with the backbone of Ala312. The N7 nitrogen of 6 forms a
hydrogen bond with the side chain of Asn112. The glutamate side
chains of 6 and PMX are oriented similarly.
4. Conclusions
In this paper, we reported the design, synthesis, biological
evaluation and molecular modeling of
a novel series of 6-
substituted benzoyl and non-benzoyl straight chain pyrrolo[2,3-d]
pyrimidines as potential antitumor agents targeting both thymi-
dylate and purine nucleotide biosynthesis. The newly synthesized

R. Xing et al. / European Journal of Medicinal Chemistry 139 (2017) 531e541
539
Fig. 10. Molecular modeling of 6 with TS. Docked pose of 6 (red) with PMX (green) in human TS (PDB ID 1JU6). (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
compounds showed antiproliferative potencies against a panel of
tumor cell lines including KB, SW620 and MCF7, whereas most
compounds of this series exhibited nanomolar to subnanomolar
inhibitory activities toward KB tumor cells. Particularly, compound
1, the most potent compounds of this series, has an IC50 of 0.078 nM
against KB cells in culture, which is about 140-fold and 1000-fold
more potent than that of the positive control MTX (IC50 of 10 nM
against KB tumor cells) and PMX (IC50 of 70 nM against KB tumor
cells), respectively. The results also established that the side chain
phenyl ring of a classical antifolate was not necessary and could be
replaced by a straight chain with even enhanced antitumor activ-
ities. A optimal distance of the alkyl amide linker between the core
of peak areas agreed with those expected for the assigned struc-
tures. 13C NMR spectra were recorded on a Bruker Avance II
100 MHz NMR spectrometer. Low resolution mass spectra (LRMS)
were determined on an ABI 3200 Qtrap mass spectrometer, and
high resolution mass spectra (HRMS) were determined on an AB
SCIEX Triple TOF 5600þ mass spectrometer. Thin-layer chroma-
tography (TLC) was carried out on Silica Gel GF254 plates, and the
spots were visualized under 254 and 365 nm illumination. Column
chromatography was taken on 230e400 mesh silica gel. Analytical
HPLC was performed on an Agilent 1200 equipped with a Diode
Array Detector (DAD). All solvents and chemicals were purchased
from Aldrich Chemical Co. and Fisher Scientific and were used as
received. Purities of all assayed compounds were determined by
analytical HPLC and were found to be ꢂ 95% pure unless otherwise
specified.
structure of pyrrolo[2,3-d]pyrimidine and the L-glutamate moiety is
essential. Consistent with the results of nucleoside protection as-
says, molecular modeling studies demonstrated that inhibition of
cell proliferation was attributable to multitargeted inhibition of
folate-dependent enzymes TS, GARFTase and AICARFTase. Growth
inhibition induced distinct early apoptosis and cell cycle arrest at S-
phase, leading to cell death. The subnanomolar antitumor activities
and structural simplicity of our non-benzoyl straight chain com-
pounds provide an excellent starting point for further study,
including rational design of additional potent multitargeted anti-
folates to optimize inhibition of folate metabolizing enzymes to-
ward discovering new generation of antitumor agents.
5.1.1. General procedure for the synthesis of target compounds 1e6
To a solution of previously synthesized intermediates 12a-12f
(1.0 mmol) in anhydrous DMF (10 mL) was added N-methyl-
morpholine (182 mg, 1.8 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-
triazine (317 mg, 1.8 mmol). The resulting mixture was stirred at
room temperature for 2 h. To this mixture was added N-methyl-
morpholine (182 mg, 1.8 mmol) and -glutamate diethyl ester hy-
L
drochloride (360 mg, 1.5 mmol). The reaction mixture was stirred
for an additional 4 h at room temperature. Silica gel (600 mg) was
then added, and the solvent was evaporated under reduced pres-
sure. The resulting plug was loaded on to a silica gel column
(1.5 ꢀ 15 cm) with 5% CHCl3 in MeOH as the eluent. Fractions that
showed the desired spot (TLC) were pooled, and the solvent
evaporated to dryness to afford a residue. To this residue was added
MeOH (10 mL) and 1 N NaOH (10 mL), then the mixture was stirred
under N2 at room temperature for 16 h. TLC showed the disap-
pearance of the starting material (Rf ¼ 0.15) and one major spot at
the origin (CHCl3/MeOH 10:1). The reaction mixture was then
evaporated to dryness under reduced pressure. The residue was
dissolved in water (10 mL), the resulting solution was cooled in an
ice bath, and the pH was adjusted to 3e4 with dropwise addition of
1 N HCl. The resulting suspension was frozen in a dry ice-acetone
5. Experimental section
5.1. Chemistry
All evaporations were carried out in vacuo with a rotary evap-
orator. Analytical samples were dried in a Buchi B-585 vacuum
(0.2 mmHg) drying oven over P2O5. Melting points were deter-
mined on a Digital Melting Point apparatus (Buchi M-560) and
were uncorrected. 1H NMR spectra were recorded on a Bruker
Avance III 400 MHz NMR spectrometer. The chemical shift values
were expressed in ppm (parts per million) relative to tetrame-
thylsilane as internal standard; s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quartet, m ¼ multiplet, br ¼ broad singlet. The relative integrals

540
R. Xing et al. / European Journal of Medicinal Chemistry 139 (2017) 531e541
bath, thawed to 4e5 ꢃC in the refrigerator, and filtered. The residue
was washed with a small amount of cold water and dried in vacuum
using P2O5 to afford 1e6 as white to yellow powder.
