Tumor-targeting with novel non-benzoyl 6-substituted straight chain pyrrolo[2,3-d ]pyrimidine antifolates via cellular uptake by folate receptor α and inhibition of de novo purine nucleotide biosynthesis

Article abstract of DOI:10.1021/jm401139z

A new series of 6-substituted straight side chain pyrrolo[2,3-d]pyrimidines 3a-d with varying chain lengths (n = 5-8) was designed and synthesized as part of our program to provide targeted antitumor agents with folate receptor (FR) cellular uptake specificity and glycinamide ribonucleotide formyltransferase (GARFTase) inhibition. Carboxylic acids 4a-d were converted to the acid chlorides and reacted with diazomethane, followed by 48% HBr to generate the α-bromomethylketones 5a-d. Condensation of 2,4-diamino-6-hydroxypyrimidine 6 with 5a-d afforded the 6-substituted pyrrolo[2,3-d]pyrimidines 7a-d. Hydrolysis and subsequent coupling with diethyl l-glutamate and saponification afforded target compounds 3a-d. Compounds 3b-d showed selective cellular uptake via FRα and -β, associated with high affinity binding and inhibition of de novo purine nucleotide biosynthesis via GARFTase, resulting in potent inhibition against FR-expressing Chinese hamster cells and human KB tumor cells in culture. Our studies establish, for the first time, that a side chain benzoyl group is not essential for tumor-selective drug uptake by FRα.

Full text of DOI:10.1021/jm401139z

Article
pubs.acs.org/jmc
Tumor-Targeting with Novel Non-Benzoyl 6‑Substituted Straight
Chain Pyrrolo[2,3‑d]pyrimidine Antifolates via Cellular Uptake by
Folate Receptor α and Inhibition of de Novo Purine Nucleotide
Biosynthesis
Yiqiang Wang,†,⊥,# Christina Cherian,‡,§,# Steven Orr,‡,§ Shermaine Mitchell-Ryan,§ Zhanjun Hou,‡,§
Sudhir Raghavan,† Larry H. Matherly,*,‡,§,∥,# and Aleem Gangjee*,†,#
†Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue,
Pittsburgh, Pennsylvania 15282, United States
‡Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, Michigan 48201,
United States
§Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan 48201, United States
∥Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, United States
S
* Supporting Information
ABSTRACT: A new series of 6-substituted straight side chain
pyrrolo[2,3-d]pyrimidines 3a−d with varying chain lengths
(n = 5−8) was designed and synthesized as part of our program
to provide targeted antitumor agents with folate receptor
(FR) cellular uptake specificity and glycinamide ribonucleotide
formyltransferase (GARFTase) inhibition. Carboxylic acids
4a−d were converted to the acid chlorides and reacted with
diazomethane, followed by 48% HBr to generate the α-bromomethylketones 5a−d. Condensation of 2,4-diamino-6-
hydroxypyrimidine 6 with 5a−d afforded the 6-substituted pyrrolo[2,3-d]pyrimidines 7a−d. Hydrolysis and subsequent coupling
with diethyl L-glutamate and saponification afforded target compounds 3a−d. Compounds 3b−d showed selective cellular uptake
via FRα and -β, associated with high affinity binding and inhibition of de novo purine nucleotide biosynthesis via GARFTase,
resulting in potent inhibition against FR-expressing Chinese hamster cells and human KB tumor cells in culture. Our studies
establish, for the first time, that a side chain benzoyl group is not essential for tumor-selective drug uptake by FRα.
normal tissues, although high levels of PCFT are present in the
duodenum and jejunum, as well as in the liver and kidney.5
Folate-dependent biosynthetic pathways are important
therapeutic targets for cancer chemotherapy.6−8 Clinically
relevant antifolate drugs for cancer include potent inhibitors of
dihydrofolate reductase [methotrexate (MTX), pralatrexate
(PDX)], thymidylate synthase [raltitrexed (RTX), pemetrexed
(PMX)], and the purine nucleotide biosynthetic enzymes,
β-glycinamide ribonucleotide (GAR) formyltransferase (GARF-
Tase) [PMX] and 5-aminoimidazole-4-carboxamide (AICA)
ribonucleotide formyltransferase (AICARFTase) [PMX]6−8
(Figure 1). Although all these agents are transported by RFC,1,9
the expression of RFC in both normal and tumor cells presents
a potential obstacle to antitumor selectivity. Further, loss of RFC
is frequently associated with antifolate resistance.1,8,10 Thus, it is
of interest to design tumor-targeted antifolates that are specific
substrates for transporters other than RFC with limited
expression and/or transport into normal tissues compared with
tumors. If these drugs also inhibit targets other than dihydrofolate
INTRODUCTION
■
Folates are essential for cell growth and tissue regeneration.
Membrane transport of extracellular folates is required because
mammalian cells cannot synthesize folates de novo. Three major
folate uptake systems have been reported.1−5 (i) The reduced
folate carrier (RFC or SLC19A1) is an anion antiporter that is
ubiquitously expressed in tissues and tumors and is the major
membrane transport system for folates in mammalian cells at
physiologic pH.1 (ii) Folate receptors (FRs) α and β are glycosyl
phosphatidylinositol-anchored proteins that mediate cellular
uptake of folates by receptor-mediated endocytosis.2,3 High
levels of FRs are expressed in a number of malignancies, including
ovarian and endometrial cancers (FRα) and in myeloid leukemias
(FRβ).2,3 However, FRs show a more restricted tissue distribution
than RFC. In normal tissues where they are expressed, FRs are
either inaccessible to circulating folate cofactors (FRα in renal
tubules) or are nonfunctional (FRβ in normal hematopoietic
cells).2,3 (iii) The proton-coupled folate transporter (PCFT;
SLC46A1) is a recently discovered proton-folate symporter that
functions optimally at acidic pH by coupling the downhill flow
of protons to the uphill transport of folates.4,5 PCFT is widely
expressed in human solid tumors4 and in modest levels in most
Received: July 26, 2013
Published: October 10, 2013
© 2013 American Chemical Society
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Figure 1. Structures of clinically relevant antifolates transported by RFC.
Figure 2. 6-Substituted non-benzoyl straight chain compounds 3a−d, based on lometrexol (LMTX) and compounds 1a−c, showing replacement of the
phenyl ring in compounds 2a−2b by 2−5 methylene groups.
reductase and thymidylate synthase, this would afford further
benefit by circumventing resistance due to increased levels or
mutated forms of these enzyme targets. This reasoning was a
major impetus to develop novel agents with selective uptake into
tumors by FRs or PCFT over RFC and which specifically target de
novo purine nucleotide biosynthesis.5,6,11−16
We previously reported a series of 6-substituted pyrrolo[2,3-d]-
pyrimidine phenyl antifolates 2a−d.11,13 (Figure 2) A highly active
compound of this series was the 3-carbon bridge analog (2a)
which was a targeted agent selectively transported into certain
tumor cells by FRs and PCFT but not RFC.11,13 Once intra-
cellular, compound 2a inhibited GARFTase in de novo purine
nucleotide biosynthesis, resulting in depletion of purine
nucleotides.11,13 Further, compound 2a was highly active in
vitro toward both KB and IGROV1 tumors.11 To further explore
the structure−activity relationships (SAR) for GARFTase inhibi-
tion and non-RFC targeted transport specificity, we synthesized
and tested several series of related analogues with modifications
of the aromatic rings and aliphatic linkers.5,6,12−16
A series of LMTX analogues, 1a−c, was reported in which the
phenyl ring in the bridge was replaced by a methylene bridge
of variable length19,20 (Figure 2). Interestingly, replacement of
the phenyl ring of LMTX by two, three, or four carbon atom
chains substantially preserved both binding to GARFTase19 and
polyglutamylation by folylpolyglutamate synthetase (FPGS).20
However, these analogues were not tested for their membrane
transport by the major folate transporters or for their capacities
to inhibit cell proliferation.
In the present work, we designed an analogous series of
6-substituted pyrrolo[2,3-d]pyrimidine analogues to examine
the impact of an alkyl-for-benzoyl ring replacement on folate
transporter specificity, GARFTase inhibition, and antiprolifer-
ative activities. Straight chain compounds 3a−d were designed
with replacement of the phenyl ring of the lead compound 2a
by a methylene bridge of variable length (n = 2−5) (Figure 2).
The precursor acids, 7e−h (Scheme 1), were also biologically
evaluated to determine the importance of the L-glutamate moiety
to their biological effects. Collectively, our results establish that
aliphatic replacement of the benzoyl ring in the 6-substituted
pyrrolo[2,3-d]pyrimidine series preserves substantial selective
FR-directed cellular uptake and GARFTase inhibition, with
potent inhibition of tumor cell proliferation, as long as there is
Lometrexol (LMTX) is an early generation GARFTase
inhibitor17 that was tested in a phase I clinical trial and was
found to be unacceptably toxic.18 This failure was likely due, at
least in part, to its membrane transport into normal cells by RFC.
