Synthesis and biological activity of a novel series of 6-substituted thieno[2,3-d]pyrimidine antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors over the reduced folate carrier and proton-coupled folate transpo

Article abstract of DOI:10.1021/jm8011323

A series of seven 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines with bridge length variations (from 2 to 8 carbon atoms) were synthesized as selective folate receptor (FR) α and β substrates and as antitumor agents. The syntheses were accomplished

Full text of DOI:10.1021/jm8011323

2940
J. Med. Chem. 2009, 52, 2940–2951
Synthesis and Biological Activity of a Novel Series of 6-Substituted Thieno[2,3-d]pyrimidine
Antifolate Inhibitors of Purine Biosynthesis with Selectivity for High Affinity Folate Receptors
over the Reduced Folate Carrier and Proton-Coupled Folate Transporter for Cellular Entry†
Yijun Deng,‡ Xilin Zhou,§ Sita Kugel Desmoulin,‡ Jianmei Wu, Christina Cherian, Zhanjun Hou, Larry H. Matherly,†,*,‡,|,
and Aleem Gangjee†,*,§
Graduate Program in Cancer Biology, Wayne State UniVersity School of Medicine, Detroit, Michigan 48201, DiVision of Medicinal Chemistry,
Graduate School of Pharmaceutical Sciences, Duquesne UniVersity, 600 Forbes AVenue, Pittsburgh, PennsylVania 15282, Department of
Pharmacology, Wayne State UniVersity School of Medicine, Detroit, Michigan 48201, DeVelopmental Therapeutics Program, Barbara Ann
Karmanos Cancer Institute, 110 East Warren AVe, Detroit, Michigan 48201
ReceiVed September 10, 2008
A series of seven 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines with bridge length variations (from 2 to 8
carbon atoms) were synthesized as selective folate receptor (FR) R and ꢀ substrates and as antitumor agents. The
syntheses were accomplished from appropriate allylalcohols and 4-iodobenzoate to afford the aldehydes, which were
converted to the appropriate 2-amino-4-carbethoxy-5-substituted thiophenes 23-29. Cyclization with chloroformamidine
afforded the thieno[2,3-d]pyrimidines 30-36, which were hydrolyzed and coupled with diethyl-L-glutamate, followed
by saponification, to give the target compounds 2-8. Compounds 3-6 were potent growth inhibitors (IC50 4.7-334
nM) of human tumor cells (KB and IGROV1) that express FRs. In addition, compounds 3-6 inhibited the growth of
Chinese hamster ovary (CHO) cells that expressed FRs but not the reduced folate carrier (RFC) or proton-coupled
folate transporter (PCFT). However, the compounds were inactive toward CHO cells that lacked FRs but contained
either the RFC or PCFT. By nucleoside and 5-amino-4-imidazole carboxamide (AICA) protection studies, along with
in vitro and in situ enzyme activity assays, the mechanism of antitumor activity was identified as the dual inhibition of
glycinamide ribonucleotide formyltransferase and, likely, AICA ribonucleotide formyltransferase. The dual inhibitory
activity of the active thieno[2,3-d]pyrimidine antifolates and the FR specificity represent unique mechanistic features
for these compounds distinct from all other known antifolates. The potent inhibitory effects of compounds 3-6 toward
cells expressing FRs but not PCFT provide direct evidence that cellular uptake of this series of compounds by FRs
does not depend on the presence of PCFT and argues that direct coupling between these transporters is not obligatory.
Introduction
mechanism of folates at physiologic pH.3 The folate receptors
(FRs) R and ꢀ are glycosylphosphoinositol-anchored proteins
that transport folates by receptor-mediated endocytosis.4 The
proton-coupled folate transporter (PCFT) is a proton-folate
symporter that functions optimally at low pH.5 It has been
reported that cellular uptake of (anti)folates by FR involves
a functional “coupling” in which (anti)folates bind to surface
FRs, followed by internalization and vesicle (endosome)
formation, followed by extravesicular export by PCFT.6
Antifolates, typified by methotrexate (MTX), pemetrexed
(PMX), and raltitrexed (RTX) (Figure 1) are structurally similar
to folic acid and typically bind to folate-dependent enzymes to
inhibit folate-dependent pathways.7 MTX continues to be an
integral component of the chemotherapeutic arsenal for several
cancers including pediatric acute lymphoblastic leukemia,
osteogenic sarcoma, lymphoma, and breast cancer.8 RTX is used
outside of the US for advanced colorectal cancer.3 PMX was
approved in 2004 for pleural mesothelioma in the US9 and,
subsequently, as a second line treatment for non-small cell lung
cancer.10
The biological importance of reduced folates derives from
their essential roles in one-carbon transfer, leading to
thymidylate, purine nucleotides, serine, and methionine, and
in biological methylation reactions from S-adenosylmethion-
ine.1 Because mammalian cells are unable to synthesize
folates de novo, internalization of extracellular folates is
essential. Three major folate uptake systems have been
described.2 The ubiquitously expressed reduced folate carrier
(RFCa) is an anion exchanger and is the primary transport
* To whom correspondence should be addressed. For L.H.M.: phone, 313-
578-4280; fax, 313-578-4287; e-mail, matherly@kci.wayne.edu. For A.G.:
phone, 412-396-6070; fax, 412-396-5593; e-mail, gangjee@duq.edu.
†
These authors contributed equally to this work.
Graduate Program in Cancer Biology, Wayne State University School of
‡
Medicine.
§
Division of Medicinal Chemistry, Graduate School of Pharmaceutical
Sciences, Duquesne University.
|
Department of Pharmacology, Wayne State University School of Medicine.
Developmental Therapeutics Program, Barbara Ann Karmanos Cancer
Institute.
MTX is a potent inhibitor of dihydrofolate reductase, whereas
PMX and RTX derive their primary cytotoxic effects by
inhibiting thymidylate synthase. Lometrexol (LMX) (Figure 1)
was introduced in 198511 as a targeted antipurine antifolate. The
notion of selectively targeting de novo purine nucleotide
biosynthesis has roots in early studies of azaserine12 or
thiopurines13 and is based on the assumption that depletion of
purine nucleotide pools can limit nucleotides for DNA synthesis
and repair while also impacting ATP and GTP stores important
for cellular energetics.14 This effect could be even more acute
a Abbreviations: AICA, 5-amino-4-imidazolecarboxamide; AICARFTase,
5-amino-4-imidazole-carboxamide ribonucleotide formyltransferase; CHO,
Chinese hamster ovary; DPBS, Dulbecco’s phosphate-buffered saline; EI,
electron impact; GARFTase, glycinamide ribonucleotide formyltransferase;
FGAR, formyl glycinamide ribonucleotide; FR, folate receptor; GAR,
glycinamide ribonucleotide; HBSS, Hank’s balanced salts solution; HEPES,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HBS, HEPES-buffered
saline; HRMS, high-resolution mass spectra; IC50, 50% inhibitory concentra-
tion; LCV, leucovorin; LMX, Lometrexol; MTX, methotrexate; MES, 2-(N-
morpholino)ethanesulfonic acid; PCFT, proton-coupled folate transporter;
PMX, Pemetrexed; RTX, Raltitrexed; RFC, reduced folate carrier; TLC,
thin layer chromatography.
10.1021/jm8011323 CCC: $40.75
2009 American Chemical Society
Published on Web 04/16/2009

6-Substituted Thieno[2,3-d]pyrimidine Antifolate Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2941
Figure 1. Folate and antifolate structures. Structures are shown for folic acid, depicting the pteridine, p-aminobenzoate, and glutamate motifs,
along with structures for classical antifolates, MTX, PMX, RTX, LMX, 1a, and 1b and the nonclassical antifolate, trimetrexate (TMQ).
in several cancer cells that lack enzymes involved in purine
salvage.15 Although there have been reports that antipurine
antifolates such as LMX were cytostatic rather than cytotoxic,16
in other reports, antipurine antifolates were distinctly cytotoxic.17,18
While the aforementioned antifolates can be envisaged to
possess limited selectivity for tumor cells over normal prolifera-
tive tissues such as bone marrow and gastrointestinal mucosa,
interest in the development of antifolates remains particularly
high for agents that target high affinity folate receptors (FRs).19
This reflects the restricted patterns of tissue expression for FRs,
including the vast majority of ovarian and endometrial cancers
for FRR and myeloid leukemias for FRꢀ.20 There are other
factors that account for tumor selectivity of FR-targeted
therapies, including the apical localization for FRR in normal
epithelia such as renal tubules or choroid plexus where it is
inaccessible to the circulation, and synthesis of nonfunctional
FRꢀ in normal hematopoietic cells.4
Ample literature documents applications of FRs for tumor
targeting with folic acid as the targeting agent. For instance,
cytotoxins (e.g., mitomycin C), liposome-encapsulated drugs (e.g.,
doxorubicin), or radionuclides have been conjugated to folic acid
for targeting FR-expressing tumors.21-23 There are at least two
potential complications of this approach. These include: (i) instabil-
ity in plasma versus that within tumor cells such that the folate
conjugate may be prematurely cleaved and release the cytotoxic
agent prior to reaching the tumor, resulting in toxicity to normal
cells, thus precluding selectivity, and (ii) the possibility that free
folic acid released upon cleavage within the tumor could provide
a growth-sustaining nutrient detrimental to tumor inhibition.
Another approach involves a targeting ligand that itself is
cytotoxic. Unfortunately, most folate-based therapeutics such
as classical antifolates (including RTX, PMX, and LMX) that
are substrates for FRs are also substrates for the ubiquitously
expressed RFC, resulting in decreased tumor selectivity with
these agents.24 Indeed, the lack of continued clinical develop-
ment of LMX can be directly traced to the severe thrombocy-
topenia encountered in a phase 1 clinical trial,25 at least partly
due to its excellent substrate activity for RFC uptake and
polyglutamylation by normal tissues. Although classical anti-
folates can be transported by PCFT,5 the role of this transporter
in chemotherapy is still emerging.
Clearly, a specific FR-targeted agent that also possesses
cytotoxic activity without transport by RFC would circumvent
many of the drawbacks of targeting FRs with folic acid-
conjugated cytotoxic agents and the lack of selectivity often
associated with clinically used antifolates. Indeed, FR selective
cytotoxic agents can be envisaged to provide highly selective
antitumor agents against tumors expressing FR with little or no
host toxicity. Ideally, analogues could be identified that are
selective substrates for FRs over RFC. One possibility, 1a
(Figure 1), was reported nearly 30 years ago,26,27 however its
modest cell growth inhibitory potency and significant toxicity
profile limited its further clinical development and led to the
introduction of RTX.28,29 More recently, Jackman and colleagues
described novel cyclopenta[g]quinazoline antifolates as FR-
targeted agents,30,31 which like 1a and RTX are potent inhibitors
ofhumanthymidylatesynthase.Oneofthesecyclopenta[g]quinazo-
lines was evaluated in mice and showed no systemic toxicity,
as reflected in no weight loss or macroscopic signs of toxicity
to major organs, consistent with its selective targeting of FRs.
Most recently, we18 described a novel series of 6-substituted
classical pyrrolo[2,3-d]pyrimidine antifolates, differing only in
the lengths of the carbon bridge (3-6 carbons), characterized
by potent and selective substrate activities for FRR and FRꢀ,
and negligible substrate activity for RFC. The intracellular
enzyme target of the 6-substituted pyrrolo[2,3-d]pyrimidines was
identified as glycinamide ribonucleotide formyltransferase
(GARFTase), the first folate-dependent reaction in the de novo
purine synthesis pathway.
In this study, we extend this focus to a novel isosteric series of
6-substituted thieno[2,3-d]pyrimidine antifolates [compounds 2-8
(Figure 2)], with a 2-8 carbon bridge between the thieno[2,3-
d]pyrimidine and the benzoyl moiety, respectively. Isosteric
replacement of the pyrrolo ring with a thieno ring for this series
provides an increase in ring size that more closely approximates
the pteridine, 6-6 fused ring system of the natural cofactor. In

2942 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Deng et al.
the lack of PCFT in the hamster sublines, we screened CHO
sublines derived from R2 cells by RT-PCR, using PCR primers
to regions in hamster PCFT completely homologous to the
human PCFT. While PCFT transcripts were detected in RNA
from MDA-MB-231 and HepG2 human cells, endogenous
PCFT transcripts were not detected in any of the CHO sublines
in spite of 98% identical sequence to human PCFT (100% for
the primer binding sites) (Figure 1S, Supporting Information).
