Tumor Targeting with Novel 6-Substituted Pyrrolo [2,3-d] Pyrimidine Antifolates with Heteroatom Bridge Substitutions via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesis

Article abstract of DOI:10.1021/acs.jmedchem.6b00594

Targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in 1 by N (4), O (8), or S (9), or with N-substituted formyl (5), acetyl (6), or trifluoroacetyl (7) moieties, were synthesized and tested for selective cellular uptake by folate receptor (FR) α and β or the proton-coupled folate transporter. Results show increased in vitro antiproliferative activity toward engineered Chinese hamster ovary cells expressing FRs by 4-9 over the CH₂ analogue 1. Compounds 4-9 inhibited de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase). X-ray crystal structures for 4 with FRα and GARFTase showed that the bound conformations of 4 required flexibility for attachment to both FRα and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, 4 was highly efficacious. Our results establish that heteroatom substitutions in the 3-atom bridge region of 6-substituted pyrrolo[2,3-d]pyrimidines related to 1 provide targeted antifolates that warrant further evaluation as anticancer agents.

Full text of DOI:10.1021/acs.jmedchem.6b00594

Subscriber access provided by STEPHEN F AUSTIN STATE UNIV
Article
Tumor Targeting with Novel 6-Substituted Pyrrolo [2,3-d] Pyrimidine
Antifolates with Heteroatom Bridge Substitutions Via Cellular
Uptake by Folate Receptor # and the Proton-coupled Folate
Transporter and Inhibition of De Novo Purine Nucleotide Biosynthesis
Lalit Kumar Golani, Adrianne Wallace-Povirk, Siobhan M. Deis, Jennifer E. Wong,
Jiyuan Ke, Xin Gu, Sudhir Raghavan, Mike R. Wilson, Xinxin Li, Lisa A. Polin, Parker
W. de Waal, Kathryn White, Juiwanna Kushner, Carrie E O'Connor, Zhanjun Hou, H.
Eric Xu, Karsten Melcher, Charles E. Dann III, Larry H. Matherly, and Aleem Gangjee
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00594 • Publication Date (Web): 26 Jul 2016
Downloaded from http://pubs.acs.org on July 27, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

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Tumor Targeting with Novel 6ꢀSubstituted Pyrrolo [2,3ꢀd] Pyrimidine Antifolates
with Heteroatom Bridge Substitutions Via Cellular Uptake by Folate Receptor α
and the Protonꢀcoupled Folate Transporter and Inhibition of De Novo Purine
Nucleotide Biosynthesis
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£ϕ
llϕ
¥
¥
€
Lalit K. Golani , Adrianne WallaceꢀPovirk , Siobhan M. Deis , Jennifer E. Wong , Jiyuan Ke ,
€
£
‡
£
ll‡
€
Xin Gu , Sudhir Raghavan , Mike R. Wilson , Xinxin Li , Lisa Polin , Parker W. de Waal ,
ll‡
ll‡
ll
ll ‡
€,ꢁ
Kathryn White , Juiwanna Kushner , Carrie O’Connor , Zhanjun Hou , H. Eric Xu , Karsten
€
¥
ll ‡§θ
£θ
Melcher , Charles E. Dann III *, Larry H. Matherly
*, and Aleem Gangjee *
£*
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne
University, 600 Forbes Avenue, Pittsburgh, PA 15282
ll
Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 110 East Warren
Ave, Detroit, MI 48201
‡
Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201
§
Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201
¥
Department of Chemistry, Indiana University, Bloomington, IN 47405
€
Laboratory of Structural Sciences and Laboratory of Structural Biology and Biochemistry, Van
Andel Research Institute, 333 Bostwick Ave NE, Grand Rapids, MI 49503
ꢁ
Key Laboratory of Receptor Research, VARIꢀSIMM Center, Center for Structure and Function
of Drug Targets, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China
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ϕθ
These authors contributed equally to this work.
*
To whom correspondence should be addressed.
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AUTHOR INFORMATION
Corresponding Authors
Aleem Gangjee, PhD, 412ꢀ396ꢀ6070; 412ꢀ396ꢀ5593 fax; gangjee@duq.edu
Larry H. Matherly, PhD, 313ꢀ578ꢀ4280; 313ꢀ578ꢀ4287 fax; matherly@karmanos.org
Charles E. Dann III, PhD, 812ꢀ856ꢀ1704; 812ꢀ856ꢀ5710 fax; cedann@indiana.edu
Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED:
5ꢀaminoimidazoleꢀ4ꢀcarboxamide (AICA); 5ꢀaminoimidazoleꢀ4ꢀcarboxamide ribonucleotide
formyltransferase (AICARFTase); Chinese hamster ovary (CHO); fetal bovine serum (FBS);
Dulbecco’s minimal essential medium (DMEM); Dulbecco’s phosphateꢀbuffered saline (DPBS);
folate receptor (FR); N10ꢀformyl tetrahydrofolate (10ꢀCHOTHF); glycinamide ribonucleotide
(GAR); glycinamide ribonucleotide formyltransferase (GARFTase); Hank’s balanced salts
solution (HBSS); HEPESꢀbuffered saline (HBS); lometrexol (LMTX); leucovorin (LCV);
methotrexate (MTX); minimal essential media (MEM); pemetrexed (PMX); polyethylene glycol
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(
(
PEG); pralatrexate (PDX); protonꢀcoupled folate transporter (PCFT); polyethylene glycol
PEG); raltitrexed (RTX); reduced folate carrier (RFC); severeꢀcombined immunodeficient
(SCID); tumorꢀassociated macrophages (TAMs); trichloroacetic acid (TCA); thin layer
chromatography (TLC).
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ABSTRACT
Targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in 1
by N (4), O (8), or S (9), or with Nꢀsubstituted formyl (5), acetyl (6), or trifluoroacetyl (7)
moieties, were synthesized and tested for selective cellular uptake by folate receptor (FR) α and
β or the protonꢀcoupled folate transporter. Results show increased in vitro antiꢀproliferative
activity toward engineered Chinese hamster ovary cells expressing FRs by 4ꢀ9 over the CH2
analog 1. Compounds 4ꢀ9 inhibited de novo purine biosynthesis and glycinamide ribonucleotide
formyltransferase (GARFTase). Xꢀray crystal structures for 4 with FRα and GARFTase showed
that the bound conformations of 4 required flexibility for attachment to both FRα and
GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, 4 was highly efficacious. Our
results establish that heteroatom substitutions in the 3ꢀatom bridge region of 6ꢀsubstituted
pyrrolo[2,3ꢀd]pyrimidines related to 1 provide targeted antifolates that warrant further evaluation
as anticancer agents.
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INTRODUCTION
1
Reduced folates are essential cofactors for biosynthesis of purines and pyrimidines. Since
humans do not synthesize folate, it is necessary to obtain these cofactors from dietary sources.