5.1.1.5. (S)-2-{6-[2-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d]
pyrimidin-6-yl)-acetylamino]-hexanoylamino}-pentanedioic acid (5).
Compound 5 was prepared using the general method described for
the preparation of 1e6 from 12e (321 mg, 1.0 mmol) to give 5 as a
off-white powder (375 mg, 83.3%); mp: 148 ꢃC; 1H NMR (400 MHz,
5.1.1.1. (S)-2-{2-[2-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d]
pyrimidin-6-yl)-acetylamino]-acetylamino}-pentanedioic acid (1).
Compound 1 was prepared using the general method described for
the preparation of 1e6 from 12a (265 mg, 1.0 mmol) to give 1 as a
off-white powder (184 mg, 46.7%); mp: 215 ꢃC; 1H NMR (400 MHz,
DMSO-d6):
d
¼ 1.18e1.26 (m, 2H, CH2), 1.36e1.41 (m, 2H, CH2),
1.45e1.50 (m, 2H, CH2), 1.71e1.99 (m, 2H, CH2), 2.09e2.13 (t, 2H,
J ¼ 7.2 Hz, CH2), 2.26e2.29 (t, 2H, J ¼ 7.2 Hz, CH2), 3.00e3.04 (m, 2H,
CH2), 3.33 (s, 2H, CH2), 4.16e4.23 (m, 1H, CH), 5.95 (s, 1H, 5-H), 6.17
(s, 2H, 2-NH2), 7.88e7.90 (t, 1H, J ¼ 4.8 Hz, CONH), 8.07e8.09 (d, 1H,
J ¼ 7.6 Hz, CONH), 10.31 (s, 1H, 3-H), 10.86 (s, 1H, 7-H), 12.35 (br,
DMSO-d6):
d
¼ 1.76e1.96 (m, 2H, CH2), 2.24e2.27 (m, 2H, CH2),
3.60e3.74 (m, 4H, CH2), 4.16e4.21 (m, 1H, CH), 6.00 (s, 1H, 5-H),
6.19 (s, 2H, 2-NH2), 8.08e8.12 (m, 2H, CONH), 10.38 (s, 1H, 3-H),
10.83 (s, 1H, 7-H), 11.93 (br, 2H); 13C NMR (100 MHz, DMSO-d6):
2H); 13C NMR (100 MHz, DMSO-d6):
d
¼ 24.91, 25.97, 26.30, 28.77,
30.13, 34.96, 35.04, 38.62, 51.05, 99.75, 99.83, 125.10, 151.08, 152.12,
158.46, 168.79, 172.31, 173.41, 173.66; ESI-MS (m/z): 449.2 [M - H]-;
HRMS (ESI): calcd for C19H25N6O7 [M - H]-, 449.1790; found,
449.1783.
d
¼ 26.84, 30.30, 34.88, 42.03, 51.64, 99.84, 100.18, 124.64, 149.91,
151.19, 152.30, 158.58, 168.67, 169.40, 173.97; ESI-MS (m/z): 393.1
[M - H]-; HRMS (ESI): calcd for C15H17N6O7 [M - H]-, 393.1164;
found, 393.1181.
5.1.1.6. (S)-2-{4-[2-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d]
pyrimidin-6-yl)-acetylamino]-benzoylamino}-pentanedioic acid (6).
