Discovery of amide-bridged pyrrolo[2,3-d]pyrimidines as tumor targeted classical antifolates with selective uptake by folate receptor α and inhibition of de novo purine nucleotide biosynthesis

Article abstract of DOI:10.1016/j.bmc.2019.115125

We previously showed that classical 6-substituted pyrrolo[2,3-d]pyrimidine antifolates bind to folate receptor (FR) α and the target purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFTase) with different cis and trans conformations. In this study, we designed novel analogs of this series with an amide moiety in the bridge region that can adopt both the cis and trans lowest energy conformations. This provides entropic benefit, by restricting the number of side-chain conformations of the unbound ligand to those most likely to promote binding to FRα and the target enzyme required for antitumor activity. NMR of the most active compound 7 showed both cis and trans amide bridge conformations in ~1:1 ratio. The bridge amide group in the best docked poses of 7 in the crystal structures of FRα and GARFTase adopted both cis and trans conformations, with the lowest energy conformations predicted by Maestro and evidenced by NMR within 1 kcal/mol. Compound 7 showed ~3-fold increased inhibition of FRα-expressing cells over its non-restricted parent analog 1 and was selectively internalized by FRα over the reduced folate carrier (RFC), resulting in significant in vitro antitumor activity toward FRα-expressing KB human tumor cells. Antitumor activity of 7 was abolished by treating cells with adenosine but was incompletely protected by 5-aminoimidazole-4-carboxamide (AICA) at higher drug concentrations, suggesting GARFTase and AICA ribonucleotide formyltransferase (AICARFTase) in de novo purine biosynthesis as the likely intracellular targets. GARFTase inhibition by compound 7 was confirmed by an in situ cell-based activity assay. Our results identify a “first-in-class” classical antifolate with a novel amide linkage between the scaffold and the side chain aryl L-glutamate that affords exclusive selectivity for transport via FRα over RFC and antitumor activity resulting from inhibition of GARFTase and likely AICARFTase. Compound 7 offers significant advantages over clinically used inhibitors of this class that are transported by the ubiquitous RFC, resulting in dose-limiting toxicities.

Full text of DOI:10.1016/j.bmc.2019.115125

Journal Pre-proofs
Discovery of amide-bridged pyrrolo[2,3-d]pyrimidines as tumor targeted clas-
sical antifolates with selective uptake by folate receptor α and inhibition of de
novo purine nucleotide biosynthesis
Weiguo Xiang, Aamod Dekhne, Arpit Doshi, Carrie O'Connor, Zhanjun Hou,
Larry H. Matherly, Aleem Gangjee
PII:
S0968-0896(19)31111-3
https://doi.org/10.1016/j.bmc.2019.115125
BMC 115125
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
1 July 2019
17 September 2019
Please cite this article as: Xiang, W., Dekhne, A., Doshi, A., O'Connor, C., Hou, Z., Matherly, L.H., Gangjee, A.,
Discovery of amide-bridged pyrrolo[2,3-d]pyrimidines as tumor targeted classical antifolates with selective uptake
by folate receptor α and inhibition of de novo purine nucleotide biosynthesis, Bioorganic & Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.bmc.2019.115125
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Discovery of amide-bridged pyrrolo[2,3-d]pyrimidines as tumor targeted classical
antifolates with selective uptake by folate receptor α and inhibition of de novo purine
nucleotide biosynthesis
Weiguo Xiang£, Aamod Dekhne‡, Arpit Doshi£, Carrie O’Connor‡, Zhanjun Hou‡ll,
Larry H. Matherly‡§ll€*, Aleem Gangjee£€*
£*Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University,
600 Forbes Avenue, Pittsburgh, PA 15282
llMolecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 421 E. Canfield Street,
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
€These authors contributed equally to this work.
*To whom correspondence should be addressed.
AUTHOR INFORMATION
Corresponding Authors
Aleem Gangjee, Ph.D., Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences,
Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282. Phone: 412-396-6070; fax: 412-396-
5593; gangjee@duq.edu
1

Larry H. Matherly, Ph.D., Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 421
East Canfield Street, Detroit, MI 48201. Phone: 313-578-4280; fax: 313-578-4287;
matherly@karmanos.org
Keywords: classical antifolates; folate receptor; selective uptake; pyrrolo[2,3-d]pyrimidines,
amide bridge
Abbreviations used:
FR, Folate Receptor; GAR, Glycinamide Ribonucleotide; GARFTase, Glycinamide Ribonucleotide
Formyltransferase; RFC, Reduced Folate Carrier; AICA, 5-Aminoimidazole-4-Carboxamide;
AICARFTase, 5-Aminoimidazole-4-Carboxamide Ribonucleotide Formyltransferase; MTX,
Methotrexate; PDX, Pralatrexate; RTX, Raltitrexed; PMX, Pemetrexed; PCFT, Proton-Coupled Folate
Transporter
2

Abstract
We previously showed that classical 6-substituted pyrrolo[2,3-d]pyrimidine antifolates bind to
folate receptor (FR)  and the target purine biosynthetic enzyme glycinamide ribonucleotide
formyltransferase (GARFTase) with different cis and trans conformations. In this study, we designed novel
analogs of this series with an amide moiety in the bridge region that can adopt both the cis and trans lowest
energy conformations. This provides entropic benefit, by restricting the number of side-chain
conformations of the unbound ligand to those most likely to promote binding to FRα and the target enzyme
required for antitumor activity. NMR of the most active compound 7 showed both cis and trans amide
bridge conformations in ~1:1 ratio. The bridge amide group in the best docked poses of 7 in the crystal
structures of FR and GARFTase adopted both cis and trans conformations, with the lowest energy
conformations predicted by Maestro and evidenced by NMR within 1 kcal/mol. Compound 7 showed ~3-
fold increased inhibition of FRα-expressing cells over its non-restricted parent analog 1 and was selectively
internalized by FR over the reduced folate carrier (RFC), resulting in significant in vitro antitumor activity
toward FRexpressing KB human tumor cells. Antitumor activity of 7 was abolished by treating cells
3