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Scheme 1
sufficient distance between the bicyclic ring system and the
terminal L-glutamate.
of isogenic Chinese hamster ovary (CHO) sublines previously
engineered to individually express the major (anti)folate
cellular uptake systems including RFC (PC43-10),21 FRα and
β (RT16 and D4, respectively),11 or PCFT (R2/PCFT4).12,13
Additional testing was performed with KB human nasophar-
engeal carcinoma cells which express RFC, FRα, and PCFT.11,12
Experiments were performed in standard RPMI 1640/10%
dialyzed fetal bovine serum (dFBS) (PC43-10) and in folate-
defined media {folate-free RPMI1640/10% dFBS, supplemented
with 2 nM (RT16, D4, KB) or 25 nM leucovorin [(6R,S)5-
formyl tetrahydrofolate (LCV)] (R2/PCFT4)}. The impact of
the drug treatments on cell proliferation was measured after 96 h,
and results were compared to negative controls including RFC-,
FR-, and PCFT-null MTXRIIOuaR2−4 (R2) CHO cells11,12,21
[either the parental R2 subline or vector control R2(VC) cells].
For FR-expressing CHO and KB cells, parallel cultures were
treated with drugs along with excess folic acid (200 nM) to
block drug binding and cellular uptake by FRs. Internalization of
exogenous folic acid by FRs would also increase intracellular
folate pools. Results were compared with those for the lead
6-substituted pyrrolo[2,3-d]pyrimidine benzoyl analogues 2a−d
and are summarized in Table 1.
The data in Table 1 clearly establish that the compounds
(7e−h) lacking the L-glutamate are not inhibitory, regardless of
the cell line model tested. Compounds 3a−d were also inactive
up to 1000 nM toward RFC-expressing PC43-10 cells and
PCFT-expressing R2/PCFT4 CHO cells. Compounds 3a−d
were inhibitory toward CHO cells engineered to express FRα
and FRβ, as well as FRα-expressing KB tumor cells. Growth
inhibition was abolished in the presence of excess (200 nM) folic
acid. The most potent inhibitor was 3c (7-carbon atom chain),
followed by 3d (8-carbon chain) and 3b (6-carbon chain). The
5-carbon chain analogue, 3a, was a much poorer inhibitor of cell
CHEMISTRY
■
Compounds 3a−d (n = 5−8) were synthesized using an
α-bromomethyl ketone condensation with 2,4-diamino-4-oxo-
pyrimidine 6 as the key step outlined in Scheme 1. Commercially
available carboxylic acids 4a−d were converted to the acid
chlorides and immediately reacted with diazomethane, followed
by 48% HBr to give the desired α-bromomethylketones 5a−d.11
Condensation of 2,4-diamino-6-hydroxypyrimidine 6 with
5a−d afforded the 6-substituted pyrrolo[2,3-d]pyrimidines
7a−d. Hydrolysis of 7a−d afforded 7e−h. Subsequent coupling
with diethyl L-glutamate using N-methyl morpholine and 2,4-
dimethoxy-6-chlorotriazine as the activating agents afforded the
diesters 8a−d. Final saponification of the diesters gave the target
compounds 3a−d (n = 5−8).
BIOLOGICAL EVALUATION AND DISCUSSION
■
Non-benzoyl 6-Carbon Chain Pyrrolo[2,3-d]pyrimidine
Antifolates Are Inhibitors of Cell Proliferation, Correlat-
ing with Expression of Folate Receptors. Our goal was
to examine the role of the side chain phenyl ring and the
terminal L-glutamate of the pyrrolo[2,3-d]pyrimidine antifolates
as determinants of folate transporter selectivity, GARFTase
inhibitory activity, and antiproliferative activity based on prior
studies of compounds 2a−d. We previously showed that the
intramolecular distance between the bicyclic scaffold and the
L-glutamate moiety of the latter series was an important
determinant of inhibitory potency toward FR-expressing cell
lines,10,11,13−15 so this was also examined.
Compounds 3a−d (Figure 2) and 7e−h (Scheme 1) were
tested in cell proliferation assays with a well-characterized cohort
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Table 1. IC50s (in nM) for 6-Substituted Pyrrolo[2,3-d]pyrimidine Antifolates and Classical Antifolates in RFC-, PCFT-, and
a
FR-Expressing Cell Lines
hRFC
PC43-10
hFRα
RT16 (+FA)
hFRβ
hPCFT
D4 (+FA) R2/hPCFT4
hRFC/FRα/hPCFT
antifolate
R2
RT16
D4
R2(VC)
KB
KB (+FA)
2a
648.6(38.1)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
216(8.7)
819(94)
894(93)
>1000
4.1(1.6)
6.3(1.6)
54(21)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>100
5.6(1.2)
10(2)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
275(101)
211(43)
ND
23.0(3.3)
213(28)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
1.7(0.4)
1.9(0.7)
13(7.2)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
31(7)
2b
2c
>1000
80(9)
2d
>1000
162(18)
>1000
198(34)
>1000
>1000
>1000
>1000
214(62)
>1000
23(12)
7e
>1000
>1000
>1000
7f
>1000
>1000
>1000
>1000
7g
>1000
>1000
>1000
>1000
7h
>1000
>10000
>1000
>1000
>1000
3a
>1000
>1000
>1000
>1000
>10000
188(41)
461(62)
>1000
388(68)
>1000
>1000
200.3(34.6)
7.9 (0.72)
1.10(0.88)
1.48(0.04)
1.2(0.6)
6.0(0.6)
0.47(0.20)
68(12)
3b
>1000
47.1(16.5)
8.54(1.53)
12.31 (3.14)
12(8)
10.55(3.69)
0.87(0.12)
3.99(0.57)
2.6(1.0)
106(11)
ND
>1000
3c
>1000
>1000
3d
>1000
>1000
LMTX
MTX
PDX
PMX
RTX
12(2.3)
12(1.1)
0.69(0.07)
138(13)
6.3(1.3)
38.0(5.3)
121(17)
57(12)
13.2(2.4)
99.5(11.4)
114(31)
168(50)
42(9)
20(2.4)
1.94(0.28)
327(103)
22(5)
60(8)
254(78)
746(138)
974.0(18.1)
>1000
15(5)
22(10)
5.9(2.2)
a
Growth inhibition assays were performed for CHO sublines engineered to express human RFC (PC43-10), FRs α and β (RT16 and D4,
respectively), or PCFT (R2/PCFT4), for comparison with transporter-null [R2, R2(VC)] CHO cells and for the KB human tumor subline
(expresses RFC, FRα, and PCFT), as described in the Experimental Section. For the FR experiments, growth inhibition assays were performed in the
presence and the absence of 200 nM folic acid (FA). The data shown are mean values from 3−10 experiments (plus/minus SEM in parentheses).
Results are presented as IC50 values, corresponding to the concentrations that inhibit growth by 50% relative to cells incubated without drug. Data
for MTX, PMX, RTX, and LMTX and for compounds 2a−d were previously published.11,13 The structures for the classical antifolate drugs are
shown in Figure 1 and those for compounds 7e−h and 3a−d are shown in Figure 2 and Scheme 1. ND: not determined.
proliferation. Growth inhibitions, as reflected in the IC50 values,
were similar with FRα-expressing sublines (KB, RT16) for
compounds 2a, 2b, 3c, and 3d. Whereas sensitivities of FRα-
expressing RT16 cells were similar to those for FRβ-expressing
D4 cells for compounds 2a and 2b, D4 cells were distinctly
more sensitive to the non-benzoyl compounds 3b−3d. The FR
specificity for the active analogues of this series contrasts with
results with classic antifolates including MTX, RTX, PMX, PDX,
and LMTX, for which antiproliferative activities for RFC- and
PCFT-expressing cells exceeded those for FR-expressing cells
(Table 1).
Binding of Non-Benzoyl Pyrrolo[2,3-d]pyrimidine
Antifolates to FRα and β. Compounds 3a−d are all inhibitors
of proliferation for cell lines that express FRα (RT16, KB) and
FRβ (D4) (Table 1). The inhibitory effects of these active
compounds were blocked by excess folic acid, consistent with
their surface binding and internalization by FRs.