Figure 2. Structures of novel 6-substituted thieno[2,3-d]pyrimidine
We used R2 CHO cells as the parental subline for transfection
with human PCFT cDNA. Following isolation of a stable clonal
subline, R2/PCFT4, cells were assayed for transport and PCFT
expression by RT-PCR and results were compared to those for
R2 cells transfected with empty pCDNA3.1 plasmid (R2/VC)
(negative control) (Supporting Information). While transport of
[3H]MTX in R2/VC cells was insignificantly different at pH
5.5 (transport by PCFT is maximal at pH 5.5)5 from that at pH
7.2 (PCFT is essentially inactive at this pH)5 [0.56 ( 0.061
(SEM) and 0.466 ( 0.076 pmol/mg/2 min, respectively; p )
0.4130, n ) 8], transport was markedly elevated in R2/PCFT4
cells [6.96 ( 0.43 pmol/mg/2 min (p ) 0.0001; n ) 8)] (Figure
3).
antifolates.
Figure 3. PCFT-mediated transport of [3H]methotrexate at pH 5.5.
R2/PCFT4 and R2/VC cells were assayed for uptake of [3H]MTX (0.5
µM) at pH 5.5 in MES-buffered saline at 37 °C over 2 min. Results
are presented as mean values ( SEM (n ) 8).
Identification of 6-Substituted Thieno[2,3-d]pyrimidine An-
tifolates as Potent Growth Inhibitors for Cells Expressing
FRs, But Not for Cells Expressing Exclusively RFC or PCFT.
Compounds 2-8, containing 2-8 methylenes in the bridge
region, were initially evaluated for their growth inhibitory effects
during a 4-day exposure against the series of isogenic CHO
sublines expressing the invidual folate transporters including
FRR (RT16), FRꢀ (D4), RFC (PC43-10), or PCFT (R2/
PCFT4). Growth inhibitory effects were compared to those for
the classical antifolate inhibitors MTX, LMX, PMX, and RTX.
For PC43-10 and R2/PCFT4 cells, results were compared to
those for R2 or R2/VC cells, respectively, with identical results,
whereas FR-effects in RT16 and D4 cells were established from
parallel incubations in the presence of excess (200 nM) folic
acid, which blocked FR-mediated cellular uptake.18 An identical
series of experiments was performed with RFC-, PCFT-, and
FRR-expressing KB and IGROV1 human tumor cells. The FR-
binding levels and RFC transport activities for all these models
were previously reported.18 The PCFT levels in KB and
IGROV1 human solid tumor cell lines are significant and will
be reported elsewhere (S. Kugel Desmoulin, Y. Wang, C.
Cherian, A. Gangjee, and L.H. Matherly; manuscript in prepara-
tion).
All of the classical antifolates (MTX, LMX, PMX, and RTX)
were active against CHO sublines expressing RFC-, PCFT-, or
FRs (Table 1). In RT16 and D4 cells, growth inhibitory effects
were abolished by excess folic acid. For KB and IGROV1 cells,
folic acid had at most a marginal effect on drug activity because
the antifolates could still enter cells via RFC, which is not
blocked by folic acid.
Compound 2 (2-carbon bridge) was uniformly inactive (IC50
> 1000 nM) in our assays. Compounds 3-6 (3- 6-carbon
bridge) were inhibitors of proliferation for CHO cells expressing
either FRR or FRꢀ but not with RFC-expressing PC43-10,
PCFT-expressing R2/PCFT4 cells, or R2 or R2/VC cells without
detectable FR, RFC, or PCFT (Table 1). The most potent
analogues of the series, compounds 3 and 4, with 3- and
4-methylene groups in the bridge region, respectively, showed
activities similar to those for the most active classical inhibitors,
LMX and RTX, toward FR-expressing cells but were somewhat
more active than either PMX or MTX. There was a progressive
diminution of the growth inhibitory effects for the 5-, 6-, 7-,
and 8-methylene analogues (compounds 5-8, respectively).
addition, replacement of the NH of the pyrrole with a S also allows
for comparison of the relative importance of a hydrogen bond donor
(NH) with a hydrogen bond acceptor (S). Our biological results
establish the cytotoxic effects of the active compounds directly
reflect potent inhibitory activities against GARFTase and cellular
uptake capacities by FRs over RFC and PCFT. From nucleoside
protection studies, lesser inhibitory effects of a second enzyme
target, most likely 5-amino-4-imidazole carboxamide ribonucleotide
formyltransferase (AICARFTase), was strongly implied. The potent
cytotoxicity in cells expressing FRs but not PCFT provides direct
evidence that cellular uptake of this series of compounds by FRs
does not depend on the presence of PCFT and argues that direct
coupling between these transporters is not obligatory.
Chemistry
Target compounds 2-8 were synthesized as shown in Scheme
1. The appropriate allyl alcohols 9-15 were treated with palladium
diacetate, ethyl 4-iodobenzoate, LiCl, LiOAc, and Bu4NCl in DMF
to afford the aldehydes 16-22 (80-85% yield).32 A modified
reaction temperature of 80 °C from that reported in the literature
improved the yield. Aldehydes 16-22 were then reacted with
sulfur, ethyl cyanoactetate, and morpholine in EtOH for 24 h at
room temperature under Gewald reaction conditions to afford
23-29.33 Cyclization of 23-29 with chloroformamidine hydro-
chloride afforded the thieno[2,3-d]pyrimidines 30-36 in 55-80%
yield. Hydrolysis of the ethyl esters of 30-36 with 1 N NaOH in
ethanol followed by acid workup gave the corresponding acids
37-43. With 2-Cl-4,6-dimethoxy-1,3,5-triazine and 4-methylmor-
pholine as the coupling reagents, acids 37-43 were coupled with
diethyl-L-glutamate hydrochloride to afford compounds 44-50
(69-77%). Hydrolysis of 44-50 in 1 N NaOH, followed by acid
workup, gave target compounds 2-8 in 96-98% yield.
Biological Evaluation and Discussion
Confirmation of the Lack of PCFT in Chinese Hamster
Ovary Sublines and Generation of PCFT Stable Transfec-
tant. We previously described a panel of Chinese hamster ovary
(CHO) cell lines engineered from RFC- and FR-null MTXRII-
OuaR2-4 (R2) cells to express human FRs (R and ꢀ; designated
RT16 and D4, respectively) or RFC (PC43-10).18 To confirm

6-Substituted Thieno[2,3-d]pyrimidine Antifolate Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2943
Scheme 1. Reagents and conditions: (a) 10%Pd(AcO)2, Bu4NCl, LiCl, LiOAc, DMF, 80 °C, 12 h; (b) S, CN-CH2-COOEt,
morpholine, RT, 24 h; (c) chlorformamidine hydrochloride, DMSO2, 140 °C, 4 h; (d) 4-methylmorpholine,
2-Cl-4,6-dimethoxy-1,3,5-triazine, DMF, diethyl L-glutamate hydrochloride, 10 h
Table 1. IC50s (nM) for Thienopyrimidine Compounds 2-8 in Cell Proliferation Inhibition of RFC-, PCFT-, and FR-Expressing cell Lines
RFC
antifolate PC43-10
PCFT
R2/PCFT4 R2/VC
>1000
FRR
FRꢀ
D4 (+FA)
>1000
RFC/FRR
RFC/FRR
R2
RT16 RT16 (+FA)
D4
KB
KB (+FA) IGROV1 IGROV1 (+FA)
2
3
4
5
6
7
8
>1000 >1000 >1000
>1000 >1000 >1000
>1000 >1000 >1000
>1000 >1000 >1000
>1000 >1000 >1000
>1000 >1000 >1000
>1000 >1000 >1000
>1000
13(3.4)
9(2.9)
56(9.8)
108(17)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
23(5.5)
>1000
>1000
>1000
4.7(1.9)
5.9(1.9)
25(1.7)
334(59)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
112(12) >1000
20(3.9) >1000
>1000
364(46) >1000
4.9(1.3) >1000
132(23) >1000
310(54) >1000
56(13)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
methotrexate 12(1.1) 216(8.7) 120.5(16.8) >1000
pemetrexed 138(13) 894(93) 13.2(2.4)
114(31)
974.0(18.1) 42(9)
461(62)
388(68)
>1000
188(41)
>1000
106(11) 211(43)
6.0(0.6) 20(2.4)
68(12)
746(138) 5.9(2.2) 22(5)
21(3.4)
327(103) 102(25)
22(2.1)
200(18)
20(4.3)
16(6)
60(8)
254(78)
raltitrexed
lometrexol
1b
6.3(1.3) >1000 99.5(11.4) >1000
12(2.3) >1000 248.0(18.2) >1000
11(3.3) >1000 >1000 >1000
15(5)
12(8)
277(81)
22(10)
12.6(3.3)
3.1(0.9)
5.2(1.7)
2.6(1.0) 275(101) 1.2(0.6) 31(7)
52(12) >1000 5.8(3.5) 32(15)
6.9(1.6)
Growth inhibition assays were performed as described in the Experimental Section. For the FR experiments, growth inhibition assays were performed
in the absence and presence of 200 nM folic acid (FA). The data shown are mean values from three experiments (plus/minus SEM in parentheses). For RFC-
and FR-expressing cells, IC50 data of classical antifolate compounds, methotrexate, pemetrexed, raltitrexed, lometrexol, and 1b, were previously published
from our laboratory.18 ND, not determined.
Analogous results were obtained with KB and IGROV1 human
tumor cells (Table 1). For compounds 3-6, growth inhibitory
effects were abolished with 200 nM folic acid, indicating a
requirement for FR uptake for these compounds.
Similar, albeit slightly less potent effects of compounds 3-5
and MTX were detected by colony-forming assays with KB
cells over long-term (10 day) continuous exposures to drug
(Figure 4). For the thieno[2,3-d]pyrimidine antifolates (but not
MTX), the effects were completely reversed by an excess of
folic acid, establishing a requirement of FRs for transport of
the thieno[2,3-d]pyrimidines. These results establish that com-
pounds 3-5 are cytotoxic toward KB human tumor cells.
Protection from the Growth Inhibitory Effects of Thieno[2,3-
Figure 4. Colony-forming inhibition assay. KB cells were plated into
60 mm dishes with a density of 100 cells per dish in the presence or
absence of different concentrations of antifolates (compounds 3-5 and
MTX) from 1 to 1000 nM. The highest concentration (1000 nM) of
d]pyrimidine Antifolates with Nucleosides. To localize the
probable enzyme target(s) for the thieno[2,3-d]pyrimidine series
that results in suppression of cell proliferation, we tested the
growth inhibitory effects of compound 4, the most potent of
our analogues, against KB cells in the presence of adenosine
(60 µM) or thymidine (10 µM). Thymidine (10 µM) did not
alter the growth inhibitory effects of compound 4, whereas
adenosine (60 µM) was completely protective (Figure 5). This
establishes the de novo purine biosynthetic pathway as the
primary metabolic target.
inhibitors was also evaluated in the presence or absence of folic acid
(100 µM). Colonies were enumerated after 10 days using a cutoff of
50 cells/colony. Data were calculated as percent of controls treated in
an identical fashion but without drugs.
we tested the protection by 5-amino-4-imidazole (AICA) (320
µM), which can be converted to 5-amino-4-imidazolecarboxa-
mide ribonucleotide (AICAR), an intermediate metabolite
between GARFTase and AICARFTase that circumvents the
GARFTase step.18,34 AICA (320 µM) almost completely
protected KB cells against the toxicity of compound 4 at low
and medium concentrations (e50 nM), whereas growth was still
Because there are two folate-dependent enzymes in the de
novo purine synthesis pathway, GARFTase and AICARFTase,

2944 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Deng et al.
