Synthesis and biological activity of 6-substituted pyrrolo[2,3-d ]pyrimidine thienoyl regioisomers as inhibitors of de novo purine biosynthesis with selectivity for cellular uptake by high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier

Article abstract of DOI:10.1021/jm201688n

We previously reported the selective transport of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl-for-benzoyl-substituted side chain and a three- (3a) and four-carbon (3b) bridge. Compound 3a was more potent than 3b against tumor cells. While 3b was completely selective for transport by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) over the reduced folate carrier (RFC), 3a was not. To determine if decreasing the distance between the bicyclic scaffold and l-glutamate in 3b would preserve transport selectivity and potency against human tumor cells, 3b regioisomers with [1,3] (7 and 8) and [1,2] (4, 5, and 6) substitutions on the thienoyl ring and with acetylenic insertions in the four-atom bridge were synthesized and evaluated. Compounds 7 and 8 were potent nanomolar inhibitors of KB and IGROV1 human tumor cells with complete selectivity for FRα and PCFT over RFC.

Full text of DOI:10.1021/jm201688n

Article
pubs.acs.org/jmc
Synthesis and Biological Activity of 6-Substituted
Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Inhibitors
of de Novo Purine Biosynthesis with Selectivity for Cellular
Uptake by High Affinity Folate Receptors and the Proton-Coupled
Folate Transporter over the Reduced Folate Carrier
Lei Wang,†,# Christina Cherian,‡,∥,# Sita Kugel Desmoulin,§,∥ Shermaine Mitchell-Ryan,§ Zhanjun Hou,‡,∥
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
‡Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, Michigan 48201,
United States
∥
§Cancer Biology Graduate Program, Department of Oncology, and ⊥Department of Pharmacology, Wayne State University School
of Medicine, Detroit, Michigan 48201, United States
S
* Supporting Information
ABSTRACT: We previously reported the selective transport of classical 2-amino-4-
oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl-for-benzoyl-substituted
side chain and a three- (3a) and four-carbon (3b) bridge. Compound 3a was more
potent than 3b against tumor cells. While 3b was completely selective for transport
by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) over
the reduced folate carrier (RFC), 3a was not. To determine if decreasing the distance between the bicyclic scaffold and
L-glutamate in 3b would preserve transport selectivity and potency against human tumor cells, 3b regioisomers with [1,3]
(7 and 8) and [1,2] (4, 5, and 6) substitutions on the thienoyl ring and with acetylenic insertions in the four-atom bridge were
synthesized and evaluated. Compounds 7 and 8 were potent nanomolar inhibitors of KB and IGROV1 human tumor cells with
complete selectivity for FRα and PCFT over RFC.
non-small-cell lung carcinomas, PCFT is highly expressed.7 PCFT
INTRODUCTION
Membrane transport of antifolate therapeutics such as metho-
■
exhibits an acidic pH optimum,4,6 which is compatible with the
low pH microenvironments of the small intestine and many solid
trexate MTX is integral to chemotherapy efficacy in treating a
variety of malignancies and nonmalignant diseases.1 The major
tumors. While PCFT is modestly expressed in most other normal
tissues, for those in which PCFT is expressed they are unlikely to
present the low pH conditions optimal for membrane transport
by this mechanism.3
membrane transporters include the reduced folate carrier
(RFC), the proton-coupled folate transporter (PCFT), and
the high affinity folate receptors (FRs) α and β.1−4 While all
systems transport folate cofactors and classical antifolates such
as MTX, raltitrexed (RTX), and pemetrexed (PMX), they differ
in terms of mechanism and substrate specificities. Whereas both
RFC and PCFT are integral membrane proteins that act as
facilitative transporters, FRs are glycosyl phosphatidylinositol-
modified proteins that mediate cellular uptake of (anti)folates
by receptor-mediated endocytosis.
The major folate transporters also differ in terms of their
tissue distributions. For instance, RFC is ubiquitously expressed
in tumors and tissues and is the primary uptake mechanism for
folate cofactors.1 FRs are expressed abundantly in certain
malignancies, most notably as the FRα isoform in ovarian
carcinomas, and in a limited number of normal epithelial tissues
such as renal tubules.2 Major sites of PCFT expression include the
upper small intestine (e.g., jejunum) and the liver and kidney.3,5,6
In solid tumors such as hepatomas, ovarian carcinomas, and
Membrane transport of classical antifolates by RFC in itself
would likely result in limited drug selectivity toward tumors over
normal proliferative tissues such as bone marrow, since these
normal tissues all express RFC. The concept of selectively
targeting FR-expressing tumors with folate-based antitumor
agents has been tested. Examples of folate-based therapeutics
include 1 [(2R)-2-((4R)-4-carboxy-5-(4-((2-(hydroxymethyl)-4-
oxo-4,6,7,8-tetrahydro-3H-cyclopenta[g]quinazolin-6-yl)(prop-2-
yn-1-yl)amino)phenyl)-5-oxopentanamido)pentanedioic acid]
[ONX0801 (BGC945)], a small molecule antifolate licensed
by Onyx Pharmaceuticals which is selectively transported by FRs
and inhibits thymidylate synthase,8 and vincaleukoblastin-23-oic
acid, O4-deacetyl-, 2-[(2-mercaptoethoxy)carbonyl]hydrazide,
Received: December 14, 2011
Published: January 13, 2012
© 2012 American Chemical Society
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Figure 1. Structures of 6-substituted pyrrolo[2,3-d]pyrimidines 3a and 3b and the thienoyl regioisomers 4−13.
disulfide with N-[4-[[(2-amino-1,4-dihydro-4-oxo-6-pteridinyl)-
methyl]amino]benzoyl]-L-γ-glutamyl-L-α-aspartyl-L-α-aspartyl-L-
cysteine 2 [EC-145 (Endocyte)], a desacetylvinblastine mono-
hydrazide−folic acid complex.9 Compound 1 is in clinical trials in
the U.K. A randomized phase II trial was performed in which
2 with doxorubicin was compared to doxorubicin alone for the
treatment of platinum-resistant ovarian cancer (ClinicalTrials.gov
given the low pH microenvironments of many solid tumors that
approximate the pH optimum for PCFT.15−17 The pyrrolo[2,3-d]-
pyrimidine thienoyl analogues 3a and 3b (Figure 1) with three
and four carbons, respectively, in the bridge between the
pyrrolo[2,3-d]pyrimidine ring and the thiophene ring were the
most potent of this series against cells expressing FRs and/or
PCFT.12,13 Cytotoxicity was attributed to the potent inhibition
of β-glycinamide ribonucleotide (GAR) formyltransferase
(GARFTase), which catalyzes the first folate-dependent
reaction in de novo purine nucleotide biosynthesis.
Our finding that compound 3a was more potent than 3b
toward FR- and PCFT-expressing cells suggested that the distance
between the bicyclic scaffold and the L-glutamate may be a critical
determinant of drug activity.13 However, in engineered hamster
cells, transport selectivity for FRα and PCFT over RFC with
compound 3a was less complete than for 3b. We hypothesized
identifier: NCT00722592).
We recently identified 6-substituted pyrrolo- and thieno[2,3-d]-
pyrimidine antifolates with selective membrane transport by
PCFT and/or FRs over RFC, resulting in selective inhibition of
proliferation of cells expressing these systems including human
tumors.7,10−14 FR targeting to tumors is based on patterns of
expression and basolateral membrane localization unique to
tumor cells. In terms of PCFT, the notion of using this transport
mechanism to selectively deliver chemotherapy drugs is appealing
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a
Scheme 1
a
Reagents and conditions: (a) CuI, Pd(0)(PPh3)4, Et3N, DMF, room temp, 12 h; (b) (i) 1 N NaOH, MeOH, CHCl3, room temp, 6 h; (ii) 1 N HCl;
(c) 10% Pd/C, H2, 55 psi, 4 h.
a
that structurally engineered substitutions on the thiophene ring
of 3a would afford distances between the bicyclic scaffold and
the L-glutamate midway between those for the three-atom
bridge 3a and the four-atom bridge 3b. We predicted that this
would preserve the potency of 3a along with the selectivity for
FRα and PCFT as in 3b.
Scheme 2
We synthesized 3b regioisomers, differing in positions of
substitution of the four-atom carbon bridge and the L-glutamic
acid on the thiophene ring (compounds 4−8) and in the
presence of a linear, shorter acetylenic linkage (compounds
9−13 and 20) in the carbon bridge region (Figure 1). These
modifications decreased the distances between the bicyclic
pyrrolo[2,3-d]pyrimidine and the L-glutamate moieties in order
to systematically determine their impact on drug selectivity
for FRs and PCFT over RFC and on potency against human
tumor cells. In this report, we describe the synthesis and
biologic properties of compounds 4−13 (Figure 1) and 20
(Scheme 3).
a
Reagents and conditions: (a) N-methylmorpholine, 2-chloro-4,6-
dimethoxy-1,3,5-triazine, L-glutamate diethyl ester hydrochloride,
DMF, room temp, 12 h.
CHEMISTRY
■
The synthesis of target compounds 4−8 (Scheme 1) started
from the reported intermediate 16.12 A Sonogashira coupling
of 16 with various bromo-substituted thiophene carboxylates
15a−e (Scheme 2) afforded 17a−e in 50−56% yield. Sub-
sequent hydrogenation and saponification of 17a−e afforded
target compounds 4−8. Direct hydrolysis of intermediates
17a−e provided conformationally restricted analogues 9−13.
Compounds 15a−e (Scheme 2) required in Scheme 1 were
synthesized by a peptide coupling of the commercially avail-
able bromo-substituted thiophene carboxylic acids 14a−e
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a
Scheme 3
a
Reagents and conditions: (a) (i) 1 N NaOH; (ii) 1 N HCl.
(Scheme 2) with L-glutamate diethyl ester hydrochloride in 89−
91% yield.