Surface FR binding of cytotoxic folate analogues is an essential
step for drug internalization by this mechanism and for inhibition
of intracellular enzyme targets.11,12,14−16 To establish a SAR
for FRα and -β binding of this series, in particular the impact of
alkyl chain length (5−8 carbons) for the 6-substituted analogues
and of the terminal L-glutamate on FR binding, we performed
competitive binding assays with compounds 3a−d and 7e−h at
pH 7.4. In these experiments, unlabeled pyrrolo[2,3-d]-
pyrimidine compounds were tested for their abilities to compete
for surface FR binding with [3H]folic acid. FR binding of unknown
ligands was compared to those for positive (compounds 2a
and 2b, also folic acid) and negative (MTX) controls, with
documented FR-binding characteristics. The CHO sublines
expressing FRα (RT16) or FRβ (D4) were initially rinsed with
acid-buffered saline to release FR-bound folates, followed by
incubation with [3H]folic acid (at pH 7.4) in the presence of
unlabeled (anti)folate competitors over a range of concentra-
tions.11,12,14−16 After further washing the cells (pH 7.4), FR-bound
[3H]folic acid was quantitated and normalized to cell protein
and results calculated as pmol [3H]folic acid bound to FRs per mg
cell protein. Relative affinities were expressed as inverse molar
ratios of (anti)folates required to decrease bound [3H]folic acid
by 50% normalized to the affinity for unlabeled folic acid in each
experiment (assigned a value of 1.0).11,12,14−16 The results are
expressed as mean values standard errors and are presented in
Figure 3.
With the 6-substituted analogues that included terminal
L-glutamates (3a−d), FR binding increased with increasing
substituent chain length (3d > 3c > 3b > 3a) for both FRα and -β,
with compound 3d exhibiting relative binding affinities approxi-
mating those for compounds 2a and 2b and slightly less than
for folic acid (Figure 3). The lack of a terminal L-glutamate
(compounds 7e−h) had a deleterious effect on FR binding,
consistent with the results for the cell proliferation experiments
(Table 1). For FRα (but not FRβ), compound 7h with the
8-carbon chain showed detectable binding. Interestingly, binding
of the active linear analogues (compounds 3b, 3c, and 3d) was
consistently reduced for FRβ compared to FRα in spite of their
increased antiproliferative effects toward FRβ-expressing D4
cells over FRα-expressing RT16 cells (Table 1).
These results demonstrate that the presence of a phenyl side
chain ring system for the 6-substituted pyrrolo[2,3-d]pyrimidine
analogues is not obligatory for binding to FRα or -β. Further,
whereas FR binding is clearly critical to cellular uptake of
cytotoxic folate analogues, this parameter shows at best an inexact
correlation with antiproliferative activities of the non-benzoyl
pyrrolo[2,3-d]pyrimidine series of analogues. Nonetheless, FR
binding for both FR α and -β was greatest for the compounds
(3c and 3d) with the most potent antiproliferative effects.
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Figure 3. Binding of non-benzoyl 6-substituted pyrrolo[2,3-d]pyrimidine analogues (3a−d and 7e−h) to FRα (RT16) and FRβ (D4), compared to
compounds 2a and 2b. Data are shown for the effects of the unlabeled ligands with FRα-expressing RT16 and FRβ-expressing D4 CHO cells. Binding
affinities for assorted folate/antifolate substrates were determined over a range of ligand concentrations and were calculated as the inverse molar ratios of
unlabeled ligands required to inhibit [3H]folic acid binding by 50%. Relative binding affinities for individual experiments were normalized to that for
unlabeled folic acid which was assigned a value of 1.0. For the various ligands, results are presented as mean values standard errors from three
experiments. Details for the binding assays are provided in the Experimental Section. Undefined abbreviations: FA, folic acid. The results for compounds
2a and 2b were previously published.11
Figure 4. Protection of KB cells from growth inhibition by non-benzoyl 6-substituted pyrrolo[2,3-d]pyrimidine analogues 3b−d in the presence of
nucleosides and 5-aminoimidazole-4-carboxamide (AICA). Proliferation inhibition was measured for KB cells over a range of concentrations of
compounds 3b−d, as shown, in complete folate-free RPMI1640 with 2 nM LCV in the absence of other additions (labeled “No Additions”), or in the
presence of 200 nM folic acid (labeled “Folate Added”), adenosine (60 μM), thymidine (10 μM), adenosine plus thymidine, or AICA (320 μM). Cell
densities were measured with CellTiter Blue fluorescence dye and a fluorescence plate reader. Results were normalized to cell densities in the absence of
drug. Results shown are representative data of experiments performed in triplicate.
Determination that GARFTase Is the Major Cellular
Target for Compounds 3b, 3c, and 3d. Previous reports of
non-benzoyl analogues of LMTX, 1a−c, in which the phenyl ring
was replaced by 2−4 methylene groups (Figure 2), established
that the aromatic B ring is not essential for binding to the
GARFTase active site, as these novel antifolate compounds were
potently inhibitory toward purified murine GARFTase.19 Further,
compounds 1a−c were all excellent substrates for polyglutamy-
lation by murine FPGS.20
To establish the targeted pathway for compounds 3b−d (i.e., de
novo thymidylate versus de novo purine nucleotide biosyn-
thesis), exogenous thymidine and adenosine were tested for
their capacities to reverse their growth inhibitory effects toward
KB cells (Figure 4).11−17 AICA, a precursor of the AICARFTase
substrate was added to circumvent the step catalyzed by
GARFTase so as to distinguish inhibition of GARFTase from
AICARFTase.11−17
For non-benzoyl pyrrolo[2,3-d]pyrimidine analogues 3b−d,
thymidine (10 μM) had no impact on the extent of inhibition
of cell proliferation, indicating that thymidylate biosynthesis
(i.e., thymidylate synthase) was not being targeted. Conversely,
By analogy to compounds 1a−c and the parent 6-substituted
pyrrolo[2,3-d]pyrimidines 2a and 2b, we hypothesized that the
non-benzoyl analogues 3b−d were also GARFTase inhibitors.
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both adenosine (60 μM) and AICA (320 μM) completely
reversed the inhibitory effects of this series, establishing that de
novo purine nucleotide biosynthesis in general and GARFTase in
particular were thelikelyintracellulartargets (Figure 4). Essentially
identical results were previously published for compounds 2a
and 2b.11 In addition, in experiments with recombinant DHFR
and TS, compounds 3b−3d were not inhibitory (data not shown).
We used an in situ activity assay to directly measure cellular
GARFTase activity in KB cells treated with the novel anti-
folates.11−17 Cells were incubated with [14C]glycine as a radiotracer
for 15 h in the presence of compounds 3b−d under conditions and
at concentrations approximating those used in the cell prolifera-
tion experiments (Table 1). In this metabolic assay, [14C]glycine
is incorporated into the GARFTase substrate [14C]GAR and
subsequently into [14C]formyl GAR (by GARFTase), which
accumulates in the presence of azaserine. Following protein
precipitation with trichloroacetic acid, the acid-soluble metabolites
are extracted and fractionated by ion-exchange chromatography,
permitting quantitation of [14C]formyl GAR normalized to cellular
protein.
(Figure 5). Calculated IC50 values for GARFTase inhibition
varied within a 3-fold range from 2.89 for compound 3b to
9.62 nM for compound 3d. By comparison, the IC50s for the
3- and 4-carbon benzoyl analogues 2a and 2b were 18 and
6.8 nM, respectively.11
These results unambiguously demonstrate that the absence of
a side chain benzoyl ring system in the 6-substituted pyrrolo[2,3-d]-
pyrimidine series is not required to potently inhibit intracellular
GARFTase, analogous to results for the non-benzoyl analogues
of LMTX 1a−c (Figure 1).
Molecular Modeling: Docking Studies of Compound
3c with Human GARFTase. Figure 6 shows the docked pose of
3c (n = 7) in the human GARFTase (PDB ID: 1NJS)22 active
site. The cofactor binding pocket of GARFTase is located at the
interface between the N-terminal mononucleotide binding domain
and the C-terminal half of the structure. The binding site for the
folate cofactor moiety consists of three parts: the pteridine binding
cleft, the benzoylglutamate region, and the formyl transfer region.22
The docked pose shows the pyrrolo[2,3-d]pyrimidine scaffold of
3c to be buried deep in the active site and occupies the same
location as the diaminopyrimidine ring in the native crystal
structure ligand 10-trifluoroacetyl-5,10-dideaza-acyclic-5,6,7,8-tet-
rahydrofolic acid (10-CF3CO-DDACTHF). This orientation of
the scaffold permits the 2-amino moiety of 3c to form hydrogen
bonds to Glu141 and the backbone of Leu92. The N1 nitrogen
interacts with the backbone of Leu92 to form a hydrogen bond.
The 4-oxo moiety forms a hydrogen bond with Asp144 and forms
water-mediated hydrogen bonds with Asp142 and Ala140. The
molecule is oriented in a manner that aids the N7-nitrogen to
form a hydrogen bond with Arg90. As is shown with 10-CF3CO-
DDACTHF (Figure 6), several hydrophobic residues flank the
pocket which holds the pyrrolo[2,3-d]pyrimidine scaffold. The
hydrophobic pocket consists of Leu85, Ile91, Leu92, Val97 (not
shown), and the folate binding loop residues 141−146. The amide
NH of the L-glutamate forms a hydrogen bond with Met89. The
7-carbon side chain of 3c shifts the glutamate side chain away from
the corresponding glutamate side chain of 10-CF3CO-DDACTHF.