with adenosine but was incompletely protected by 5-aminoimidazole-4-carboxamide (AICA) at higher drug
concentrations, suggesting GARFTase and AICA ribonucleotide formyltransferase (AICARFTase) in de
novo purine biosynthesis as the likely intracellular targets. GARFTase inhibition by compound 7 was
confirmed by an in situ cell-based activity assay. Our results identify a “first-in-class” classical antifolate
with a novel amide linkage between the scaffold and the side chain aryl L-glutamate that affords exclusive
selectivity for transport via FR over RFC and antitumor activity resulting from inhibition of GARFTase
and likely AICARFTase. Compound 7 offers significant advantages over clinically used inhibitors of this
class that are transported by the ubiquitous RFC, resulting in dose-limiting toxicities.
1. INTRODUCTION
Antifolates that inhibit one-carbon biosynthetic enzymes such as dihydrofolate reductase and
thymidylate synthase are important agents for anticancer chemotherapy.1,2 Clinically useful antifolates
include methotrexate (MTX), pralatrexate (PDX), raltitrexed (RTX) and pemetrexed (PMX) (Figure 1)
with applications for both hematologic malignancies and solid tumors.2 Unfortunately, dose-limiting
toxicities plague the clinical utility of these drugs and are due, at least in part, to non-specific drug
accumulation resulting in inhibition of critical enzyme targets and downstream pathways in both tumors
and normal tissues without selectivity.
4

Figure 1. Clinically used antifolate drugs for cancer.
The principal (anti)folate transport system in tissues and tumors is the ubiquitously expressed
reduced folate carrier (RFC).3 Notably, all the aforementioned antifolates (Figure 1) are excellent substrates
for transport by RFC and accumulate in normal tissues, as well as in tumor cells, precluding selectivity.1,3
Uptake systems with greater tumor specificity than RFC have been described and include the proton-
coupled folate transporter (PCFT)4-7 and folate receptor (FR) α.8,9 PCFT is a proton symporter that is
expressed in a wide range of solid tumors, including ovarian cancer, non-small cell lung cancer, and
malignant pleural mesothelioma.10-13 Moreover, PCFT is active at acidic pH characterizing the
microenvironments of solid tumors.6,14 Although certain normal tissues such as the proximal small intestine,
liver, and kidney also express PCFT, the tissue microenvironments of most normal tissues are unlikely to
be sufficiently acidic to support PCFT transport,7 except the jejunum where the pH is acidic and conducive
of folate transport.
FRα is expressed on the surface of epithelial ovarian, non-small cell lung, kidney, endometrial,
colorectal and breast cancers where it comes into contact with the systemic circulation.8,9,15 FRα is also
expressed in a subset of normal tissues including kidney, lung, choroid plexus and placenta. However, in
contrast to tumors, in normal tissues, FRα localizes to the luminal membranes without exposure to the
systemic circulation. 8,9,15. Thus, there is a compelling rationale for developing FR- and PCFT-based
therapies for cancer, particularly for solid tumors.
Clinically tested FRα-targeted therapies include a FRα monoclonal antibody [Farletuzumab
(Morphotech)16,17], a FRα-targeting antibody-drug conjugate [IMGN853 (ImmunoGen)18], and cytotoxic
folic acid (FA) conjugates [Vintafolide, EC1456 and EC1788 (Endocyte)] 9,19. Although an FR-targeted
antifolate
N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta
[g]quinazolin-6-yl] amino] benzoyl]-L-γ-glutamyl-D-glutamic acid (ONX0801) was described20,21 and
advanced to a phase 1 clinical trial,22 no FRα-targeted antifolate has yet been FDA-approved for cancer.
5

Figure 2. 6-Substituted pyrrolo[2,3-d]pyrimidine antifolates 1-6.
We discovered novel 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl L-glutamate antifolates with 3- or 4-
bridge carbons (CH2) (1 and 2, respectively) (Figure 2) as selective transport substrates for FRs and/or
PCFT over RFC.23,24 Following internalization by tumor cells, these compounds inhibited glycinamide
ribonucleotide (GAR) formyltransferase (GARFTase), the first folate-dependent step in de novo purine
biosynthesis. Side-chain replacement of the phenyl moiety of 1 with thienyl regioisomers (3-5) (Figure 2)
increased the inhibitory effects toward FRα-expressing cells and with PCFT-expressing cells, as well.25,26
N-heteroatom substitution in the bridge region as in 6 (Figure 2) increased FRα-targeting at the expense of
RFC and PCFT uptake; however, 6 did not show absolute selectivity for FR or PCFT over RFC.27
Cytotoxic drugs generally bind to different cellular targets in distinct conformations. For anionic
compounds, a requisite step in drug activity involves mediated uptake into tumor cells (ideally via a tumor-
selective uptake process), whereupon they inhibit intracellular target enzymes. In principle, these
compounds could adopt distinct conformations, including one that strongly favors mediated uptake into
cells and another favoring binding to an intracellular enzyme target(s) that ultimately effects a cytotoxic
response. We previously reported that the pyrrolo[2,3-d]pyrimidine antifolate 3 adopts different poses when
bound to FR versus when bound to its target enzyme GARFTase (Figure 3).27,28 As an extension
of this concept, since amides assume distinct conformations as cis and trans rotamers with the lowest energy
conformers,29 we reason that an analog with a unique amide linkage in the bridge region would afford a
6

somewhat restricted three atom bridge, compared to a more flexible three carbon (CH2) bridge. For the
former, this could derive significant entropic benefit and increased target selectivity by restricting the
number of side-chain conformations in both unbound and bound states. Thus, analogs that do not need to
significantly alter their conformations to adopt the bound conformation at their cellular target would likely
bind more effectively to target proteins than analogs that exhibit different low energy conformations
compared to their bound conformations.30
7

Figure 3. Compound 3 and its two different poses in FR and GARFTase, as well as newly designed compounds 7-
12.
To test this concept and to begin developing new targeted inhibitors as selective cytotoxic agents
for cancer, we designed a novel series of amide-bridge 6-substituted pyrrolo[2,3-d]pyrimidine antifolates
with different energy barriers between the cis and trans conformers. It was of interest to determine the
effect of inserting an amide in the bridge region on biological activity and transport selectivity for FRα
and/or PCFT over RFC for three C-atom-bridge compounds such as 1. In this report, we describe the
synthesis and biological activities of the unique amide-substituted pyrrolo[2,3-d]pyrimidine compounds 7-
12 (Figure 3). In designing this series, we also introduced an N-methyl group on the amide nitrogen to
augment the metabolic stability of the amide linkage from possible enzymatic hydrolysis to make these
more generally useful.31 Our results establish these compounds as potent and highly selective FR-targeted
inhibitors of de novo purine biosynthesis at GARFTase, and likely at 5-aminoimidazole-4-carboxamide
(AICA) ribonucleotide formyltransferase (AICARFTase) (Figure 3).
2. RESULTS AND DISCUSSION
2.1.Rationale for compound design
It was of interest to determine if introducing an amide into the 3-atom bridge of 6-substituted
pyrrolo[2,3-d]pyrimidine compounds (7-12) would decrease the number of low energy accessible
conformations, compared to the corresponding 3-atom carbon chain analogs (1, 3-5). By molecular
modeling in Maestro,32 we determined that the amide bridge analogs afforded a reduced number of
conformations compared to their corresponding 3-carbon atom bridge analogs (Table 1). This suggests that
amide group insertion into the side-chain as in 7-12 should provide a distinct entropic advantage that should
favor binding to cellular targets.
8