In mammals, three specialized systems exist that mediate membrane transport of folates and
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antifolates across biological membranes. These include the reduced folate carrier (RFC), folate
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4, 5
receptor (FRs) α and β, and the protonꢀcoupled folate transporter (PCFT).
RFC and PCFT
are facilitative folate transporters, whereas FRs are glycosylphosphatidylinositolꢀlinked proteins
that mediate uptake of folates into cells by receptorꢀmediated endocytosis. RFC is ubiquitously
2
expressed and is the major tissue transporter for folate cofactors. FRα and FRβ, as well as
PCFT, exhibit narrower patterns of tissue expression and likely serve more specialized
3, 5
physiologic roles. For instance, in the proximal tubules of the kidney, FRα contributes to
3
reabsorption of folate from the urine. Notably, FRs in normal tissues are either inaccessible to
3
circulating folates (e.g., FRα in renal tubules) or are nonꢀfunctional (FRβ in thymus). PCFT is
expressed in the upper gastrointestinal tract where it functions at acidic pH as the major
6
ꢀ8
intestinal transporter for absorption of dietary folates. Although appreciable PCFT can be
5
detected in a number of other normal tissues (e.g., liver, kidney), given the requirement for an
4
, 8
acidic pH (pH < 7, optimum at pH 5ꢀ5.5) for activity, PCFT transport should be limited in most
normal tissues.
Classical antifolates continue to serve a key role in the therapy of cancer, as well as for other
9ꢀ13
diseases.
Clinically important antifolates include methotrexate (MTX), pemetrexed (PMX),
pralatrexate (PDX) and raltitrexed (RTX) (Figure 1). MTX and PDX are both inhibitors of
1
0
dihydrofolate reductase. PMX and RTX inhibit thymidylate synthase as their primary
intracellular target, although for PMX, secondary targets have been described, including 5ꢀ
aminoimidazoleꢀ4ꢀcarboxamide (AICA) ribonucleotide formyltransferase (AICARFTase) and
glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) in de novo purine nucleotide
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14ꢀ16
biosynthesis, and dihydrofolate reductase.
Notably, all these compounds are excellent
substrates for the RFC and, to varying degrees, are also substrates for FRs α and β and for
2, 5, 10
PCFT.
Transport by RFC confers limited tumor selectivity, as both tumors and normal
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tissues express this transporter. Interestingly, a substantial cohort of solid tumors (e.g.,
1
7, 18 19
ovarian, nonꢀsmall cell lung cancer) also expresses PCFT, often in concert with FRα.
In
tumors (unlike normal tissues), FRα is accessible to the circulation, thus facilitating tumor
20, 21
targeting via this mechanism.
FRβ is expressed in hematologic malignancies such as acute
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myeloid leukemia and in white blood cells of the myeloid lineage, including tumorꢀassociated
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macrophages (TAMs). PCFT is active at the acidic pHs typically associated with the tumor
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microenvironment which confers an additional element of tumor selectivity.
Figure 1. Structures of classic antifolate drugs, including methotrexate (MTX), pemetrexed (PMX),
pralatrexate (PDX), and raltitrexed (RTX).
In response to the patterns of expression and function of FRs and PCFT in tumors and normal
tissues, new cytotoxic agents are being developed for selective tumor targeting by virtue of their
specificities for FRs and/or PCFT. FRβꢀpositive TAMs may play an important role in the tumor
microenvironment in relation to tumor metastasis and angiogenesis by releasing proangiogenic
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factors (e.g., vascular endothelial growth factor, matrix metalloproteinase), suggesting that
TAMs may constitute an additional potential therapeutic target in cancer for FRβꢀtargeted
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agents.
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Figure 2. Structures of 6ꢀpyrrolo[2,3ꢀd]pyrimidine antifolates, 1, 2 and 3.
Folate and pteroate conjugates have been used to deliver cytotoxic agents to FRꢀexpressing
21, 24, 25
tumors including ovarian cancer and nonꢀsmall cell lung cancer (NSCLC).
For example,
a folic acidꢀvindesine conjugate (vintafolide) was developed which is internalized by FRs, then
24, 25
cleaved in endosomes to release the cytotoxic vinca alkaloid.
Based on its success in a
26
randomized Phase II clinical trial with platinum resistant ovarian cancer, vintafolide was
25, 27
advanced to a Phase III clinical trial. More recently, a folic acidꢀtubulysin conjugate,
was
introduced into Phase I clinical trials. Nꢀ[4ꢀ[2ꢀpropynꢀ1ꢀyl[(6S)ꢀ4,6,7,8ꢀtetrahydroꢀ2ꢀ
(
hydroxymethyl)ꢀ4ꢀoxoꢀ3Hꢀcyclopenta[g]quinazolinꢀ6ꢀyl]amino]benzoyl]ꢀLꢀγꢀglutamylꢀDꢀglutamic
acid (ONX0801) is a small molecule antifolate with selective substrate activity toward FRs over
28
RFC that is an inhibitor of thymidylate synthase. This compound is currently being tested in a
Phase I clinical trial.
We previously described novel 6ꢀsubstituted pyrrolo[2,3ꢀd]pyrimidine benzoyl antifolates with
selective cellular uptake by FRs and/or PCFT over RFC, resulting in potent inhibitory activity
18, 29ꢀ35
toward human tumor cells.
The most active analog of this series, 1, included a 3 carbon
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bridge (Figure 2) and showed high level activity toward KB human tumors (IC = 1.7 nM)
50
expressing FRα and PCFT. Compound 2 has a 4ꢀcarbon bridge and showed a better selectivity
than 1 for both FRα and PCFT over RFC. An analog of compound 1 with a sideꢀchain thienoylꢀ
forꢀbenzoyl replacement (compound 3) (Figure 2) showed even greater FRꢀ and PCFTꢀtargeted
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3
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activity. Although 1 and 3 were not effective substrates for RFC, an apparent nonꢀmediated
uptake process, as reflected in growth inhibition of both RFC and transportꢀnull cells, was
29, 31
detected.
Compounds 1, 2 and 3 were identified as potent inhibitors of cellular GARFTase
29, 31
and de novo purine nucleotide biosynthesis.
