Asymmetric synthesis of inhibitors of glycinamide ribonucleotide transformylase

Article abstract of DOI:10.1021/jm800555h

Glycinamide ribonucleotide transformylase (GAR Tfase) catalyzes the first of two formyl transfer steps in the de novo purine biosynthetic pathway that require folate cofactors and has emerged as a productive target for antineoplastic therapeutic intervention. The asymmetric synthesis and evaluation of the two diastereomers of 10-methylthio-DDACTHF (10R-3 and 10S-3) and related analogues as potential inhibitors of GAR Tfase are reported. This work, which defines the importance of the C10 stereochemistry for this class of inhibitors of GAR Tfase, revealed that both diastereomers are potent inhibitors of rhGAR Tfase (10R-3 K_i = 210 nM, 10S-3 K_i = 180 nM) that exhibit effective cell growth inhibition (CCRF-CEM IC₅₀ = 80 and 50 nM, respectively), which is dependent on intracellular polyglutamation by folylpolyglutamate synthetase (FPGS) but not intracellular transport by the reduced folate carrier.

Full text of DOI:10.1021/jm800555h

J. Med. Chem. 2008, 51, 5441–5448
5441
Asymmetric Synthesis of Inhibitors of Glycinamide Ribonucleotide Transformylase
Jessica K. DeMartino, Inkyu Hwang, Stephen Connelly, Ian A. Wilson, and Dale L. Boger*
Departments of Chemistry, Molecular Biology, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed May 13, 2008
Glycinamide ribonucleotide transformylase (GAR Tfase) catalyzes the first of two formyl transfer steps in
the de novo purine biosynthetic pathway that require folate cofactors and has emerged as a productive
target for antineoplastic therapeutic intervention. The asymmetric synthesis and evaluation of the two
diastereomers of 10-methylthio-DDACTHF (10R-3 and 10S-3) and related analogues as potential inhibitors
of GAR Tfase are reported. This work, which defines the importance of the C10 stereochemistry for this
class of inhibitors of GAR Tfase, revealed that both diastereomers are potent inhibitors of rhGAR Tfase
(10R-3 Ki ) 210 nM, 10S-3 Ki ) 180 nM) that exhibit effective cell growth inhibition (CCRF-CEM IC50
) 80 and 50 nM, respectively), which is dependent on intracellular polyglutamation by folylpolyglutamate
synthetase (FPGS) but not intracellular transport by the reduced folate carrier.
Introduction
Glycinamide ribonucleotide transformylase (GAR Tfase)a is
a folate-dependent enzyme central to the de novo purine
biosynthetic pathway.1,2 GAR Tfase utilizes the cofactor (6R)-
N10-formyltetrahydrofolic acid (10-formyl-THF) to transfer a
formyl group to the primary amine of its substrate, ꢀ-glycina-
mide ribonucleotide (ꢀ-GAR) (Figure 1). This one carbon
transfer provides the C-8 carbon of the purines and is the first
of two formyl transfer reactions enlisted in the biosynthesis of
purines. Inhibitors of folate dependent enzymes including GAR
Tfase have provided important compounds for cancer chemo-
therapy as a result of their inhibition of the biosynthesis of
nucleic acid precursors.3,4 Validation of GAR Tfase as a useful
anticancer target emerged with the discovery of the first potent
and selective inhibitor, 5,10-dideaza-5,6,7,8-tetrahydrofolic acid
(DDATHF).5 Its selective toxicity has been attributed in part
to the reliance of sensitive tumor cells on de novo purine
synthesis, whereas the salvage pathway is an available and
primary source of purines in normal untransformed cells.6,7
In previous studies, we reported the synthesis and biological
evaluation of two unique folate-based inhibitors of GAR Tfase.
10-Formyl-DDACTHF8 (1) and 10-CF3CO-DDACTHF9 (2),
bearing a nontransferable formyl or trifluoroacetyl group, proved
to be potent inhibitors of GAR Tfase (1, Ki ) 0.014 µM against
rhGAR Tfase; 2, Ki ) 0.015 µM against rhGAR Tfase). Both
inhibitors have been shown through X-ray and NMR studies to
bind GAR Tfase as the gem-diols.9,10 The formation of the gem-
Figure 1. Cofactor and representative inhibitors of folate-dependent
diol mimics the formyl transfer reaction tetrahedral intermediate
and provides strong stabilizing interactions between the inhibitor
and the active site catalytic residues of the protein. In both
instances, the inhibitors C10 diastereomers rapidly interconvert,
preventing independent assessment of the individual diastereomers.
enzymes.
binding interactions, we reported the synthesis and biological
activity of its precursor 3, 10-methylthio-DDACTHF, which
proved to be a surprisingly potent inhibitor of GAR Tfase (IC50
) 100 nM, CCRF-CEM; Ki ) 250 nM, rhGAR Tfase).11
Although the activity of 3 was unexpected because it does not
contain a C10 tetrahedral intermediate mimic, the thiomethyl
moiety does incorporate a potential hydrogen bond acceptor and
presents a soft hydrophobic substituent potentially stabilizing
active site binding. The introduction of sulfur within inhibitors
of GAR Tfase is well established. The known inhibitors
LY309887 (4), AG2034 (5), and AG2037 (6) all incorporate
sulfur into their structures although none do so in a manner
analogous to 3 (Figure 2),12-14 and it has been reported that
In a more recent study designed to explore the ability of 10-
methanesulfonyl-DDACTHF to also mimic such active site
* To whom correspondence should be addressed. Phone:858-784-7522.
Fax: 858-784-7550. E-mail: boger@scripps.edu.
a Abbreviations: GAR Tfase, glycinamide ribonucleotide transformylase;
rhGAR Tfase, recombinant human GAR Tfase; FPGS, folypolyglutamate
synthetase; 10-formyl-THF, 10-formyltetrahydrofolic acid; MTX, methotr-
exate; AICAR, aminoimidazole carboxamide ribonucleotide transformylase;
rhAICAR Tfase, recombinant human AICAR Tfase; DDACTHF, dideaza-
acyclotetrahydrofolic acid.
10.1021/jm800555h CCC: $40.75
2008 American Chemical Society
Published on Web 08/08/2008