Table 1. Conformational search results using Maestro 11.8. Target compounds (blue) and lead
compounds (black)
Compound (Ar)
Number of conformations within 5
kcal/mol of minimum energy
conformation
1 (1,4-phenyl)
7 (1,4-phenyl)
8 (1,3-phenyl)
12 (1,4-phenyl)
3 (2,5-thienyl)
9 (2,5-thienyl)
4 (2,4-thienyl)
10 (2,4-thienyl)
5 (3,5-thienyl)
11 (3,5-thienyl)
219
143
152
121
192
134
148
97
131
83
From our previous studies, the side-chain aromatic ring is an important determinant of drug potency
and transport selectivity of 6-substituted pyrrolo[2,3-d]pyrimidine inhibitors.25,33,34 Thus, different
regioisomers involving a side-chain phenyl ring and its replacement with thienyl regioisomers profoundly
impacted (generally improved) compound potency. Accordingly, we designed amide-bridge analogs, with
side-chain 1,4- and 1,3-disubstituted phenyl (7 and 8), and 2,5-, 2,4- and 3,5-di-substituted thienyl moieties
(9-11) (Figure 3), based on the parent molecules (1, 3-5), in order to determine the optimal side-chains for
selective cellular uptake by FRα vis á vis RFC and for inhibition of intracellular target enzymes (i.e.
GARFTase) resulting in antitumor activity. Finally, we designed a sulfonamide bridge compound 12
(Figure 3) as a bioisosteric replacement of the amide in 7.32
To determine the rotational barrier around the amide (N(CH3)-C(O)) bond, we performed a
coordinate scan35 of target compounds 7 - 12 around the bridge amide bond, and of the parent compounds
(1, 3-5) around the C-C bond (Maestro 11.8). The resulting plot of relative energy (∆E, kcal/mol) versus
the dihedral angle (°) is shown in Figure 4. For the amide compounds (7-12), a coordinate scan predicted
two distinct minimum energy conformations with trans and cis conformations of the amide bond and a high
9

energy barrier (~8-20 kcal/mol) between them. In contrast, a torsional scan of the carbon bridge analogs (1,
3-5) predicted three distinct minimum energy conformations with a low energy barrier among them (~5
kcal/mol). This demonstrates the conformational restriction associated with the amide bridge. Based on
these results, we propose that reducing the number of low energy conformations (Table 1) and increasing
the energy barrier between bridge amide rotamers (Figure 4) in the target compounds (7-12) could enhance
drug activity, reflecting uptake via FRα and inhibition of intracellular enzymes such as GARFTase.
Figure 4. Plots of rotational barrier (relative energy, kcal/mol) around the carbon-carbon bridge of compounds 1, 3,
4 and 5 (left) and the bridged amide bond of compounds 7-12 (right). Coordinate Scan panel of Maestro 11.8 was used
to determine and plot the rotational energy barrier for both the amide bridge and lead compounds; the settings were
as follows: Force Field, OPLS3e; Solvent, water; Coordinate to Scan, dihedral angles of amide bridge for target
compounds and carbon bridge for lead compounds (the rest of the settings were kept at default).32
We docked the amide-bridge compounds (Figure 3) in the FRPDB: 5IZQ27 and GARFTase
(PDB: 4ZZ134) crystal structures. Figures 5 and 6 show the docked poses of compound 7 in FR and
GARFTase, respectively. The amide bridge in 7 adopts both cis and trans conformations within 1 kcal/mol
(docking score) for both the FRand GARFTase structures. The major binding interactions of 7 with the
target proteins are similar to those for the co-crystallized ligands 6 (FRα)27 and 5 (GARFTase)34. The
10

oxygen atom of the bridge amide group of 7 in both rotamers can form an ion-dipole interaction (distance:
2.78 Å and 2.67 Å, Figure 6) with the terminal amino group of the substrate GAR in the GARFTase binding
site. Compound 7 shows better docking scores (FRα = -14.77 kcal/mol and GARFTase = -15.23 kcal/mol)
than compound 1 (FRα = -13.33 kcal/mol and GARFTase = -14.43 kcal/mol), suggesting its more avid
binding to these target proteins and predicting greater potency. The docked poses of 8 (FRα = -14.27
kcal/mol and GARFTase = -14.72 kcal/mol) and 9 (FRα = -15.23 kcal/mol and GARFTase = -14.75
kcal/mol) mimic those for 7 in FRα and GARFTase.
Compounds 10 (side-chain 2,4-thienyl) and 11 (side-chain 3,5-thienyl) were designed to vary the
distance between the amide bridge and the glutamyl side-chain, and hence their conformations in
comparison to the 2,5-thienyl side-chain analog 9.34 These changes introduce additional conformational
restrictions and further reduce the number of possible low energy conformations even further (to 97 for 10
and 83 for 11, compared to the 134 possible conformations for the 2,5-thienyl side-chain analog 9; Table
1). Compound 12 is designed to explore the bioisosteric replacement of the amide group in compound 7.
The sulfonamide moiety in compound 12 further reduces the number of possible low energy conformations
compared to 7 (Table 1). Compounds 10 (FRα = -12.71 kcal/mol and GARFTase = -13.31 kcal/mol), 11
(FRα = -12.75 kcal/mol and GARFTase = -12.87 kcal/mol) and 12 (FRα = -13.42 kcal/mol and GARFTase
= -13.53 kcal/mol) show comparable docking scores to compounds 1 (FRα = -13.33 kcal/mol and
GARFTase = -14.43 kcal/mol) and 3 (FRα = -13.67 kcal/mol and GARFTase = -14.12 kcal/mol) for both
FRα and GARFTase. Moderate docking scores of the compounds 10, 11 and 12 suggest that despite the
conformational restriction, the nature of the aromatic side-chain and the bridge linker may dictate the
binding affinity for the proposed compounds.
Based on our in silico studies, we predicted that compounds 7-12 with the amide group in the bridge
should exhibit significant cellular uptake by FRα, resulting in GARFTase inhibition and antitumor efficacy.
On the basis of the better docking scores in FRα and GARFTase, 7, 8 and 9 are predicted to be more potent
inhibitors of tumor growth compared to 10-12 and the lead compounds 1-6.
11