The carbonyl group of the glutamate side chain of 3c interacts with
Arg64, which is not seen with the corresponding carbonyl group of
the side chain of 10-CF3CO-DDACTHF. The γ-carboxylic acid
of 3c interacts with Arg64. The α-carboxylic acid of the glutamate
side chain of 3c interacts with Arg90. Thus, the interaction pattern
of the α-and γ-carboxylic acid moieties of 3c are reversed compared
The results show that in KB cells, compounds 3b−d were all
potent GARFTase inhibitors at extracellular drug concentra-
tions approximating those required to inhibit cell proliferation
Figure 5. In situ GARFTase inhibition assay. For the in situ assays,
incorporation of [14C]glycine into [14C]formyl GAR was measured in
KB tumor cells cultured for 15 h in complete folate-free RPMI 1640 plus
2 nM LCV. Details are described in the Experimental Section. Results
are presented as a percent of control treated without drugs for KB cells
treated with nanomolar concentrations of 3b−d. Results are presented
as mean IC50 values
calculated as 2.89 ( 0.62) nM for 3b, 5.49 ( 1.36) nM for 3c, and 9.62
0.98) nM for 3d. For comparison, IC50s for compounds 2a and 2b
were 18 ( 2) nM and 6.8 ( 0.9) nM, respectively.11
standard errors. Mean IC50s ( SEs) were
(
Figure 6. Stereoview overlay of the docked pose of 3c (white) with 10-CF3CO-DDACTHF (purple) in human GARFTase (PDB ID: 1NJS).22
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Figure 7. Stereoview overlay of the docked pose of compound 3c (white) with folic acid (blue) in human FRα (PDB ID: 4LRH).23
to the corresponding α- and γ-carboxylic acids of 10-CF3CO-
DDACTHF and could represent an alternate conformation of the
side chain. These interactions could easily permit binding and
potent inhibition of 3c against GARFTase, as demonstrated by the
results of our in situ GARFTase assays (Figure 5).
Molecular Modeling: Docking Studies of Compound
3c with Human FRα. The X-ray crystal structure of human
FRα with folic acid was recently published.23 Accordingly,
we determined the docked structure of 3c (a prototype of the
non-benzoyl series of 6-substituted pyrrolo-[2,3-d]pyrimidine
analogues) with FRα.
over classical antifolates such as RTX or PMX that are
transported by all systems.1,5
In previous studies, we began establishing a SAR for FR and
́
PCFT binding and cellular uptake vis a vis RFC and of GARFTase
inhibition in de novo purine nucleotide biosynthesis.5,6,11−16
Hence, (i) the 6-substituted pyrrolo[2,3-d]pyrimidine ring
system, (ii) a thienoyl-for-benzoyl B ring replacement, and (iii)
optimal spacing between the pyrrolo[2,3-d]pyrimidine system
and the terminal L-glutamate all favored FR and PCFT cellular
uptake and GARFTase inhibition, resulting in potent anti-
proliferative activity.5,6,11−16
Figure 7 shows the docked pose of compound 3c in the human
FRα (PDB ID: 4LRH)23 binding site. The long and open folate-
binding pocket of FRα contains a predominantly negatively
charged pocket where the pteroate group of folic acid binds and
a positively charged entrance of the binding pocket which is
occupied by the glutamate moiety of folic acid. In its docked pose,
the pyrrolo[2,3-d]pyrimidine scaffold of 3c binds similar to the
pterin scaffold of folic acid. The 2-NH2 moiety of 3c interacts with
the side chain carboxylic acid of Asp81. Additional hydrogen
bonds are formed between N3 and the side chain hydroxyl of
Ser174 and between the 4-oxo group and the side chain NH of
Arg103 and His135. The pyrrolo[2,3-d]pyrimidine scaffold is
sandwiched between the side chains of Tyr60 and Tyr171, similar
to the pteridine ring seen in the bound conformation of folic acid.
The pyrrole NH of 3c does not interact with the binding pocket.
The flexible C6-substituted chain in 3c enables the glutamate
moiety to be oriented similarly to the corresponding groups
in folic acid. The α-carboxylic acid of 3c can form hydrogen
bonds with the backbone NH of Gly137 and Trp138, while the
γ-carboxylic acid interacts with the backbone NH of Lys136 and
the side chain NH of Trp102. The hydrophobic C6-linker of 3c
can interact in the linker region with surrounding hydrophobic
amino acids such as Tyr60, Phe62, Trp102, Trp134, and His135.
The docking score of 3c with FRα was −46.4850.
In this report, we continued our systematic study of structural
determinants of cellular uptake by RFC, PCFT, and FRs α and -β.
A novel series of classical 6-substituted straight chain pyrrolo-
[2,3-d]pyrimidine antifolates 3a−d was designed and synthe-
sized as analogues of 2a with replacement of the phenyl ring by a
linear carbon chain of variable lengths (n = 2−5) with or without
the terminal L-glutamate. Our results demonstrate, for the first
time, that the presence of the phenyl side chain ring is not
essential for binding and cellular uptake by FRs. There is an
absolute requirement for a terminal L-glutamate, and there is an
optimal distance for the alkyl linker separating the pyrrolo[2,3-
d]pyrimidine ring system and the L-glutamate moiety. The lack of
a requirement of the phenyl side chain for FR-mediated cellular
uptake is consistent with structural data for FRα23 and our
docking results with compound 3c and FRα, which show that this
region of the molecule interacts with FRα only via hydrophobic
interactions.
There were modest differences in surface binding to FRα
and -β at neutral pH between the active analogues, 3b−d, with
binding to FRα exceeding that for FRβ. However, this did not
translate into corresponding differences in in vitro drug efficacies
toward isogenic CHO cell line models expressing one or the
other transport system. Rather, inhibition of proliferation of
FRβ-expressing CHO cells exceeded that for FRα-expressing
CHO cells. This apparent discrepancy may reflect differences in
relative affinities of bound substrates at the acidic pH conditions
of the endosome for FR α and -β. FRβ but not FRα was found
to show (by isothermal titration calorimetry) a pH-dependent
decrease in binding affinities for number of classic antifolates
(MTX, aminopterin, PMX) at acidic versus neutral pH.24 By
analogy, for the linear 6-substituted pyrrolo[2,3-d]pyrimidine
CONCLUSIONS
■
There is growing interest in tumor targeting of cytotoxic folate-
based therapeutics via their selective cellular uptake by FRs or
PCFT over RFC.5,6,11−16 Specific uptake by FRs or PCFT but
not RFC would afford these analogues selectivity for tumor cells
expressing FRα and -β and/or PCFT and significant advantages
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N-nitroso-N-methylurea) over 10 min. To this solution was added
48% HBr (1.5 mL). The resulting mixture was refluxed for 1.5 h. After
the mixture was cooled to room temperature, the organic layer was
separated and the aqueous layer was extracted with ethyl ether (20 mL× 3).
The combined organic layers were washed with two portions of 10%
Na2CO3 solution and dried over Na2SO4. The solvent was evaporated to
afford 0.38 g 5a: yield 79% as white crystals, mp 68−69 °C, Rf = 0.50
(hexane/EtOAc, 5:1). 1H NMR (CDCl3) δ 1.24−1.27 (t, 3 H, CH3, J =
7.5 Hz), 1.32−1.37 (m, 2 H, CH2), 1.62−1.65 (m, 4 H, 2 CH2), 2.28−2.31
(t, 2 H, CH2, J = 7.5 Hz), 2.65−2.69 (m, 2 H, CH2), 3.87 (s, 2H, CH2Br),
4.10−4.14 (q, 2 H, CH2, J = 7.5 Hz).
Methyl 9-Bromo-8-oxononanoate (5b). Compound 5b was
synthesized as described for 5a: yield 83% as light-yellow crystals, mp
70−71 °C, Rf = 0.51 (hexane/EtOAc, 5:1). 1H NMR (CDCl3) δ 1.32−
1.33 (m, 4 H, 2 CH2), 1.60−1.63 (m, 4 H, 2 CH2), 2.28−2.31 (t, 2 H,
CH2, J = 7.5 Hz), 2.63−2.66 (t, 2 H, CH2, J = 7.5 Hz), 3.66 (s, 3H, CH3),
3.87 (s, 2H, CH2Br).