Figure 6. Competitive inhibition of FR binding activities by 6-sub-
stituted thieno[2,3-d]pyrimidines. Data are shown for the effects of the
thieno[2,3-d]pyrimidine antifolates 2-8 with FRR-expressing RT16
CHO cells and FRꢀ-expressing D4 CHO cells. Relative binding
affinities for assorted folate/antifolate substrates were determined over
a range of ligand concentrations and were calculated as the inverse
molar ratio of unlabeled ligands required to inhibit [3H]folic acid binding
by 50%. By definition, the relative affinity of folic acid (FA) is 1. The
data for MTX, PMX, LMX, LCV, and folic acid (FA) were previously
published.18
Figure 5. Protection of cell growth inhibition by nucleosides, AICA,
and folic acid. Cell proliferation inhibition by thieno[2,3-d]pyrimidine
compound 4 was measured on 96-well plates with 4000 KB cells per
well initially and a range of antifolate concentrations (1-1000 nM) in
the presence or absence of folic acid (200 nM), adenosine (60 µM),
thymidine (10 µM), or AICA (320 µM). Cell densities were measured
with CellTiter Blue fluorescence dye and a fluorescence plate reader.
Results were normalized to cell density in the absence of drug. Results
shown are representative data of experiments performed in triplicate.
Table 2. IC50s for Thieno[2,3-d]pyrimidines Compounds 2-6 in in
Vitro and in Situ GARFTase Inhibition Assaysa
significantly inhibited at higher concentrations of compound 4
(above 100 nM) (Figure 5), even in the presence of AICA.
IC50
Thus, the pattern of nucleoside protection indicates a potent
inhibition by the thieno[2,3-d]pyrimidine antifolate 4 on de novo
purine nucleotide biosynthesis. GARFTase appears to be the
major enzyme target leading to growth inhibition of FR-
expressing KB cells, although a secondary target, most likely
AICARFTase, also seems likely at higher doses of drug.
Analogous results were obtained with compound 3. These results
suggest a unique mechanistic feature of the thieno[2,3-d]pyri-
midines 3 and 4, distinguishing this series from the pyrrolo[2,3-
d]pyrimidines reported previously,18 which were only GARFTase
inhibitors.
antifolate
in vitro (µM)
in situ (nM)
2
3
4
5
>20
32.3 (9.5)
13.8 (9.5)
13.3 (5.5)
23.6 (6.2)
26.6 (7.5)
30 (7.7)
8.52 (2.71)
5.51 (0.82)
>20
6
>20
>20
0.78 (0.08)
pemetrexed
lometrexol
14 (5.6)
a GARFTase inhibition assays, both in vitro and in situ, were performed
as described in the Experimental Section. The IC50 data shown are mean
values from three (in vitro; SEM in parentheses) or two (in situ; range in
parentheses) experiments. IC50 data of classical antifolate compounds,
pemetrexed and lometrexol, were previously published.18
Molecular Mechanisms of 6-Substituted Thieno[2,3-d]pyri-
midine Antifolates. The results of the above experiments
strongly indicate that the 6-substituted thieno[2,3-d]pyrimidine
antifolates 3-8 are (i) substrates for FR-mediated cellular uptake
but not for RFC or PCFT (Table 1), and (ii) inhibitors of de
novo purine biosynthesis, reflecting primary inhibition of
GARFTase (Figure 5). Additional mechanistic experiments were
performed to validate these conclusions.
The most potent of the 6-substituted thieno[2,3-d]pyrimidine
series, compounds 2-6, were tested for their inhibition of
GARFTase using an in vitro assay with recombinant mouse
GARFTase, which followed the absorbance changes at 295 nm
accompanying one-carbon transfer from 10-formyl-5,8-didea-
zafolic acid to ꢀ-glycinamideribonucleotide (ꢀGAR), forming
5,8-dideazafolic and formyl GAR (FGAR). Table 2 summarizes
the IC50 values for in vitro GARFTase inhibitions by compounds
2-6 for comparison with those for LMX and PMX. Similar to
our cytotoxicity results, compounds 3 and 4 were the most potent
of the series, with IC50s less than 10 µM. However, by in vitro
assay, compounds 3 and 4 were less inhibitory than was LMX
(Table 2). Compounds 2, 5, and 6, along with PMX, all showed
IC50s above 20 µM, the upper limit of the in vitro activity assay
(Table 2).
For FRs, substrate binding is a good reflection of FR-mediated
uptake.24 Accordingly, competition by compounds 2-8 with
[3H]folic acid for binding to FRs R and ꢀ (in RT16 and D4
cells, respectively) was measured and the results compared to
those with the classical antifolates MTX, LMX, and PMX and
the folate cofactor leucovorin (LCV). Cells were washed at pH
3.5 to remove bound folate and then treated at neutral pH with
50 nM [3H]folic acid in the presence of a range of inhibitor
concentrations. After additional washing (at neutral pH), binding
of [3H]folic acid was measured. Relative affinities were
determined over a range of concentrations of unlabeled ligands
and were calculated as the inverse molar ratios of unlabeled
ligands required to inhibit 50% of [3H]folic acid binding.
To assess the inhibitory effects of compounds 2-6 in cells,
in situ experiments were performed in which KB cells were
metabolically labeled with [14C]glycine in the presence of
azaserine, and the accumulations of [14C]FGAR were measured
following ion exchange fractionation. Again, compounds 3 and
4 showed the lowest IC50s of the series (Table 2) and were
equipotent with LMX but slightly more active than PMX and
compounds 2, 5, and 6. While for compounds 3-6 (but not
compound 2), the results of the in situ GARFTase assays
generally paralleled their relative in vitro growth inhibitions
(Table 1), this was less than complete and likely further reflects
Using relative binding of folic acid set to a value of 1, MTX
showed negligible binding (<0.05), whereas LMX showed high
(∼0.8) and comparable affinities toward both FRR and ꢀ (Figure
6). Similarly, relative binding affinities of compounds 3-8 were
high (above 0.8) to both FRR and FRꢀ. Compound 2 resembled
PMX and LCV in its moderate binding affinity for FRR
(∼0.25-0.45) and poor binding affinity for FRꢀ (<0.01). This
establishes a clear bridge length preference for binding FRR
and FRꢀ.

6-Substituted Thieno[2,3-d]pyrimidine Antifolate Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2945
at least a partial effect of the thieno[2,3-d]pyrimidines at an
intracellular target other than GARFTase.
literature of antifolates that afford this exquisite selectivity for
cellular uptake by FRs over RFC and PCFT that can be exploited
for selective inhibition of tumors that express FRs.
Conclusions
Experimental Section
Taken together, these results establish that the thieno[2,3-
d]pyrimidines are potent and selective inhibitors of cell prolif-
eration in cells that express FRs R and ꢀ and that, with the
exception of compound 2, there is no appreciable difference in
affinity for the FR isoforms for these analogues. In contrast, by
growth inhibition assays, the thieno[2,3-d]pyrimidine antifolates
2-8 are all exceedingly poor substrates for both RFC and PCFT.
Compounds 3 and 4 were consistently the most potent inhibitors
of proliferation in cells expressing FRs R and ꢀ, and this was
significantly associated with inhibition of GARFTase. By both
in vitro and in situ GARFTase assays, the bridge length with
3- and 4-methylenes is optimal for GARFTase binding and
inhibition, analogous to our previous findings, with the 6-pyr-
rolo[2,3-d]pyrimidine antifolates.18 The dramatic differences in
inhibition potencies by in situ (nM) versus in vitro (µM) assays
of GARFTase (Table 2) likely reflect increased enzyme affinities
by drug polyglutamates within cells. The possibility of a
secondary enzyme target, most likely AICARFTase, suggested
by our protection studies with AICA and in situ GARFTase
assays, raises the intriguing possibility of a dual inhibition at
high concentrations of these novel agents. The presence of two
molecular targets would likely reduce the acquisition of
resistance due to alterations in enzyme affinities for the cytotoxic
drugs.
The thieno[2,3-d]pyrimidine antifolates 2-8 are unique from
all the other classical antifolates evaluated, including pyr-
rolo[2,3-d]pyrimidine (PMX), quinazoline (RTX), and pteridine
(MTX) antifolates in that they are neither substrates for RFC
nor PCFT. This characteristic results in selective targeting of
FR-expressing cells, while exerting no cytotoxic effects toward
cells that express these other transporters in the absence of FRs.
Given that PCFT has been suggested to function in tandem with
FRs in mediating folate uptake,6 our finding of exclusive FR-
selective activity in FR-expressing CHO cells that do not express
detectable PCFT is of further interest in that they establish that
that the presence of PCFT is not obligatory to FR-mediated
cellular uptake, at least for the thieno[2,3-d]pyrimidine anti-
folates described herein.
All evaporations were carried out in vacuo with a rotary
evaporator. Analytical samples were dried in vacuo (0.2 mmHg)
in a Chem-Dry drying apparatus over P2O5 at 55 °C. Melting points
were determined on a Mel-Temp II melting point apparatus with
Fluke 51 K/J electronic thermometer and are uncorrected. Nuclear
magnetic resonance spectra for proton (1H NMR) were recorded
on a Bruker WH-300 (300 MHz) or a Bruker 400 MHz/52 MM
(400 MHz) spectrometer. The chemical shift values are expressed
in ppm (parts per million) relative to tetramethylsilane as internal
standard: s, singlet; d, doublet; t, triplet; q, quartet; m, mutiplet;
br, broad singlet. The relative integrals of peak areas agreed with
those expected for the assigned structures. High-resolution mass
spectra (HRMS), using electron impact (EI), were recorded on a
VG Autospec (Fisons Instruments) micromass (EBE Geometry)
double focusing mass spectrometer. Thin-layer chromatography
(TLC) was performed on Whatman Sil G/UV254 silica gel plates
with fluorescent indicator, and the spots were visualized under 254
and 366 nm illumination. Proportions of solvents used for TLC
are by volume. Column chromatography was performed on a
230-400 mesh silica gel (Fisher, Somerville, NJ) column. Elemen-
tal analyses were performed by Atlantic Microlab, Inc. Norcross,
GA. Element compositions are within (0.4% of the calculated
values. Fractional moles of water or organic solvents frequently
found in some analytical samples of antifolates could not be
prevented despite 24-48 h of drying in vacuo and were confirmed
where possible by their presence in the 1H NMR spectra. All
solvents and chemicals were purchased from Aldrich Chemical Co.
or from Fisher Scientific and were used as received.
General Procedure for the Synthesis of Compounds 16-22. To
a 250 mL round-bottom flask, fitted with a magnetic stir bar, were
placed palladium diacetate (0.269 g, 1.2 mmol), the appropriate
allyl alcohol (20 mmol), ethyl 4-iodobenzoate (5.52 g, 20 mmol),
LiCl (0.848 g, 20 mmol), LiOAc (3.3 g, 50 mmol) and Bu4NCl
(11.12 g, 40 mmol), and DMF (40 mL). The mixture was stirred
vigorously at 70 °C for 24 h. The reaction mixture was cooled to
room temperature, and 80 mL of water was added and then 100
mL of ethyl acetate. The ethyl acetate layer was separated, washed
with brine (30 mL × 3), dried over anhydrous sodium sulfate, and
concentrated under reduced pressure to afford a brown oil. The
residue was loaded on a silica gel column and eluted with 5% ethyl
acetate in hexane. The fractions containing the desired product
(TLC) were pooled and evaporated to afford the product.
To summarize, this report documents our continued efforts
to identify novel antifolates with potential selectivity for tumors
expressing high affinity FRs over normal cells that express RFC
and PCFT. We describe a novel series of 6-substituted thieno[2,3-
d]pyrimidine antifolates 2-8 that differ in the lengths of the
methylene bridge from 2 to 8 carbons. These analogues, like
our previous series of 6-substituted pyrrolo[2,3-d]pyrimidine
antifolates, are excellent substrates for FRs and potently inhibit
de novo purine nucleotide biosynthesis rather than thymidylate
synthesis. Further, as with the pyrrolo[2,3-d]pyrimidine anti-
folates, GARFTase appeared to be the primary enzyme target
for the 6-substituted thieno[2,3-d]pyrimidine analogues, although
our results strongly suggested a secondary enzyme target, most
likely AICARFTase, may also be important. Thus, unlike the
pyrrolo[2,3-d]pyrimidines, the thieno[2,3-d]pyrimidines appear
to be dual acting agents and likely inhibit both GARFTase and
AICARFTase in the de novo purine nucleotide biosynthetic
pathway at higher drug concentrations. In addition, the thieno[2,3-
d]pyrimidines afford structural specificity for transport via FRs
and are not stubstrates for RFC or PCFT. Thus, the thieno[2,3-
d]pyrimidines provide a scaffold that allows selectivity of uptake
by FRs over RFC and PCFT. This is the only example in the
Ethyl 4-(4-Oxobutyl)benzoate (16). Using the general procedure,
16 was obtained as a colorless liquid (4.11 g, 87.2%); Rf 0.70
(hexane/EtOAc 3:1). 1H NMR (CDCl3): δ 1.37-1.41 (3 H, t,
COOCH2CH3), 1.96-2.01 (2 H, m, CH2CH2CH2CHO), 2.36-2.49
(2 H, q, CH2CH2CH2CHO), 2.69-2.76 (2 H, t, CH2CH2CH2CHO),
4.36-4.38 (2 H, q, COOCH2CH3), 7.23-7.98 (4 H, dd, C6H4), 9.77
(1 H, s, CHO). This compound was unstable and could not be
analyzed by HRMS and was used directly for the next step.