A
“Trans conformation”
B
“Cis conformation”
Figure 5. Two alternate docked poses of 7 in FR(PDB ID: 5IZQ). A) Cyan: compound 7, and pink: 6 (co-
crystallized ligand). The amide linker in 7 adopted a trans conformation. Docking score: -14.77 kcal/mol. B) The
amide linker in 7 adopted a cis conformation. Docking score: -13.89 kcal/mol. Maestro 11.8.
12

A
“Trans conformation”
“Cis conformation”
B
Figure 6. Two alternate docked poses of 7 in GARFTase (PDB ID: 4ZZ1). A) Purple, compound 7, gray 5 (co-
crystallized ligand), and red, GAR (endogenous substrate). The amide linker in 7 adopted a trans conformation.
Docking score: -15.23 kcal/mol. B) Purple, compound 7. The amide linker in 7 adopted a cis conformation. Docking
score: -14.79 kcal/mol. Maestro 11.8.
13

3. Chemistry
Scheme 1. Two current synthesis methods for antifolate 15.
There are two synthetic methods available for 6-substituted classical antifolates 15 (Scheme
1).34,36 The first method uses a Sonogashira coupling to install an alkyne linker on 6-halogenated
pyrrolo[2,3-d]pyrimidines 13 and provides key intermediates 14. This approach is not suitable for
heteroatom side chain compounds.36 The second method is to cyclize an -bromoketone 17 with 2,6-
diamino-4-hydroxy pyrimidine 18, which affords the pteroic ester precursors 19. In this approach,
diazomethane is an indispensable, yet dangerous, starting material.37 Hence, a third synthetic method was
devised for our proposed amide compounds to avoid the diazomethane reaction.
Scheme 2. Synthesis of N-monosubstituted 6-amiomethyl pyrrolo[2,3-d]pyrimidines 23.
14

Our synthesis for the 6-aminomethyl pyrrolo[2,3-d]pyrimidines is shown in Scheme 2.
Pivaloylation of the 2-amino moiety of the pyrrolo[2,3-d]pyrimidine 20 provided 21 (95% yield). The
purpose of this step was to decrease the polarity and increase the solubility of 20. The product 21 was
directly precipitated from the reaction mixture by adding a mixture of hexane and ethyl acetate (5:1).
Subsequently, a Mannich reaction with 21 afforded 22 in 78% yield. Finally, Pd-catalyzed debenzylation
of 22 provided 23 in 90% yield.
Figure 7. HMBC spectrum of 23. a) HMBC spectrum and interpretation. The diagnostic 3JCH couplings are circled
in red, and 2JCH couplings are circled in green; b) Highlighted diagnostic HMBC signals. 3J CH signals are highlighted
with red arrows and 2J CH signals are highlighted with green arrows.
15

Since the Mannich reaction38-41 could afford 5- and/or 6- position substituted regioisomers of the
pyrrolo[2,3-d]pyrimidine, we analyzed the regioselectivity of the reaction from 21 to 22 with compound 23
using NOESY42 and HMBC (Figure 7). A weak NOE signal of N7-H and 6’-H was observed. The weak
NOE signal is explained by the quadrupole N7-H. In HMBC (Figure 7), the signal of 3J(C4-H5), as well as
3J(C5-H7 and C9-H3), confirmed the structure as 23 with the aminomethyl substitution at the 6-position.
This demonstrated that under our Mannich reaction conditions, the 6-substituted regioisomer was the
predominant product. In addition, our synthesis provided a secondary amine in 23, which was amenable to
functionalization.
Scheme 3. Synthesis of 24a-f.
Amide or sulfonamide coupling of 23 (Scheme 3) with the corresponding acid 25a, 26b-c, 27a-b
or methyl 4-(chlorosulfonyl)benzoate 27c afforded 24a-f in 69-82% yield. Compounds 26b-c were, in turn,
synthesized by periodic acid oxidation of the corresponding aldehydes 25b-c in 56-63% yields.
16

Scheme 4. Synthesis of target compounds 7-12.
Depivaloylation and hydrolysis of the methyl esters in 24a-f (Scheme 4) with sodium carbonate
afforded pteroic acids 28a-f in 78-91% yields. CDMT auxiliary amide coupling of 28a-f with dimethyl L-
glutamate afforded 29a-f. Sodium carbonate hydrolysis of 29a-f provided the target classical antifolates 7-
12 in 56-74% yields over two steps.
The 1H NMR of 7 (Figure 8) exhibited two sets of peaks of the NCH3, NCH2 and 5-H, for a mixture
of the cis and trans rotamers, with all other protons at the same chemical shift. In each rotamer, the NCH3,
NCH2 and 5-H have an integration ratio of 3 to 2 to 1, which was used to assign peaks to the two different
sets. The chemical shifts of protons for the cis conformation of the amide oxo moiety are larger than those
of the trans conformation.43 As a result, the red set (Figure 8) are assigned to the cis while the blue set are
the peaks for the trans. This NMR result corroborates the two lowest energy rotamers of 7, as predicted by
molecular modeling.
17

Figure 8. 1H NMR (DMSO-d6) of 7 at 400 MHz, the proton of NCH3, NCH2 and 5-H in the cis and trans rotamers
are illustrated as red and blue, respectively. Red: 2.81 (1.82H, cis NCH3), 4.58 (1.15H, cis NCH2), 6.17 (0.53H, cis
5-H); Blue: 2.94 (1.3H, trans NCH3), 4.58 (0.9H, trans NCH2), 6.17 (0.48H, trans 5-H).
4. Biological evaluation
The bridge amide-substituted pyrrolo[2,3-d]pyrimidine compounds 7–12 were initially tested in
cell proliferation assays with a unique panel of isogenic Chinese hamster ovary (CHO) cell lines engineered
to individually express human RFC (PC43-10), PCFT (R2/PCFT4), or FRα (RT16).23,24,44 The results with
the amide series were compared to previous 6-substituted pyrrolo[2,3-d]pyrimidine analogs with an alkyl
bridge linked to side-chain phenyl (1)23 or thienyl (3-5)25,34 rings, as appropriate, and to standard antifolates
18