Compound 19 (Scheme 3) was previously reported as a
precursor to 3b (Figure 1).12 For comparison to 3b, it was also
of interest to synthesize and evaluate the classical analogue 20
derived from 19. Synthesis of 20 from 19 is shown in Scheme 3.
BIOLOGICAL EVALUATION AND DISCUSSION
■
6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Re-
gioisomers as Inhibitors of Cell Proliferation. The distance
between the bicyclic scaffold and L-glutamate portions of a series
of pyrrolo- and thieno[2,3-d]pyrimidine antifolates seems to
dictate both selectivity for cellular uptake by FRs and PCFT over
RFC and cytotoxic potencies against tumor cells.10−14 Thus,
decreasing the bridge lengths from eight carbons to four carbons
was accompanied by substantially increased antiproliferative
activities, reflecting transport by PCFT and/or FRs and inhibition
of intracellular GARFTase. Decreasing the bridge length from
four to three carbons further increased drug potencies. However,
for the pyrrolo[2,3-d]pyrimidine thienoyl antifolate series, this
appeared to be accompanied by some loss of absolute transporter
specificity for FRs and/or PCFT over other modes of uptake.13
Finally, for all series, analogues with bridge lengths less than three
carbons showed substantially decreased drug activities.
Figure 2. Molecular modeling of the thiophene ring (MOE 2008.10)
and angles and distances of the regioisomers of 3b.
conformational rigidity in the four-carbon atom chain and
decreases the length of the carbon bridge (3.52 Å), compared
to the saturated analogues (3.87 Å) 3b−8.
Compounds 4−13 and 20 were initially screened for growth
inhibition in a series of cell lines with defined expression patterns
of the major (anti)folate transporters for comparison with
compound 3b. The experimental models were a panel of isogenic
Chinese hamster ovary (CHO) sublines derived from RFC-, FR-,
and PCFT-null MTXRIIOuaR2-4 CHO cells engineered to
express FRα (RT16),10 human RFC (PC43-10),18 or human
PCFT (R2/hPCFT4).5,11 Additional growth inhibition studies
were performed with KB and IGROV1 human tumor cells that
express FRα, RFC, and PCFT. We previously showed that during
cell outgrowth, the pH of the culture medium decreased from pH
7.2 to ∼6.8 14 such that all three transporters would be active in
drug transport.
For measurements of inhibitory effects on cell proliferation,
the cell lines were cultured with the antifolates over a range of
concentrations, as described in the Experimental Section. With
RT16 CHO, KB, and IGROV1 cells, FRα-mediated drug uptake
was established with a parallel culture treated with the cytotoxic
drugs and excess (200 nM) folic acid to block FRs. For the
PC43-10 and R2/hPCFT4 CHO sublines, results were
compared to those for parental R2 cells or to vector-control
R2 cells transfected with empty pcDNA3.1 vector [designated
R2(VC)]. Patterns of drug sensitivity or resistance for the novel
pyrrolo[2,3-d]pyrimidine analogues were compared to those for
compounds 3a and 3b and to those previously reported for
classic antifolates (e.g., MTX, PMX, LMTX, RTX).10−14 Results
of the cell proliferation assays are summarized in Table 1.
MTX, PMX, RTX, and LMTX were all active (albeit
variably) toward RFC-, PCFT-, or FRα-expressing CHO cells.
The 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl analogues
with contiguous thiophene substitutions [2′, 3′ (4 and 10), 4′, 5′
(5 and 11), 3′, 4′ (6 and 12)] were completely inactive toward
the RFC- and PCFT-expressing CHO sublines. Compounds 7, 8,
13, and 20 were likewise inert toward PC43-10 cells, although
In the present study, we used the pyrrolo[2,3-d]pyrimidine
thienoyl platform with a four-carbon bridge. We systematically
assessed the effects on cell proliferation for an expanded series
of pyrrolo[2,3-d]pyrimidine thienoyl regioisomers of 3b with a
four-carbon bridge and thienoyl ring substitutions, 2′,3′ (4),
4′,5′ (5), 3′,4′ (6), 3′,5′ (7), and 2′,4′ (8) (Figure 1), as an
alternative approach for decreasing the distance between the
bicyclic pyrrolo[2,3-d]pyrimidine and the L-glutamate portions.
Hence, in analogues 4−6, the 1,2-substitution pattern on the
thiophene forces the bicyclic scaffold and L-glutamate closer
together than in the parent four-atom bridge compound 3b
which includes a 1,3-pattern on the thiophene ring.
Compounds 7 and 8, and also 1,3-substitution on the thio-
phene ring, have an intervening smaller carbon compared to
the larger sulfur in 3b. Further, the C−C−C (112°) bond
angle of both 7 and 8 is larger than the C−S−C (91.8°) bond
angle of 3b, and the lengths of the C−S (1.71 Å) and C−C
(1.38 Å) bonds are also different (Figure 2, MOE 2008.10).18
Molecular modeling (Figure 2, MOE 2008.10) shows that the
distances from the α-carbon of the bridge to the amide carbonyl
are shorter in compounds 7 and 8 (5.10 and 5.11 Å,
respectively) than that in 3b (5.33 Å). The distances from
the bicyclic scaffold to the L-glutamate for compounds 7 and 8
are designed to be between those for 3b and 3a.
Compounds 9−13 and 20 are analogues of 3b−8 and
contain a single conformational restriction in the four-carbon
atom bridge, in the form of a linear acetylenic linkage instead of
the saturated C−C linkage of 3b−8. This also provides some
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Table 1. IC50 (in nM) for 6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers 3b, 4−13, and 20, Compared to IC50
a
for 3a and Classical Antifolates in RFC, PCFT, and FR-Expressing Cell Lines
RFC
hFRα
PCFT
RFC/FRα/PCFT
RFC/FRα/PCFT
RT16
(+FA)
IGROV1
(+FA)
antifolate
PC43-10
R2
RT16
R2/hPCFT4
R2(VC)
KB
KB (+FA)
IGROV1
3a
101.0(16.6)
>1000
273.5(49.1)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
216(8.7)
894(93)
>1000
>1000
0.31 (0.14)
1.82(0.28)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
461(62)
388(68)
>1000
188(41)
3.34 (0.26)
43.4(4.1)
>1000
288(12)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
974.0(18.1)
>1000
>1000
0.26(0.03)
0.55(0.10)
171(36)
267(33)
35.3(4.4)
0.17(0.02)
0.27(0.07)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
20(2.4)
327(103)
22(5)
0.55(0.10)
0.97(0.12)
ND
>1000
>1000
ND
3b
4
>1000
5
>1000
>1000
>1000
ND
ND
6
>1000
147(53)
2.54(0.52)
2.60(0.59)
>1000
>1000
102.9(25.9)
0.48(0.08)
1.4(0.5)
ND
>1000
>1000
>1000
ND
7
>1000
41.54(13.09)
63.82(16.23)
>1000
8
>1000
9
>1000
10
>1000
>1000
>1000
>1000
ND
ND
11
>1000
>1000
>1000
>1000
ND
ND
12
>1000
13.48(2.42)
8.26 (1.86)
12.07(5.02)
114(31)
42(9)
>1000
>1000
>1000
>1000
>1000
>1000
22(2.1)
200(18)
20(4.3)
16(6)
13
>1000
>1000
>1000
>1000
20
>1000
>1000
>1000
>1000
MTX
PMX
RTX
LMTX
12(1.1)
138(13)
6.3(1.3)
12(2.3)
120.5(16.8)
13.2(2.4)
99.5(11.4)
38.0(5.3)
6.0(0.6)
68(12)
21(3.4)
102(25)
12.6(3.3)
3.1(0.9)
15(5)
5.9(2.2)
1.2(0.6)
12(8)
31(7)
a
Growth inhibition assays were performed for CHO sublines engineered to express RFC (PC43-10), FRα (RT16), or PCFT (R2/hPCFT4), for
comparison with transporter-null [R2, R2(VC)] CHO cells, and for the KB and IGROV1 tumor sublines (express 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 2−10 experiments (plus/minus SEM in parentheses). Results are presented as IC50 (in nM),
corresponding to the concentration that inhibit growth by 50% relative to cells incubated without drug. Data for MTX, PMX, RTX, and LMTX were
previously published.10−14. ND: not determined
both compounds 7 and 8 (but not conformationally rigid 13
and 20) inhibited proliferation of R2/hPCFT4 cells (IC50s
of 41.5 and 63.8 nM for 7 and 8, respectively), essentially
equivalent to compound 3b (IC50 of 43.4 nM). This establishes
cellular uptake of compounds 7 and 8 by PCFT. Drug
sensitivities for 7 and 8 with R2/hPCFT4 cells exceeded those
for MTX and RTX and were comparable to those for LMTX.
However, 7 and 8 were less potent than PMX (∼3- and
∼5-fold, respectively) and 3a (∼12- and ∼19-fold, respec-
tively). None of the 6-substituted pyrrolo[2,3-d]pyrimidine
regioisomers (3b, 4−13, and 20) had any impact on prolifera-
tion of R2 or R2(VC) cells.
For FRα-expressing RT16 cells, compounds 4, 5, 10, and 11
were all inert at drug concentrations up to 1000 nM.
Compounds 6, 7, 8, 12, 13, and 20, like 3b, all showed some
level of antiproliferative activity toward RT16 cells. The active
drugs followed an order of potency, 7 ∼ 8 ∼ 3b > 12 ∼ 13 >
20 ≫ 6. FR-mediated growth inhibitions for the most active
analogues (i.e., 3b, 7, and 8) exceeded those for standard
antifolates (Table 1), and for all the active analogues, inhibitions
were abolished with 100-fold excess folic acid.
KB and IGROV1 human tumor cells express all three transport
systems7 and in these models, 7 and 8 were the most potent
drugs in our series (Table 1). Inhibitory activities for 7 and 8
were similar to those for 3a and 3b in both KB and IGROV1
cells. In KB cells, compounds 4, 5, and 6 were all substantially less
active than 7 and 8, in order of decreasing potency, 6 ≫ 5 ∼ 4.