Notably, these compounds were entirely
unique from previously reported GARFTase inhibitors including lometrexol [(6R)5,10ꢀ
36
dideazatetrahydrofolate; LMTX], (2S)ꢀ2ꢀ((5ꢀ(2ꢀ((6R)ꢀ2ꢀaminoꢀ4ꢀoxoꢀ 5,6,7,8ꢀtetrahydroꢀ1Hꢀ
3
6
pyrido[2,3ꢀd]pyrimidinꢀ6ꢀyl)ethyl)thiopheneꢀ2ꢀcarbonyl)amino) pentanedioic acid (LY309887),
and (2S)ꢀ2ꢀ((5ꢀ(2ꢀ((6S)ꢀ2ꢀaminoꢀ4ꢀoxoꢀ1,6,7,8ꢀtetrahydropyrimido[5,4ꢀb][1,4]thiazinꢀ6ꢀyl)ethyl)
37
thiopheneꢀ2ꢀcarbonyl)amino) pentanedioic acid (AG2034), as all these latter antifolates are
excellent substrates for RFC. In Phase I clinical trials with these earlier antifolates, toxicities
38ꢀ40
were doseꢀlimiting,
likely due at least in part to their cellular uptake by RFC and their
metabolism to polyglutamates in normal tissues.
Figure 3. Structure of N10ꢀformyl tetrahydrofolate.
The natural substrate for GARFTase, N10ꢀformyl tetrahydrofolate (10ꢀCHOTHF, Figure 3), is a
6
ꢀsubstituted pteridine with a –CH ꢀNꢀ twoꢀatom bridge. The N10 is substituted with a formyl
2
moiety which forms a hydroxylated, tetrahedral intermediate prior to transfer of the oneꢀcarbon
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group. Thus, replacement of the C10ꢀbenzylic CH of the 6ꢀsubstituted pyrrolo[2,3ꢀ
2
d]pyrimidine 1 with a NH, NꢀCOCH , NꢀCHO, and NꢀCOCF [4, 5, 6, and 7, respectively; Figure
3
3
4
] would afford “mimics” of the natural substrate with a threeꢀatom rather than twoꢀatom bridge.
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We hypothesized that these 6ꢀsubstituted threeꢀatom bridged NH and Nꢀsubstituted analogs
would function as potent inhibitors of human GARFTase rather than as substrates. It was further
anticipated that these N10ꢀH (4) and N10ꢀsubstituted compounds (5ꢀ7) would provide additional
hydrogen bonding with the target enzyme GARFTase that would be absent with the threeꢀ
carbon bridge analog 1.
Compound
X
4
5
6
7
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9
NH
NCHO
NCOCH
3
NCOCF
3
O
S
Figure 4. Structures of 6ꢀsubstituted pyrrolo[2,ꢀ3ꢀd]pyrimidine antifolates with heteroatom bridge
substitutions.
We previously reported that in the carbonꢀbridged analogs the length of the sideꢀchain plays an
1
8, 29
important role in determining potency and transport selectivity
such that the most transport
selective analogs have a fourꢀcarbon bridge (e.g., 2) and the most potent GARFTase and tumor
inhibitors have a three carbon bridge (e.g., 1). Thus, it was of interest to compare the effects of
replacing the benzylic CH of 1 with NH (compound 4), O (compound 8) and S (compound 9)
2
42
(
Figures 4 and 5). The CꢀS bond length is 1.81 Å, compared to the CꢀC bond length of 1.54 Å.
Replacing the benzylic CH of the threeꢀatom chain of 1 with larger sulfur would afford a chain
2
length between those for the 3ꢀcarbon and 4ꢀcarbon bridges of 1 and 2, respectively. In
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addition to the increased bond length, the CꢀSꢀC bond angle is 99° compared to a CꢀCꢀC bond
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angle of 109° (Figure 5). This provides a slightly different conformational orientation of the
benzene ring, and hence Lꢀglutamate orientation. Similarly, replacement of the benzylic CH2
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with NH or an O would afford subtle variations in bond length (CꢀO = 1.43 Å) and bond angle
(CꢀOꢀC = 111°, Figure 5). Collectively, these structural alterations could impact transport
selectivity, as well as GARFTase inhibition and antitumor efficacy.
In this study, we report the synthesis and biological activities of analogs of 1 with isosteric
heteroatom replacements, including S (9), O (8) and N (4) vicinal to the side chain phenyl ring,
along with additional Nꢀsubstituted analogs including formyl (5), acetyl (6) and trifluoroacetyl (7)
moieties.
Compound
X
Distance (d)
2.63 Å
Angle (a)
109.5°
126.1°
111°
3
4
8
9
CH
2
NH
O
2.52 Å
2.42 Å
S
2.88 Å
99°
Figure 5. Distances and bond angle variations predicted by the nature of the bridge at the benzylic
42
position (X). Bond angles for X = CH , O and S obtained from literature. Distances and angles for X =
2
43
NH were measured using energyꢀminimized conformations of compounds with MOE 2014.09.
MOLECULAR MODELING
To provide a rationale for our proposed analogs, molecular modeling studies were initially
18
carried out using our Xꢀray crystal structure of human GARFTase bound to 3 (PDB: 4ZYW).
Figure 6A shows the docked pose of the lead compound 1 (green) in the GARFTase active site
as a representative example of docking the target molecules. Compound 4 (orange) is also
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docked in Figure 6A. The pyrrolo[2,3ꢀd]pyrimidine scaffold of 1 binds in the region occupied by
18
the bicyclic scaffold of 3 in the GARFTase crystal structure (PDB: 4ZYW , 3 not shown for
clarity). Hydrogen bonds between the N1 nitrogen of 1 and the backbone of Leu899, 2ꢀNH of 1
2
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and the carbonyls of Glu948 and Leu899, N3 of 1 and the amide of Ala947, and 4ꢀoxo of 1 and
the amide of Asp951 stabilize the scaffold. Additionally, the N7ꢀnitrogen of 1 forms a hydrogen
bond with the carbonyl of Arg897. The pyrrolo[2,3ꢀd]pyrimidine scaffold of 1 forms hydrophobic
interactions with Leu892, Ile898, Leu899, and Val904, and the folate binding loop residues 948ꢀ
951. The amide NH of the Lꢀglutamate of 1 forms a hydrogen bond with the carbonyl of Met896.
The Lꢀglutamyl moiety of 1 is oriented similar to 3, with the αꢀcarboxylate interacting with
Arg897.
18
Figure 6. Molecular modeling studies with human GARFTase (PDB: 4ZYW). (Panel A)
Superimposition of docked poses of 1 (green) and 4 (orange) (Panel B) Docked pose of 7 (purple). In
both panels, GAR is shown in pink.
The docked poses of 4 (Figure 6A, orange) and 7 (Figure 6B) retain the hydrogen bonding and
hydrophobic interactions seen in the docked pose of the lead compound 1. In addition, the
docked pose of 4 at the N10ꢀH forms hydrogen bonds with the substrate GAR and with Asn913,
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His915 and Asp951 via a bridging water molecule (Figure 6A). Similar water molecules are
18, 44
seen in other reported Xꢀray crystal structures of GARFTase.