(e.g., MTX) without transporter selectivity. The negative control for these experiments was the RFC-, FR-,
and PCFT-null MTXRIIOuaR2-4 (R2) CHO cell line.45 For the FRα-expressing CHO cells, additional
control involved treatment with excess folic acid (200 nM) to selectively block cellular uptake by FR (not
shown). Proliferation experiments were also performed with KB human tumor cells that express highly
elevated FRα, along with RFC and PCFT. These results are summarized in Table 2.
As previously reported, the non-amide pyrrolo[2,3-d]pyrimidine compounds (1, 3-5) were potent
inhibitors of CHO cells expressing FRα or PCFT and are some of the most active analogs identified for this
structural platform;4,23,25,34 however, this activity was not selective for FRα and/or PCFT as these
compounds were taken up by a non-specific (i.e., non-mediated) process that results in equivalent growth
inhibition toward RFC-expressing (PC43-10) and transporter-null (R2) cells23,25,34 (Table 2). Notably, the
amide-substituted compounds, 7, 8 and 9 were inert toward RFC-expressing PC43-10 cells. Compounds 7,
8 and 9 all preserved FRα activities, reflected in growth inhibition of RT16 and KB tumor cells (Table 2).
Thus, compounds 7, 8 and 9 were completely selective for FRα over RFC. For 7, inhibition of RT16 cells
(FRα) significantly exceeded (~3-fold) that for the corresponding parent analog (1) (p<0.05), consistent
with the notion that the presence of the amide bridge favors a conformation optimal for FR binding and
internalization. While regioisomer 8 (meta substitution on the side chain phenyl ring) was equally potent
as 7 toward FRα-expressing RT16 cells, 8 (meta-substituted on the side-chain phenyl) was ~4-fold less
active than 7 (para substituted on the side chain phenyl ring) toward KB tumor cells (Table 2). Although
these results may reflect factors unrelated to FRα internalization, it nonetheless suggests that that decreased
distance between the scaffold and the L-glutamate moiety as in 8 compared to 7 is detrimental to antitumor
activity, as previously described.27 Isosteric replacement of the phenyl side chain in 7 with a thiophene ring
resulted in modestly decreased growth inhibition (for 9), or a substantial reduction (11) or complete loss
(10) of FRα-targeted inhibition of RT16 and/or KB cells. The sulfonamide analog 12 was inactive
regardless of the expressed transporter (Table 2).
19

Table 2. Structures of 1-12 and the IC50 values for inhibition of proliferation of transporter null (R2), FRα (RT16), PCFT
(R2/PCFT4), and RFC (PC43-10)-expressing CHO cells, and KB human tumor cells (expresses FRα, RFC, and PCFT) in culture.
Results are expressed as mean values (+ standard errors) from 3-10 experiments. Abbreviations: MTX, methotrexate; PMX,
pemetrexed; RTX, raltitrexed. *These data were previously published (see reference).
IC50 (nM)
RT16
KB
Compound
X
Reference
PC43-10
(RFC)
R2/PCFT4
(PCFT)
R2
(FRα)
(FRα/RFC/PCFT)
1.7(0.4)
46
-
304 (89)
>1000
448 (78)
>1000
4.1(1.6)
1.4(0.24)
1.78 (0.17)
23.0(3.3)
48.60(16.40)
747 (156)
1*
7
-
-
1.40(0.24)
6.36 (1.00)
>1000
>1000
8
-
>1000
>1000
>1000
>1000
669 (112)
12
20

25
0.26 (0.03)
4.23(0.57)
0.09(0.02)
-
-
101.0 (16.6)
>1000
273.0(49.1)
>1000
0.31 (0.14)
7.78(1.63)
0.61(0.11)
3.34 (0.26)
>1000
3
9
-
34
189(51)
290((8)
6.51(1.30)
4*
-
34
-
>1000
>1000
>1000
>1000
>1000
10
5*
0.17(0.05)
-
197(49)
355(10)
0.33(0.15)
5.39(1.27)
69.8(16.1)
>1000
>1000
867(147)
>1000
11
6.0(0.6)
68(12)
-
-
-
-
12(1.1)
138(13)
6.3(1.3)
216(8.7)
894(93)
>1000
114(31)
42(9)
121(17)
13.292.4)
99.5(11.4)
MTX*
PMX*
RTX*
-
5.9(2.2)
-
15(5)
21

While compounds 7-12 were all designed to inhibit GARFTase in de novo purine biosynthesis
following internalization by FRα, this was directly tested for the compounds 7 and 9 with side-chain phenyl
and 2,5-thiophene moieties analogous to parent compounds 1 and 3. For these experiments, we determined
the extent of growth inhibition of KB tumor cells by these compounds in the presence of thymidine (10 µM)
or adenosine (60 µM).25,27,33,34,36 We also tested the protective effects of glycine (130 μM) to explore the
potential inhibition of mitochondrial C1 metabolism.47 Results for the nucleoside/glycine protection
experiments are shown in Figure 9A. Adenosine completely reversed the drug effects of all the compounds,
whereas thymidine and glycine were completely ineffective. Thus, neither thymidylate synthase nor
mitochondrial C1 metabolism are involved in the mechanism of action of 7 or 9. The metabolite AICA (320
µM) is metabolized to AICA ribonucleotide (ZMP), the AICARFTase substrate which circumvents the
GARFTase step. 27,28,33,34,36,44 AICA completely reversed the inhibitory effects of compounds 1 and 3 up to
1 μM; however, at higher concentrations of 3, AICA was less protective. By analogy with published results
for related compounds,27,28,33,34,36,44,46 our results suggest that GARFTase is the primary intracellular target
for this series, although at higher inhibitor concentrations, targeting of AICARFTase also contributes.
To further confirm GARFTase as the principal cellular target for our most potent analogs
(compounds 7 and 9), GARFTase was directly assayed in situ in KB cells. For these experiments, KB cells
were incubated with a range of concentrations of compounds 7 or 9, or of compounds 1 and 3 as controls,
under conditions closely approximating those for the cell proliferation assays (Table 1). Cells were
incubated with [14C]glycine, which is incorporated into [14C]GAR and [14C]FGAR (the GARFTase product).
The latter accumulates in the presence of azaserine (inhibits FGAR amidotransferase) and is isolated by
anion exchange fractionation for quantitation and calculation of IC50 values for GARFTase inhibition. All
compounds inhibited GARFTase with IC50 values approximately one-order of magnitude higher than that
for inhibition of KB proliferation. Collectively, these results establish that de novo purine biosynthesis is
the principal targeted pathway by the active amide-substituted pyrrolo[2,3-d]pyrimidine compounds 7 and
9 at GARFTase, and likely AICARFTase, as well.
22