For the active analogues, growth inhibition was again reversed by
excess folic acid, establishing that, like compound 3b, FRα was
the major mechanism of uptake in these cells despite the presence
of RFC and PCFT. Thus, for the 7 and 8 regioisomers of 3b, we
confirmed our hypothesis that by varying the distance between
the bicyclic scaffold and the L-glutamate, we preserved both the
absolute selectivity of 3b and the high antiproliferative potencies
of 3a and 3b.
Interestingly, none of the acetylenic compounds (9−13 and
20) were inhibitory toward FRα-expressing human KB cells, in
striking contrast to the results with RT16 CHO cells with
compounds 12, 13, and 20. This suggests that for 12, 13, and
20, there must be differences in cellular metabolism, drug
binding to intracellular targets, and/or substrate activity for
folylpolyglutamate synthetase between the human and hamster
cells. When several of these analogues (3b, 6, 12, 13, and 20,
compared to 7 and 8) were tested in IGROV1 cells, results
analogous to those with KB cells were obtained (Table 1).
Additional experiments used colony-forming assays with KB
tumor cells continuously exposed to 3b, 7, or 8 for up to
14 days, after which colonies were stained with 1% methylene
blue and counted. Compounds 7 and 8 inhibited KB cell colony
formation (Supporting Information, Figure 1S) with IC50s (0.39
and 0.16 nM, respectively) similar to that for compound 3b
(IC50 = 0.20 nM) and substantially lower than that for MTX
(IC50 = 8.5 nM).
These results establish that (i) none of the regioisomers 3b,
4−13, and 20 are RFC substrates. (ii) For the saturated bridge
analogues (3b, 4−8), a 1,3-relationship between the carbon
bridge domain and the L-glutamate moiety is an important
determinant of transport substrate activity with FRα or PCFT.
(iii) Compounds 7 and 8, like 3b, were substantially less
inhibitory toward PCFT-expressing cells than compound 3a.
However, this difference was much reduced in human tumor
cells for which FRα was the primary mode of drug delivery. The
results with compounds 4, 5, and 6 and FRα-expressing cells
suggest that decreasing distance between the bicyclic ring and
the L-glutamate beyond a point has adverse effects on drug
activity, analogous to those resulting from decreasing bridge
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Figure 3. Growth inhibition of KB cells by the 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers 3b, 7, and 8 in the presence of
physiologic concentrations of reduced folate. Cell proliferation inhibition was measured over a range of concentrations of the antifolates in complete
folate-free RPMI1640 in the presence of LCV at 2 and 50 nM. Cell densities were measured as in the Experimental Section with a fluorescence dye
and a fluorescence plate reader. Results were normalized to cell density in the absence of drug. Results shown are average values plus/minus ranges
for duplicate experiments.
lengths to less than three atoms. (iv) None of the conforma-
tionally restricted acetylenic analogues (9−13 and 20) were
substrates for PCFT. (v) For this series, the [1,3] acetylenic
analogues (12, 13, and 20) are substrates for FRα, although
other factors unrelated to membrane transport seem likely to
account for the marked differences in their antiproliferative
activities between the human and hamster sublines.
5-amino-4-imidazolecarboxamide (AICA) (320 μM) was also
protective. Since AICA is metabolized to AICA ribonucleotide
(AICAR), the substrate for AICAR formyltransferase (AI-
CARFTase, the second folate-dependent reaction after
GARFTase),10,19 7 and 8 must derive their growth inhibitory
effects by inhibiting GARFTase. This was further tested below.
Cellular Pharmacology of Cytotoxic 6-Substituted
Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers. Results
of our cell proliferation assays in engineered CHO and human
tumor cells suggested that 7 and 8 are bona fide transport
substrates for FRα and PCFT. From results of nucleoside and
AICA protection studies, 7 and 8, like 3b, following inter-
nalization, inhibit de novo purine nucleotide biosynthesis
apparently at the level of GARFTase. Interestingly, whereas the
acetylenic bridge analogues 12, 13, and 20 inhibited prolifera-
tion of FRα-expressing RT16 CHO cells, these compounds
were inactive toward human tumor cells.
Using inhibition of human tumor cell proliferation as a
primary metric, we selected compounds 3b and 4−8 for testing
FRα binding, reflected in competitive inhibition of [3H]folic
acid binding to FRα in RT16 CHO cells. For these experiments,
RT16 cells were washed with acid-buffered saline (pH 3.5),
treated (at neutral pH) with 50 nM [3H]folic acid, along with a
range of concentrations of the pyrrolo[2,3-d]pyrimidines 3b and
4−8, folic acid, LMTX, PMX, or MTX. After additional washes
(neutral pH), cells were solubilized (in 0.5 N NaOH). FRα-
bound [3H]folic acid was measured and normalized to cellular
protein, and relative inhibitor affinities were calculated as inverse
molar ratios of the concentrations of unlabeled ligands which
decreased [3H]folic acid binding by 50%.
Impact of Elevated Leucovorin and Nucleosides on
Antitumor Activities of Compounds 7 and 8. Since our
proliferation assays with FR-expressing cells routinely use
limiting (2 nM) concentrations of extracellular leucovorin
[(6R,S)5-formyl tetrahydrofolate, a chemically stable reduced
folate] (LCV), additional experiments were performed with KB
cells to assess the impact of elevated (50 nM) LCV [for which
the active (6S) stereoisomer approximates the concentration of
5-methyl tetrahydrofolate in serum] on the growth inhibitory
effects of the most potent analogues (compounds 7 and 8) of
our series compared to compound 3b (Figure 3). In KB cells, the
impact of 50 nM LCV was uneven and ranged from ∼7-fold for
3b to ∼20-fold for 7 and >38-fold for 8. For the data in Figure 3,
IC50 values in the presence of 50 nM LCV were 2.2 nM for 3b,
3.4 nM for 7, and >20 nM for 8. Thus, both compounds 3b and
7 maintained substantial antitumor potencies even in the
presence of elevated physiologic levels of reduced folate.
As previously reported for 3b,12 growth inhibitory effects of 7
and 8 toward KB (Figure 4) and IGROV1 cells (not shown)
were completely reversed when drug exposures were performed
in the presence of adenosine (60 μM) but not thymidine
(10 μM). This established that the effects of 7 and 8 on cell
proliferation were the consequence of inhibiting the de novo
purine nucleotide biosynthetic pathway. For both 7 and 8,
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Figure 4. Protection of KB cells from growth inhibition by the 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers 7 and 8 in the presence of
nucleosides and 5-amino-4-imidazolecarboxamide (AICA). Proliferation inhibition was measured for KB cells over a range of concentrations of compounds
7 and 8, 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), or AICA (320 μM). Cell densities were measured as in the
Experimental Section with a 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. Analogous results were obtained with IGROV1 cells (not shown).
a
In these experiments, compounds 7 and 8 were all high
affinity ligands (relative affinities of ≥1) for FRα, essentially
equivalent to 3b and folic acid and substantially better than
LMTX (Figure 5). Like MTX, compounds 4−6 did not bind to
Table 2. Ki for PCFT Substrates
Ki (μM)
substrate
pH 5.5
pH 6.8
3b
7
0.23 0.06 (n = 7)
0.39 0.10 (n = 4)
0.42 0.11 (n = 4)
7.23 2.23 (n = 7)
28.1 5.3 (n = 4)
30.7 8.5 (n = 4)
2.54 0.31 (n = 8)
8
PMX
0.094 0.006 (n = 8)
a
Ki values for PCFT substrates were determined by Dixon plots with
[3H]MTX as substrate and a range of inhibitor concentrations in R2/
hPCFT4 cells. Ki values were calculated from the slopes of the Dixon
plots and kinetic constants (Kt and Vmax) for MTX. Results are
presented as mean values standard errors from multiple experiments
(numbers shown in parentheses).
and approached the Ki for PMX. At pH 6.8, Ki values for PMX
and 3b were likewise similar (∼27- and 31-fold higher,
respectively, than the values at pH 5.5). In comparison, Ki
values for 7 and 8 were further increased at pH 6.8 (∼70-fold
greater) over the Ki values recorded at pH 5.5.
Collectively, these experiments confirm the high affinity
binding for the [1,3] thienoyl regioisomers 3b, 7, and 8 to FRα
and PCFT, suggested from their potent antiproliferative and
cytotoxic effects toward FRα- and PCFT-expressing CHO and
human tumor cells. While compounds 7 and 8, like 3b, are
potent inhibitors of PCFT at pH 5.5, binding of 7 and 8 to
PCFT (as reflected in Ki) appears to be more sensitive to pH
than for either 3b or PMX.
Figure 5. Binding of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl
regioisomers (3b, 4−8) to FRα. Data are shown for the effects of the
unlabeled ligands with FRα-expressing RT16 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 ratios of unlabeled ligands required to inhibit
[3H]folic acid binding by 50%. By definition, the relative affinity of
folic acid is 1. Details for the binding assays are provided in the
Experimental Section. Results are presented as average values and
ranges from two to three experiments. Undefined abbreviations are as
follows: FA, folic acid.
FRα in this assay, in spite of detectable (albeit lower) levels of
FRα-directed activity in our cell proliferation experiments (with
KB cells, Table 1). The detectable antiproliferative activities
with 4, 5, and 6 in KB cells (but not RT16 CHO cells) likely
reflect markedly elevated levels of FRα in this human tumor
cell line, as previously reported.10
Since compounds 7 and 8 inhibited proliferation of R2/
hPCFT4 cells, these analogues were tested as competitive
inhibitors of PCFT transport of [3H]MTX in R2/hPCFT4 cells.