This suggests that 4 with the
N10ꢀH hydrogen bond would possibly bind better than 1 and 2 with C10ꢀH. Docking scores of 4
(ꢀ56.31 kJ/mol) were better than those of 1 (ꢀ51.26 kJ/mol) and 2 (ꢀ52.57 kJ/mol). Similar
interactions are predicted to occur between the heteroatom bridges of 8 (oxygen) and 9 (sulfur)
and the conserved water molecule, with the heteroatoms functioning as hydrogen bond
acceptors (not shown).
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The N10ꢀCOCF moiety in 7 (Figure 6B) is oriented similar to the N10ꢀH of 4, but binds deeper
3
into the formyl transfer region that is occupied by the formyl group of the natural GARFTase
substrate, 10ꢀCHOTHF. The increased bulk at the N10ꢀposition of 7 displaces the water
molecule involved in Hꢀbonding with the N10ꢀH of 4, and affords direct interaction between the
trifluoromethyl of the NCOCF moiety of 7 and the terminal amine of substrate GAR. The
3
docking score of 7 in GARFTase was ꢀ53.31 kJ/mol. Similar interactions involving the terminal
amine of GAR are also observed with the carbonyl of the formyl group of 5 and the carbonyl
group of the acetyl of 6 (not shown).
Figure 7 shows the results of molecular modeling studies in the folate binding cleft of FRα
45
(
PDB: 4LRH) for lead compounds 1 (panel A), 2 (panel B) and 4 (panel C). All three
compounds display similar interactions in the pocket, maintaining important protein contacts
between the bicyclic scaffolds and benzoyl glutamate tail, as also seen in the crystal structure
45
ligand folic acid. In the docked pose of 4 (Figure 7C), the 2ꢀNH of 4 interacts with Asp103
2
(
81) (for FR, numbering is that of the fullꢀlength gene product with mature protein numbering in
parentheses). The 4ꢀoxo moiety forms hydrogen bonds with the side chain hydroxyl of Ser196
174) and the side chain nitrogens of Arg125 (103) and His157 (135). The pyrrolo[2,3ꢀ
d]pyrimidine scaffold is stacked between the side chains of Tyr82 (60) and Trp193 (171), similar
(
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45
to that seen with the pteroyl ring of folic acid in its bound conformation. The Lꢀglutamate
45
moiety of 4 is oriented similar to the corresponding Lꢀglutamate in folic acid. The αꢀcarboxylic
acid of 4 forms a network of hydrogen bonds involving the backbone NH of Gly159 (137) and
Trp160 (138), while the γꢀcarboxylic acid of 4 interacts with the amine of Lys158 (136) and the
side chain NH of Trp124 (102). The bridge of 4 forms hydrophobic interactions with Tyr82 (60),
Phe84 (62), Trp124 (102) and His157 (135).
0
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Figure 7: Molecular modeling studies using the human FRα crystal structure (PDB: 4LRH). Panel
A, superimposition of the docked pose of 1 (green) with the crystal structure of folic acid (black). Panel B,
superimposition of the docked pose of 2 (pink) with crystal structure of folic acid (black) (C) Docked pose
of 4 (orange).
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Comparing the docked poses of 1 (Figure 7A)and 2 (Figure 7B) with the crystal structure of
45
folic acid in FRα indicates that the 3ꢀ and 4ꢀatom bridges of 1 and 2 are accommodated within
a similar space as the 2ꢀatom bridge in folic acid. Since the bridge NH in folic acid does not form
hydrogen bonds with the binding pocket, its replacement with an allꢀcarbon bridge (1 and 2)
could, perhaps, better utilize the hydrophobic nature of the pocket for binding interactions.
Introduction of heteroatom bridges in the target compounds was not expected to affect FRα
transport capabilities of these compounds, reflected by their highly similar docked pose (e.g.,
compound 4, Figure 7C) compared to lead 1 and folic acid. The docking score of 4 was ꢀ43.87
kJ/mol, similar to the docked scores of 1 (ꢀ44.29 kJ/mol) and 2 (ꢀ46.99 kJ/mol).
10
11
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CHEMISTRY
Compounds 10 and 11 (Scheme I) were subjected to Michael addition to afford the βꢀketo
amine 12. Compound 12 was then selectively brominated at the α carbon under acidic
conditions using 33% HBr in acetic acid/Br to give 13. Protection of the amine in 13 using
2
trifluoroacetic anhydride and subsequent condensation with 2,6ꢀdiaminoꢀ3Hꢀpyrimidinꢀ4ꢀone in
DMF at room temperature for 3 days afforded the 2ꢀaminoꢀ4ꢀoxoꢀ6ꢀsubstitutedꢀpyrrolo[2,3ꢀ
d]pyrimidine 14. Hydrolysis of 14 afforded the corresponding free acid 15. Subsequent coupling
with Lꢀglutamate diꢀtertꢀbutyl ester using 2ꢀchloroꢀ4,6ꢀdimethoxyꢀ1,3,5ꢀtriazine as the activating
agent afforded the diester 16. Deprotection of the diꢀtertꢀbutyl ester afforded the corresponding
acid 4. Compound 4 was then reacted with formic acid and acetic anhydride, acetic anhydride or
trifluoroacetic anhydride to afford target compounds 5, 6 and 7, respectively.
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Scheme I
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Reagents and conditions. (a) Ethanol, reflux, 5 h, 59% ;(b) 33% HBr in CH COOH, Br , rt, 2.5 h, 33%;
3
2
(
c) (CF CO) O, rt, overnight, 76%; (d) 2,6ꢀdiaminopyrimidinꢀ4(3H)ꢀone, DMF, rt, 3 d, 25%; (e) 1N NaOH,
3 2
rt, 10 h, 88%; (f) Nꢀmethylmorpholine, 2ꢀchloroꢀ4,6ꢀmethoxyꢀ1,3,5ꢀtriazine, LꢀGlutamate diꢀtertꢀbutyl,
DMF, 12 h, 60%; (g) CF COOH, CH Cl , rt, 2 h, 74%; (h) (CH CO) O, rt, 12 h, 47% or (CF CO) O, rt, 4 h,
3
2
2
3
2
3
2
76% or (CH CO) O, HCOOH, 1 h, reflux, 35%.
3 2
Carboxylic acid 19 (Scheme II) was obtained from βꢀpropiolactone 17 and methyl 4ꢀ
hydroxybenzoate 18 using NaOH at 50 °C for 30 min. The carboxylic acid 19 was then
converted to the acid chloride and immediately reacted with diazomethane, followed by 48%
HBr in water, to give the αꢀbromomethylketone 20. Condensation of 2,6ꢀdiaminoꢀ3Hꢀpyrimidinꢀ4ꢀ
one with 20 in DMF at room temperature for 3 days afforded the 2ꢀaminoꢀ4ꢀoxoꢀ6ꢀsubstitutedꢀ
pyrrolo[2,3ꢀd]pyrimidine 21. Hydrolysis of 21 afforded the corresponding free acid 22.