Methyl 10-Bromo-9-oxodecanoate (5c). Compound 5c was
synthesized as described for 5a: yield 78% as yellow crystals, mp 123−
124 °C, Rf = 0.51 (hexane/EtOAc, 5:1). 1H NMR (CDCl3) δ 1.31−1.33
(m, 6 H, 3 CH2), 1.60−1.62 (m, 4 H, 2 CH2), 2.28−2.31 (t, 2 H, CH2,
J = 7.5 Hz), 2.63−2.66 (t, 2 H, CH2, J = 7.5 Hz), 3.66 (s, 3H, CH3), 3.87
(s, 2H, CH2Br).
Methyl 11-Bromo-10-oxoundecanoate (5d). Compound 5d
was synthesized as described for 5a: yield 72% as yellow crystals, mp
92−93 °C, Rf = 0.52 (hexane/EtOAc, 3:1). 1H NMR (CDCl3) δ 1.31−
1.33 (m, 8 H, 4 CH2), 1.59−1.62 (m, 4 H, 2 CH2), 2.28−2.31 (t, 2 H,
CH2, J = 7.5 Hz), 2.62−2.65 (t, 2 H, CH2, J = 7.5 Hz), 3.66 (s, 3H, CH3),
3.87 (s, 2H, CH2Br).
Ethyl 6-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)hexanoate (7a). To a suspension of 2,4-diamino-6-
hydroxypyrimidine 6 (1.26 g, 10.0 mmol) in anhydrous DMF (40 mL)
was added 5a (2.64 g, 10.0 mmol). The resulting mixture was stirred
under N2 at room temperature for 3 days. TLC showed the dis-
appearance of starting materials and the formation of one major spot.
After evaporation of solvent, CH3OH (20 mL) was added followed by
silica gel (5 g). Evaporation of the solvent afforded a plug, which was
loaded onto a silica gel column (3.5 cm × 15 cm) and eluted initially with
CHCl3 followed by 10% MeOH in CHCl3 and then 15% MeOH in
CHCl3. Fractions showing Rf = 0.39 were pooled and evaporated to
afford 1.20 g 7a: yield 41% as a yellow solid, mp 189−190 °C, Rf = 0.39
(CHCl3/MeOH, 5:1). 1H NMR (DMSO-d6) δ 1.15−1.17 (t, 3 H, CH3,
J = 7.5 Hz), 1.26−1.31 (m, 2 H, CH2), 1.52−1.57 (m, 4 H, 2 CH2),
2.25−2.28 (t, 2 H, CH2, J = 7.5 Hz), 2.44−2.47 (t, 2 H, CH2, J = 7.5 Hz),
4.01−4.06 (m, 2 H, OCH2), 5.84 (s, 1 H, CH), 5.94 (s, 2 H, 2-NH2),
10.10 (s, 1 H, 3-NH), 10.77 (s, 1H, 7-NH).
analogues, in spite of their slightly decreased binding affinities for
FRβ at pH 7.4, bound drugs may be more effectively released in
the endosome than drugs bound to FRα.
For both FRα and -β, the most active compounds of the
present series, compounds 3c and 3d, bound with the highest
affinities and inhibited proliferation of FR-expressing CHO and
KB tumor cells with nanomolar potencies approaching those
of compounds 2a and 2b, among the most potent 6-substituted
pyrrolo[2,3-d]pyrimidine analogues previously reported by us,5,11
albeit without any evidence of PCFT transport. Likewise, there
was no indication of RFC membrane transport, as an engineered
CHO cell line expressing ample human RFC and established in
vitro cytotoxicities to RFC antifolate substrates was completely
unaffected by the most potent non-benzoyl analogues.
Our results establish that although the phenyl side chain is
not essential for FR binding and cellular uptake, it appears to
be absolutely necessary for PCFT transport. Inhibition of cell
proliferation was attributable to potent inhibition of GARFTase
in de novo purine nucleotide bioynthesis at concentrations
approximating those which inhibited cell proliferation. Thus,
our results are entirely consistent with previous reports that
the absence of a side chain phenyl ring in a series of non-benzoyl
analogues of LMTX analogues preserved substantial catalytic
activity for FPGS and inhibition of isolated GARFTase.19,20
The structural simplicity and extraordinary antitumor activities of
the 6-substituted straight chain compounds described herein
provide an excellent starting point for future rational design
efforts to further optimize selective cellular uptake by FRs and
GARFTase inhibition as an important step toward discovering
additional potent targeted antitumor agents.
EXPERIMENTAL SECTION
■
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 80 °C. Melting points were determined on a
MEL-TEMP II melting point apparatus with a FLUKE 51 K/J electronic
thermometer and are uncorrected. Nuclear magnetic resonance spectra
for proton (1H NMR) were recorded on either a Bruker WH-400
(400 MHz) spectrometer or a Bruker WH-500 (500 MHz) spectrometer.
The chemical shift values are expressed in ppm (parts per million) relative
to tetramethylsilane as an internal standard: s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; br, broad singlet. Mass spectra were recorded
on a VG-7070 double-focusing mass spectrometer or in a LKB-9000
instrument in the electron ionization (EI) mode. Chemical names follow
IUPAC nomenclature. Thin-layer chromatography(TLC) was performed
on Whatman Sil G/UV254 silica gel plates with a fluorescent indicator,
and the spots were visualized under 254 and 365 nm illumination. All
analytical samples were homogeneous on TLC in three different solvent
systems. Proportions of solvents used for TLC are by volume. Column
chromatography was performed on a 230−400 mesh silica gel (Fisher,
Somerville, NJ) column. Elemental analyses were performed by Atlantic
Microlab, Inc., Norcross, GA. Element compositions are within 0.4% of
the calculated values. Fractional moles of water frequently found in the
analytical sample of antifolates could not be prevented in spite of 24−48 h
of drying in vacuo and were confirmed where possible by the presence in
Methyl 7-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)heptanoate (7b). Compound 7b was synthesized as
described for 7a: yield 39% as a yellow solid, mp 178−179 °C, Rf = 0.39
1
(CHCl3/MeOH, 5:1). H NMR (DMSO-d6) δ 1.24−1.29 (m, 4 H,
2 CH2), 1.49−1.56 (m, 4 H, 2 CH2), 2.27−2.30 (t, 2 H, CH2, J = 7.5 Hz),
2.44−2.47 (m, 2 H, CH2), 3.57 (s, 3 H, OCH3), 5.83 (s, 1 H, CH), 5.94
(s, 2 H, 2-NH2), 10.11 (s, 1 H, 3-NH), 10.77 (s, 1H, 7-NH).
Methyl 8-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)octanoate (7c). Compound 7c was synthesized as
described for 7a: yield 43% as a yellow solid, mp 162−163 °C, Rf = 0.41
1
(CHCl3/MeOH, 5:1). H NMR (DMSO-d6) δ 1.21−1.30 (m, 6 H, 3
CH2), 1.49−1.56 (m, 4 H, 2 CH2), 2.27−2.29 (t, 2 H, CH2, J = 7.5 Hz),
2.43−2.47 (t, 2 H, CH2, J = 7.5 Hz), 3.57 (s, 3 H, OCH3), 5.84 (s, 1 H,
CH), 6.00 (s, 2 H, 2-NH2), 10.16 (s, 1 H, 3-NH), 10.79 (s, 1H, 7-NH).
Methyl 9-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)nonanoate (7d). Compound 7d was synthesized as
described for 7a: yield 42% as a yellow solid, mp 165−166 °C, Rf = 0.41
1
the H NMR spectra. All solvents and chemicals were purchased from
Aldrich Chemical Co. or Fisher Scientific and were used as received. For
all the compounds submitted for biological evaluation, elemental analysis
(C, H, N) was performed to confirm >95% purity.
Ethyl 8-Bromo-7-oxooctanoate (5a). To a solution of 7-methoxy-
7-oxoheptanoic acid (4a) (0.32 g, 1.5 mmol) in a 50 mL flask was added
oxalyl chloride (1.5 mL) and anhydrous CH2Cl2 (10 mL). The resulting
solution was refluxed for 1 h and then cooled to room temperature. After
evaporation of solvent under reduced pressure, the residue was dissolved
in ethyl ether (20 mL). The resulting solution was added dropwise to an
ice-cold ether solution of diazomethane (generated in situ from 1.4 g
1
(CHCl3/MeOH, 5:1). H NMR (DMSO-d6) δ 1.21−1.30 (m, 8 H,
4 CH2), 1.49−1.56 (m, 4 H, 2 CH2), 2.26−2.29 (t, 2 H, CH2, J = 7.5 Hz),
2.44−2.47 (t, 2 H, CH2, J = 7.5 Hz), 3.57 (s, 3 H, OCH3), 5.85 (s, 1 H,
CH), 6.03 (s, 2 H, 2-NH2), 10.20 (s, 1 H, 3-NH), 10.81 (s, 1H, 7-NH).