5442 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
DeMartino et al.
Scheme 2
Figure 2. Inhibitors of GAR Tfase that contain sulfur atoms.
Scheme 3
Scheme 1
based on the well-established stereochemical preferences of the
reagent16 and confirmed in X-ray structures of both 10R-3 and
10S-3 bound to recombinant human GAR Tfase. The details of
these latter studies will be disclosed elsewhere. Similarly, the
S-Me-CBS catalyst was utilized to provide the enantiomer of
14 (not shown) and characterization data for this enantiomeric
series is provided in the Supporting Information.
The preformed sodium salt of ethyl cyanoacetate was
alkylated with S-14 to give the common intermediate S-15 for
all three optically active inhibitors. Treatment of S-15 with
thiolacetic acid under modified Mitsunobu conditions provided
R-16 with inversion of C10 stereochemistry (Scheme 2).
Compound R-16 was subsequently deprotected and the free thiol
was methylated to yield R-18. Intermediate R-18 was treated
with the free base of guanidine under basic conditions to form
the pyrimidine R-19, and this intermediate was subsequently
saponified to give R-20. Compound R-20 was coupled with di-
tert-butyl L-glutamate and subsequently deprotected using
trifluoroacetic acid to provide final inhibitor 10R-3. The C10
diastereomer 10S-3 was prepared using an analogous route by
employing the enantiomeric R-Me-CBS catalyst to provide R-14
(See Supporting Information).
analysis of the GAR Tfase active site using the program GRID
suggested that sulfur atoms should have particular affinity for
two regions of the folate cofactor binding site.13
The original synthesis of 3 was racemic, and the derivative
was evaluated as an equimixture of C10-diastereomers. How-
ever, and unlike 1 and 2, the individual C10-diastereomers of
3 would not be expected to easily interconvert and could be
anticipated to be individually evaluated revealing the importance
of the C10 stereochemistry to the properties of the candidate
inhibitors. Herein, we report an asymmetric synthesis of 10-
methylthio-DDACTHF (3), permitting this investigation into the
importance of the C10 stereochemistry and report the biological
properties of each C10-diastereomer of 10-thiomethyl-DDACTHF
(3) along with that of a series of key analogues that further
define the importance of the C10 substituent itself: 10-hydroxy-
DDACTHF (10R-7 and 10S-7) and 10-methoxy-DDACTHF
(10R-8 and 10S-8) and C10 substituted dithiane 9.
The inhibitor 10S-7 bearing a C10-hydroxyl substituent was
prepared from the common intermediate S-15 (Scheme 3). Com-
pound S-15 was treated with guanidine hydrochloride to give the
corresponding pyrimidone S-22, which subsequently was saponified
using lithium hydroxide to afford S-23. Intermediate S-23 was then
coupled with dimethyl L-glutamate and deprotected under basic
conditions to provide the final inhibitor 10S-7.
A key compound for comparison is the C10-methoxy
derivative 8, where an oxygen replaces the sulfur of 3 as a probe
for its unique importance. The synthesis of inhibitor 10S-8,
bearing the C10-methoxy substituent, was accomplished by
methylation of the common intermediate S-15 using TMSCHN2
under acidic conditions17 to give S-25 (Scheme 4). Intermediate
S-25 was carried forward by employing an analogous route,
starting with formation of pyrimidone S-26 and saponification
to give S-27. Compound S-27 was coupled with di-tert-butyl
Inhibitor Synthesis. Starting from methyl 4-formylbenzoate,
the aldehyde was converted to dithiane 11 upon reaction with
1,3-propanedithiol (Scheme 1). The resulting dithiane was
alkylated with 1-chloro-3-iodopropane to give 12, and subse-
quent removal of the dithiane using bis(trifluoro)acetoxyiodo-
benzene (PIFA) provided 13. Ketone 13 was reduced using the
R-Me-CBS15 catalyst in high yield (97%) and high enantiomeric
excess (99% ee) to yield alcohol S-14. The absolute stereo-
chemistry of S-14 and its subsequent derivatives were assigned