Subsequent coupling with the Lꢀglutamate dimethyl ester using 2ꢀchloroꢀ4,6ꢀdimethoxyꢀ1,3,5ꢀ
triazine as the activating agent afforded the diester 23. Saponification of the diester gave the
target compound 8.
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Scheme II
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Reagents and conditions: (a) NaOH, H O, 30 min, 80 °C, 52%; (b) Oxalyl chloride, CH Cl , reflux, 1 h,
2
2
2
7
9%; (c) Diazomethane, (CH CH ) O, rt, 1h; (d) 48% HBr in water, 80 °C, 2 h, 94%; (e) 2,6ꢀ
3 2 2
diaminopyrimidinꢀ4(3H)ꢀone, DMF, rt, 3 d, 31%; (f) 1N NaOH, rt, 12 h, 74%; (g) Nꢀmethylmorpholine 2ꢀ
chloroꢀ4, 6ꢀmethoxyꢀ1,3,5ꢀtriarzine, Lꢀglutamate dimethyl, DMF, 12 h, 42%; (h) 1N NaOH, rt, 4 h, 88%.
The 6ꢀsubstituted pyrrolo[2,3ꢀd]pyrimidine 27 (Scheme III) was synthesized by condensation of
2,6ꢀdiaminoꢀ4ꢀoxopyrimidine 25 with ethyl 4ꢀchloroꢀ3ꢀoxobutanoate 26. Reduction of the ester
group of 27 to the alcohol 28 and subsequent mesylation of the alcohol gave 29. Nucleophilic
displacement with 4ꢀmercaptoꢀbenzoic acid methyl ester under basic conditions gave the
thiopheneꢀlinked ester 30. Hydrolysis of the ester moiety of 30 to acid 31 and coupling with Lꢀ
glutamate diethyl ester using 2ꢀchloroꢀ4,6ꢀdimethoxyꢀ1,3,5ꢀtriazine as the activating agent
afforded the diester 32. Saponication of the diester moieties gave target 9.
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Scheme III
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Reagents and conditions: (a) NaOAc, H O, reflux, 18 h, 54%; (b) LiEt BH, THF, 0 °C, 30 min, 75%; (c)
2
3
CH SO Cl, DMF, Et N, 0 °C, 2 h, 80%; (d) 4ꢀmercaptoꢀbenzoic acid methyl ester, K CO , DMF, rt, 12 h,
3
2
3
2
3
37%; (e) 1N NaOH, MeOH, rt, 95ꢀ99%; (f) Lꢀglutamic acid diethyl ester hydrochloride, Nꢀmethyl
morpholine, 2ꢀchloroꢀ4,6ꢀdimethoxyꢀ1,3,5ꢀtriazine, DMF, rt, 6 h, 60%.
BIOLOGICAL EVALUATION AND DISCUSSION
Antiꢀproliferative effects of 6ꢀsubstituted pyrrolo[2,3ꢀd]pyrimidine benozyl analogs with
heteroatom bridge substitutions in relation to mechanisms of folate transport. The goal of
this study was to explore the impact of various isosteric heteroatom substitutions including N, O,
and S (designated compounds 4, 8, and 9, respectively) at position 10 in the bridge region
connecting the bicyclic scaffold and the phenyl ring of 6ꢀpyrrolo[2,3ꢀd]pyrimidine antifolates
(Figure 4). Additional Nꢀsubstituted analogs related to 4 with formyl (5), acetyl (6), and
trifluoroacetyl (7) substitutions were also synthesized and tested.
As a primary screen of antiproliferative activities for these novel agents, we initially used a panel
29
of isogenic Chinese hamster ovary (CHO) sublines individually expressing FRα (RT16 cells),
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FRβ (D4), RFC (PC43ꢀ10), or PCFT (R2/PCFT4) . These engineered CHO cell lines were
R
48
developed from RFCꢀ, FRꢀ and PCFTꢀnull MTXRIIOua 2ꢀ4 CHO cells (hereafter, referred to
as R2) by stably transfecting the human transporters and have been documented for their
2
9, 46, 47, 49
10
11
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transport characteristics.
To assess the inhibitory potentials of the 6ꢀsubstituted
pyrrolo[2,3ꢀd]pyrimidine analogs (or classical antifolate inhibitors such as PMX, for comparison),
the CHO sublines were cultured in the presence of a range of drug concentrations for up to 96
hours and cell viabilities were measured as a fluorescence readꢀout. IC values, corresponding
50
to the drug concentrations that inhibit growth by 50%, were calculated. Results for the
engineered CHO sublines were compared to those for R2 CHO cells.
The 6ꢀsubstituted pyrrolo[2,3ꢀd]pyrimidine analogs with heteroatom bridge replacements, 4 (N),
8
(O), and 9 (S), all showed potent growth inhibition toward FRαꢀexpressing RT16 cells and
toward FRβꢀexpressing D4 cells with IC values equal or less than those previously reported
50
29, 49
for 1
(Table 1). The impacts of Nꢀsubstitutions including formyl (5), acetyl (6) or
trifluoroacetyl (7) on FRꢀtargeted activity compared to Nꢀunsubstituted 4 were modest. Notably,
the Nꢀheteroatom analogs including unsubstituted (4) and Nꢀsubstituted (5 and 6) forms were
significantly more potent toward D4 than toward RT16 cells (<0.003) (Table 1). While 7 was
also more active toward D4 cells, the difference from RT16 was not statistically significant.
These results suggest an increased FRβ selectivity over FRα for the N heteroatom series of 6ꢀ
substituted pyrrolo[2,3ꢀd]pyrimidine analogs.
With PCFTꢀexpressing R2/PCFT4 CHO cells, antiꢀproliferative activities were reduced for 4 (N),
8 (O) and 9 (S), compared to that for compound 1 (Table 1). Nꢀsubstitution of 4 (i.e., 5, 6, and
7) resulted in no significant increase in growth inhibition of R2/PCFT4 cells compared to the
nonꢀsubstituted compound (4).
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Toward RFCꢀexpressing PC43ꢀ10 cells, very minor inhibitory effects were detected for all the
heteroatom compounds, with IC s which were far in excess of the inhibition recorded for FRαꢀ,
50
FRβꢀ or PCFTꢀexpressing CHO cells (Table 1). The modest inhibitions of the heteroatom
29
31
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analogs were likewise less than those previously reported toward PC43ꢀ10 cells for 1 or 3 .
Thus, the selectivity for this series (as reflected in the relative IC s for FRα or FRβ versus RFCꢀ
50
29, 31
expressing CHO cells) is greater than previously reported for compounds 1, 2 or 3.