Experiments were performed at pH 5.5, approximating the pH
optimum for PCFT, and at pH 6.8, which is commonly achieved
during cell outgrowth.14 Ki values were calculated over a range
of inhibitor concentrations from Dixon plots, and results were
compared to those for 3b and PMX (Table 2). At pH 5.5, Ki
values for 3b, 7, and 8 were all similar (within a factor of ∼2)
Results of nucleoside/AICA protection experiments with KB
(Figure 4) and IGROV1 tumor cells (not shown) suggested
that GARFTase in the de novo purine nucleotide biosynthetic
pathway was the primary intracellular target of 7 and 8,
analogous to 3b,12 thus explaining the potent antiproliferative
activities of these agents. To confirm GARFTase inhibition
with compounds 7 and 8, we used an in situ GARFTase activity
assay in which intact cells were incubated with the antifolate
drugs along with [14C]glycine as a radiotracer. In the presence
of azaserine, [14C]glycine is incorporated into GAR, accumulat-
ing [14C]formyl GAR which can be isolated by ion exchange
chromatography, and radioactivity is determined.10−13,19
Data are shown in Figure 6 for the effects of compounds 3b,
7, and 8 on in situ GARFTase activity in IGROV1 cells,
although completely analogous results were obtained with KB
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distinctly (and selectively) inhibitory toward FRα-expressing
RT16 CHO cells. This further supports our conclusion that
only the [1,3] substituted thiophene is amenable to high affinity
FRα binding and cellular uptake and that species differences in
intracellular drug metabolism and/or target enzyme binding
must account for the lack of activity of these acetylenic analogues
with human tumor cells.
Thus, our present and published results establish that the
pyrrolo[2,3-d]pyrimidine scaffold, along with a thienoyl side
chain, is particularly amenable to FR and PCFT transport in the
absence of RFC transport and to potent GARFTase inhibition.
Our new finding that [1,3] substituted regioisomers of this series
are preferred over the [1,2] analogues is consistent with the
notion of an optimal distance between the bicyclic ring system
and L-glutamate moiety for maximal antiproliferative effects.
However, among [1,3] compounds 3b, 7, and 8, or even
including 3a, there was no consistent relationship between
relative drug sensitivities toward FRα- and PCFT-expressing cells
and these intramolecular distances. Indeed, compounds 7 and 8
showed in vitro potencies that were generally comparable to
those for compound 3b or even 3a, although this was cell line-
dependent. This may reflect differential impacts of extra- and/or
intracellular reduced folates on efficacies of the various analogues,
combined with cell-specific differences in folate transport (i.e.,
RFC levels) or metabolism and, for PCFT, differences in relative
binding affinities between drugs at pH ∼6.8.
Of course, the most distinguishing feature of the three active
regioisomers 3b, 7, and 8 is their complete lack of RFC-
mediated growth inhibition (presumably reflecting their lack of
uptake), which is key to their potential therapeutic utility, as this
would obviate toxicity to normal susceptible tissues for which
drug transport by FRs or PCFT should be nominal. Clearly, the
profound selectivity of these novel 6-substituted pyrrolo[2,3-
d]pyrimidine[1,3]thienoyl antifolates for FRs and PCFT over
RFC offers a paradigm for selective tumor targeting, based on
established patterns of FR and PCFT expression in human
tumors7 and optimal PCFT membrane transport under acidic
conditions characteristic of the solid tumor microenvironment.
Figure 6. In situ GARFTase inhibition assay. For the in situ assays,
incorporation of [14C]glycine into [14C]formyl GAR was measured in
IGROV1 tumor cells cultured for 15 h in complete folate-free RPMI
plus 2 nM LCV. Details are described in the Experimental Section.
Results are presented as a percent of controls treated without drugs for
IGROV1 cells treated with nanomolar concentrations of 3b, 7, 8, or
LMTX. LMTX was not tested at 5 nM (labeled “ND” for not
determined). Results are presented as average values plus/minus
ranges. Mean IC50 values were calculated as 3.46 nM for 3b, 1.79 nM
for 7, 2.03 nM for 8, and 5.77 nM for LMTX.
cells (not shown). In this assay, 3b, 7, and 8, like LMTX, were
all potent inhibitors at low nanomolar concentrations.
CONCLUSIONS
■
A lack of selectivity for folate transporters is a major cause of
toxicity and chemotherapy failure with clinically used
antifolates. We previously reported potent inhibition of tumor
cell proliferation by a classical 2-amino-4-oxo-6-substituted
pyrrolo[2,3-d]pyrimidine with a thienoyl-for-benzoyl-substi-
tuted side chain and a four-carbon bridge (compound 3b).7,12
Compound 3b was selective for transport by FRs and PCFT
over RFC and inhibited GARFTase in de novo purine
biosynthesis.7,12 An analogue of this series with a three-carbon
bridge (compound 3a) was even more active, suggesting that
the distance between the bicyclic scaffold and L-glutamate could
be an important determinant of drug activity, although this may
be partly at the expense of absolute transport selectivity.13
Accordingly, we pursued other approaches for altering this
feature, without appreciably impacting drug potency toward
PCFT- and FRα-expressing cells while preserving selectivity for
transport by non-RFC uptake mechanisms over RFC.
EXPERIMENTAL SECTION
■
All evaporations were carried out in vacuum with a rotary evaporator.
Analytical samples were dried in vacuo (0.2 mmHg) in a CHEM-DRY
drying apparatus over P2O5 at 60 °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 Bruker
Avance II 400 (400 MHz) and 500 (500 MHz) spectrometers. 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. 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 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. The
amount (weight) of silica gel for column chromatography was in the
range of 50−100 times the amount (weight) of the crude compounds
being separated. Columns were dry-packed unless specified otherwise.
Elemental analyses were performed by Atlantic Microlab, Inc.,
Norcross, GA. Element compositions are within 0.4% of the cal-
culated 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. Elemental analysis was used
to determine the purities of the final compounds 4−13. All solvents
We now describe the synthesis and biological character-
ization of regioisomers of 3b (compounds 4−8), differing in
positions of thienoyl ring substitution to vary the distance and
orientation between the bicyclic scaffold and the L-glutamate.
Additional conformationally restricted analogues of 3b and 4−8
(designated 20 and 9−13, respectively) were also studied in
which a linear acetylenic linkage was inserted into the bridge
region. Of this expanded series, analogues with a saturated
bridge and with a [1,3] substitution pattern on the thienoyl ring
(compounds 3b, 7, and 8) were the best inhibitors of KB and
IGROV1 human tumor cell growth and clonogenicity. This was
associated with a potent inhibition of GARFTase in de novo
purine nucleotide biosynthesis. Whereas compounds 3b, 7,
and 8 all showed FRα- and PCFT-mediated effects on cell
proliferation, neither 3b nor compounds 4−8 showed activity
toward CHO cells expressing only RFC. Further, none of the
conformationally restricted linear acetylenic bridge analogues
9−13 and 20 inhibited human tumor cell growth, although the
[1,3] thiophene-substituted compounds 12, 13, and 20 were
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and chemicals were purchased from Aldrich Chemical Co. and Fisher
Scientific and were used as received. Purities of the final compounds
4−13 were >95% and were determined by elemental (C, H, N, S)
analysis.
and gas inlet, was added a mixture of tetrakis(triphenylphosphine)-
palladium(0) (277 mg, 0.24 mmol), triethylamine (1.52 g, 15 mmol),
15a−e (882 mg, 2.25 mmol), and anhydrous DMF (20 mL). To the
stirred mixture, under N2, were added copper(I) iodide (46 mg,
0.24 mmol) and 2-amino-6-but-3-ynyl-3,7-dihydropyrrolo[2,3-d]-
pyrimidin-4-one, 16 (303 mg, 1.5 mmol). The reaction mixture was
stirred at room temperature overnight (17−18 h). Silica gel (0.5 g) was
then added, and the solvent was evaporated under reduced pressure.
The resulting plug was loaded onto a silica gel column (1.5 cm ×
12 cm) and eluted with CHCl3 followed by 3% MeOH in CHCl3 and
then 5% MeOH in CHCl3. Fractions with desired Rf (TLC) were
pooled and evaporated to afford 17a−e.
General Procedure for the Synthesis of Compounds 15a−e.
To a 250 mL round-bottomed flask was added a mixture of bromo-
substituted thiophenecarboxylic acid 14a−e (1.04 g, 5 mmol), N-
methylmorpholine (909 mg, 9 mmol), 2-chloro-4,6-dimethoxy-1,3,5-
triazine (1.58 g, 9 mmol), and anhydrous DMF (20 mL). The mixture
was stirred for 1.5 h at room temperature. N-Methylmorpholine
(909 mg, 9 mmol) and L-glutamic acid diethyl ester hydrochloride
(1.8 g, 7.5 mmol) were added to the flask, and the reaction mixture
was then stirred at room temperature for 12 h. After evaporation of
solvent under reduced pressure, MeOH (20 mL) was added followed
by silica gel (2.5 g). The resulting plug was loaded onto a silica gel
column (2.5 cm × 12 cm) and eluted with 50% EtOAc in hexane.
Fractions with the desired Rf (TLC) were pooled and evaporated to
afford 15a−e as colorless liquid.
(S)-2-({2-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)but-1-ynyl]thiophene-3-carbonyl}amino)-
pentanedioic Acid Diethyl Ester (17a). Compound 17a was
prepared using the general method described for the preparation of
17a−e, from 16 (303 mg, 1.5 mmol) and 15a (882 mg, 2.25 mmol) to
give 412 mg (53%) of 17a as a brown powder. Mp 81−82 °C. TLC
1
Rf = 0.53 (CHCl3/MeOH, 5:1). H NMR (DMSO-d6): δ 1.16−1.22
(S)-2-[(2-Bromothiophene-3-carbonyl)amino]pentanedioic
Acid Diethyl Ester (15a). Compound 15a was prepared using
the general method described for the preparation of 15a−e, from
2-bromo-thiophene-3-carboxylic acid, 14a (1.04 g, 5 mmol), to give
1.76 g (90%) of 15a as a colorless liquid. TLC Rf = 0.44 (hexane/
EtOAc, 1:1). 1H NMR (CDCl3-d1): δ 1.24−1.35 (m, 6H,
COOCH2CH3), 2.09−2.56 (m, 4H, β-CH2, γ-CH2), 4.10−4.17 (m,
2H, COOCH2CH3), 4.23−4.29 (m, 2H, COOCH2CH3), 4.78−4.86
(m, 1H, α-CH), 7.20−7.22 (d, J = 7.2 Hz, 1H, CONH, exch), 7.26−
7.27 (d, J = 5.6 Hz, 1H, Ar), 7.36−7.37 (d, J = 5.6 Hz, 1H, Ar).