Ethyl 4-(5-Oxopentyl)benzoate (17). Using the general procedure,
17 was obtained as a colorless liquid (4.36 g, 87.9%); Rf 0.71
(hexane/EtOAc 3:1). 1H NMR (CDCl3): δ 1.33-1.36 (3 H, t,
COOCH2CH3), 1.62-1.64 (4 H, m, CH2CH2CH2CH2CHO), 2.38-
2.42 (2 H, m, CH2CH2CH2CH2CHO), 2.66-2.71 (2 H, m,
CH2CH2CH2CH2CHO),4.32-4.37(2H,q,COOCH2CH3),7.21-7.95
(4 H, dd, C6H4), 9.79 (1 H, s, CHO). HRMS (ESI, pos mode) m/z
[M + Na]+ calcd for C14H18O3, 257.1154; found, 257.1144.
Ethyl 4-(6-Oxohexyl)benzoate (18). Using the general procedure,
18 was obtained as a colorless liquid (3.25 g, 65.5%); Rf 0.73
(hexane/EtOAc 3:1). 1H NMR (CDCl3): δ 1.22-1.27 (5 H, t,
COOCH2CH3 and CH2CH2CH2CH2CH2CHO), 1.47-1.57 (4 H, m,
CH2CH2CH2CH2CH2CHO), 2.26-2.31 (2 H, m, CH2CH2CH2CH2-
CH2CHO), 2.51-2.56 (2 H, m, CH2CH2CH2CH2CH2CHO),
4.19-4.26 (2 H, q, COOCH2CH3), 7.11-7.84 (4 H, dd, C6H4), 9.62

2946 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Deng et al.
(1 H, s, CHO). This compound was unstable and could not be
analyzed by HRMS and was used directly for the next step.
Ethyl 4-(7-Oxoheptyl)benzoate (19). Using the general procedure,
19 was obtained as a colorless liquid (4.3 g, 82.06%); Rf 0.74
CH2CH2CH2), 2.59-2.64 (2 H, t, C6H4-CH2CH2CH2), 2.69-2.74
(2 H, t, C6H4-CH2CH2CH2), 4.23-4.30 (2 H, q, COOCH2CH3),
4.34-4.41 (2 H, q, COOCH2CH3), 5.80 (2 H, s, NH2 exch), 6.65
(1 H, s, 4-H), 7.22-8.00 (4 H, dd, C6H4). HRMS (ESI, pos mode)
m/z [M + Na]+ calcd for C19H23NO4S, 384.1245; found, 384.1281.
1
(hexane/EtOAc 3:1). H NMR (CDCl3): δ 1.17-1.27 (7 H, m,
COOCH2CH3 and CH2CH2CH2CH2CH2CH2CHO), 1.44-1.55 (4
H, m,CH2CH2CH2CH2CH2CH2CHO), 2.25-2.31 (2 H, m,CH2CH2-
CH2CH2CH2CH2CHO), 2.49-2.54 (2 H, t, CH2CH2CH2CH2-
CH2CH2CHO), 4.19-4.26 (2 H, q, COOCH2CH3), 7.08-7.85 (4
H, dd, C6H4), 9.61 (1 H, s, CHO). This compound was unstable
and could not be analyzed by HRMS and was used directly for the
next step.
Ethyl 2-Amino-5-{4-[4-(ethoxycarbonyl)phenyl]butyl}thiophene-
3-carboxylate (25). Compound 25 (2.99 g, 66.73%) was obtained
as an orange liquid by reacting 18 (2.96 g, 11.92 mmol), sulfur
(0.38 g, 11.92 mmol), and ethyl cyanoactetate (1.35 g, 11.92 mmol)
as described in the general procedure above; Rf 0.71 (hexane/EtOAc
3:1). 1H NMR (CDCl3): δ 1.27-1.32 (3 H, t, COOCH2CH3),
1.33-1.37 (3 H, t, COOCH2CH3) 1.57-1.69 (4 H, m,C6H4-
CH2CH2CH2CH2), 2.54-2.59 (2 H, t,C6H4-CH2CH2CH2CH2),
2.62-2.67 (2 H, t, C6H4-CH2CH2CH2CH2) 4.18-4.25 (2 H, q,
COOCH2CH3), 4.29-4.36 (2 H, q, COOCH2CH3), 5.73 (2 H, s,
NH2 exch), 6.58 (1 H, s, 4-H), 7.18-7.93 (4 H, dd, C6H4). HRMS
(ESI, pos mode) m/z [M + Na]+ calcd for C20H25NO4S, 398.1402;
found, 398.1392.
Ethyl 4-(8-Oxooctyl)benzoate (20). Using the general procedure,
20 was obtained as a colorless liquid (4.63 g, 88.3%); Rf 0.76
1
(hexane/EtOAc 3:1). H NMR (CDCl3): δ 1.32-1.42 (9 H, m,
COOCH2CH3 and CH2CH2CH2CH2CH2CH2CH2CHO), 1.50-1.68
(4 H, m,CH2CH2CH2CH2CH2CH2CH2CHO), 2.13-2.45 (2 H,
m,CH2CH2CH2CH2CH2CH2CH2CHO), 2.59-2.68 (2 H, m, CH2CH2-
CH2CH2CH2CH2CH2CHO), 4.34-4.41 (2H, q, COOCH2CH3),
7.22-7.98 (4H, dd, C6H4), 9.36 (1H, s, CHO). This compound was
unstable and could not be analyzed by HRMS and was used directly
for the next step.
Ethyl 2-Amino-5-{5-[4-(ethoxycarbonyl)phenyl]pentyl}thiophene-
3-carboxylate (26). Compound 26 (2.62 g, 63.17%) was obtained
as an orange liquid by reacting 19 (2.8 g, 10.67 mmol), sulfur (0.34
g, 10.67 mmol), and ethyl cyanoactetate (1.20 g, 10.67 mmol) as
described in the general procedure above; Rf 0.71 (hexane/EtOAc
3:1). 1H NMR (CDCl3): δ 1.37-1.47 (8 H, m, 2COOCH2CH3 and
C6H4-CH2CH2CH2CH2 CH2), 1.62-1.76 (4 H, m, C6H4-CH2CH2-
CH2CH2CH2), 2.60-2.65 (2 H, t, C6H4-CH2CH2CH2CH2CH2),
2.69-2.74 (2 H, t, C6H4-CH2CH2CH2CH2CH2), 4.28-4.35 (2 H,
q, COOCH2CH3), 4.39-4.46 (2 H, q, COOCH2CH3), 5.85 (2 H, s,
NH2 exch), 6.66 (1 H, s, 4-H), 7.28-8.03 (4 H, dd, C6H4). HRMS
(ESI, pos mode) m/z [M + Na]+ calcd for C21H27NO4S, 412.1559;
found, 412.1537.
Ethyl 4-(9-Oxononyl)benzoate (21). Using the general procedure,
21 was obtained as a colorless liquid (4.53 g, 78.1%); Rf 0.76
1
(hexane/EtOAc 3:1). H NMR (CDCl3): δ 1.34-1.53 (11 H, m,
COOCH2CH3andCH2CH2CH2CH2CH2CH2CH2CH2CHO),1.60-1.63
(4 H, m, CH2CH2CH2CH2CH2CH2CH2CH2CHO), 2.33-2.41 (2 H,
m,CH2CH2CH2CH2CH2CH2CH2CH2CHO), 2.62-2.65 (2 H, t,
CH2CH2CH2CH2CH2CH2 CH2CH2CHO), 4.33-4.37 (2 H, q,
COOCH2CH3), 7.21-7.96 (4 H, dd, C6H4), 9.35 (1 H, s, CHO).
HRMS (ESI, pos mode) m/z [M + K]+ calcd for C18H26O3,
329.1519; found, 329.1498.
Ethyl 2-Amino-5-{6-[4-(ethoxycarbonyl)phenyl]hexyl}thiophene-
3-carboxylate (27). Compound 27 (3.62 g, 68.86%) was obtained
as an orange liquid by reacting 20 (3.6 g, 13.03 mmol), sulfur (0.42
g,13.03 mmol), and ethyl cyanoactetate (1.47 g,13.03 mmol) as
described in the general procedure above; Rf 0.73 (hexane/EtOAc
Ethyl 4-(10-Oxodecyl)benzoate (22). Using the general procedure,
22 was obtained as a colorless liquid (5.05 g, 83.5%); Rf 0.77
1
(hexane/EtOAc 3:1). H NMR (CDCl3): δ 1.29-1.38 (13 H, m,
COOCH2CH3 and CH2CH2CH2CH2CH2CH2CH2CH2CH2CHO),
1.52-1.56 (4 H, m, CH2CH2CH2CH2CH2CH2CH2CH2CH2CHO),
2.23-2.30 (2 H, m, CH2CH2CH2CH2CH2CH2CH2CH2CH2CHO),
2.52-2.55 (2 H, t, CH2CH2CH2CH2CH2CH2CH2CH2CH2CHO),
4.23-4.27 (2 H, q, COOCH2CH3), 7.12-7.85 (4 H, dd, C6H4), 9.56
(1 H, s, CHO). HRMS (ESI, pos mode) m/z [M + Na]+ calcd for
C19H28O3, 327.1936; found, 327.1950.
1
3:1). H NMR (CDCl3): δ 1.31-1.41 (10 H, m, 2COOCH2CH3
and C6H4-CH2CH2CH2CH2 CH2CH2), 1.46-1.66 (4 H, m,C6H4-
CH2CH2CH2CH2CH2CH2), 2.53-2.58 (2 H, t,C6H4-CH2CH2-
CH2CH2CH2CH2), 2.62-2.69 (2 H, t, C6H4-CH2CH2CH2CH2-
CH2CH2), 4.22-4.29 (2 H, q, COOCH2CH3), 4.33-4.40 (2 H, q,
COOCH2CH3), 5.77 (2 H, s, NH2 exch), 6.62 (1 H, s, 4-H),
7.22-7.97 (4 H, dd, C6H4). HRMS (ESI, pos mode) m/z [M +
Na]+ calcd for C22H29NO4S, 426.1715; found, 426.1688.
General Procedure for the Synthesis of Compounds 23-29. A
mixture of sulfur (1 mmol), the appropriate aldehyde (1 mmol),
ethyl cyanoactetate (1 mmol), and EtOH (5 mL) were placed in a
round-bottom flask and warmed to 45 °C and treated dropwise with
morpholine (1 mmol) over 15 min. The mixture was stirred for 5 h
at 45 °C and 24 h at room temperature. Unreacted sulfur was
removed by filtration, and the filtrate was concentrated under
reduced pressure to afford an orange oil. The residue was loaded
on a silica gel column and eluted with 10% ethyl acetate in hexane.
The fractions containing the desired product (TLC) were pooled
and evaporated to afford the products.
Ethyl 2-Amino-5-{2-[4-(ethoxycarbonyl)phenyl]ethyl}thiophene-
3-carboxylate (23). Compound 23 (3.68 g, 67.65%) was obtained
as an orange liquid by reacting 16 (3.7 g, 15.67 mmol) with sulfur
(0.5 g, 15.67 mmol) and ethyl cyanoactetate (1.78 g, 15.67 mmol)
as described in the general procedure above; Rf 0.68 (hexane/EtOAc
3:1). 1H NMR (CDCl3): δ 1.30-1.34 (3 H, t, COOCH2CH3),
1.36-1.41 (3 H, t, COOCH2CH3), 2.88-2.97 (4 H, m, C6H4-
CH2CH2), 4.21-4.27 (2 H, q, COOCH2CH3), 4.33-4.40 (2 H, q,
COOCH2CH3), 5.79 (2 H, s, NH2 exch), 6.63 (1 H, s, 4-H),
7.23-7.97 (4 H, dd, C6H4). HRMS (ESI, pos mode) m/z [M +
Na]+ calcd for C18H21NO4S, 370.1089; found, 370.1112.