The results with the engineered CHO sublines were generally recapitulated in KB human
18
nasopharengeal carcinoma cells, which express FRα and PCFT along with RFC , although
there were some differences, with 4 and 8 showing potencies essentially equivalent to 3 in this
tumor model (Table 1). In KB tumor cells, 4 was significantly more active than 5, 6, 7, and 9
(
p<0.05), with the antiproliferative effects for the entire series substantially reversed in the
presence of 200 nM folic acid. This is consistent with a FRꢀmediated cellular uptake process,
18
although nonꢀFR uptake (i.e., PCFT) manifested (as reflected by a lack of folic acid protection )
at higher concentrations of 4, 6, 7 and 8 (Figure 10 shows results for KB cells).
The inhibitory potencies measured for the 6ꢀsubstituted pyrrolo[2,3ꢀd]pyrimidine heteroatom
compounds 4, 5, 6, 7, 8, and 9 toward the engineered CHO and human tumor cell lines
exceeded those for standard antifolates including MTX, PMX, RTX and LMTX (Table 1).
Further, there was limited selectivity for FR and PCFT over RFC for the standard agents.
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Table 1. IC50s (in nM) for 6ꢀsubstituted pyrrrolo[2,3ꢀd]pyrimidine benzoyl antifolates with heteroatom replacements, and classical antifolates in RFCꢀ,
PCFTꢀ and FRꢀexpressing cell lines. Proliferation assays were performed for CHO sublines engineered to express human RFC (PC43ꢀ10), FRα (RT16), FRβ
2
9, 46ꢀ48
(D4) or PCFT (R2/PCFT4), and transporterꢀnull (R2) CHO cells,
and KB human tumor cells (express RFC, FRα, and PCFT). For the experiments measuring
FRꢀmediated effects, assays were performed in the presence or absence of 200 nM folic acid (results are shown only for KB cells). Results are presented as IC50
values, corresponding to the concentrations that inhibit growth by 50% relative to cells incubated without drug. The data are mean values from 5ꢀ16 experiments
2
9ꢀ31, 34, 47, 49
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47
48
49
(+/ꢀ standard errors in parentheses). Some of the data for 1, 2, 3, MTX, PDX, PMX and LMTX have been previously published.
Results are also
summarized for KB cells for the protective effects of adenosine (60 ꢂM), thymidine (10 ꢂM), or 5ꢀaminoimidazoleꢀ4ꢀcarboxamide (320 ꢂM). For compounds 4, 5, 6,
7, 8, and 9, folic acid and nucleoside/AICA protection results are shown in Figure 10. Methods are summarized in the Experimental Section. Undefined
abbreviations: Ade, adenosine; AICA, 5ꢀaminoimidazoleꢀ4ꢀcarboxamide; FA, folic acid; ND, not determined; Thd, thymidine. *Significantly different from compound
4 with RT16 or KB cells, as appropriate (p<0.05); ϕθ¥, significantly different from RT16 with 4 (p=0.0006), 6 (p=0.0026), and 5 (p=0.0001).
RFC
FRα
FRβ
D4
PCFT
RFC/FRα/PCFT
Antifolate
KB +
Ade/Thd/AICA
PC43ꢀ10
R2
RT16
R2/PCFT4
R2
KB
KB (+FA)
1
2
3
4
5
6
7
8
9
649(38)
>1000
>1000
>1000
4.1(1.6)
6.3(1.6)
5.6(1.2)
10(2)
23.0(3.3)
213(28)
>1000
>1000
288(12)
>1000
>1000
>1000
>1000
>1000
1.7(0.4)
1.7(0.4)
>1000
>1000
101(7)
666(46)
>1000
>1000
924(40)
704(73)
>1000
Ade/AICA
Ade/AICA
Ade/AICA
Ade/AICA
Ade/AICA
Ade/AICA
Ade/AICA
Ade/AICA
Ade/AICA
101.0 (16.6) 273.5 (49.1)
0.31 (0.14)
3.04 (0.71)
3.01 (0.57)
4.87 (0.81)
1.08 (0.69)
1.89 (0.85)
0.51 (0.10)*
0.17 (0.03)
3.34(0.26)
87.4(9.9)
88.5(13.4)
81.7(91)
109 (26)
57.5(5.8)
267(19)
0.26(0.03)
0.32(0.05)
2.09(0.72)*
2.57(0.60)*
0.56(0.05)*
0.34(0.05)
1.66(0.26)*
ϕ
510(90)
642(239)
808(124)
783(109)
641(140)
>1000
>1000
>1000
>1000
>1000
940(60)
>1000
0.62 (0.20)
0.53 (0.11)
1.57 (0.53)
¥
θ
0.45 (0.11)
2.59 (1.17)
0.34 (0.02)
>1000
>1000
974 (18)
>1000
MTX
PMX
RTX
12(1.1)
138(13)
6.3(1.3)
12(2.3)
114(31)
42(9)
15(5)
114 (31)
42 (9)
15 (5)
106 (11)
60 (8)
22 (10)
2.6 (1.0)
121 (17)
13.2(2.4)
99.5(11.4)
38.0(5.3)
6.00(0.60)
68(12)
5.90(2.20)
1.20(0.60)
20(2.4)
327(103)
22(5)
Thd/Ade
Thd/Ade
Thd
LMTX
12(8)
12 (8)
>1000
31(7)
Ade/ AICA
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1
1
1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
T
ransport characteristics of 6ꢀsubstituted pyrrolo[2,3ꢀd]pyrimidine benozyl analogs with
heteroatom bridge substitutions. The growth inhibition data with the engineered CHO cell
lines in Table 1 strongly imply that the heteroatom analogs 4ꢀ9 are internalized by FRα and
FRβ, as well as by PCFT.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. FRα and FRβ binding affinities for compounds 4ꢀ9, compared to folic acid, MTX and
compound 1. Results are shown for the relative binding affinities of the (anti)folates with FRαꢀexpressing
RT16 and FRβꢀexpressing D4 CHO cells. Relative binding affinities were determined over a range of
(
anti)folate concentrations and were calculated as the inverse molar ratios of the unlabeled ligands
3
required to inhibit [ H]folic acid binding by 50%. By this definition, the relative affinity of folic acid is 1.
Results are shown as mean values plus/minus standard errors from 3ꢀ4 experiments. Detailed
experimental methods are provided in the Experimental Section. Abbreviation: FA, folic acid.
The binding affinity for folic acid, as reflected in the dissociation constant (K ) value measured
d
50
by isothermal calorimetry, was greater for FRα (K =0.01 nM) than for FRβ (K =2.7 nM). To
d
d
determine relative binding affinities individually for FRα and FRβ compared to folic acid for this
3
series, we incubated RT16 and D4 cells with [ H]folic acid at 4 °C in the presence of a range of
concentrations of the unlabeled pyrrolo[2,3ꢀd]pyrimidine antifolates 1 and 4ꢀ9 (0ꢀ1000 nM).