6-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-
6-yl)hexanoic Acid (7e). To a solution of the ester 7a (100 mg,
0.26 mmol) was added 1N NaOH (5 mL), and the mixture was stirred
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under N2 at room temperature for 1 h. TLC showed the disappearance
of the starting material and formation of one major spot at the origin
(CHCl3/MeOH, 5:1). The resulting solution was cooled in an ice bath,
and the pH was adjusted to 3−4 with dropwise addition of 1N HCl or
acetic acid. The resulting suspension was frozen in a dry ice/acetone
bath, thawed in a refrigerator to 4−5 °C, and filtered. The residue was
washed with a small amount of cold water, dichloromethane, and ethyl
acetate and dried in vacuo using P2O5 to afford 81 mg 7e: yield 93% as a
yellow powder, mp 188−189 °C decomposed, Rf = 0.06 (CHCl3/
MeOH, 5:1). 1H NMR (DMSO-d6) δ 1.20−1.30 (m, 2 H, CH2), 1.47−
1.56 (m, 4 H, 2 CH2), 2.17−2.20 (t, 2 H, CH2, J = 7.5 Hz), 2.43−2.47
(t, 2 H, CH2, J = 7.5 Hz), 5.83 (s, 1 H, CH), 5.94 (s, 2 H, 2-NH2), 10.11
(s, 1 H, 3-NH), 10.77 (s, 1H, 7-NH), 11.95 (s, 1H, COOH). Anal.
(C12H16N4O3·0.2 CH3COOC2H5·0.2CH3COOH) C, H, N.
7-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-
6-yl)heptanoic Acid (7f). Compound 7f was synthesized as described
for 7e: yield 91% as a brown powder, mp 200−201 °C decomposed, Rf =
0.08 (CHCl3/MeOH, 5:1). 1H NMR (DMSO-d6) δ 1.20−1.30 (m, 4 H,
2 CH2), 1.47−1.56 (m, 4 H, 2 CH2), 2.17−2.20 (t, 2 H, CH2, J = 7.5 Hz),
2.44−2.47 (t, 2 H, CH2, J = 7.5 Hz), 5.83 (s, 1 H, CH), 5.94 (s, 2 H,
2-NH2), 10.11 (s, 1 H, 3-NH), 10.77 (s, 1H, 7-NH), 11.95 (s, 1H,
COOH). Anal. (C13H18N4O3·0.6CH3OH) C, H, N.
(S)-Diethyl 2-(8-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-6-yl)octanamido)pentanedioate (8c). Compound 8c
was synthesized as described for 8a: yield 68% as a yellow syrup, Rf =
0.64 (CHCl3/MeOH, 5:1). 1H NMR (DMSO-d6) δ 1.16−1.19 (t, 6 H,
2 CH3, J = 7.0 Hz), 1.22−1.32 (m, 6 H, 3 CH2), 1.47−1.57 (m, 4 H,
2 CH2), 1.76−1.82 (m, 1 H, CH), 1.92−1.99 (m, 1 H, CH), 2.09−2.12
(t, 2 H, CH2, J = 7.0 Hz), 2.34−2.37 (m, 2 H, CH2), 2.45−2.48 (m, 2 H,
CH2), 4.02−4.08 (m, 4 H, 2 CH2), 4.20−4.24 (m, 1 H, CH), 5.84 (s,
1 H, CH), 5.94 (s, 2 H, 2-NH2), 8.15 (d, 1 H, CONH, J = 3.8 Hz), 10.10
(s, 1 H, 3-NH), 10.76 (s, 1H, 7-NH).
(S)-Diethyl 2-(9-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-6-yl)nonanamido)pentanedioate (8d). Compound
8d was synthesized as described for 8a: yield 66% as a yellow syrup, Rf =
0.64 (CHCl3/MeOH, 5:1). 1H NMR (DMSO-d6) δ 1.15−1.18 (t, 6 H,
2 CH3, J = 7.0 Hz), 1.22−1.32 (m, 8 H, 4 CH2), 1.47−1.57 (m, 4 H,
2 CH2), 1.76−1.82 (m, 1 H, CH), 1.92−1.99 (m, 1 H, CH), 2.09−2.12
(t, 2 H, CH2, J = 7.0 Hz), 2.34−2.37 (m, 2 H, CH2), 2.45−2.48 (m, 2 H,
CH2), 4.02−4.08 (m, 4 H, 2 CH2), 4.20−4.24 (m, 1 H, CH), 5.83 (s,
1 H, CH), 5.94 (s, 2 H, 2-NH2), 8.15 (d, 1 H, CONH, J = 3.8 Hz), 10.10
(s, 1 H, 3-NH), 10.77 (s, 1H, 7-NH).
(S)-2-(6-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)hexanamido)pentanedioic Acid (3a). To a sol-
ution of the diester 8a (100 mg, 0.22 mmol) was added 1N NaOH
(5 mL), and the mixture was stirred under N2 at room temperature for
1 h. TLC showed the disappearance of the starting material and forma-
tion of one major spot at the origin (CHCl3/MeOH, 5:1). The resulting
solution was cooled in an ice bath, and the pH was adjusted to 3−4 with
dropwise addition of 1N HCl. The resulting suspension was frozen in a
dry ice/acetone bath, thawed in a refrigerator to 4−5 °C, and filtered.
The residue was washed with a small amount of cold water and ethyl
acetate and dried in vacuo using P2O5 to afford 78 mg 3a: yield 90% as a
white powder, mp 145−146 °C decomposed, Rf = 0.08 (CHCl3/MeOH,
5:1). 1H NMR (DMSO-d6) δ 1.24−1.30 (m, 2 H, CH2), 1.48−1.58 (m,
4 H, 2 CH2), 1.71−1.79 (m, 1 H, CH), 1.88−1.95 (m, 1 H, CH), 2.09−
2.12 (t, 2 H, CH2, J = 7.5 Hz), 2.24−2.27 (m, 2 H, CH2, J = 7.5 Hz),
2.44−2.47 (m, 2 H, CH2), 4.16−4.21 (m, 1 H, CH), 5.84 (s, 1 H, CH),
5.95 (s, 2 H, 2-NH2), 7.99 (d, 1 H, CONH, J = 4.0 Hz), 10.10 (s, 1 H,
3-NH), 10.76 (s, 1H, 7-NH), 12.50 (br, 2 H, 2 COOH). Anal.
(C17H23N5O6·2.0H2O) C, H, N.
8-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-
6-yl)octanoic Acid (7g). Compound 7g was synthesized as described
for 7e: yield 89% as a brown powder, mp 211−212 °C decomposed, Rf =
0.06 (CHCl3/MeOH, 5:1). 1H NMR (DMSO-d6) δ 1.20−1.30 (m, 6 H,
3 CH2), 1.47−1.56 (m, 4 H, 2 CH2), 2.17−2.20 (t, 2 H, CH2, J = 7.5 Hz),
2.43−2.47 (t, 2 H, CH2, J = 7.5 Hz), 5.83 (s, 1 H, CH), 5.94 (s, 2 H,
2-NH2), 10.10 (s, 1 H, 3-NH), 10.76 (s, 1H, 7-NH), 11.94 (s, 1H,
COOH). Anal. (C14H20N4O3·0.4H2O) C, H, N.
9-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-
6-yl)nonanoic Acid (7h). Compound 7h was synthesized as described
for 7e: yield 95% as a brown powder, mp 186 °C decomposed, Rf = 0.09
1
(CHCl3/MeOH, 5:1). H NMR (DMSO-d6) δ 1.20−1.30 (m, 8 H,
4 CH2), 1.45−1.56 (m, 4 H, 2 CH2), 2.17−2.20 (t, 2 H, CH2, J = 7.5 Hz),
2.44−2.47 (t, 2 H, CH2, J = 7.5 Hz), 5.83 (s, 1 H, CH), 5.97 (s, 2 H,
2-NH2), 10.13 (s, 1 H, 3-NH), 10.77 (s, 1H, 7-NH), 11.95 (s, 1H,
COOH). Anal. (C15H22N4O3·0.46CH2Cl2) C, H, N.
(S)-Diethyl 2-(6-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-6-yl)hexanamido)pentanedioate (8a). To a suspen-
sion of 7e (81 mg, 0.34 mmol) in anhydrous DMF (5 mL) was
added 6-chloro-2,4-dimethoxy-1,3,5-triazine (72 mg, 0.42 mmol) and
N-methylmorpholine (43 mg, 0.42 mmol). After the mixture was stirred
at rt for 2 h, N-methylmorpholine (43 mg, 0.42 mmol) and diethyl
L-glutamate hydrochloride (120 mg, 0.51 mmol) were added all at once.