Ethyl 2-Amino-5-{7-[4-(ethoxycarbonyl)phenyl]heptyl}thiophene-
3-carboxylate (28). Compound 28 (1.94 g, 63.5%) was obtained as
an orange liquid by reacting 21 (2.1 g, 7.2 mmol), sulfur (0.23 g,
7.2 mmol), and ethyl cyanoactetate (0.81 g, 7.2 mmol) as described
1
in the general procedure above; Rf 0.72 (hexane/EtOAc 3:1). H
NMR (CDCl3): δ 1.29-1.41 (12 H, m, 2COOCH2CH3 and C6H4-
CH2CH2CH2CH2CH2 CH2CH2), 1.46-1.66 (4 H, m,C6H4-CH2CH2-
CH2CH2CH2CH2CH2), 2.51-2.58 (2 H, t,C6H4-CH2CH2CH2-
CH2CH2CH2CH2), 2.60-2.71 (2 H, t, C6H4-CH2CH2CH2CH2-
CH2CH2CH2), 4.20-4.27 (2 H, q, COOCH2CH3), 4.31-4.38 (2
H, q, COOCH2CH3), 5.86 (2 H, s, NH2 exch), 6.60 (1 H, s, 4-H),
7.16-8.01 (4 H, dd, C6H4). HRMS (ESI, pos mode) m/z [M +
Na]+ calcd for C23H31NO4S, 440.1872; found, 440.1837.
Ethyl 2-Amino-5-{8-[4-(ethoxycarbonyl)phenyl]octyl}thiophene-
3-carboxylate (29). Compound 29 (4.77 g, 62.4%) was obtained as
an orange liquid by reacting 22 (5.4 g, 17.7 mmol), sulfur (0.57 g,
17.7 mmol), and ethyl cyanoactetate (2.0 g, 17.7 mmol) as described
1
in the general procedure above; Rf 0.74 (hexane/EtOAc 3:1). H
NMR (CDCl3): δ 1.30-1.43 (14 H, m, 2COOCH2CH3 and C6H4-
CH2CH2CH2CH2CH2CH2 CH2CH2), 1.47-1.66 (4 H, m,C6H4-
CH2CH2CH2CH2CH2CH2CH2CH2), 2.53-2.58 (2H, t,C6H4-CH2CH2-
CH2CH2CH2CH2CH2CH2), 2.62-2.67 (2 H, t, C6H4-CH2CH2-
CH2CH2CH2CH2CH2CH2), 4.21-4.28 (2 H, q, COOCH2CH3),
4.32-4.39 (2 H, q, COOCH2CH3), 5.77 (2 H, s, NH2 exch), 6.61
(1 H, s, 4-H), 7.20-7.96 (4 H, dd, C6H4). HRMS (ESI, pos mode)
m/z [M + Na]+ calcd for C24H33NO4S, 454.2028; found, 454.2016.
Ethyl 2-Amino-5-{3-[4-(ethoxycarbonyl)phenyl]propyl}thiophene-
3-carboxylate (24). Compound 24 (1.49 g, 65.32%) was obtained
as an orange liquid by reacting 17 (1.58 g, 6.31 mmol), sulfur (0.20
g, 6.31 mmol), and ethyl cyanoactetate (0.71 g, 6.31 mmol) as
described in the general procedure above; Rf 0.69 (hexane/EtOAc
3:1). 1H NMR (CDCl3): δ 1.32-1.36 (3 H, t, COOCH2CH3),
1.37-1.42 (3 H, t, COOCH2CH3) 1.88-1.98 (2 H, p, C6H4-

6-Substituted Thieno[2,3-d]pyrimidine Antifolate Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2947
General Procedure for the Synthesis of Compounds 30-36. A
mixture of appropriate thiophene and chloroformamidine hydro-
chloride (1:4) in DMSO2 was heated at 140 °C for 4 h. The mixture
was cooled to room temperature, and 15 mL of water was added
and ammonium hydroxide was used to neutralize the suspension.
The brown solid, obtained by filtration, was washed with water
and dried over P2O5 vacuum. The solid was dissolved in methanol,
and silica gel was added. A dry silica gel plug was obtained after
evaporation of the solvent. The plug was loaded on to a silica gel
column and eluted with 5% methanol in chloroform. The fractions
containing the desired product (TLC) were pooled and evaporated
to afford the products.
(1.4 g, 12 mmol); mp 190.2-192.1 °C; Rf 0.55 (MeOH/CHCl3,
1
1:6). H NMR (DMSO-d6): δ 1.16-1.33 (9 H, m, COOCH2CH3
andC6H4-CH2CH2CH2CH2CH2CH2CH2),1.56(4H,m,C6H4-CH2CH2-
CH2CH2CH2CH2CH2), 2.50-2.69 (4 H, m,C6H4-CH2CH2CH2-
CH2CH2CH2CH2CH2), 4.25-4.32 (2 H, q, COOCH2CH3), 6.46 (2
H, s, 2-NH2 exch), 6.76 (1 H, s, 5-H), 7.30-7.87 (4 H, dd, C6H4),
10.83 (1 H, s, 3-NH exch). HRMS (ESI, pos mode) m/z [M +
Na]+ calcd for C22H27N3O3S, 436.5266; found, 436.5239.
Ethyl 4-[8-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-
6-yl)octyl]benzoate (36). Using the general procedure above,
compound 36 (1.52 g, 70.3%) was obtained as a yellow solid by
reacting 29 (2.18 g, 5.05 mmol) and chloroformamidine hydro-
chloride (2.3 g, 20.2 mmol); 274.5-276.2 °C; Rf 0.54 (MeOH/
CHCl3, 1:6). 1H NMR (DMSO-d6): δ 1.13-1.33 (11H, m, COOCH2-
CH3 and C6H4-CH2CH2CH2 CH2CH2CH2CH2CH2), 1.55 (4 H,
m,C6H4-CH2CH2CH2CH2CH2CH2CH2CH2), 2.50-2.72 (4 H, m,C6H4-
CH2CH2CH2CH2CH2CH2CH2CH2), 4.25-4.32 (2 H, q,
COOCH2CH3), 6.46 (2 H, s, 2-NH2 exch), 6.78 (1 H, s, 5-H),
7.31-7.87 (4 H, dd, C6H4), 10.84 (1 H, s, 3-NH exch). HRMS
(ESI, pos mode) m/z [M + Na]+ calcd for C23H29N3O3S, 450.1827;
found, 450.1790.
Ethyl 4-[2-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-
6-yl)ethyl]benzoate (30). Using the general procedure above,
compound 30 (0.73 g, 71.51%) was obtained as a yellow solid by
reacting 23 (0.7 g, 2.96 mmol) and chloroformamidine hydrochlo-
ride (1.30 g, 11.85 mmol); mp 274.6-275.9 °C; Rf 0.53 (MeOH/
1
CHCl3, 1:6). H NMR (DMSO-d6): δ 1.28-1.33 (3 H, t, COO-
CH2CH3), 2.98-3.04 (4 H, m, C6H4-CH2CH2), 4.25-4.32 (2 H, q,
COOCH2CH3), 6.46 (2 H, s, 2-NH2 exch), 6.77 (1 H, s, 5-H),
7.37-7.88 (4 H, dd, C6H4), 10.81 (1 H, s, 3-NH exch). Anal.
(C17H17N3O3S) C, H, N, S.
General Procedure for the Synthesis of Compounds 37-43. To
a solution of 30-36 in ethanol (10-50 mL) was added aq 1 N
NaOH and the reaction mixture stirred at room temperature for
12 h. The ethanol was evaporated under reduced pressure, and the
residue was dissolved in water (5-10 mL). The solution was
carefully acidified to pH 3 with the dropwise addition of 1 N HCl.
The resulting suspension was left at 0 °C for an hour, and then the
residue was collected by filtration, washed with water (5 mL), and
dried over P2O5/vacuum at 50 °C to afford the free acids 37-43.
4-[2-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)eth-
yl]benzoic Acid (37). Using the general procedure described above,
compound 37 (0.529 g, 95.0%) was obtained as a white solid from
30 (0.61 g, 1.77 mmol) by hydrolysis in ethanol (50 mL) and 1 N
NaOH (25 mL); mp >300 °C; Rf 0.55 (MeOH/CHCl3, 1:6 + 1
Ethyl 4-[3-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-
6-yl)propyl]benzoate (31). Using the general procedure above,
compound 31 (0.43 g, 81.13%) was obtained as a yellow solid by
reacting 24 (0.54 g, 1.49 mmol) and chloroformamidine hydro-
chloride (0.68 g, 5.9 mmol); mp 224.4-225.3 °C; Rf 0.53 (MeOH/
CHCl3, 1:6). 1H NMR (DMSO-d6): δ 1.28-1.33 (3 H, t,
COOCH2CH3), 1.86-1.96 (2 H, p, C6H4-CH2CH2CH2), 2.68-2.74
(4 H, m, C6H4-CH2CH2CH2) 4.26-4.33 (2 H, q, COOCH2CH3),
6.46 (2 H, s, 2-NH2 exch), 6.82 (1 H, s, 5-H), 7.35-7.89 (4 H, dd,
C6H4), 10.81 (1 H, s, 3-NH exch). Anal. (C18H19N3O3S·0.4CH3OH)
C, H, N, S.
Ethyl 4-[4-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-
6-yl)butyl]benzoate (32). Using the general procedure above,
compound 32 (1.05 g, 76.51%) was obtained as a yellow solid by
reacting 25 (1.40 g, 3.71 mmol) and chloroformamidine hydro-
chloride (1.70 g, 14.84 mmol); mp 273.5-275 °C; Rf 0.54 (MeOH/
CHCl3, 1:6). 1H NMR (DMSO-d6): δ 1.27-1.32 (3 H, t,
COOCH2CH3), 2.47-2.52 (4 H, m, C6H4-CH2CH2CH2CH2), 2.65-
2.73 (4 H, m, C6H4-CH2CH2CH2CH2), 4.24-4.32 (2 H, q,
COOCH2CH3), 6.44 (2 H, s, 2-NH2 exch), 6.78 (1 H, s, 5-H),
7.32-7.87 (4 H, dd, C6H4), 10.84 (1 H, s, 3-NH exch). Anal.
(C19H21N3O3S·0.2H2O) C, H, N, S.
1
drop of gl HOAc). H NMR (DMSO-d6): δ 2.97-2.99 (2 H, t,
C6H4-CH2CH2), 3.02-3.41 (2 H, t, C6H4-CH2CH2), 6.50 (2 H, s,
2-NH2 exch), 6.78 (1 H, s, 5-H), 7.35-7.86 (4 H, dd, C6H4), 10.85
(1 H, s, 3-NH exch), 12.76 (1 H, s, -COOH exch). Anal.
(C15H13N3O3S·0.6HCl) C, H, N, S, Cl.
4-[3-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)pro-
pyl]benzoic Acid (38). Using the general procedure described above,
compound 38 (0.18 g, 92.6%) was obtained as a white solid from
31 (0.22 g, 0.60 mmol) under hydrolysis in ethanol (20 mL) and 1
N NaOH (10 mL); mp 292.7-293.4 °C; Rf 0.52 (MeOH/CHCl3,
1:6 + 1 drop of gl HOAc). 1H NMR (DMSO-d6): δ 1.85-1.95 (2
H, m, C6H4-CH2CH2CH2), 2.66-2.73 (4 H, m, C6H4-CH2CH2CH2),
6.58 (2 H, s, 2-NH2 exch), 6.82 (1 H, s, 5-H), 7.32-7.90 (4 H, dd,
C6H4), 10.94 (1 H, s, 3-NH exch), 12.79 (1H, s, -COOH exch).
Anal. (C16H15N3O3S·0.7CH3OH) C, H, N, S.