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2
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4
5
6
7
8
9
Controls for these experiments included a comparable concentration range of nonꢀradioactive
folic acid (positive control) and MTX (negative control). Relative binding affinities are reflected in
3
the extents to which the analogs compete with [ H]folic acid for binding to FR, with FRꢀbound
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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31
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[ H]folic acid normalized to total cell proteins. Relative FR binding affinities were expressed as
inverse molar ratios of the unlabeled compounds required to reduce the level of FRꢀbound
3
[
H]folic acid by 50%, with the folic acid affinity assigned a value of 1.
A
C
K (ꢁM)
i
Antifolate Mean
SEM
N
PMX
0.094
0.006
8
Cpd 1
Cpd 4
Cpd 5
Cpd 6
Cpd 7
Cpd 8
Cpd 9
0
.22
0.17
0.10
3
3
0.85
0.73
1.68
0.80
0.64
1.48
0.04
0.11
0.08
0.058
0.27
4
3
3
3
3
B
Figure 9. Transport competition by 6ꢀsubstituted pyrrolo[2,3ꢀd]pyrimidine analogs for PCFT in
R2/PCFT4. Panels A and B: Data are shown for the inhibitory effects of the unlabeled ligands (1 or 10
3
nM) on [ H]MTX uptake (5 min) with human PCFTꢀexpressing R2/PCFT4 CHO cells at pH 5.5 and pH
6.8. The extent of inhibition in this assay is a reflection of relative PCFT binding affinities for assorted
antifolate substrates. Results are presented as average values plus/minus ranges for 2ꢀ4 experiments.
Results are compared to those for normalized rates in the absence of any additions (“No additions”) and
to PCFTꢀnull R2 cells without additions. Panel C: In the table are shown K values for inhibition of
i
3
[
H]MTX uptake at pH 5.5, as measured by Dixon analysis for 3ꢀ8 separate experiments. Experimental
details are provided in the Experimental Section. Cpd, compound.
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1
1
1
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In this assay, with both FRαꢀ and FRβꢀexpressing cells, relative binding profiles for 4, 8, and 9
were similar and ~60ꢀ110% of the affinity for folic acid (Figure 8). These results indicate that
heteroatom substitution for the benzylic CH in 1 preserves binding affinity for both FRα (RT16)
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and FRβ (D4). For FRα, the Nꢀformylꢀsubstituted 5 and the Nꢀacetylꢀ and Nꢀtrifluoroacetylꢀ
substituted 6 and 7, respectively, all showed binding that was substantially reduced (<15% of
that for folic acid and for 4). A similar pattern was detected with FRβꢀexpressing D4 cells and 4,
5, 6, and 7, in spite of increased in vitro drug efficacies toward D4 over RT16 cells (Table 1).
Thus, relative FR binding affinities were only partly reflected in the differences in in vitro drug
efficacies toward RT16 or D4 cells (Table 1), suggesting that factors unrelated to FR binding
are necessary to explain the differential drug sensitivities at low nanomolar or subnanomolar
concentrations of these novel antifolates.
Since the 6ꢀsubstituted pyrrolo[2,3ꢀd]pyrimidine heteroatom compounds were transported by
PCFT in R2/PCFT4 CHO cells (based on patterns of growth inhibition; Table 1), we determined
relative binding affinities for this series compared to PMX in order to assess a possible basis for
differences in relative PCFTꢀtargeted activities. We compared the effects of the heteroatom
3
compounds (at 1 or 10 ꢃM) on PCFTꢀmediated transport of [ H]MTX (0.5 ꢃM) over 5 min at pH
5.5 (the pH optimum of PCFT) and at pH 6.8 (approximating the pH of tumor microꢀ
5
environment) (Figure 9). Results were compared to those for PMX, among the best PCFT
4, 5
substrates, and for PT523 (N(alpha)ꢀ(4ꢀaminoꢀ4ꢀdeoxypteroyl)ꢀN(delta)ꢀhemiphthaloylꢀLꢀ
30
ornithine), an antifolate substrate for RFC with no significant transport by PCFT. At pH 5.5,
3
PMX potently inhibited [ H]MTX uptake (> 90% at both 1 and 10 ꢃM), whereas 4, 5, 6, 7, 8, and
9, like 1 were somewhat less inhibitory (80ꢀ90% inhibition at 10 ꢃM and 30ꢀ60% inhibition at 1
ꢃM). At pH 6.8, inhibitions for all compounds were decreased, although appreciable transport
inhibitions (~40ꢀ60%) compared to controls were detected at 10 ꢂM for compounds 1, 4, 7 and
3
8
. K values were calculated from the extent of [ H]MTX transport inhibition at pH 5.5 over a
i
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8
9
range of drug concentrations by Dixon analysis (Table, Figure 9). K values ranged from ~7ꢀfold
i
(for 8) to ~18ꢀfold (for 6) higher than for PMX. Thus, the patterns of growth inhibition toward
PCFTꢀexpressing R2/PCFT4 cells are only partially reflected in analog binding to PCFT.
10
11
12
13
14
15
16
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 10. Growth inhibition of KB human tumor cells by 6ꢀsubstituted pyrrolo[2,3ꢀd]pyrimidine
antifolates 1, 4, 5, 6, 7, 8 and 9, and protective effects of excess folic acid, nucleosides, or 5ꢀ
aminoimidazoleꢀ4ꢀcarboxamide (AICA). KB cells were plated (4000 cells/well) in folateꢀfree RPMI 1640
medium with 10% dialyzed FBS, antibiotics, Lꢀglutamine, and 2 nM LCV with a range of drug
concentrations, in presence of folic acid (200 nM), adenosine (60 ꢂM), thymidine (10 ꢂM), or AICA (320
ꢂM). Cell proliferation was assayed with a fluorescenceꢀbased assay. Data are representative of at least
triplicate experiments. These results are summarized in Table 1. The methods are described in the
Experimental Section. Abbreviation: Cpd, compound.
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1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Identification of GARFTase as the intracellular enzyme target of 6ꢀsubstituted pyrrolo
[
2,3ꢀd]pyrimidine antifolates with heteroatom bridge substitutions. To identify the targeted
pathway, we determined the effects of nucleoside additions on the growth inhibitory effects of
compounds 4ꢀ9 toward KB tumor cells. The results are shown in Figure 10 and are summarized
in Table 1. The antiproliferative effects of all compounds were abolished by adenosine (60 ꢃM)
but not by thymidine (10 ꢃM), identifying de novo purine nucleotide rather than thymidylate
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
29
biosynthesis as the targeted pathway, analogous to 1. Since AICA (320 ꢃM), a precursor of
AICA ribonucleotide (ZMP) which circumvents the step catalyzed by GARFTase (by providing
downstream substrate for AICARFTase, the 2nd folateꢀdependent reaction) also protected
(
Figure 10), the likely enzyme target is GARFTase.