The mixture was stirred at rt for 4 h. TLC showed the formation of
one major spot at Rf = 0.62 (CHCl3/MeOH, 5:1). The reaction mixture
was evaporated to dryness under reduced pressure. The residue was
dissolved in a minimum amount of CHCl3/MeOH, 5:1 and chromato-
graphed on a silica gel column (2 cm ×15 cm) with 4% MeOH in CHCl3
as the eluent. Fractions that showed the desired single spot at Rf = 0.62
were pooled and evaporated to dryness to afford 8a 102 mg: yield 67% as
a yellow syrup, Rf = 0.62 (CHCl3/MeOH, 5:1). 1H NMR (DMSO-d6) δ
1.15−1.18 (t, 6 H, 2 CH3, J = 7.0 Hz), 1.23−1.29 (m, 2 H, CH2), 1.48−
1.58 (m, 4 H, 2 CH2), 1.76−1.82 (m, 1 H, CH), 1.92−1.98 (m, 1 H,
CH), 2.09−2.12 (t, 2 H, CH2, J = 7.0 Hz), 2.33−2.37 (m, 2 H, CH2),
2.44−2.47 (m, 2 H, CH2), 4.02−4.07 (m, 4 H, 2 CH2), 4.19−4.24 (m,
1 H, CH), 5.84 (s, 1 H, CH), 5.94 (s, 2 H, 2-NH2), 8.16 (d, 1 H, CONH,
J = 3.8 Hz), 10.10 (s, 1 H, 3-NH), 10.76 (s, 1H, 7-NH).
(S)-2-(7-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)heptanamido)pentanedioic Acid (3b). Com-
pound 3b was synthesized as described for 3a: yield 91% as a pale-
yellow powder, mp 116−117 °C decomposed, Rf = 0.08 (CHCl3/MeOH,
5:1). 1H NMR (DMSO-d6) δ 1.22−1.32 (m, 4 H, 2 CH2), 1.45−1.58 (m,
4 H, 2 CH2), 1.71−1.79 (m, 1 H, CH), 1.91−1.98 (m, 1 H, CH), 2.09−
2.12 (t, 2 H, CH2, J = 7.5 Hz), 2.24−2.28 (m, 2 H, CH2, J = 7.5 Hz), 2.44−
2.47 (m, 2 H, CH2), 4.16−4.21 (m, 1 H, CH), 5.84 (s, 1 H, CH), 5.94 (s,
2 H, 2-NH2), 8.02 (d, 1 H, CONH, J = 4.0 Hz), 10.10 (s, 1 H, 3-NH),
10.76 (s, 1H, 7-NH), 12.12 (br, 1 H, 1 COOH), 12.52 (br, 1 H, 1 COOH).
Anal. (C18H25N5O6·0.5H2O) C, H, N.
(S)-2-(8-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)octanamido)pentanedioic Acid (3c). Compound
3c was synthesized as described for 3a: yield 95% as a yellow powder, mp
1
135−136 °C decomposed, Rf = 0.08 (CHCl3/MeOH, 5:1). H NMR
(DMSO-d6) δ 1.20−1.32 (m, 6 H, 3 CH2), 1.45−1.58 (m, 4 H, 2 CH2),
1.71−1.79 (m, 1 H, CH), 1.90−1.98 (m, 1 H, CH), 2.08−2.12 (t, 2 H,
CH2, J = 7.5 Hz), 2.24−2.28 (m, 2 H, CH2, J = 7.5 Hz), 2.44−2.47 (t,
2 H, CH2, J = 7.5 Hz), 4.16−4.21 (m, 1 H, CH), 5.84 (s, 1 H, CH), 5.94
(s, 2 H, 2-NH2), 8.02 (d, 1 H, CONH, J = 3.8 Hz), 10.11 (s, 1 H, 3-NH),
10.76 (s, 1H, 7-NH). Anal. (C19H27N5O6·0.5H2O) C, H, N.
(S)-Diethyl 2-(7-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-6-yl)heptanamido)pentanedioate (8b). Compound
8b was synthesized as described for 8a: yield 70% as a yellow syrup, Rf =
0.62 (CHCl3/MeOH, 5:1). 1H NMR (DMSO-d6) δ 1.15−1.18 (m, 6 H,
2 CH3), 1.23−1.30 (m, 4 H, 2 CH2), 1.48−1.58 (m, 4 H, 2 CH2), 1.76−
1.82 (m, 1 H, CH), 1.92−1.99 (m, 1 H, CH), 2.09−2.12 (t, 2 H, CH2, J =
7.0 Hz), 2.33−2.37 (m, 2 H, CH2), 2.44−2.47 (m, 2 H, CH2), 4.02−4.08
(m, 4 H, 2 CH2), 4.20−4.24 (m, 1 H, CH), 5.83 (s, 1 H, CH), 5.93 (s,
2 H, 2-NH2), 8.15 (d, 1 H, CONH, J = 3.8 Hz), 10.10 (s, 1 H, 3-NH),
10.77 (s, 1H, 7-NH).
(S)-2-(9-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)nonanamido)pentanedioic Acid (3d). Compound
3d was synthesized as described for 3a: yield 90% as a pale-yellow
powder, mp 123−124 °C, decomposed, Rf = 0.08 (CHCl3/MeOH, 5:1).
1H NMR (DMSO-d6) δ 1.20−1.32 (m, 8 H, 4 CH2), 1.42−1.58 (m, 4 H,
2 CH2), 1.71−1.79 (m, 1 H, CH), 1.90−1.98 (m, 1 H, CH), 2.08−2.11
(t, 2 H, CH2, J = 7.5 Hz), 2.24−2.28 (m, 2 H, CH2, J = 7.5 Hz), 2.44−
2.47 (t, 2 H, CH2, J = 7.5 Hz), 4.16−4.21 (m, 1 H, CH), 5.84 (s, 1 H,
CH), 5.94 (s, 2 H, 2-NH2), 8.01 (d, 1 H, CONH, J = 3.8 Hz), 10.11 (s,
1 H, 3-NH), 10.76 (s, 1H, 7-NH). Anal. (C20H29N5O6·0.5 H2O) C, H, N.
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Molecular Modeling and Computational Studies. The X-ray
crystal structure of human GARFTase at 1.98 Å resolution (PDB ID:
1NJS)22 was obtained from the protein database. The human GARFTase
crystal structure contains GARFTase complexed with the hydrolyzed
form of 10-trifluoroacetyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic
acid (10-CF3CO-DDACTHF). Molecular modeling studies with FRα
were carried out using the 2.80 Å crystal structure of human FRα
cocrystallized with folic acid (PDB: 4LRH).23
1640 (contains 2.3 μM folic acid) with 10% dFBS and antibiotics for
experiments with R2 and PC43-10 cells. For RT16 and KB cells, cells
were cultured in folate-free RPMI media with 10% dFBS and antibiotics
supplemented with 2 nM LCV and 2 mM L-glutamine. The requirement
for FR-mediated drug uptake in these assays was established in parallel
incubations including 200 nM folic acid. For R2/hPCFT4 cells, the
medium was folate-free RPMI 1640 (pH 7.2) containing 25 nM LCV,
supplemented with 10% dFBS, antibiotics, and L-glutamine. Cells
were routinely incubated for up to 96 h, and metabolically active cells (a
measure of cell viability) were assayed with CellTiter-blue cell viability
assay (Promega, Madison, WI), with fluorescence measured (590 nm
emission, 560 nm excitation) using a fluorescence plate reader.11−16 Raw
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.11−16
For some of the in vitro growth inhibition studies, the inhibitory
effects of the antifolate inhibitors on de novo thymidylate biosynthesis
(i.e., thymidylate synthase) and de novo purine nucleotide biosynthesis
(GARFTase and AICARFTase) were tested by coincubations with
thymidine (10 μM) and adenosine (60 μM), respectively.11−17 For de
novo purine nucleotide biosynthesis, additional protection experiments
used AICA (320 μM) to distinguish inhibitory effects at GARFTase
from those at AICARFTase.11−17
Docking studies were performed using LeadIT 2.1.3.25 The
protonation state of the proteins and the ligands were calculated using
the default settings. Water molecules in the active site were permitted to
rotate freely. The active site was defined by a sphere of 6.5 Å from the
native ligand in the crystal structure. Ligands for docking were built
using the molecule builder function in MOE 2010.1026 and energy
minimized using the MMF94X forcefield 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
exported and visualized in MOE.
To validate the docking software for docking the proposed com-
pounds, the native ligands for GARFTase (10-CF3CO-DDACTHF)
and FRα (folic acid) were built using the molecule builder function in
MOE, energy minimized and docked using LeadIT 2.1.3 as described
above. RMSDs of the docked poses were calculated using an SVL code
obtained from the MOE web site (www.chemcomp.com) and compared
to the conformation of the crystal structure ligands. The best docked
pose for 10-CF3CO-DDACTHF in the human GARFTase crystal
structure had an RMSD of 1.0437 Å (docking score: −40.0853), while
the best docked pose for FRα had an RMSD of 0.633 Å (docking score:
−55.5940). Thus, LeadIT 2.1.3 was validated for our docking purposes
in GARFTase and FRα.