C p d
3
C p d
1
A
B
140
120
100
80
140
120
100
80
N o A d d itio n s
200 nM F olic A cid
100  M A d en osine
10  M Thym idine
130  M G lycine
60
60
40
40
A d en o s in e
+
T h ym id in e
320  M A IC A
20
20
0
0
0.01
0.1
1
10
100
1000 10000
0.01
0.1
1
10
100
1000 10000
In h ib ito r C o n c e n tra tio n (n M )
In h ib ito r C o n c e n tra tio n (n M )
C p d
9
C p d
7
C
D
E
140
100
80
60
40
20
0
140
120
100
80
120
100
80
60
40
20
0
60
40
20
0
0.01
0.1
1
10
100
1000 10000
1
7
9
0.01
0.1
1
10
100
1000 10000
3
d
d
p
d
p
d
p
p
In h ib ito r C o n c e n tra tio n (n M )
In h ib ito r C o n c e n tra tio n (n M )
C
C
C
C
Figure 9. Identification of the intracellular target by protection by nucleosides, glycine and AICA, and in situ
GARFTase assays. A-D) KB cells were incubated with drugs in folate-, nucleoside-, and glycine-free RPMI 1640
medium with 10% dialyzed FBS, antibiotics, L-glutamine, and 2 nM leucovorin with a range of drug concentrations
in the presence of folic acid (200 nM), adenosine (60 μM), thymidine (10 μM), glycine (130 μM) or AICA (320 μM).
Cell proliferation was assayed with Cell Titer Blue (Promega) using a fluorescence plate reader. Data are
representative of at least triplicate experiments. Error bars represent the standard errors. E) Inhibition of GARFTase
activity in KB cells by the 6-substituted pyrrolo[2,3-d]pyrimidine compounds including compounds 1, 3, 7 and 9. KB
cells were treated with the drugs under conditions approximating those for the cell outgrowth experiments (Table 2).
Incorporation of [14C(U)]glycine into [14C]formyl GAR was used as an in situ measure of endogenous GARFTase
activity. Experimental details are summarized in the Experimental Section. IC50 values (nM) were calculated as mean
values +/- standard errors for triplicate experiments.
5. Conclusion
In this study, we used molecular modeling and docking studies to rationally design a series of
amide-bridged pyrrolo[2,3-d]pyrimidine antifolates with cis and trans conformations as the two lowest
energy conformations. These conformations were supported by 1H NMR analysis. We previously described
carbon-bridge analogs of this series (1 - 6) that are not selective for FR and PCFT over other uptake
mechanisms including RFC23,25,36 that allows the accumulation of the analogs in normal tissues and
precludes tumor selectivity. Based on proliferation studies in isogenic CHO cell lines exclusively
expressing human FRα, PCFT or RFC, we determined that the active amide compounds 7-9 are transported
23

by FRα with or without PCFT, but not by RFC, and thus should possess absolute tumor selectivity. This
indicates that the amide bridge is highly conducive to tumor transport selectivity for 7-9. Based on the
inhibitory results toward KB human tumor cells, compounds 7 and 9 were tested to identify the likely
targeted pathway and enzyme(s) in comparison to compounds 1 and 3, previously identified as exclusive
inhibitors of GARFTase in de novo purine nucleotide biosynthesis.23,25 Whereas compounds 7 and 9 were
confirmed as GARFTase inhibitors by nucleoside/AICA protection, a secondary target, most likely
AICARFTase, was also implicated.
Our original hypothesis was that conformational restriction caused by the amide bridge in the
analogs 7-12 would favor targeting FRand possibly GARFTase by our analogs. Consistent with our
hypothesis, compound 7 was ~3-fold more potent than 1 toward CHO cells exclusively expressing FRα,
although 7 and 1 are equipotent toward KB cells that express RFC, FRand PCFT. This suggests that the
conformational restriction in 7 promotes FRtargeting of our analogs, although at least for this analog,
uptake by PCFT is preserved, as well, likely contributing to drug effects in KB cells. In the absence of
crystal structures for RFC and PCFT, the information from this study can guide the design of
conformationally restricted compounds that are exclusively transported by FRα-expressingcancer cells to
afford highly selective, non-toxic cancer chemotherapeutic agents.
6. Experimental Section
Analytical samples were dried in vacuum (0.2 mmHg) in a CHEM-DRY drying apparatus over
P2O5 at 50 °C. Melting points were determined on a digital MEL-TEMP II melting point apparatus with
FLUKE 51K/J electronic thermometer and are uncorrected. Nuclear magnetic resonance spectra for protons
(1H NMR, 13C NMR and HMBC) were recorded on a Bruker Avance II 400 (400 MHz) or on a 500 (500
MHz) NMR systems. 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; quint, quintet; m,
multiplet; br, broad singlet. Thin-layer chromatography (TLC) was performed on Whatman Sil G/UV254
24