(S)-2-[(3-Bromothiophene-2-carbonyl)amino]pentanedioic
Acid Diethyl Ester (15b). Compound 15b was prepared using
the general method described for the preparation of 15a−e, from
3-bromo-thiophene-2-carboxylic acid, 14b (1.04 g, 5 mmol), to give
1.8 g (91%) of 15b as a colorless liquid. TLC Rf = 0.45 (hexane/
EtOAc, 1:1). 1H NMR (CDCl3-d1): δ 1.24−1.36 (m, 6H,
COOCH2CH3), 2.13−2.55 (m, 4H, β-CH2, γ-CH2), 4.10−4.16 (m,
2H, COOCH2CH3), 4.25−4.30 (m, 2H, COOCH2CH3), 4.83−4.88
(m, 1H, α-CH), 7.07−7.08 (d, J = 5.6 Hz, 1H, Ar), 7.48−7.49 (d, J =
5.6 Hz, 1H, Ar), 7.73−7.75 (d, J = 7.2 Hz, 1H, CONH, exch).
(S)-2-[(4-Bromothiophene-3-carbonyl)amino]pentanedioic
Acid Diethyl Ester (15c). Compound 15c was prepared using
the general method described for the preparation of 15a−e, from
4-bromo-thiophene-3-carboxylic acid, 14c (1.04 g, 5 mmol), to give
1.74 g (89%) of 15c as a colorless liquid. TLC Rf = 0.45 (hexane/
EtOAc, 1:1). 1H NMR (CDCl3-d1): δ 1.24−1.35 (m, 6H,
COOCH2CH3), 2.07−2.56 (m, 4H, β-CH2, γ-CH2), 4.11−4.17 (m,
2H, COOCH2CH3), 4.23−4.30 (m, 2H, COOCH2CH3), 4.78−4.87
(m, 1H, α-CH), 7.35−7.36 (d, J = 3.6 Hz, 1H, Ar), 7.36−7.38 (d, J =
7.2 Hz, 1H, CONH, exch), 8.07−8.08 (d, J = 3.6 Hz, 1H, Ar).
(S)-2-[(4-Bromothiophene-2-carbonyl)amino]pentanedioic
Acid Diethyl Ester (15d). Compound 15d was prepared using
the general method described for the preparation of 15a−e, from
4-bromo-thiophene-2-carboxylic acid, 14d (1.04 g, 5 mmol), to give
1.8 g (91%) of 15d as a colorless liquid. TLC Rf = 0.44 (hexane/
EtOAc, 1:1). 1H NMR (CDCl3-d1): δ 1.25−1.34 (m, 6H,
COOCH2CH3), 2.13−2.51 (m, 4H, β-CH2, γ-CH2), 4.14−4.17 (m,
2H, COOCH2CH3), 4.23−4.28 (m, 2H, COOCH2CH3), 4.71−4.76
(m, 1H, α-CH), 7.04−7.06 (d, J = 7.2 Hz, 1H, CONH, exch), 7.42−
7.43 (d, J = 1.6 Hz, 1H, Ar), 7.16−7.47 (d, J = 1.6 Hz, 1H, Ar).
(S)-2-[(5-Bromothiophene-3-carbonyl)amino]pentanedioic
Acid Diethyl Ester (15e). Compound 15e was prepared using
the general method described for the preparation of 15a−e, from
5-bromo-thiophene-3-carboxylic acid, 14e (1.04 g, 5 mmol), to give
1.76 g (90%) of 15e as a colorless liquid. TLC Rf = 0.45 (hexane/
EtOAc, 1:1). 1H NMR (CDCl3-d1): δ 1.25−1.34 (m, 6H,
COOCH2CH3), 2.12−2.53 (m, 4H, β-CH2, γ-CH2), 4.12−4.18 (m,
2H, COOCH2CH3), 4.23−4.28 (m, 2H, COOCH2CH3), 4.70−4.75
(m, 1H, α-CH), 6.96−6.98 (d, J = 7.2 Hz, 1H, CONH, exch), 7.40−
7.41 (d, J = 1.6 Hz, 1H, Ar), 7.83−7.84 (d, J = 1.6 Hz, 1H, Ar).
General Procedure for the Synthesis of Compounds 17a−e.
To a 250-mL round-bottomed flask, equipped with a magnetic stirrer
(m, 6H, COOCH2CH3), 1.92−2.14 (m, 2H, β-CH2), 2.43−2.46 (t, J =
8 Hz, 2H, γ-CH2), 2.80 (m, 4H, CH2CH2), 4.01−4.52 (q, J = 7 Hz,
2H, COOCH2CH3), 4.10−4.16 (m, 2H, COOCH2CH3), 4.43−4.48
(m, 1H, α-CH), 5.99 (s, 1H, C5-CH), 6.01 (s, 2H, 2-NH2, exch),
7.35−7.36 (d, J = 5.5 Hz, 1H, Ar), 7.53−7.54 (d, J = 5.5 Hz, 1H, Ar),
8.34−8.36 (d, J = 7.5 Hz, 1H, CONH, exch), 10.16 (s, 1H, 3-NH,
exch), 10.88 (s, 1H, 7-NH, exch).
(S)-2-({3-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)but-1-ynyl]thiophene-2-carbonyl}amino)-
pentanedioic Acid Diethyl Ester (17b). Compound 17b was
prepared using the general method described for the preparation of
17a−e, from 16 (303 mg, 1.5 mmol) and 15b (882 mg, 2.25 mmol) to
give 423 mg (55%) of 17b as a brown powder. Mp 77−78 °C. TLC
1
Rf = 0.52 (CHCl3/MeOH, 5:1). H NMR (DMSO-d6): δ 1.16−1.22
(m, 6H, COOCH2CH3), 1.92−2.18 (m, 2H, β-CH2), 2.39−2.43 (m,
2H, γ-CH2), 2.84 (m, 4H, CH2CH2), 3.99−4.03 (q, J = 7 Hz, 2H,
COOCH2CH3), 4.14−4.18 (q, J = 7 Hz, 2H, COOCH2CH3), 4.53−
4.57 (m, 1H, α-CH), 6.00 (s, 1H, C5-CH), 6.01 (s, 2H, 2-NH2, exch),
7.16−7.17 (d, J = 5.0 Hz, 1H, Ar), 7.81−7.82 (d, J = 5.0 Hz, 1H, Ar),
8.19−8.20 (d, J = 7.5 Hz, 1H, CONH, exch), 10.16 (s, 1H, 3-NH,
exch), 10.91 (s, 1H, 7-NH, exch).
(S)-2-({4-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)but-1-ynyl]thiophene-3-carbonyl}amino)-
pentanedioic Acid Diethyl Ester (17c). Compound 17c was
prepared using the general method described for the preparation of
17a−e, from 16 (303 mg, 1.5 mmol) and 15c (882 mg, 2.25 mmol) to
give 433 mg (56%) of 17c as a brown powder. Mp 80−81 °C. TLC
1
Rf = 0.53 (CHCl3/MeOH, 5:1). H NMR (DMSO-d6): δ 1.16−1.22
(m, 6H, COOCH2CH3), 1.92−2.14 (m, 2H, β-CH2), 2.42−2.46 (m,
2H, γ-CH2), 2.69−2.78 (m, 4H, CH2CH2), 4.01−4.05 (q, J = 7 Hz,
2H, COOCH2CH3), 4.11−4.16 (m, 2H, COOCH2CH3), 4.45−4.50
(m, 1H, α-CH), 5.99 (s, 1H, C5-CH), 6.00 (s, 2H, 2-NH2, exch),
7.16−7.17 (d, J = 3.5 Hz, 1H, Ar), 8.08−8.09 (d, J = 3.5 Hz, 1H, Ar),
8.40−8.41 (d, J = 7.5 Hz, 1H, CONH, exch), 10.16 (s, 1H, 3-NH,
exch), 10.88 (s, 1H, 7-NH, exch).
(S)-2-({4-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)but-1-ynyl]thiophene-2-carbonyl}amino)-
pentanedioic Acid Diethyl Ester (17d). Compound 17d was
prepared using the general method described for the preparation of
17a−e, from 16 (303 mg, 1.5 mmol) and 15d (882 mg, 2.25 mmol) to
give 406 mg (53%) of 17d as a brown powder. Mp 79−80 °C. TLC
1
Rf = 0.53 (CHCl3/MeOH, 5:1). H NMR (DMSO-d6): δ 1.16−1.22
(m, 6H, COOCH2CH3), 1.92−2.12 (m, 2H, β-CH2), 2.42−2.45 (t, J =
7.5 Hz, 2H, γ-CH2), 2.71−2.78 (m, 4H, CH2CH2), 4.03−4.07 (q, J = 7
Hz, 2H, COOCH2CH3), 4.09−4.13 (q, J = 6.5 Hz, 2H,
COOCH2CH3), 4.36−4.40 (m, 1H, α-CH), 6.00 (s, 1H, C5-CH),
6.02 (s, 2H, 2-NH2, exch), 7.87−7.88 (m, 2H, Ar), 8.76−8.77 (d, J =
7.5 Hz, 1H, CONH, exch), 10.17 (s, 1H, 3-NH, exch), 10.90 (s, 1H,
7-NH, exch).