Compound 6 was prepared using the general method described for
the preparation of 1e6 from 12f (327 mg, 1.0 mmol) to give 6 as a
off-white powder (264 mg, 57.8%); mp: 204 ꢃC; 1H NMR (400 MHz,
5.1.1.2. (S)-2-{3-[2-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d]
pyrimidin-6-yl)-acetylamino]-propionylamino}-pentanedioic
acid
(2). Compound was prepared using the general method
2
described for the preparation of 1e6 from 12b (280 mg, 1.0 mmol)
DMSO-d6):
d
¼ 1.92e2.13 (m, 2H, CH2), 2.34e2.37 (t, 2H, J ¼ 7.2 Hz,
to give 2 as a off-white powder (175 mg, 43%); mp: 280 ꢃC; 1H NMR
CH2), 3.66 (s, 2H, CH2), 4.35e4.41 (m, 1H, CH), 6.04 (s, 1H, 5-H), 6.30
(s, 2H, 2-NH2), 7.75e7.87 (dd, 4H, benzene), 8.52e8.53 (d, 1H,
J ¼ 7.2 Hz, CONH), 10.42 (s, 1H, CONH), 10.80 (s, 1H, 3-H), 11.08 (s,
(400 MHz, DMSO-d6):
d
¼ 1.74e1.92 (m, 2H, CH2), 2.24e2.31 (m, 4H,
CH2), 3.19e3.28 (m, 2H, CH2), 3.33 (s, 2H, CH2), 4.15e4.21 (m, 1H,
CH), 5.95 (s, 1H, 5-H), 6.08 (s, 2H, 2-NH2), 7.93e7.96 (t, 1H,
J ¼ 5.2 Hz, CONH), 8.03e8.04 (d, 1H, J ¼ 7.6 Hz, CONH), 10.25 (s, 1H,
3-H), 10.86 (s, 1H, 7-H), 12.93 (br, 2H); 13C NMR (100 MHz, DMSO-
1H, 7-H), 12.39 (br, 2H); 13C NMR (100 MHz, DMSO-d6):
d
¼ 25.90,
30.46, 36.13, 51.92, 99.79, 100.30, 118.09, 124.23, 128.24, 142.18,
151.14, 152.35, 158.41, 165.99, 168.43, 173.42, 173.82; ESI-MS (m/z):
455.1 [M - H]-; HRMS (ESI): calcd for C20H19N6O7 [M - H]-, 455.1321;
found, 455.1330.
d6):
d
¼ 28.10, 32.67, 39.64, 40.79, 41.54, 56.01, 99.73, 123.41, 124.71,
147.98, 148.58, 151.11, 152.02, 158.43, 167.48, 173.98; ESI-MS (m/z):
407.1 [M - H]-; HRMS (ESI): calcd for C16H19N6O7 [M - H]-, 407.1321;
found, 407.1333.
5.2. Biological evaluation
5.1.1.3. (S)-2-{4-[2-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d]
pyrimidin-6-yl)-acetylamino]-butyrylamino}-pentanedioic acid (3).
Compound 3 was prepared using the general method described for
the preparation of 1e6 from 12c (294 mg, 1.0 mmol) to give 3 as a
yellow powder (187 mg, 44.4%); mp: 217 ꢃC; 1H NMR (400 MHz,
5.2.1. Cell culture
KB cells were obtained from American Type Culture Collection
(ATCC). SW620 and MCF7 cells were obtained from Type Culture
Collection of the Chinese Academy of Sciences (Shanghai, China).
KB, SW620 and MCF7 cells were routinely cultured in folate-free
RPMI1640 medium, supplemented with 10% fetal bovine serum,
DMSO-d6):
d
¼ 1.59e1.66 (m, 2H, CH2), 1.72e1.94 (m, 2H, CH2),
penicillin-streptomycin solution, and 2 mM
with 5% CO2.
L
-glutamine at 37 ꢃC
2.11e2.15 (t, 2H, J ¼ 7.6 Hz, CH2), 2.25e2.28 (t, 2H, J ¼ 7.6 Hz, CH2),
3.00e3.09 (m, 2H, CH2), 3.33 (s, 2H, CH2), 4.15e4.20 (m, 1H, CH),
5.95 (s, 1H, 5-H), 6.08 (s, 2H, 2-NH2), 7.91e7.93 (t, 1H, J ¼ 5.2 Hz,
CONH), 8.02e8.04 (d, 1H, J ¼ 7.6 Hz, CONH), 10.24 (s, 1H, 3-H), 10.85
5.2.2. Growth inhibition assays
(s,1H, 7-H),12.57 (br, 2H); 13C NMR (100 MHz, DMSO-d6):
d
¼ 18.53,
To evaluate antiproliferative properties of the synthesized
compounds, the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenylte-
trazolium bromide (MTT) assay was used. The cell lines were
assessed by trypsinizing each cell line and seeding 4 ꢀ 103 cells per
well into 96-well plates. Cells were grown for 24 h and then treated
25.38, 26.71, 32.70, 34.99, 38.37, 51.31, 55.99, 99.84, 125.02, 133.21,
151.11, 152.03, 158.46, 168.88, 173.51, 173.86; ESI-MS (m/z): 421.2 [M
- H]-; HRMS (ESI): calcd for C17H21N6O7 [M - H]-, 421.1477; found,
421.1471.