Ethyl 4-[5-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-
6-yl)pentyl]benzoate (33). Using the general procedure above,
compound 33 (0.56 g, 54.37%) was obtained as a yellow solid by
reacting 26 (1.08 g, 2.76 mmol) and chloroformamidine hydro-
chloride (1.58 g, 13.8 mmol); mp 219.3-220.1 °C; Rf 0.55 (MeOH/
1
CHCl3, 1:6). H NMR (DMSO-d6): δ 1.27-1.31 (5 H, t,COO-
CH2CH3 and C6H4-CH2CH2CH2CH2CH2), 1.54-1.64 (4 H, m,C6H4-
CH2CH2CH2CH2CH2), 2.59-2.69 (4 H, m, C6H4-CH2CH2-
CH2CH2CH2), 4.24-4.31 (2 H, q, COOCH2CH3), 6.45 (2 H, s,
2-NH2 exch), 6.78 (1 H, s, 5-H), 7.32-7.87 (4 H, dd, C6H4), 10.86
(1 H, s, 3-NH exch). Anal. (C20H23N3O3S) C, H, N, S.
Ethyl 4-[6-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-
6-yl)hexyl]benzoate (34). Using the general procedure above,
compound 34 (0.78 g, 67.24%) was obtained as a yellow solid by
reacting 27 (1.18 g, 2.91 mmol) and chloroformamidine hydro-
chloride (1.30 g, 11.64 mmol); mp 196.8-197.4 °C; Rf 0.53
(MeOH/CHCl3, 1:6). 1H NMR (DMSO-d6): δ 1.27-1.32 (7 H, m,
COOCH2CH3 and C6H4-CH2CH2CH2CH2CH2CH2), 1.50-1.61 (4
H, m,C6H4-CH2CH2CH2CH2CH2CH2), 2.50-2.71 (4 H, m,C6H4-
CH2CH2CH2 CH2CH2CH2), 4.25-4.32 (2 H, q, COOCH2CH3),
6.53 (2 H, s, 2-NH2 exch), 6.85 (1 H, s, 5-H), 7.31-7.87 (4 H,
dd, C6H4), 10.82 (1 H, s, 3-NH exch). Anal.
(C21H25N3O3S · 0.2H2O) C, H, N, S.
4-[4-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)bu-
tyl]benzoic Acid (39). Using the general procedure described above,
compound 39 (0.138 g, 93.6%) was obtained as a yellow solid 32
(0.16 g, 0.43 mmol) under hydrolysis in ethanol (15 mL) and 1 N
NaOH (7 mL); mp >300 °C; Rf 0.52 (MeOH/CHCl3, 1:6 + 1 drop
1
of gl HOAc). H NMR (DMSO-d6): δ 1.59-1.62 (4 H, m, C6H4-
CH2CH2CH2CH2), 2.65-2.75 (4 H, m, C6H4-CH2CH2 CH2CH2),
6.46 (2 H, s, 2-NH2 exch), 6.79 (1 H, s, 5-H), 7.29-7.86 (4 H, dd,
C6H4), 10.83 (1 H, s, 3-NH exch), 12.77 (1 H, s, -COOH exch).
Anal. (C17H17N3O3S·0.5H2O) C, H, N, S.
4-[5-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)-
pentyl]benzoic Acid (40). Using the general procedure described
above, compound 40 (0.199 g, 93.3%) was obtained as a white
solid from 33 (0.23 g, 0.60 mmol) under hydrolysis in ethanol (20
mL) and 1 N NaOH (10 mL); mp 259.4-260 °C; Rf 0.52 (MeOH/
CHCl3, 1:6 + 1 drop of gl HOAc). 1H NMR (DMSO-d6): δ
1.28-1.38 (2 H, m, C6H4-CH2CH2CH2CH2CH2), 1.56-1.66 (4 H,
m, C6H4-CH2CH2 CH2CH2CH2), 2.61-2.71 (4 H, p, C6H4-
CH2CH2CH2CH2CH2), 6.45 (2 H, s, 2-NH2 exch), 6.79 (1 H, s,
5-H), 7.29-7.85 (4 H, dd, C6H4), 10.82 (1 H, s, 3-NH exch), 12.77
(1 H, s, -COOH exch). Anal. (C18H19N3O3S·0.4H2O) C, H, N, S.
Ethyl 4-[7-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-
6-yl)heptyl]benzoate (35). Using the general procedure above,
compound 35 (0.67 g, 67.7%) was obtained as a yellow solid by
reacting 28 (1 g, 2.4 mmol) and chloroformamidine hydrochloride

2948 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Deng et al.
4-[6-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)-
hexyl]benzoic Acid (41). Using the general procedure described
above, compound 41 (0.141 g, 95.2%) was obtained as a yellow
solid from 34 (0.16 g, 0.40 mmol) and ethanol (15 mL) and 1 N
NaOH (7 mL); mp 282-283.8 °C; Rf 0.52 (MeOH/CHCl3, 1:6 +
1 drop of gl HOAc). 1H NMR (DMSO-d6): δ 1.31-1.32 (4 H, m,
C6H4-CH2CH2CH2CH2CH2CH2), 1.51-1.60 (4 H, m, C6H4-
CH2CH2CH2CH2CH2CH2), 2.59-2.69 (4 H, p, C6H4-CH2CH2CH2-
CH2CH2CH2), 6.45 (2 H, s, 2-NH2 exch), 6.78 (1 H, s, 5-H),
7.28-7.85 (4 H, dd, C6H4), 10.82 (1 H, s, 3-NH exch), 12.76 (1
H, s, -COOH exch). Anal. (C19H21N3O3S) C, H, N, S.
4-[7-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)-
heptyl]benzoic Acid (42). Using the general procedure described
above, compound 42 (0.22 g, 94.9%) was obtained as a white solid
from 35 (0.25 g, 0.60 mmol) and ethanol (20 mL) and 1 N NaOH
(10 mL); mp 233.8-235 °C; Rf 0.51 (MeOH/CHCl3, 1:6 + 1 drop
of gl HOAc). 1H NMR (DMSO-d6): δ 1.29 (6 H, m, C6H4-
CH2CH2CH2CH2CH2CH2CH2), 1.53-1.58 (4 H, m, C6H4-CH2CH2-
CH2CH2CH2CH2CH2), 2.60-2.69 (4 H, m, C6H4-CH2CH2CH2
CH2CH2CH2CH2), 6.46 (2 H, s, 2-NH2 exch), 6.78 (1 H, s, 5-H),
7.29-7.85 (4 H, dd, C6H4), 10.83 (1 H, s, 3-NH exch), 12.74 (1H,
s, -COOH exch). HRMS (ESI, pos mode) m/z [M + Na]+ calcd
for C20H23N3O3S, 408.1358; found, 408.1380.
dd, C6H4), 8.64-8.66 (1 H, d, CONH exch), 10.86 (1 H, s, 3-NH
exch). Anal. (C25H30N4O6S·0.2H2O) C, H, N, S.
Diethyl N-{4-[4-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyri-
midin-6-yl)butyl]benzoyl}-L-glutamate (46). Using the general
procedure described above, compound 46 (0.134 g, 67.0%) was
obtained as a white solid from 39 (0.13 g, 0.38 mmol); mp
1
166.8-167.3 °C; Rf 0.64 (MeOH/CHCl3, 1:6). H NMR (DMSO-
d6): δ 1.13-1.20 (6 H, m, 2COOCH2CH3), 1.57-1.62 (4 H, m,
C6H4-CH2CH2CH2CH2), 1.95-2.12 (2 H, m, Gluꢀ-CH2), 2.40-2.45
(2 H, t, Gluγ-CH2), 2.65-2.75 (4 H, m, C6H4-CH2CH2CH2CH2),
4.00-4.13 (4 H, m, 2COOCH2CH3), 4.38-4.45 (1 H, m, GluR-
CH), 6.46 (2 H, s, 2-NH2 exch), 6.79 (1 H, s, 5-H), 7.28, 7.31-7.78,
7.80 (4H, dd, C6H4), 8.63-8.65 (1 H, d, CONH exch), 10.85 (1H,
s, 3-NH exch). Anal. (C26H32N4O6S) C, H, N, S.
Diethyl N-{4-[5-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyri-
midin-6-yl)pentyl]benzoyl}-L-glutamate (47). Using the general
procedure described above, compound 47 (0.108 g, 68.7%) was
obtained as a white solid from 40 (0.10 g, 0.29 mmol); mp
1
144.2-144.8 °C; Rf 0.64 (MeOH/CHCl3, 1:6). H NMR (DMSO-
d6): δ 1.14-1.21 (8 H, m, 2COOCH2CH3 and C6H4-CH2CH2-
CH2CH2CH2), 1.57-1.66 (4 H, p, C6H4-CH2CH2CH2CH2CH2),
1.93-2.17 (2 H, m, Gluꢀ-CH2), 2.41-2.46 (2 H, t, Gluγ-CH2),
2.61-2.72 (4 H, p, C6H4-CH2CH2CH2CH2CH2), 4.01-4.14 (4 H,
m, 2COOCH2CH3), 4.38-4.46 (1 H, m, GluR-CH), 6.46 (2 H, s,
2-NH2 exch), 6.79 (1 H, s, 5-H), 7.29, 7.31-7.78, 7.79 (4 H, dd,
C6H4), 8.63-8.66 (1 H, d, CONH exch), 10.83 (1 H, s, 3-NH exch).
HRMS (ESI, pos mode) m/z [M + Na]+ calcd for C27H34N4O6S,
565.2097; found, 565.2134.
4-[8-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)oc-
tyl]benzoic Acid (43). Using the general procedure described above
compound 43 (0.22 g, 92.8%) was obtained as a yellow solid from
36 (0.26 g, 0.60 mmol) and ethanol (20 mL) and 1 N NaOH (9
mL); Rf 0.53 (MeOH/CHCl3, 1:6 + 1 drop of gl HOAc). 1H NMR
(DMSO-d6): δ 1.29 (8 H, m, C6H4-CH2CH2CH2CH2CH2CH2-
CH2CH2), 1.56 (4 H, m, C6H4-CH2CH2CH2CH2CH2CH2CH2CH2),
2.60-2.69 (4 H, m, C6H4-CH2CH2CH2CH2 CH2CH2CH2CH2), 6.46
(2 H, s, 2-NH2 exch), 6.78 (1 H, s, 5-H), 7.30-7.87 (4 H, dd, C6H4),
10.83 (1 H, s, 3-NH exch), 12.75 (1 H, s, -COOH exch). HRMS
(ESI, pos mode) m/z [M + Na]+ calcd for C21H25N3O3S, 422.1514;
found, 422.1553.
General Procedure for the Synthesis of Compound 44-50. To
a solution of 37-43 (0.1 mmol) in anhydrous DMF (5-10 mL)
was added N-methylmorpholine (0.12 mmol) and 2-chloro-4,6-
dimethoxy-1,3,5-triazine (0.12 mmol). The resulting mixture was
stirred at room temperature for 2 h. N-Methylmorpholine (0.12
mmol) and diethyl-L-glutamate hydrochloride (0.1 mmol) were
added to the mixture. The reaction mixture was stirred for an
additional 3 h at room temperature, and silica gel was added to
this solution and the suspension evaporated under reduced pressure.
The plug obtained was loaded on a silica gel column and eluted
with 2% methanol in chloroform. The fractions containing the
desired product (TLC) were pooled and evaporated to afford the
products.
Diethyl N-{4-[6-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyri-
midin-6-yl)hexyl]benzoyl}-L-glutamate (48). Using the general
procedure described above, compound 48 (0.170 g, 80.57%) was
obtained as a white solid from 41 (0.14 g, 0.38 mmol); mp
1
153.7-154.4 °C; Rf 0.64 (MeOH/CHCl3, 1:6). H NMR (DMSO-
d6): δ 1.12-1.19 (6 H, m, 2COOCH2CH3), 1.30-1.32 (4 H,
m,C6H4-CH2CH2CH2CH2CH2CH2), 1.54-1.58 (4 H, m,C6H4-
CH2CH2CH2 CH2CH2CH2), 1.94-2.14 (2 H, m, Gluꢀ-CH2),
2.39-2.44 (2 H, t, Gluγ-CH2), 2.58-2.69 (4 H, m, C6H4-
CH2CH2CH2CH2CH2CH2), 3.99-4.12 (4 H, m, 2COOCH2CH3),
4.36-4.45 (1 H, m, GluR-CH), 6.45 (2 H, s, 2-NH2 exch), 6.78 (1
H, s, 5-H), 7.27, 7.30-7.77, 7.80 (4 H, dd, C6H4), 8.63-8.65 (1
H, d, CONH exch), 10.86 (1 H, s, 3-NH exch). Anal. (C28H36N4O6S)
C, H, N, S.