(S)-2-({5-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)but-1-ynyl]thiophene-3-carbonyl}amino)-
pentanedioic Acid Diethyl Ester (17e). Compound 17e was
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prepared using the general method described for the preparation of
17a−e, from 16 (303 mg, 1.5 mmol) and 15e (882 mg, 2.25 mmol) to
give 387 mg (50%) of 17e as a brown powder. Mp 81−82 °C. TLC
(S)-2-({5-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)butyl]thiophene-3-carbonyl}amino)pentanedioic
Acid Diethyl Ester (18e). Compound 18e was prepared using the
general method described for the preparation of 18a−e, from 17e
(200 mg, 0.39 mmol) to give 183 mg (91%) of 18e as a light yellow
1
Rf = 0.53 (CHCl3/MeOH, 5:1). H NMR (DMSO-d6): δ 1.16−1.22
(m, 6H, COOCH2CH3), 1.92−2.12 (m, 2H, β-CH2), 2.41−2.44 (t, J =
7.5 Hz, 2H, γ-CH2), 2.78 (m, 4H, CH2CH2), 4.03−4.07 (q, J = 7 Hz,
2H, COOCH2CH3), 4.09−4.13 (m, 2H, COOCH2CH3), 4.36−4.41
(m, 1H, α-CH), 6.00 (s, 1H, C5-CH), 6.01 (s, 2H, 2-NH2, exch),
7.58−7.59 (d, J = 1.5 Hz, 1H, Ar), 8.12−8.13 (d, J = 1.5 Hz, 1H, Ar),
8.56−8.58 (d, J = 7.5 Hz, 1H, CONH, exch), 10.16 (s, 1H, 3-NH,
exch), 10.88 (s, 1H, 7-NH, exch).
General Procedure for the Synthesis of Compounds 18a−e.
To a Parr hydrogenation flask was added 17a−e (200 mg, 0.39 mmol),
10% palladium on activated carbon (100 mg), and MeOH (50 mL).
Hydrogenation was carried out at 55 psi of H2 for 4 h. The reaction
mixture was filtered through Celite, washed with MeOH (100 mL),
and concentrated under reduced pressure to give 18a−e.
1
powder. Mp 82−83 °C. TLC Rf = 0.54 (CHCl3/MeOH, 5:1). H
NMR (DMSO-d6): δ 1.15−1.18 (m, 6H, COOCH2CH3), 1.64
(m, 4H, CH2CH2), 1.93−2.12 (m, 2H, β-CH2), 2.41−2.44 (t, J =
7.5 Hz, 2H, γ-CH2), 2.55 (m, 2H, CH2), 2.81 (m, 2H, CH2), 4.03−
4.07 (q, J = 7.0 Hz, 2H, COOCH2CH3), 4.08−4.12 (q, J = 7.0 Hz, 2H,
COOCH2CH3), 4.36−4.40 (m, 1H, α-CH), 5.96 (s, 3H, C5-CH,
2-NH2, exch), 7.26 (s, 1H, Ar), 7.98 (s, 1H, Ar), 8.46−8.48 (d, J = 8.0
Hz, 1H, CONH, exch), 10.76 (s, 1H, 3-NH, exch), 11.22 (s, 1H,
7-NH, exch).
General Procedure for the Synthesis of Compounds 4−13.
To a solution of 18a−e (100 mg, 0.19 mmol) or 17a−e (100 mg, 0.19
mmol) in 10 mL of MeOH/CHCl3 (1:1) or MeOH/CH2Cl2 (1:1)
was added 1 N NaOH (5 mL), and the mixture was stirred under N2
at room temperature for 16 h. TLC showed the disappearance of the
starting material and one major spot at the origin. The reaction
mixture was evaporated to dryness under reduced pressure. The
residue was dissolved in water (10 mL). The resulting solution was
cooled in an ice bath, and the pH was adjusted to 3−4 with dropwise
addition of 1 N HCl. The resulting suspension was frozen in a dry
ice−acetone bath, thawed to 4−5 °C in the refrigerator, and filtered.
The residue was washed with a small amount of cold water and dried
in vacuum using P2O5 to afford 4−13.
(S)-2-({2-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)butyl]thiophene-3-carbonyl}amino)pentanedioic
Acid (4). Compound 4 was prepared using the general method
described for the preparation of 4−13, from 15a (100 mg, 0.19 mmol)
to give 83 mg (95%) of 4 as a light yellow powder. Mp 176−177 °C.
1H NMR (DMSO-d6): δ 1.61 (m, 4H, CH2CH2), 1.87−2.10 (m, 2H,
β-CH2), 2.34−2.37 (t, J = 7.5 Hz, 2H, γ-CH2), 2.47 (m, 2H, CH2),
3.09−3.11 (m, 2H, CH2), 4.32−4.37 (m, 1H, α-CH), 5.86 (s, 1H,
C5-CH), 5.98 (s, 2H, 2-NH2, exch), 7.33−7.34 (d, J = 5.0 Hz, 1H, Ar),
7.35−7.36 (d, J = 5.0 Hz, 1H, Ar), 8.28−8.29 (d, J = 7.5 Hz, 1H,
CONH, exch), 10.16 (s, 1H, 3-NH, exch), 10.80 (s, 1H, 7-NH, exch),
12.36 (br, 2H, COOH, exch). Anal. (C20H23N5O6S·1.85H2O) C,
H, N, S.
(S)-2-({3-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)butyl]thiophene-2-carbonyl}amino)pentanedioic
Acid (5). Compound 5 was prepared using the general method
described for the preparation of 4−13, from 18b (100 mg, 0.19 mmol)
to give 83 mg (95%) of 5 as a light yellow powder. Mp 159−160 °C.
1H NMR (DMSO-d6): δ 1.56 (m, 4H, CH2CH2), 1.88−2.11 (m, 2H,
β-CH2), 2.33−2.36 (t, J = 7.5 Hz, 2H, γ-CH2), 2.48 (m, 2H, CH2),
2.86−2.88 (m, 2H, CH2), 4.31−4.36 (m, 1H, α-CH), 5.85 (s, 1H, C5-
CH), 5.98 (s, 2H, 2-NH2, exch), 7.01−7.02 (d, J = 5.0 Hz, 1H, Ar),
7.58−7.59 (d, J = 5.0 Hz, 1H, Ar), 8.24−8.26 (d, J = 7.5 Hz, 1H, CONH,
exch), 10.16 (s, 1H, 3-NH, exch), 10.79 (s, 1H, 7-NH, exch), 12.38 (br,
2H, COOH, exch). Anal. (C20H23N5O6S·1.0H2O) C, H, N, S.
(S)-2-({4-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)butyl]thiophene-3-carbonyl}amino)pentanedioic
Acid (6). Compound 6 was prepared using the general method
described for the preparation of 4−13, from 18c (100 mg, 0.19 mmol)
to give 83 mg (95%) of 6 as a light yellow powder. Mp 195−196 °C.
1H NMR (DMSO-d6): δ 1.56 (m, 4H, CH2CH2), 1.87−2.10 (m, 2H,
β-CH2), 2.35−2.38 (t, J = 8.0 Hz, 2H, γ-CH2), 2.47−2.48 (m, 2H,
CH2), 2.75−2.81 (m, 2H, CH2), 4.32−4.36 (m, 1H, α-CH), 5.85 (s, 1H,
C5-CH), 5.96 (s, 2H, 2-NH2, exch), 7.19−7.20 (d, J = 3.0 Hz, 1H, Ar),
7.92−7.93 (d, J = 3.0 Hz, 1H, Ar), 8.40−8.41 (d, J = 8.0 Hz, 1H, CONH,
exch), 10.14 (s, 1H, 3-NH, exch), 10.78 (s, 1H, 7-NH, exch), 12.31 (br,
2H, COOH, exch). Anal. (C20H23N5O6S·0.4CHCl3) C, H, N, S.
(S)-2-({4-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)butyl]thiophene-2-carbonyl}amino)pentanedioic
Acid (7). Compound 7 was prepared using the general method
described for the preparation of 4−13, from 18d (100 mg, 0.19 mmol)
to give 83 mg (95%) of 7 as a light yellow powder. Mp 174−175 °C.
1H NMR (DMSO-d6): δ 1.61 (m, 4H, CH2CH2), 1.88−2.11 (m, 2H,
(S)-2-({2-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)butyl]thiophene-3-carbonyl}amino)pentanedioic
Acid Diethyl Ester (18a). Compound 18a was prepared using the
general method described for the preparation of 18a−e, from 17a
(200 mg, 0.39 mmol) to give 191 mg (95%) of 18a as a light yellow
1
powder. Mp 81−82 °C. TLC Rf = 0.54 (CHCl3/MeOH, 5:1). H
NMR (DMSO-d6): δ 1.15−1.17 (m, 6H, COOCH2CH3), 1.61 (m,
4H, CH2CH2), 1.91−2.12 (m, 2H, β-CH2), 2.41−2.44 (t, J = 7.0 Hz,
2H, γ-CH2), 2.51−2.53 (m, 4H, CH2CH2), 4.03−4.07 (q, J = 7 Hz,
2H, COOCH2CH3), 4.08−4.13 (m, 2H, COOCH2CH3), 4.35−4.40
(m, 1H, α-CH), 5.96 (s, 3H, C5-CH, 2-NH2, exch), 7.35 (s, 2H, Ar),
8.40−8.42 (d, J = 7.5 Hz, 1H, CONH, exch), 10.99 (s, 1H, 3-NH,
exch), 11.35 (s, 1H, 7-NH, exch).
(S)-2-({3-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)butyl]thiophene-2-carbonyl}amino)pentanedioic
Acid Diethyl Ester (18b). Compound 18b was prepared using the
general method described for the preparation of 18a−e, from 17b
(200 mg, 0.39 mmol) to give 187 mg (93%) of 18b as a light yellow
1
powder. Mp 80−81 °C. TLC Rf = 0.53 (CHCl3/MeOH, 5:1). H
NMR (DMSO-d6): δ 1.15−1.20 (m, 6H, COOCH2CH3), 1.57 (m,
4H, CH2CH2), 1.93−2.12 (m, 2H, β-CH2), 2.40−2.43 (t, J = 8.0 Hz,
2H, γ-CH2), 2.51 (m, 2H, CH2), 2.87 (m, 2H, CH2), 4.03−4.07 (q, J =
7.0 Hz, 2H, COOCH2CH3), 4.08−4.14 (m, 2H, COOCH2CH3),
4.34−4.39 (m, 1H, α-CH), 5.96 (m, 3H, C5-CH, 2-NH2, exch), 7.02−
7.03 (d, J = 5.0 Hz, 1H, Ar), 7.59−7.60 (d, J = 5.0 Hz, 1H, Ar), 8.39−
8.40 (d, J = 7.5 Hz, 1H, CONH, exch), 11.09 (s, 1H, 3-NH, exch),
11.41 (s, 1H, 7-NH, exch).