silica gel plates with a fluorescent indicator, and the spots were visualized under 254 and 365 nm
illumination. Proportions of solvents used for TLC are by volume. Column chromatography was performed
on a 230−400 mesh silica gel (Fisher Scientific) column. Elemental analyses were performed by Atlantic
Microlab, Inc., Norcross, GA. Elemental compositions are within ±0.4% of the calculated values and
indicate > 95% purity of the compounds. Fractional moles of water or organic solvents found in some
analytical samples could not be prevented despite 24 h of drying in vacuum and were confirmed where
1
possible by their presence in the H NMR spectra. UPLC-MS was analyzed on Acquity system. A linear
gradient of 90% of 0.1% formic acid in water, 10% of 0.1% formic acid in acetonitrile (ACN) over 10 min,
and then 100% ACN was used for 5 min. All final compounds were > 95% purity established by CHN, or
UPLC-MS, or both CHN and UPLC-MS. All solvents and chemicals were purchased from Sigma-Aldrich
Co. or Fisher Scientific Inc. and were used as received.
2-Pivalamido-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidine (21)
2-Amino-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidine (20) (3.0 g, 20 mmol) was added to pivalic
o
anhydride (12 mL). The resulting suspension was stirring at 90 C for 3 h. The reaction was cooled to rt
and a mixture of hexane (40 mL) and EtOAc (8 mL) was added dropwise. The suspension was filtered,
washed with acetone (10 mL), and dried at 50 oC under vacuum overnight to afford 4.44 g of pale yellow
solid 21 in 95% yield. mp > 250 oC (lit48 295 oC); TLC Rf 0.55 (CHCl3: MeOH, 20:1); 1H NMR (DMSO-
d6)  1.28 (s, 9H, Piv), 6.41 (d, J = 4.2 Hz, 1H, 5-H), 6.95 (d, J = 4.2 Hz, 1H, 6-H), 10.73 (br, 1H, exch,
PivNH), 11.55 (br, 1H, exch, NH), 11.83 (br, 1H, exch, NH).
2-Pivalamido-4-oxo-6-(N-methyl-benzylaminomethyl)-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine (22)
N-Methyl benzylamine (3.63 g, 30 mmol) and paraformaldehyde (1.05 g, 35 mmol) were added to water
(20 mL). To this solution, acetic acid (20 mL) was added dropwise. The mixture was stirred at 80 oC for 3
h. Compound 21 (2.34 g, 10 mmol) and KOAc (2.94 g, 30 mmol) were added and the stirring was continued
at 80 oC overnight. The solvent was removed by a rotary evaporator and acetone (40 mL) was added. The
25

suspension was filtered and the filtrate was concentrated into a semisolid residue. This residue was purified
by a silica column, which was flushed with CHCl3 and MeOH, to provide 2.86 g of a white solid, 22, in
78% yield. mp: 181.1 - 182.5 oC; TLC Rf 0.37 (CHCl3: MeOH, 20:1); 1H NMR (DMSO-d6)  1.24 (s, 9H,
Piv), 2.09 (s, 3H, NCH3), 3.48 (s, 2H, NCH2), 3.58 (s, 2H, NCH2), 6.31 (s, 1H, 5-H), 7.32 (m, 5H, Ph),
10.85 (br, 1H, exch, PivNH), 11.55 (br, 1H, exch, NH), 11.95 (br, 1H, exch, NH); Anal. calcd. for
.
(C20H25N5O2 0.5H2O) C, H, N; UPLC-MS > 95%; m/z [M-MePhNH]+, calcd 247.12, found 247.25.
2-Pivalamido-4-oxo-6-(methylaminomethyl)-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine hydrochloric
salt (23)
Compound 22 (1.84 g, 5 mmol) was dissolved in methanol (40 mL) and chloroform (2 mL) in a Parr
hydrogenation flask. Pd/C (10%, 200 mg) was added to the above solution under Ar atmosphere and placed
on the Parr hydrogenation shaker under 50 psi hydrogen for 2 h. The catalyst was filtered through celite,
and the solvent was evaporated. To the residue was added acetone (5 mL) and the suspension was filtered,
and dried to afford 1.41 g of 23 as a white solid in 90% yield. mp > 250 oC; TLC Rf 0.13 (CHCl3: MeOH,
10:1); 1H NMR (DMSO-d6)  1.24 (s, 9H, Piv), 3.07 (s, 3H, NCH3), 4.17 (s, 2H, NCH2), 6.58 (s, 1H, 5-H),
+
9.32 (br, 2H, exch, NH2 ), 10.95 (br, 1H, exch, PivNH), 11.86 (br, 2H, exch, NH); 13C NMR (DMSO-d6) 
31.99, 44.48, 104.34, 105.09, 127.39, 148.67, 148.73, 157.11, 181.37; UPLC-MS > 95%; m/z [M-MeNH]+,
calcd 247.12, found 247.18.
General procedures for the synthesis of methoxycarbonyl thiophene carboxylic acids 26b-c
To methoxycarbonyl thiophene carboxylaldehyde 25b-c in acetonitrile solution, periodic acid (2 eq.) was
added and the reaction was stirred at rt for 1 h. To the above solution, water (10 mL) was added and mixture
was extracted with EtOAc (3 X 10 mL). The organic layers were combined, dried, concentrated, and
purified by a silica column with CHCl3 and MeOH, to provide a white solid 26b-c.
26

2-Methoxycarbonyl-thiophene-4-carboxylic acid (26b)
o
1
Yield: 59%. mp 136.9 - 137.8 C; TLC Rf 0.22 (CHCl3: MeOH, 20:1); H NMR (CDCl3)  3.93 (s, 3H,
OCH3), 8.28 (d, J = 1 Hz, 1H, ArH), 8.37 (d, J = 1 Hz, 1H, ArH); Anal. calcd. for (C7H6O4S.0.15H2O) C,
H, S.
3-Methoxycarbonyl-thiophene-5-carboxylic acid (26c)
o
1
Yield: 63%. mp 189.1 - 189.5 C; TLC Rf 0.22 (CHCl3: MeOH, 20:1); H NMR (CDCl3)  3.95 (s, 3H,
OCH3), 8.24 (s, 1H, ArH), 8.40 (s, 1H, ArH); Anal. calcd. for (C7H6O4S) C, H, S.
Methyl
4-{[N-methyl-(2-pivalamido-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-
methylamino]-carbonyl}-phenylcarboxylate (24a)
To a solution of 4-methoxycarbonyl phenyl carboxylic acid (180 mg, 1 mmol) in DMF (1 mL) was added
N-methyl morpholine (310 mg, 3 mmol), EDCI (240 mg, 1.2 mmol) and HOAt (183 mg, 1.05 mmol). The
mixture was stirred at rt for 1 h. Compound 23 (313 mg, 1 mmol) was added and stirring was continued
overnight. The solvent was removed and the residue was purified by a silica column with CHCl3 and MeOH
1
to provide 316 mg of 24a as a semi-solid in 72% yield. TLC Rf 0.16 (CHCl3/MeOH, 40:1); H NMR
(DMSO-d6)  1.25 (s, 9H, Piv), 2.84 (s, 1.8H, cis NCH3), 2.99 (s, 1.2H, trans NCH3), 3.88 (s, 3H, OCH3),
4.44 (s, 0.8H, trans NCH2), 4.70 (s, 1.2H, cis NCH2), 6.24 (s, 0.4H, trans 5-H), 6.38 (s, 0.6H, cis 5-H), 7.60
(d, J = 6.8 Hz, 2H, Ph), 8.04 (d, J = 6.8 Hz, 2H, Ph), 10.79 (br, 1H, exch, PivNH), 11.62 (br, 1H, exch, NH),
11.87 (s, 1H, exch, NH).
Methyl
3-{[N-methyl-(2-pivalamido-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-
methylamino]-carbonyl}-phenylcarboxylate (24b)
Compound 24b was synthesized following the procedure of 24a and was obtained as a semi-solid in 69%
yield. TLC Rf 0.15 (CHCl3/MeOH, 40:1); 1H NMR (DMSO-d6)  1.25 (s, 9H, Piv), 2.73 (s, 1.2H, cis NCH3),
27