with compounds at concentrations ranging from 1 nM to 10
mM and
5.1.1.4. (S)-2-{5-[2-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d]
incubated for 72 h in 200 L media. MTT reagent (20 L) in serum-
m
m
pyrimidin-6-yl)-acetylamino]-pentanoylamino}-pentanedioic
acid
free medium (5 mg/mL) was added to each well and incubated
further for 4 h. Media was removed, and the resulting formazan
(4). Compound was prepared using the general method
4
described for the preparation of 1e6 from 12d (307 mg, 1.0 mmol)
crystals were re-solubilized in 200 mL of DMSO. A490 was measured
to give 4 as a yellow powder (252 mg, 57.7%); mp: 221 ꢃC; 1H NMR
using a Thermomax Molecular Device plate reader. The experi-
ments were performed in quadruplicate and repeated at least three
times for each compound per cell line. Cells treated with 0.1% DMSO
were used as a control, while MTX and PMX were used as positive
controls.
(400 MHz, DMSO-d6):
d
¼ 1.37e1.42 (m, 2H, CH2), 1.45e1.53 (m, 2H,
CH2), 1.70e1.99 (m, 2H, CH2), 2.10e2.14 (t, 2H, J ¼ 7.2 Hz, CH2),
2.25e2.29 (t, 2H, J ¼ 8.0 Hz, CH2), 3.01e3.06 (m, 2H, CH2), 3.33 (s,
2H, CH2), 4.18e4.22 (m, 1H, CH), 5.95 (s, 1H, 5-H), 6.05 (s, 2H, 2-
NH2), 7.82e7.84 (t, 1H, J ¼ 5.2 Hz, CONH), 8.07e8.09 (d, 1H,
J ¼ 8.0 Hz, CONH), 10.21 (s, 1H, 3-H), 10.82 (s, 1H, 7-H), 12.37 (br,
5.2.3. Nucleosides protection assays
2H); 13C NMR (100 MHz, DMSO-d6):
d
¼ 22.68, 26.29, 28.62, 30.07,
The inhibitory effects of the antifolate inhibitors on de novo
thymidylate biosynthesis (i.e., TS) and de novo purine biosynthesis
(GARFTase and AICARFTase) were tested by coincubations with
34.63, 34.96, 38.47, 51.01, 99.75, 99.84,125.13,151.09,151.98,158.49,
168.76, 172.21, 173.42, 173.67; ESI-MS (m/z): 435.2 [M - H]-; HRMS
(ESI): calcd for C18H23N6O7 [M - H]-, 435.1634; found, 435.1643.
thymidine (10
mM) and adenosine (60 mM), respectively. For de

R. Xing et al. / European Journal of Medicinal Chemistry 139 (2017) 531e541
541
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novo purine biosynthesis, additional protection experiments used
AICA (320 M) as a means of distinguishing inhibitory effects at
GARFTase from those at AICARFTase.
m
5.2.4. Assessment of cell cycle distribution and apoptosis
KB cells were treated with 1
mM compound 1, 1 mM compound 6,
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m
M etoposide and 10 M PMX for 96 h in complete folate-free
m
RPMI 1640 medium/10% dFBS and 2 nM LCV. Cells were trypsi-
nized, pelleted, and washed once with ice-cold DPBS. Cells (1 ꢀ 106)
were fixed in ethanol (ꢁ20 ꢃC, overnight), then stained by resus-
pension in 0.5 ml of DPBS containing 50 mg/ml PI and 100 mg/ml
RNase type I-A (Sigma Aldrich). The cells were analyzed by flow
cytometry 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. Apoptosis was measured using the annexin
V-FITC/PI kit, as specified by the manufacturer (BD Biosciences),
with a minimum of 20,000 events (cells) analyzed for early
apoptosis (Q1-LR), late apoptosis (Q1-UR) and necrosis (Q1-UL);
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LT (v4.0) software (VeritySoftwareHouse).
5.3. Molecular modeling
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The X-ray crystal structures of human GARFTase at 2.5 Å reso-
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ID 1P4R) and human TS at 2.89 Å resolution (PDB ID 1JU6) were
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the ligands were calculated using the default settings. Water mol-
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site was defined by a sphere of 6.5 Å from the native ligand in the
crystal structure. Molecules used for the docking experiments were
prepared using MOE and energy-minimized using the MMF94ꢀ
force field to a constant of 0.05 kcal/mol. Triangle matching was
used as the placement method, and the docked poses were scored
using default settings. The docked poses were visualized using
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Acknowledgments
This work was supported by the National Natural Science
Foundation of China (21507022, 81202401, 21502041, 81473292);
Natural Science Foundation of Hebei Province, China
(H2017206291, H2014206310), Educational Commission of Hebei
Province, China (ZD2015112) and Department of Human Resources
and Social Security of Hebei Province, China (C2013003026,
C201400329).
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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