Diethyl N-{4-[7-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyri-
midin-6-yl)heptyl]benzoyl}-L-glutamate (49). Using the general
procedure described above, compound 49 (0.183 g, 66.8%) was
obtained as a semisolid from 42 (0.19 g, 0.48 mmol); Rf 0.65
(MeOH/CHCl3, 1:6). It was used directly in the next step without
further characterization.
Diethyl N-{4-[8-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyri-
midin-6-yl)octyl]benzoyl}-L-glutamate (50). Using the general
procedure described above, compound 50 (0.170 g, 68.20%) was
obtained as a yellow solid from 43 (0.16 g, 0.40 mmol); Rf 0.64
(MeOH/CHCl3, 1:6). 1H NMR (DMSO-d6): δ 1.12-1.19 (6 H, m,
2COOCH2CH3), 1.32-1.56 (8 H, m,C6H4-CH2CH2CH2CH2-
CH2CH2CH2CH2), 1.58-1.62 (4 H, m,C6H4-CH2CH2CH2CH2CH2-
CH2CH2CH2), 1.98-2.24 (2 H, m, Gluꢀ-CH2), 2.41-2.45 (2 H, t,
Gluγ-CH2), 2.59-2.69 (4 H, m, C6H4-CH2CH2CH2CH2CH2-
CH2CH2CH2), 4.01-4.12 (4 H, m, 2COOCH2CH3), 4.38-4.47 (1
H, m, GluR-CH), 6.46 (2 H, s, 2-NH2 exch), 6.78 (1 H, s, 5-H),
7.27, 7.29-7.78, 7.80 (4 H, dd, C6H4), 8.63-8.65 (1 H, d, CONH
exch), 10.83 (1 H, s, 3-NH exch). HRMS (ESI, pos mode) m/z [M
+ Na]+ calcd for C30H40N4O6S, 607.7212; found, 607.7233.
General Procedure for the Synthesis of Compounds 2-8. To a
solution of 44-50 in ethanol (5-10 mL) was added aqueous 1 N
NaOH and the reaction mixture stirred at room temperature for
3 h. The ethanol was evaporated under reduced pressure, and the
residue was dissolved in water (5-10 mL). The solution was cooled
to 0 °C and carefully acidified to pH 3 with dropwise addition of
1 N HCl. The resulting suspension was left at 0 °C for 12 h, and
the residue was collected by filtration, washed with water (5 mL),
and dried over P2O5/vacuum at 50 °C to afford the free acids 2-8.
Diethyl N-{4-[2-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyri-
midin-6-yl)ethyl]benzoyl}-L-glutamate (44). Using the general
procedure described above, compound 44 (0.114 g, 71.5%) was
obtained as a white solid from 37 (0.126 g, 0.40 mmol); mp
1
153.4-154 °C; Rf 0.63 (MeOH/CHCl3, 1:6). H NMR (DMSO-
d6): δ 1.14-1.21 (6 H, m, COOCH2CH3), 1.95-2.23 (2 H, m, Gluꢀ-
CH2), 2.41-2.46 (2 H, t, Gluγ-CH2), 2.97-3.05 (4 H, m, C6H4-
CH2CH2), 4.00-4.06 (2 H, q, COOCH2CH3), 4.08-4.14 (2 H, q,
COOCH2CH3), 4.38-4.45 (1 H, m, GluR-CH), 6.45 (2 H, s, 2-NH2
exch), 6.79 (1 H, s, 5-H), 7.33, 7.36-7.77, 7.80 (4 H, dd, C6H4),
8.63-8.66 (1 H, d, CONH exch), 10.81 (1 H, s, 3-NH exch). Anal.
(C24H28N4O6S) C, H, N, S.
Diethyl N-{4-[3-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyri-
midin-6-yl)pentyl]benzoyl}-L-glutamate (45). Using the general
procedure described above, compound 45 (0.106 g, 73.1%) was
obtained as a yellow solid from 38 (0.10 g, 0.30 mmol); mp
1
122.5-123.2 °C; Rf 0.63 (MeOH/CHCl3, 1:6). H NMR (DMSO-
d6): δ 1.15-1.21 (6 H, m, 2COOCH2CH3), 1.89-1.93 (2 H, m,
C6H4-CH2CH2CH2), 1.99-2.11 (2 H, m, Gluꢀ-CH2), 2.41-2.45 (2
H, t, Gluγ-CH2), 2.68-2.71 (4 H, m, C6H4-CH2CH2CH2), 4.00-4.13
(4 H, m, 2COOCH2CH3), 4.41-4.45 (1 H, m, GluR-CH), 6.47 (2
H, s, 2-NH2 exch), 6.82 (1 H, s, 5-H), 7.30, 7.32-7.79, 7.82 (4 H,

6-Substituted Thieno[2,3-d]pyrimidine Antifolate Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2949
N-{4-[2-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)-
ethyl]benzoyl}-L-glutamic Acid (2). Using the general procedure
described above, compound 2 (0.064 g, 97.7%) was obtained from
44 (0.075 g, 0.15 mmol) as a white solid; mp 167.7-168.4 °C; Rf
0.62 (MeOH/CHCl3, 1:6 + 1 drop of gl HOAc). 1H NMR (DMSO-
d6): δ 1.89-2.12 (2 H, m, Gluꢀ-CH2), 2.32-2.37 (2 H, t, Gluγ-
CH2), 2.97, 2.99-3.02, 3.04 (4 H, dd, C6H4-CH2CH2), 4.34-4.44
(1 H, m, GluR-CH), 6.45 (2 H, s, 2-NH2 exch), 6.79 (1 H, s, 5-H),
7.32, 7.36-7.78, 7.80 (4 H, dd, C6H4), 8.51-8.53 (1 H, d, CONH
exch), 10.81 (1 H, s, 3-NH exch), 12.40 (2 H, s, 2COOH exch).
Anal. (C20H20N4O6S·0.5H2O) C, H, N, S.
N-{4-[3-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)-
propyl]benzoyl}-L-glutamic Acid (3). Using the general procedure
described above, compound 3 (0.071 g, 96.5%) was obtained from
45 (0.083 g, 0.16 mmol) as a white solid; mp 155.5-156.1 °C; Rf
0.62 (MeOH/CHCl3, 1:6 + 1 drop of gl HOAc). 1H NMR (DMSO-
d6): δ 1.91-2.10 (4 H, m, Gluꢀ-CH2 and C6H4-CH2CH2CH2),
2.32-2.36 (2 H, t, Gluγ-CH2), 2.66-2.73 (4 H, q, C6H4-
CH2CH2CH2), 4.37-4.38 (1 H, m, GluR-CH), 6.47 (2 H, s, 2-NH2
exch), 6.81 (1 H, s, 5-H), 7.29, 7.32-7.79, 7.82 (4 H, dd, C6H4),
8.49-8.51 (1 H, d, CONH exch), 10.83 (1 H, s, 3-NH exch), 12.51
(2 H, s, 2COOH exch). Anal. (C21H22N4O6S·0.5 CH3OH) C, H,
N, S.
N-{4-[4-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)-
butyl]benzoyl}-L-glutamic Acid (4). Using the general procedure
described above, compound 4 (0.086 g, 95.8%) was obtained from
46 (0.10 g, 0.19 mmol) as a yellow solid; mp 154.5-154.9 °C; Rf
0.64 (MeOH/CHCl3, 1:6 + 1 drop of gl HOAc). 1H NMR (DMSO-
d6): δ 1.61-1.62 (4 H, m, C6H4-CH2CH2CH2CH2), 1.91-2.10 (2
H, m, Gluꢀ-CH2), 2.32-2.37 (2 H, t, Gluγ-CH2), 2.66-2.73 (4 H,
q, C6H4-CH2CH2CH2CH2), 4.36-4.41 (1 H, m, GluR-CH), 6.46
(2 H, s, 2-NH2 exch), 6.79 (1 H, s, 5-H), 7.28, 7.30-7.78, 7.81 (4
H, dd, C6H4), 8.51-8.54 (1 H, d, CONH exch), 10.83 (1 H, s, 3-NH
exch), 12.41 (2 H, s, 2COOH exch). Anal. (C22H24N4O6S·0.5H2O)
C, H, N, S.
(4 H, dd, C6H4), 8.51 (1 H, d, CONH exch) 10.82 (1 H, s, 3-NH
exch), 12.38 (2 H, s, 2COOH exch). Anal. (C25H30N4O6S·0.5H2O)
C, H, N, S.
N-{4-[8-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-
yl)octyl]benzoyl}-L-glutamic Acid (8). Using the general procedure
described, above compound 8 (0.087 g, 97.6%) was obtained from
50 (0.1 g, 0.17 mmol) as a brown solid; mp 122.2-123 °C; Rf
0.66 (MeOH/CHCl3, 1:6 + 1 drop of gl HOAc). 1H NMR (DMSO-
d6): δ 1.38 (8 H, m, C6H4-CH2 CH2CH2CH2CH2CH2CH2CH2), 1.57
(4 H, s, C6H4-CH2CH2CH2CH2CH2CH2CH2CH2), 1.92-2.11 (2 H,
m, Gluꢀ-CH2), 2.32-2.35 (2 H, m, Gluγ-CH2), 2.59-2.68 (4 H,
m,C6H4-CH2CH2CH2 CH2CH2CH2CH2CH2), 4.35-4.39 (1 H, m,
GluR-CH), 6.44 (2 H, s, 2-NH2 exch), 6.77 (1 H, s, 5-H), 7.26-7.79
(4 H, dd, C6H4), 8.50-8.51 (1 H, d, CONH exch), 10.81 (1 H, s,
3-NH exch), 12.80 (2 H, s, 2COOH exch). Anal. (C26H32-
N4O6S·0.6H2O) C, H, N, S.
Reagents for Biological Studies. [3′,5′,7-3H]MTX (20 Ci/mmol),
[3′,5′,7,9-3H] folic acid (25 Ci/mmol), and [14C(U)]-glycine (87mCi/
mmol) were purchased from Moravek Biochemicals (Brea, CA).
Unlabeled folic acid was purchased from the Sigma Chemical Co.
(St. Louis, MO). Unlabeled MTX and LCV [(6R,S) 5-formyl
tetrahydrofolate] were provided by the Drug Development Branch,
National Cancer Institute, Bethesda, MD. Both labeled and
unlabeled MTX were purified by HPLC prior to use.36 R,ꢀ-GAR
and coenzyme 10-formyl-5,8-dideazafolic acid were provided by
Dr. Richard Moran (Virginia Commonwealth University, Richmond,
VA). Other chemicals were obtained from commercial sources in
the highest available purity.
Cell Lines and Assays of Antitumor Drug Activities. RFC-,
PCFT-, and FRR-null MTXRIIOuaR2-4 (R2) CHO cells were a
gift from Dr. Wayne Flintoff (University of Western Ontario)36
and were cultured in R-minimal essential medium (MEM) supple-
mented with 10% bovine calf serum (Invitrogen, Carlsbad, CA),
penicillin-streptomycin solution, and glutamine at 37 °C with 5%
CO2.
PC43-10 cells are R2 cells transfected with human RFC.37 RT16
cells are R2 cells transfected with human FRR, and D4 cells are
R2 cells transfected with human FRꢀ. Both RT16 and D4 cells
were described in a previous publication.18 The lack of endogenous
hamster PCFT in the CHO R2, RT16, and PC43-10 sublines was
confirmed by RT-PCR of PCFT transcripts (Supporting Information)
and by transport assay at pH 5.5 and 7.2 with [3H]MTX (see
Supporting Information). R2/PCFT4 cells and R2/VC cells were
prepared by transfection with human PCFT cDNA in pCDNA3.1
vector and with empty pCDNA3.1, respectively (below). All the
R2 transfected cells (PC43-10, RT16, D4, and R2/PCFT4) were
routinely cultured in R-MEM with serum and antibiotics plus 1.5
mg/mL G418. Prior to the cytotoxicity assays (see below), RT16
and D4 cells were cultured in complete folate-free RPMI 1640
(without added folate) for three days.