(S)-2-({4-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)butyl]thiophene-3-carbonyl}amino)pentanedioic
Acid Diethyl Ester (18c). Compound 18c was prepared using
the general method described for the preparation of 18a−e, from 17c
(200 mg, 0.39 mmol) to give 193 mg (96%) of 18c as a light yellow
1
powder. Mp 79−80 °C. TLC Rf = 0.54 (CHCl3/MeOH, 5:1). H
NMR (DMSO-d6): δ 1.17−1.18 (m, 6H, COOCH2CH3), 1.56 (m,
4H, CH2CH2), 1.91−2.11 (m, 2H, β-CH2), 2.43−2.46 (t, J = 7.5 Hz,
2H, γ-CH2), 2.46−2.49 (m, 2H, CH2), 2.75−2.77 (m, 2H, CH2),
4.03−4.08 (q, J = 7.0 Hz, 2H, COOCH2CH3), 4.08−4.13 (m, 2H,
COOCH2CH3), 4.35−4.39 (m, 1H, α-CH), 5.83 (s, 1H, C5-CH),
5.98 (s, 2H, 2-NH2, exch), 7.20−7.21 (d, J = 3.5 Hz, 1H, Ar), 7.92−
7.93 (d, J = 3.5 Hz, 1H, Ar), 8.53−8.54 (d, J = 7.5 Hz, 1H, CONH,
exch), 10.13 (s, 1H, 3-NH, exch), 10.78 (s, 1H, 7-NH, exch).
(S)-2-({4-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)butyl]thiophene-2-carbonyl}amino)pentanedioic
Acid Diethyl Ester (18d). Compound 18d was prepared using the
general method described for the preparation of 18a−e, from 17d
(200 mg, 0.39 mmol) to give 185 mg (92%) of 18d as a light yellow
1
powder. Mp 81−82 °C. TLC Rf = 0.55 (CHCl3/MeOH, 5:1). H
NMR (DMSO-d6): δ 1.17−1.19 (m, 6H, COOCH2CH3), 1.62 (m,
4H, CH2CH2), 1.95−2.12 (m, 2H, β-CH2), 2.41−2.45 (t, J = 7.5 Hz,
2H, γ-CH2), 2.54−2.62 (m, 4H, CH2CH2), 4.03−4.07 (q, J = 7.0 Hz,
2H, COOCH2CH3), 4.09−4.13 (m, 2H, COOCH2CH3), 4.36−4.40
(m, 1H, α-CH), 5.99 (s, 3H, C5-CH, 2-NH2, exch), 7.41 (s, 1H, Ar),
7.76 (s, 1H, Ar), 8.67−8.68 (d, J = 7.5 Hz, 1H, CONH, exch), 11.00
(s, 1H, 3-NH, exch), 11.39 (s, 1H, 7-NH, exch).
1767
dx.doi.org/10.1021/jm201688n | J. Med. Chem. 2012, 55, 1758−1770

Journal of Medicinal Chemistry
Article
1
β-CH2), 2.33−2.36 (t, J = 7.5 Hz, 2H, γ-CH2), 2.56 (m, 4H,
CH2CH2), 4.32−4.37 (m, 1H, α-CH), 5.88 (s, 1H, C5-CH), 5.96
(s, 2H, 2-NH2, exch), 7.39 (s, 1H, Ar), 7.75 (s, 1H, Ar), 8.54−8.55
(d, J = 8.0 Hz, 1H, CONH, exch), 10.13 (s, 1H, 3-NH, exch), 10.81
(s, 1H, 7-NH, exch), 12.45 (br, 2H, COOH, exch). Anal.
(C20H23N5O6S·0.31CHCl3) C, H, N, S.
(S)-2-({5-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)butyl]thiophene-3-carbonyl}amino)pentanedioic
Acid (8). Compound 8 was prepared using the general method
described for the preparation of 4−13, from 18e (100 mg, 0.19 mmol)
to give 83 mg (95%) of 8 as a light yellow powder. Mp 175−176 °C.
1H NMR (DMSO-d6): δ 1.64 (m, 4H, CH2CH2), 1.87−2.10 (m, 2H,
β-CH2), 2.32−2.35 (t, J = 7.5 Hz, 2H, γ-CH2), 2.55 (m, 2H, CH2),
2.80−2.82 (m, 2H, CH2), 4.33−4.37 (m, 1H, α-CH), 5.88 (s, 1H,
C5-CH), 5.96 (s, 2H, 2-NH2, exch), 7.27 (s, 1H, Ar), 7.96 (s, 1H, Ar),
8.31−8.33 (d, J = 8.0 Hz, 1H, CONH, exch), 10.12 (s, 1H, 3-NH,
exch), 10.80 (s, 1H, 7-NH, exch), 12.43 (br, 2H, COOH, exch). Anal.
(C20H23N5O6S·1.5H2O) C, H, N, S.
powder. Mp 197−198 °C. H NMR (DMSO-d6): δ 1.92−2.14 (m,
2H, β-CH2), 2.33−2.37 (t, J = 8.0 Hz, 2H, γ-CH2), 2.70−2.80 (m, 4H,
CH2CH2), 4.45−4.50 (m, 1H, α-CH), 6.00 (s, 3H, C5-CH, 2-NH2,
exch), 7.74−7.75 (d, J = 3.0 Hz, 1H, Ar), 8.10−8.11 (d, J = 3.0 Hz, 1H,
Ar), 8.30−8.32 (d, J = 8.0 Hz, 1H, CONH, exch), 10.17 (s, 1H, 3-NH,
exch), 10.86 (s, 1H, 7-NH, exch), 12.44 (br, 2H, COOH, exch). Anal.
(C20H19N5O6S·0.41CH2Cl2) C, H, N, S.
(S)-2-({5-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)but-1-ynyl]thiophene-2-carbonyl}amino)-
pentanedioic Acid (20). To a solution of 19 (50 mg, 0.1 mmol) in
MeOH (10 mL) was added 1 N NaOH (5 mL), and the mixture was
stirred under N2 at room temperature for 16 h. TLC showed the
disappearance of the starting material and one major spot at the origin.
The reaction mixture was evaporated to dryness under reduced
pressure. The residue was dissolved in water (10 mL). The resulting
solution was cooled in an ice bath, and the pH was adjusted to 3−4
with dropwise addition of 1 N HCl. The resulting suspension was
frozen in a dry ice−acetone bath, thawed to 4−5 °C in the refrigerator,
and filtered. The residue was washed with a small amount of cold
water and dried in vacuum using P2O5 to afford 43 mg (95%) of 20 as
(S)-2-({2-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)but-1-ynyl]thiophene-3-carbonyl}amino)-
pentanedioic Acid (9). Compound 9 was prepared using the general
method described for the preparation of 4−13, from 17a (100 mg,
0.19 mmol, dissolved in 10 mL of MeOH/CH3COOH (9:1)) to give
1
a light yellow powder. Mp 189−190 °C. H NMR (DMSO-d6): δ
1.89−2.05 (m, 2H, β-CH2), 2.30−2.34 (t, J = 7.6 Hz, 2H, γ-CH2), 2.76
(m, 4H, CH2CH2), 4.33 (m, 1H, α-CH), 5.92 (s, 2H, 2-NH2, exch),
5.98−5.99 (d, J = 4 Hz, 1H, C5-CH), 7.19−7.20 (d, J = 4 Hz, 1H, Ar),
7.74−7.75 (d, J = 4 Hz, 1H, Ar), 8.69−8.71 (d, J = 8 Hz, 1H, CONH,
exch), 10.15 (s, 1H, 3-NH, exch), 10.86 (s, 1H, 7-NH, exch), 12.47
(br, 2H, COOH, exch). HRMS calcd for C20H19N5O6S (M + H)+,
458.1134; found, 458.1155.
1
84 mg (95%) of 9 as a light yellow powder. Mp 196−197 °C. H
NMR (DMSO-d6): δ 1.91−2.13 (m, 2H, β-CH2), 2.34−2.37 (t, J = 7
Hz, 2H, γ-CH2), 2.81 (m, 4H, CH2CH2), 4.41−4.45 (m, 1H, α-CH),
6.01 (s, 3H, C5-CH, 2-NH2, exch), 7.37−7.38 (d, J = 5.5 Hz, 1H, Ar),
7.53−7.54 (d, J = 5.5 Hz, 1H, Ar), 8.25−8.26 (d, J = 7.5 Hz, 1H,
CONH, exch), 10.19 (s, 1H, 3-NH, exch), 10.88 (s, 1H, 7-NH, exch),
12.47 (br, 2H, COOH, exch). Anal. (C20H19N5O6S·1.0CH3COOH)
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 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-
methylamino]-2-thienoyl)-L-glutamic acid], AstraZeneca Pharmaceut-
icals (Maccesfield, Cheshire, England); 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 sublines including RFC- and FRα-null
MTXRIIOuaR2-4 (R2), RFC- (pC43-10), PCFT- (R2/PCFT4), or FRα-
(RT16) expressing CHO sublines was previously described.10,11,14,18
Likewise, pDNA3.1 vector control CHO cells (R2/VC) were reported.