2.85 (s, 1.8H, trans NCH3), 3.98 (s, 3H, OCH3), 4.43 (s, 0.8H, trans NCH2), 4.69 (s, 1.2H, cis NCH2), 6.27
(s, 0.4H, trans 5-H), 6.39 (s, 0.6H, cis 5-H), 7.62 (m, 1H, Ph), 7.75 (m, 1H, Ph), 8.03 (m, 2H, Ph), 10.79 (s,
1H, exch, PivNH), 11.63 (s, 1H, exch, NH), 11.89 (s, 1H, exch, NH).
Methyl
5-{[N-methyl-(2-pivalamido-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-
methylamino]-carbonyl}-thiophene-2-carboxylate (24c)
Compound 24c was synthesized following the procedure of 24a from 25a and was obtained as a semi-solid
1
in 82% yield. TLC Rf 0.16 (CHCl3/MeOH, 40:1); H NMR (DMSO-d6)  1.24 (s, 9H, Piv), 3.05 (br, 3H,
cis and trans NCH3), 3.85 (s, 3H, OCH3), 4.69 (s, 2H, cis and trans NCH2), 6.35 (s, 1H, cis and trans 5-
H), 7.54 (s, 1H, thiophene), 7.78 (s, 1H, thiophene), 10.80 (s, 1H, exch, PivNH), 11.62 (s, 1H, exch, NH),
11.88 (s, 1H, exch, NH).
Methyl
4-{[N-methyl-(2-pivalamido-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-
methylamino]-carbonyl}-thiophene-2-carboxylate (24d)
Compound 24d was synthesized following the procedure of 24a from 26b and was obtained as a semi-solid
in 76% yield. TLC Rf 0.17 (CHCl3/MeOH, 40:1); 1H NMR (DMSO-d6)  1.25 (s, 9H, Piv), 2.89 (s, 1.5H,
cis NCH3), 2.89 (s, 1.5H, trans NCH3), 3.80 (s, 3H, OCH3), 4.69 (s, 2H, cis and trans NCH2), 6.33 (s, 1H,
cis and trans 5-H), 7.78 (s, 1H, thiophene), 8.54 (s, 1H, thiophene), 10.79 (s, 1H, exch, PivNH), 11.64 (s,
1H, exch, NH), 11.89 (br, 1H, exch, NH).
Methyl
5-{[N-methyl-(2-Pivalamido-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-
methylamino]-carbonyl}-thiophene-3-carboxylate (24e)
Compound 24e was synthesized following the procedure of 24a from 26c and was obtained as a semi-solid
in 79% yield. TLC Rf 0.16 (CHCl3/MeOH, 40:1); 1H NMR (DMSO-d6)  1.25 (s, 9H, Piv), 2.98 (s, 3H, cis
and trans NCH3), 3.92 (s, 3H, OCH3), 4.65 (br, 2H, cis and trans NCH2), 6.37 (br, 1H, cis and trans 5-H),
28

7.91 (d, J = 2.8 Hz, 1H, thiophene), 8.34 (d, J = 3.0 Hz, 1H, thiophene), 10.78 (s, 1H, exch, PivNH), 11.55
(s, 1H, exch, NH), 11.86 (s, 1H, exch, NH).
Methyl
4-{[N-methyl-(2-pivalamido-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-
methylamino]-sulfonyl}-phenylcarboxylate (24f)
To a solution of 23 (313 mg, 1 mmol) in DMF (1 mL), was added triethylamine (210 mg, 2.05 mmol),
methyl 4-chlorosulfonyl benzoate 27c (235 mg, 1 mmol) and 4-dimethylaminopyridine (10 mg). The
mixture was stirred at rt for 4 h. The solvent was removed and the residue was purified by a silica column
with CHCl3 and MeOH to afford 356 mg of 24f as a white solid in 75% yield. TLC Rf 0.13 (CHCl3/MeOH,
40:1); 1H NMR (DMSO-d6)  1.25 (s, 9H, Piv), 2.61 (s, 3H, NCH3), 3.91 (s, 3H, OCH3), 4.21 (s, 2H, NCH2),
6.28 (s, 1H, 5-H), 7.95 (d, J = 8.0 Hz, 2H, Ph), 8.18 (d, J = 8.0 Hz, 2H, Ph), 10.85 (s, 1H, exch, PivNH),
11.66 (s, 1H, exch, NH), 11.88 (s, 1H, exch, NH).
4-{[N-Methyl-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)-methylamino]-
carbonyl}-phenylcarboxylic acid (28a)
To a solution of 24a (220 mg, 0.5 mmol) in water (10 mL) and methanol (10 mL) was added Na2CO3 (0.5
o
g). The mixture was stirred at 80 C for 2h. The methanol was removed and the pH of the solution was
adjusted to 3 with 1N HCl. The suspension was filtered and the filter cake was washed with deionized water
o
and acetone to facilitate drying, and dried to afford 152 mg white solid 28a in 90% yield. mp > 250 C;
TLC Rf 0.53 (CHCl3: MeOH: AcOH, 10:1:0.5); 1H NMR (DMSO-d6)  2.78 (s, 1.8H, cis NCH3), 2.93 (s,
1.2H, trans NCH3), 4.30 (s, 0.8H, trans NCH2), 4.58 (s, 1.2H, cis NCH2), 6.01 (s, 0.4H, trans 5-H), 6.07 (s,
2H, exch, 2-NH2), 6.16 (s, 0.6H, cis 5-H), 7.58 (d, J = 6.8 Hz, 2H, Ph), 8.01 (d, J = 6.8 Hz, 2H), 10.25 (s,
.
1H, exch), 10.33 (s, 1H, exch), 13.14 (s, 1H, exch); Anal. calcd. for (C16H15N5O4 1.40H2O) C, H, N; UPLC-
MS > 95%; m/z [M+H]+, calcd 342.11, found 342.25.
29