Figure 11. In situ GARFTase assay in KB tumor cells treated with 4, 5, 6, 7, 8, and 9. Inhibition of
cellular GARFTase activity by the pyrrolo[2,3ꢀd]pyrimidine antifolates was assayed in KB tumor cells. KB
14
14
cells were treated with drug and [ C]glycine for 16 h, then [ C]formyl GAR was extracted and isolated by
1
4
anionꢀexchange chromatography for quantitation. Relative [ C]formy GAR was expressed as percent of
the untreated control as a function of drug concentrations. IC s were as follows: 2.64+/ꢀ0.24 nM, 4;
50
2.11+/ꢀ0.37 nM, 5; 5.58+/ꢀ0.40 nM, 6; 4.95+/ꢀ0.44 nM, 7; 6.62+/ꢀ1.63 nM, 8; and 5.25+/ꢀ0.60 nM, 9. For
comparison, the IC50 for GARFTase inhibitions in KB cells by compound 1, PMX and LMTX were 1.92
47
47
(+/-0.40) nM (not shown), 30 nM and 14 nM, respectively. The methods are described in the
Experimental Section. Abbreviation: Cpd, compound.
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Table 2. K
i
values for inhibition of human GARFTase by
6
ꢀpyrrolo[2,3ꢀd]pyrimidine heteroatom analogs.
GARFTase activity was determined by measuring formation
of 5,8ꢀdideazafolate from 10ꢀformylꢀ5,8ꢀdideazafolic acid
using a spectrophotometric assay (295 nm) in the presence
of the indicated antifolate. Detailed methods are described in
the Experimental Section. Results are shown as mean
values +/ꢀ standard errors from three replicate assays. The
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
8
results for compound 3 and PMX were previously reported.
Abbreviation; Cpd, compound.
Antifolate
PMX
K (nM)
i
1000 +/ꢀ 160
160 ± 17
68 +/ꢀ 11
59 ± 9
Cpd 1
Cpd 3
Cpd 4
Cpd 5
Cpd 6
Cpd 7
Cpd 8
Cpd 9
99 ± 15
62 ± 7
122 ± 14
61 ± 4
201 ± 36
We used both in situ cellꢀbased (in KB tumor cells) and in vitro GARFTase assays with
recombinant GARFTase to confirm GARFTase as the enzyme target for our analogs. The
14
14
former measures the generation of [ C]formyl GAR from [ C]glycine (and GAR) by GARFTase
in KB cells treated with azaserine. In these experiments, we incubated KB cells with a range of
14
concentrations of compounds 4ꢀ9 and [ C]glycine for 16 h, under conditions approximating
those in our proliferation experiments (Table 1). Our results demonstrated dramatically
14
decreased [ C]formyl GAR in cells in drugꢀtreated cells, with nanomolar IC values
50
approximating or slightly exceeding those for inhibition of cell proliferation (Figure 11).
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We used an in vitro spectrophotometric enzyme assay with recombinant formyltransferase
domain of human GARFTase to measure inhibition by monoglutamyl 6ꢀpyrrolo[2,3ꢀd]pyrimidine
18
antifolates, 1, 4, 5, 6, 7, 8, and 9, along with 3 and PMX from our previous study. Absorbance
was measured at 295 nm accompanying oneꢀcarbon transfer from 10ꢀformylꢀ5,8ꢀdideazafolic
acid to GAR to form 5,8ꢀdideazafolic acid and formyl GAR. Assays were performed in the
presence and absence of the inhibitors, and the initial rates were plotted versus inhibitor
concentrations and fit to a hyperbola (Figure 1S, Supporting Information) for calculating Ki
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
values (Table 2). Nanomolar K values were calculated that varied over a ~3ꢀ4ꢀfold range, with
i
the most potent GARFTase inhibition by compounds 4, 6, and 8.
Collectively, our results identify GARFTase, the first folateꢀdependent step in de novo purine
biosynthesis, as the principal intracellular target for the heteroatom series. The data in Table 2
suggest that as the monoglutamate forms, heteroatom substitution with N (4) or O (8) affords an
approximately 3ꢀfold improvement in GARFTase inhibitory potency over C (1). In addition, Nꢀ
acetyl substitution (6) maintains this inhibitory potency. The formyl (5) and trifluoroacetyl (7) Nꢀ
substitutions decrease potency somewhat against isolated GARFTase. Further, all of the
heteroatom analogs, 4ꢀ9, as well as the carbon analogs 1 and 3, are 5ꢀ to 17ꢀfold better
GARFTase inhibitors than PMX.
XꢀRay crystal structure of 6ꢀpyrrolo[2,3ꢀd]pyrimidine heteroatom antifolates with FRα and
human GARFTase. Since compound 4 is selectively transported via FRα over RFC, we
45
determined the xꢀray crystal structure of FRα bound to 4 using our published methods. We
expressed human FRα in HEK293 cells and purified FRα by affinity and sizeꢀexclusion
chromatography. To confirm functionality of the purified soluble FRα, we measured competition
3
for binding to purified FRα by folic acid or 4 with [ H]folic acid. Compound 4 showed an IC only
50
slightly higher than that of folic acid in this assay (not shown), consistent with the results of our
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FRα binding experiments in RT16 CHO cells (Figure 8). For structural determinations, FRα was
deglycosylated, stripped of bound folate, then incubated overnight with a molar excess of 4. The
complex was purified and used to set up crystallization screens. The FRα/compound 4 complex
formed crystals that permitted the determination of its structure at a resolution of 3.6 Å.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 12. Crystal structures of FRα/folic acid (Panel A) andFRα/compound 4 (Panel B).
Superimposition of the crystal structure of folic acid with FRα onto the crystal structure of 4 indicates that
the truncated 5ꢀmembered pyrrolo ring of 4 is compensated via the 3ꢀatom bridge, compared to the 2ꢀ
atom bridge of folic acid. Thus, the overall dimensions of both folic acid and 4 are comparable, permitting
all important interactions with FRα to remain unaltered. The heteroatom in the bridge does not play an
important role in binding to FRα for folic acid or 4. Figure 2S (A,B) shows electron density maps for 4 in
complex with FRα.
Compound 4 assumes a position in the pocket very similar to that of folic acid (Figures 12A and
12B) with interactions with the same 12 key residues [Tyr82 (60), Asp103 (81), Tyr107 (85),
Trp124 (102), Arg125 (103), Arg128 (106), His157 (135), Lys158 (136), Trp160 (138), Trp162
(
140), Ser196 (174), and Tyr197 (175); again, numbering for FRα is that of the fullꢀlength gene
product with mature protein numbering in parentheses]. When compound 4 was computationally
docked into the FRα pocket (Figure 7C), the position of the docked 4 was very close (RMSD
0.37 Å) to that of 4 in the experimentally determined FRα/4 coꢀcrystal (Figure 12).
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