FR Binding Assay. Competitive inhibition of [3H]folic acid binding
to FRα and FRβ using RT16 and D4 CHO cells, respectively, was used
was used to assess relative binding affinities for assorted (anti)folate
ligands.11,12,14−16 For these experiments, cells (∼1.6 × 106) were rinsed
twice with DPBS, followed by two washes an acidic buffer (10 mM
sodium acetate, 150 mM NaCl, pH 3.5) to remove FR-bound folates.
Cells were washed twice with ice-cold HEPES-buffered saline (20 mM
HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 5 mM glucose,
pH 7.4) (HBS), then 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 antifolates (over a range of concentrations) for 15 min
at 0 °C. The dishes were rinsed three times with ice-cold HBS, after
which the cells were solubilized (0.5 N NaOH) and aliquots measured
for radioactivity and protein contents. Protein concentrations were
measured with Folin phenol reagent.27 [3H]Folic acid bound to FRα
and FRβ was calculated as pmol/mg protein, and relative binding
affinities were calculated in each individual experiment as the inverse
molar ratios of unlabeled ligands required to inhibit [3H]folic acid
binding by 50%.11,12,14−16 By definition, the relative affinity of folic acid
is 1. Experiments were performed in triplicate.
In Situ GARFT Enzyme Inhibition Assay. Incorporation of
[14C]glycine into [14C]formyl GAR, as an in situ measure of endogenous
GARFTase activity, was measured.6,11−17 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. Azaserine (4 μM final concentration) was
added in the presence and absence of the antifolate inhibitors. After
30 min, L-glutamine (final concentration, 2 mM) and [14C]glycine(tracer
amounts; 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 PBS. Cell pellets were dissolved in 2 mL of 5% trichloroacetic acid
at 0 °C. The trichloracetic acid insoluble fraction was removed by
centrifugation and solubilized in 0.5N NaOH, and the cell protein
contents were measured with Folin phenol reagent.27 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 (Bio-Rad), washed with 10 mL of 0.5N formic acid and then 10 mL
of 4N formic acid, and finally eluted with 8 mL of 1N HCl. The eluates
were collected and determined for radioactivity. The accumulation of
[14C]formyl GAR 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 [14C]formyl GAR synthesis.
Experiments were performed in triplicate.
Reagents for Biological Studies. [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 Sigma Chemical Co. (St. Louis, MO). LCV [(6R,S)-5-formyl
tetrahydrofolate] was provided by the Drug Development Branch,
National Cancer Institute, Bethesda, MD. The sources of the classical
antifolate drugs were as follows: MTX, Drug Development Branch,
National Cancer Institute (Bethesda, MD); RTX [N-(5-[N-(3,4-
dihydro-2-methyl-4-oxyquinazolin-6-ylmethyl)-N-methyl-amino]-2-
thienoyl)-L-glutamic acid], AstraZeneca Pharmaceuticals (Maccesfield,
Cheshire, England); PDX ((2S)-2-[[4-[(1RS)-1-[(2, 4-diaminopter-
idin-6-yl)methyl]but-3-ynyl]benzoyl]amino]pentanedioic acid), Allos
Therapeutics (Henderson, NV); and LMTX (5,10-dideaza-5,6,7,8-
tetrahydrofolate) and PMX [N-{4-[2-(2-amino-3,4-dihydro-4-oxo-7H-
pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic acid] (Alimta),
Eli Lilly and Co. (Indianapolis, IN). Other chemicals were obtained
from commercial sources in the highest available purity.
Cell Lines and Assays of Antitumor Drug Activities. The origin
of the engineered CHO cell lines, including the RFC-, PCFT-, and
FRα-null MTXRIIOuaR2−4 (R2), and RFC- (pC43-10), PCFT-
(R2/PCFT4), or FRα- (RT16) expressing sublines, were previously
described.11,12,21 Likewise, pDNA3.1 vector control CHO cells (R2/VC)
were reported.11,12 The CHO cells were cultured in α-minimal essential
medium (MEM) supplemented with 10% bovine calf serum (Invitrogen,
Carlsbad, CA), 100 units/ml penicillin/100 μg/mL streptomycin, and
2 mM L-glutamine at 37 °C with 5% CO2. All the R2 transfected cells
[PC43-10, RT16, R2/hPCFT4, R2(VC)] were routinely cultured in
α-MEM plus 1.5 mg/mL G418. Prior to the cell proliferation assays (see
below), RT16 cells were cultured in complete folate-free RPMI 1640
(without added folate) for three days. R2/hPCFT4 and R2(VC) cells
were cultured in complete folate-free RPMI 1640 including dFBS
(Invitrogen) and 25 nM LCV with 1.5 mg/mL G418. KB human
nasopharyngeal carcinoma cells were purchased from the American Type
Culture Collection (Manassas, VA). KB cells were routinely cultured in
folate-free RPMI 1640 medium, supplemented with 10% fetal bovine
serum, penicillin−streptomycin solution, and 2 mM L-glutamine at 37 °C
with 5% CO2.
For growth inhibition assays, cells (CHO and KB) were plated in 96
well dishes (∼2500−5000 cells/well, total volume of 200 μL medium)
with a range of antifolate concentrations.11−16 The medium was RPMI
8693
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Journal of Medicinal Chemistry
Article
Systemic Compartments and Tissues. Expert Rev. Mol. Med. 2009, 11,
e4.
ASSOCIATED CONTENT
* Supporting Information
Elemental analysis. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
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Kushner, J.; Stout, M.; Hou, Z.; Cherian, C.; Gangjee, A.; Matherly, L. H.
Therapeutic Targeting of a Novel 6-Substituted Pyrrolo[2,3-d]-
pyrimidine Thienoyl Antifolate to Human Solid Tumors Based on
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d]pyrimidine Thienoyl Antifolate Inhibitors of Purine Biosynthesis with
Selectivity for High Affinity Folate Receptors and the Proton-Coupled
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Synthesis, Biological, and Antitumor Activity of a Highly Potent 6-
Substituted Pyrrolo[2,3-d] pyrimidine Thienoyl Antifolate Inhibitor
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AUTHOR INFORMATION
Corresponding Authors
■
*For L.H.M.: phone, 313-578-4280; fax, 313-578-4287; E-mail,
matherly@karmanos.org; address, Molecular Therapeutics Pro-
gram, Barbara Ann Karmanos Cancer Institute, 110 East Warren
Avenue, Detroit, Michigan 48201, United States.
*For A.G.: phone, 412-396-6070; fax, 412-396-5593; E-mail,
gangjee@duq.edu; address, Division of Medicinal Chemistry,
Graduate School of Pharmaceutical Sciences, Duquesne
University, 600 Forbes Avenue, Pittsburgh, Pennsylvania
15282, United States.
Present Address
⊥Center for Drug Discovery and Translational Research, Beth
Israel Deaconess Medical Center, Harvard Medical School,
Boston Massachusetts 02215, United States. Phone: 617-667-
1839. E-mail: ywang15@bidmc.harvard.edu.
Author Contributions
#Y.W. and C.C. contributed equally to this work. L.H.M. and
A.G. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by grants from the National
Institutes of Health, National Cancer Institute CA152316 (L.H.M.
and A.G.), CA166711 (A.G. and L.H.M.), and CA53535
(L.H.M.), and the Duquesne University Adrian Van Kaam Chair
in Scholarly Excellence (A.G.). S.M.-R. was supported by T32
CA009531 (L.H.M.) and F31 CA165853 (S.M.-R.).
ABBREVIATIONS USED
■
AICA, 5-aminoimidazole-4-carboxamide; AICARFTase, 5-ami-
noimidazole-4-carboxamide ribonucleotide formyltransferase;
CHO, Chinese hamster ovary; dFBS, dialyzed fetal bovine
serum; DPBS, Dulbecco’s phosphate-buffered saline; FR, folate
receptor; FPGS, folylpolyglutamate synthetase; GAR, glycina-
mide ribonucleotide; GARFTase, glycinamide ribonucleotide
formyltransferase; HBSS, Hank’s Balanced Salts Solution; HBS,
HEPES-buffered saline; LCV, leucovorin; LMTX, lometrexol;
MTX, methotrexate; MEM, minimal essential media; PMX,
pemetrexed; PDX, pralatrexate; PCFT, proton-coupled folate
transporter; RTX, raltiterxed; RFC, reduced folate carrier; SAR,
structure−activity relationship; 10-CF3CO-DDACTHF, 10-
trifluoroacetyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid
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