The CHO cells were cultured in α-minimal essential medium (MEM)
supplemented with 10% bovine calf serum (Invitrogen, Carlsbad, CA),
100 units/mL penicillin per 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 cytotoxicity assays (see below), RT16 cells
were cultured in complete folate-free RPMI 1640 (without added folate)
for 3 days. R2/hPCFT4 and R2(VC) cells were cultured in complete
folate-free RPMI 1640 including dialyzed fetal bovine serum (Invitrogen)
and 25 nM LCV plus 1.5 mg/mL G418. KB human nasopharyngeal
carcinoma cells were purchased from the American Type Culture
Collection (Manassas, VA), whereas IGROV1 ovarian carcinoma cells
were a gift of Dr. Manohar Ratnam (University of Toledo, OH). KB and
IGROV1 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.
(S)-2-({3-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)but-1-ynyl]thiophene-2-carbonyl}amino)-
pentanedioic Acid (10). Compound 10 was prepared using the
general method described for the preparation of 4−13, from 17b
(100 mg, 0.19 mmol) to give 83 mg (94%) of 10 as a light yellow
1
powder. Mp 179−180 °C. H NMR (DMSO-d6): δ 1.96−2.18 (m,
2H, β-CH2), 2.31−2.34 (t, J = 7.5 Hz, 2H, γ-CH2), 2.84 (m, 4H,
CH2CH2), 4.41−4.53 (m, 1H, α-CH), 6.01 (s, 3H, C5-CH, 2-NH2,
exch), 7.16−7.17 (d, J = 5.0 Hz, 1H, Ar), 7.80−7.81 (d, J = 5.0 Hz, 1H,
Ar), 8.22−8.23 (d, J = 7.5 Hz, 1H, CONH, exch), 10.20 (s, 1H, 3-NH,
exch), 10.89 (s, 1H, 7-NH, exch), 12.30 (br, 2H, COOH, exch). Anal.
(C20H19N5O6S·0.3CHCl3) C, H, N, S.
(S)-2-({4-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)but-1-ynyl]thiophene-3-carbonyl}amino)-
pentanedioic Acid (11). Compound 11 was prepared using the
general method described for the preparation of 4−13, from 17c
(100 mg, 0.19 mmol) to give 84 mg (95%) of 11 as a light yellow
1
powder. Mp 211−212 °C. H NMR (DMSO-d6): δ 1.92−2.14 (m,
2H, β-CH2), 2.33−2.37 (t, J = 8.0 Hz, 2H, γ-CH2), 2.70−2.80 (m, 4H,
CH2CH2), 4.45−4.50 (m, 1H, α-CH), 6.00 (s, 3H, C5-CH, 2-NH2,
exch), 7.74−7.75 (d, J = 3.0 Hz, 1H, Ar), 8.10−8.11 (d, J = 3.0 Hz, 1H,
Ar), 8.30−8.32 (d, J = 8.0 Hz, 1H, CONH, exch), 10.17 (s, 1H, 3-NH,
exch), 10.86 (s, 1H, 7-NH, exch), 12.44 (br, 2H, COOH, exch). Anal.
(C20H19N5O6S·0.3CHCl3) C, H, N, S.
(S)-2-({4-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)but-1-ynyl]thiophene-2-carbonyl}amino)-
pentanedioic Acid (12). Compound 12 was prepared using the
general method described for the preparation of 4−13, from 17d
(100 mg, 0.19 mmol) to give 85 mg (96%) of 12 as a light yellow
1
powder. Mp 195−196 °C. H NMR (DMSO-d6): δ 1.91−2.11 (m,
2H, β-CH2), 2.34−2.37 (t, J = 7.5 Hz, 2H, γ-CH2), 2.71−2.79 (m, 4H,
CH2CH2), 4.32−4.36 (m, 1H, α-CH), 6.01 (s, 3H, C5-CH, 2-NH2,
exch), 7.86−7.88 (m, 2H, Ar), 8.64−8.65 (d, J = 7.5 Hz, 1H, CONH,
exch), 10.16 (s, 1H, 3-NH, exch), 10.88 (s, 1H, 7-NH, exch), 12.46
(br, 2H, COOH, exch). Anal. (C20H19N5O6S·0.37CH2Cl2) C, H, N, S.
(S)-2-({5-[4-(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidin-6-yl)but-1-ynyl]thiophene-3-carbonyl}amino)-
pentanedioic Acid (13). Compound 13 was prepared using the
general method described for the preparation of 4−13, from 17e
(100 mg, 0.19 mmol) to give 84 mg (95%) of 13 as a light yellow
For growth inhibition assays, cells (CHO, KB, or IGROV1) were
plated in 96-well dishes (∼2500−5000 cells/well, total volume of
200 μL of medium) with a range of antifolate concentrations. The
medium was RPMI 1640 (contains 2.3 μM folic acid) with 10%
dialyzed fetal bovine serum and antibiotics for experiments with R2
and PC43-10 cells. For RT16, KB, and IGROV1 cells, cells were
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cultured in folate-free RPMI medium with 10% dialyzed fetal bovine
serum 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% dialyzed
fetal bovine serum, 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. 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.
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 biosyn-
thesis (GARFTase and AICARFTase) were tested by co-incubations
with thymidine (10 μM) and adenosine (60 μM), respectively. For de
novo purine nucleotide biosynthesis, additional protection experiments
used AICA (320 μM) to distinguish inhibitory effects at GARFTase from
those at AICARFTase.10−12
For assays of clonogenicity (colony formation), KB cells (500 cells)
were plated in folate-free RPMI1640 medium supplemented with 2
nM LCV, 10% dialyzed fetal bovine serum, penicillin−streptomycin
solution, and 2 mM L-glutamine, in the presence of antifolate drugs.
The dishes were incubated at 37 °C with 5% CO2 for 10−14 days. To
stain the colonies for counting, the dishes were rinsed with Dulbecco’s
phosphate-buffered saline (DPBS), 5% trichloroacetic acid, then
borate buffer (10 mM, pH 8.8), followed by 30 min of incubation
in 1% methylene blue in the borate buffer. The dishes were rinsed with
the borate buffer, and colonies were counted for calculating percent
colony-forming efficiency normalized to control.
In Situ GARFT Enzyme Inhibition Assay. Incorporation of
[14C]glycine into [14C]formyl GAR, as an in situ measure of endo-
genous GARFTase activity, was measured as described.10−12 For these
experiments, IGROV1 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. After
30 min, L-glutamine (final concentration, 2 mM) and [14C]glycine
(tracer amounts, final specific activity of 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 treated with 2 mL of 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 AG1x8 (chloride form), 100−200 mesh (Bio-Rad),
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. The eluates were
collected and determined for radioactivity. The accumulation of
radioactive formyl GAR was calculated as pmol per mg protein over a
range of inhibitor concentrations. IC50 values were calculated as the
concentrations of inhibitors that resulted in a 50% decrease in
[14C]formyl GAR synthesis.
ASSOCIATED CONTENT
* Supporting Information
■
S
Colony formation assay data and elemental analysis results.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
FR Binding Assay. Competitive inhibition of [3H]folic acid
binding to FRα with RT16 CHO cells was used to assess relative
binding affinities for assorted (anti)folate ligands.10−12 Briefly, RT16
cells (∼1.6 × 106) were rinsed twice with DPBS, followed by two
washes with 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 antifolate
(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.20 [3H]Folic acid bound to FRα was calculated as
pmol/mg protein, and relative binding affinities were calculated as the
inverse molar ratios of unlabeled ligands required to inhibit [3H]folic
acid binding by 50%. By definition, the relative affinity of folic acid is 1.
Transport Assays. For transport assays, R2/hPCFT4 CHO cells
were grown as monolayers and used to seed spinner flasks. For experi-
ments to determine the inhibitions of transport by antifolate substrates,
cells were collected from spinners, washed with DPBS, and resuspended
in 2 mL of HBS, adjusted to pH 6.8, or 4-morpholinepropanesulfonic
acid-buffered saline (20 mM MES, 140 mM NaCl, 5 mM KCl, 2 mM
MgCl2, and 5 mM glucose), adjusted to pH 5.5. For determinations
of Ki values, cells were incubated with 1 μM [3H]MTX over 2 min at
37 °C in the presence and absence of a range of concentrations of
unlabeled antifolates. Controls included a comparable amount of vehicle
(DMSO to 0.5%). Uptakes of [3H]MTX were quenched with ice-cold
DPBS. Cells were washed with ice-cold DPBS (3×) and solubilized with
0.5 N NaOH. Levels of intracellular radioactivity were expressed as
pmol/mg protein, calculated from direct measurements of radioactivity
and protein contents of cell homogenates. Protein concentrations were
measured with Folin phenol reagent.20 Ki values were calculated from
Dixon plots, and kinetic constants (Kt, Vmax) for [3H]MTX uptake were
calculated from Lineweaver−Burk plots, as previously described.14
■
*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.
Author Contributions
#These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by the National Institutes of
Health, National Cancer Institute, Grants CA125153 (A.G.),
CA152316 (L.H.M. and A.G.), and CA53535 (L.H.M.); a grant
from the Mesothelioma Applied Research Foundation, a pilot
grant from the Barbara Ann Karmanos Cancer Institute; and the
Duquesne University Adrian Van Kaam Chair in Scholarly
Excellence (A.G.). S.K.D. was supported by a Doctoral Research
Award from the Canadian Institutes of Health Research (CIHR).
S.M.-R. was supported by Grant T32 CA009531 (L.H.M.).
ABBREVIATIONS USED
■
AICA, 5-amino-4-imidazolecarboxamide; AICAR, 5-amino-4-
imidazolecarboxamide ribonucleotide; AICARFTase, 5-amino-
4-imidazolecarboxamide ribonucleotide formyltransferase;
CHO, Chinese hamster ovary; DPBS, Dulbecco’s phosphate-
buffered saline; FR, folate receptor; GAR, glycinamide
ribonucleotide; GARFTase, glycinamide ribonucleotide formyl-
transferase; HBSS, Hank’s balanced salt solution; HEPES,
N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; HBS,
N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffered
saline; IC50, 50% inhibitory concentration; LCV, leucovorin;
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