KB human cervical cancer were purchased from the American
Type Culture Collection (Manassas, VA), whereas IGROV1 ovarian
carcinoma cells were a gift of Dr. Manohar Ratnam (Medical
University of Ohio). Cells were routinely cultured in folate-free
RPMI 1640 medium, supplemented with 10% fetal bovine serum,
penicillin-streptomycin solution, and 2 mM glutamine at 37 °C
with 5% CO2. FR and RFC levels in IGROV1 and KB cells were
previously reported.18 Human PCFT levels were measured by real-
time PCR with gene specific primers, and the results will be reported
elsewhere (S. Kugel Desmoulin, Y. Wang, C. Cherian, A. Gangjee,
and L.H. Matherly manuscript in preparation).
N-{4-[5-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)-
pentyl]benzoyl}-L-glutamic Acid (5). Using the general procedure
described above, compound 5 (0.071 g, 97.9%) was obtained from
47 (0.080 g, 0.15 mmol) as a white solid; mp 132.6-133 °C; Rf
0.64 (MeOH/CHCl3, 1:6 + 1 drop of gl HOAc). 1H NMR (DMSO-
d6): δ 1.16-1.23 (2 H, m, C6H4-CH2CH2CH2CH2CH2), 1.59-1.63
(4 H, m, C6H4-CH2CH2CH2CH2CH2), 1.89-2.12 (2 H, m, Gluꢀ-
CH2), 2.32-2.37 (2 H, t, Gluγ-CH2), 2.59-2.71 (4 H, m, C6H4-
CH2CH2CH2CH2CH2), 4.37-4.39 (1 H, m, GluR-CH), 6.47 (2 H,
s, 2-NH2 exch), 6.79 (1 H, s, 5-H), 7.27, 7.30-7.77, 7.80 (4 H, dd,
C6H4), 8.49-8.52 (1 H, d, CONH exch), 10.83 (1 H, s, 3-NH exch),
12.47 (2 H, s, 2COOH exch). Anal. (C23H26N4O6S·0.5C2H5OH)
C, H, N, S.
N-{4-[6-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)-
hexyl]benzoyl}-L-glutamic Acid (6). Using the general procedure
described above compound 6 (0.086 g, 98.6%) was obtained from
48 (0.098 g, 0.17 mmol) as a solid; mp 139.7-141.1 °C; Rf 0.64
(MeOH/CHCl3, 1:6 + 1 drop of gl HOAc). 1H NMR (DMSO-d6):
δ 1.32 (4 H, s, C6H4-CH2 CH2CH2CH2CH2CH2), 1.57 (4 H, s, C6H4-
CH2CH2CH2CH2CH2CH2), 1.93-2.09 (2 H, m, Gluꢀ-CH2),
2.33-2.35 (2 H, m, Gluγ-CH2), 2.62-2.68 (4 H, m,C6H4-
CH2CH2CH2CH2CH2CH2), 4.38-4.39 (1 H, m, GluR-CH), 6.47 (2
H, s, 2-NH2 exch), 6.78 (1 H, s, 5-H), 7.27, 7.29-7.78, 7.80 (4 H,
dd, C6H4), 8.50-8.52 (1 H, d, CONH exch), 10.85 (1 H, s, 3-NH
exch), 12.40 (2 H, s, 2COOH exch). Anal. (C24H28N4O6S·0.4H2O)
C, H, N, S.
N-{4-[7-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-yl)-
heptyl]benzoyl}-L-glutamic Acid (7). Using the general procedure
described above, compound 7 (0.287 g, 97.9%) was obtained from
49 (0.326 g, 0.57 mmol) as a yellow solid; mp 134.9-135.5 °C;
Rf 0.64 (MeOH/CHCl3, 1:6 + 1 drop of gl HOAc). 1H NMR
(DMSO-d6): δ 1.35 (6 H, m, C6H4-CH2 CH2CH2CH2CH2CH2CH2),
1.59 (4 H, s, C6H4-CH2CH2CH2CH2CH2CH2CH2), 1.90-2.08 (2
H, m, Gluꢀ-CH2), 2.32-2.35 (2 H, m, Gluγ-CH2), 2.59-2.68 (4
H, m,C6H4-CH2CH2CH2CH2CH2CH2CH2), 4.36-4.40 (1 H, m,
GluR-CH), 6.44 (2 H, s, 2-NH2 exch), 6.77 (1 H, s, 5-H), 7.26-7.79
For growth inhibition assays, cells (CHO, KB, or IGROV1) were
plated in 96-well plates (∼5000 cells/well, total volume of 200 µL
of medium) with a broad range of antifolate concentrations. The
medium was RPMI 1640 (contains 2.3 µM folic acid) with 10%
dialyzed serum and antibiotics for experiments with R2 and PC43-
10 cells. For RT16, D4, KB, and IGROV1 cells, cells were cultured
in folate-free RPMI media with 10% dialyzed fetal bovine serum
(Invitrogen) and antibiotics supplemented with 2 nM LCV. The
requirement for FR-mediated drug uptake in these assays was
established in parallel incubation including 200 nM folic acid. For

2950 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Deng et al.
R2/PCFT4 cells, the medium used routine growth inhibition assays
was folate-free RPMI 1640 (pH 7.2) containing 25 nM LCV,
supplemented with 10% dialyzed fetal bovine serum and antibiotics.
For all cell lines, incubations were routinely up to 96 h; metaboli-
cally active cells (a measure of cell viability) were assayed with
CellTiter-Blue cell viability assay (Promega, Madison, WI), and
fluorescence was measured (590 nm emission, 560 nm excitation)
with a Molecular Dynamics fluorescence plate reader. Data were
exported from Softmax Pro software to an Excel spreadsheet for
analysis and determinations of IC50s, corresponding to the drug
concentrations that result in 50% loss of cell growth.
For some of the in vitro growth inhibition studies, the inhibitory
effects of the antifolate inhibitors on de novo thymidylate biosyn-
thesis (i.e., thymidylate synthase) and de novo purine biosynthesis
(GARFTase and AICARFTase) were tested by coincubations with
thymidine (10 µM) and adenosine (60 µM), respectively. For de
novo purine biosynthesis, additional protection experiments used
AICA (320 µM) as a means of distinguishing inhibitory effects at
GARFTase from those at AICARFTase.18,34,35
For assays of colony formation in the presence of the antifolate
drugs, KB cells were harvested in log phase, diluted, and 100 cells
were plated into 60 mm dishes in folate-free RPMI1640 medium
supplemented with 2 nM LCV, 10% dialyzed fetal bovine serum,
penicillin-streptomycin, and 2 mM glutamine, in the presence or
absence of the thieno[2,3-d]pyrimidine compounds and MTX (from
1 to 1000 nM). The highest concentrations (1000 nM) of antifolate
drugs were also tested in the presence of folic acid (100 µM) to
examine the protection effect. The dishes were incubated at 37 °C
with 5% CO2 for 10 days. At the end of the incubations, the dishes
were rinsed with Dulbecco’s phosphate-buffered saline (DPBS),
5% trichloroacetic acid, and borate buffer (10 mM, pH 8.8),
followed by 30 min incubation in 1% methylene blue in the borate
buffer. The dishes were rinsed with borate buffer, and colonies were
enumerated for calculating percent colony-forming efficiency
normalized to control.
Moran (Virginia Commonwealth University, Richmond, VA).40
Enzyme activity was assayed spectrophotometrically at 37 °C using
GARFTase (0.75 nM), R,ꢀ-GAR (11 µM), and coenzyme 10-
formyl-5,8-dideazafolic acid (10 µM) in HEPES buffer (75 mM,
pH 7.5) with or without antifolate inhibitor (10-20,000 nM). The
absorbance of the reaction product, 5,8-dideazafolic acid, was
monitored at 295 nm over the first minute as a measure of the initial
rate of enzyme activity. IC50s were calculated as the concentrations
of inhibitors that resulted in a 50% decrease in the initial velocity
of the GARFTase reaction.
In Situ GARFTase Enzyme Inhibition Assay. Incorporation
of [14C]glycine into [14C]FGAR as an in situ measure of endogenous
GARFTase activity was described by Beardsley et al.34 as modified
by Deng et al.18 for studies with the GARFTase inhibitor LMX.
For these experiments, KB cells were seeded in 4 mL of complete
folate-free RPMI 1640 plus 2 nM LCV in 60 mm dishes at a density
of 2 × 106 cells per dish. On the next day, the medium was replaced
with 2 mL of fresh complete folate-free RPMI 1640 plus 2 nM
LCV (without supplementing glutamine). Azaserine (4 µM final
concentration) was added in the presence and absence of the
antifolate inhibitors (0.1, 1, 10, 100, 1000 nM). After 30 min,
glutamine (final concentration, 2 mM) and [14C]glycine (final
specific activity 0.1 mCi/L) were added. Incubations were at 37
°C for 15 h, at which time cells were washed (one time) with ice-
cold folate-free RPMI 1640 plus serum. Cell pellets were dissolved
in 2 mL 5% trichloroacetic acid at 0 °C. Cell debris was removed
by centrifugation (the cell protein contents in the pellets were
measured), and the supernatants were extracted twice with 2 mL
of ice-cold ether. The aqueous layer was passed through a 1 cm
column of AG1 × 8 (chloride form), 100-200 mesh (BioRad,
Carlsbad, CA), washed with 10 mL of 0.5 N formic acid and then
10 mL of 4 N formic acid, and finally eluted with 8 mL of 1 N
HCl solution. The elutants were collected and determined for
radioactivity. The accumulation of radioactive FGAR was calculated
as pmol per mg protein over a range of inhibitor concentrations.
IC50s were calculated as the concentrations of inhibitors that resulted
in a 50% decrease in FGAR synthesis.
FR Binding Assay. [3H]Folic acid binding was used to assess
levels of surface FRs.38 Briefly, cells (e.g., RT16 or D4) (∼1.6 ×
106) were rinsed twice with DPBS, followed by a quick rinse with
an acetate buffer (10 mM sodium acetate, 150 mM NaCl, pH 3.5)
to remove FR-bound folates. Cells were washed twice with ice-
cold 2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-
buffered saline (20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2
mM MgCl2, 5 mM glucose, pH7.4) (HBS). Cells were incubated
in HBS with [3H]folic acid (50 nM, specific activity 0.5 Ci/mmol)
in the presence and absence of unlabeled folic acid or antifolate
for 15 min at 0 °C. The dishes were rinsed three times with ice-
cold HBS, after which the cells were solubilized with 0.5 N sodium
hydroxide and aliquots measured for radioactivity and protein
contents. Protein concentrations were measured with Folin phenol
reagent.39 Bound [3H]folic acid was calculated as pmol/mg protein.
Relative binding affinities for assorted folate/antifolate substrates
were determined as previously described18 and were calculated as
the inverse molar ratio of unlabeled ligands required to inhibit
[3H]folic acid binding by 50%. By definition, the relative affinity
of folic acid is 1.
Statistical Analyses. Statistical analyses were performed with
GraphPad Prism 4.0.
Acknowledgment. This work was supported in part by
grants from the National Institutes of Health, National Cancer
Institute, CA125153 (A.G.) and CA53535 (L.H.M.). We thank
Dr. Wayne Flintoff for the gift of the MTXRIIOuaR2-4 Chinese
hamser ovary cells, Dr. Manohar Ratnam for providing a culture
of IGROV1 cells, and Dr. Richard Moran for providing
recombinant mouse GARFTase and assay reagents.
Supporting Information Available: Confirmation that R2,
RT16, and PC43-10 cells are PCFT null, development of CHO
(Chinese hamster ovary) subline expressing human proton-coupled
folate transporter (PCFT), and elemental analysis high-resolution
mass spectra (HRMS) (ESI). This material is available free of charge
via the Internet at http://pubs.acs.org.
PCFT-Mediated Transport Assay. To assess PCFT transport
activity in R2/PCFT4 and R2/VC cells, transport was assayed at
pH 7.2 in HEPES-buffered saline (20 mM HEPES, 140 mM NaCl,
5 mM KCl, 2 mM MgCl2, 5 mM glucose) and at pH 5.5 in
4-morphilinopropane sulfonic acid (MES)-buffered saline (20 mM
MES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 5 mM glucose)
in 2 mL suspension (1.5 × 107 cells total). Uptake of [3H]MTX
(0.5 µM) was measured at 37 °C for 2 min then quenched with 10
mL of ice-cold DPBS. Cells were washed with ice-cold DPBS (3×)
and solubilized with 0.5 N NaOH. Levels of intracellular radioac-
tivity were expressed as pmol/mg protein, calculated from direct
measurements of radioactivity and protein contents of cell homo-
genates. Protein concentrations were measured with Folin phenol
reagent.39
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