Inhibitors of Glycinamide Ribonucleotide Transformylase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5443
Table 1. GAR and AICAR Tfase Inhibition (Ki, µM)
Scheme 4
compound
rhGAR Tfase
rhAICAR Tfase
10R-3
10S-3
10R-7
10S-7
10R-8
10S-8
9
DDACTHF
2
lometrexol
0.21
0.18
1.1
1.4
0.85
1.2
>100
>100
>100
>100
>100
>100
20
20
>100
>100
1.1
1.7
0.03
0.06a
a Ref 18.
Table 2. In Vitro Cytotoxic Activity
CCRF-CEM (IC50, µM)
compound
(-) T, (-) H
(+) T, (-) H
(-) T, (+) H
10R-3
10S-3
10R-7
10S-7
10R-8
10S-8
9
DDACTHF
2
lometrexol
0.08
0.05
2.4
0.5
0.5
0.7
100
2.7
0.016
0.2
0.08
0.07
5.7
0.6
0.7
0.9
>100
3.6
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
Scheme 5
0.017
0.2
GAR Tfase active site. Inhibitors 10R-7 and 10S-7 displayed
very modest activity (Ki ) 1060 nM and 1210 nM, respectively)
against rhGAR Tfase. This represents a 6-fold reduction in
activity relative to 3, and again both diastereomers proved active,
although it was the C10-R diastereomer that was slightly more
potent than the corresponding C10-S diastereomer. Even more
interestingly, methoxy derivatives 10R-8 and 10S-8 were found
to inhibit rhGAR Tfase with potencies nearly equivalent to the
corresponding alcohols (Ki ) 850 nM and 1240 nM, respec-
tively). Again, while both diastereomers exhibited near equiva-
lent potencies, the C10-R diastereomer was slightly more
effective and the 4-6 fold distinction relative to 3 highlights
the special nature of the thiomethyl versus methoxy interaction
at the enzyme active site. Inhibitor 9 also exhibited a reduced
binding to rhGAR Tfase (Ki ) 1060 nM), illustrating that
optimally one, but not two, thioalkyl substituents may enhance
binding at the enzyme active site. These results define a clear
role and remarkable stereochemical accommodation of the C10
substituents on DDACTHF with a unique stabilization attribut-
able to the C10-thiomethyl group.
All inhibitors were also examined for activity against rhAI-
CAR Tfase. The only compound in the series to show any
activity against the enzyme was dithiane 9 (Ki ) 20 µM). Each
diastereomer of the thiomethyl, hydroxyl, and methoxy inhibitors
was inactive against rhAICAR Tfase (Ki >100 µM), illustrating
that they disrupt de novo purine biosynthesis by selectively
targeting GAR Tfase.
The compounds were also examined for cytotoxic activity
(growth inhibition) both in the presence (+) and absence (-)
of added hypoxanthine (purine) or thymidine (pyrimidine)
against the CCRF-CEM cell line (Table 2). All the inhibitors
exhibited activity that paralleled their potency against rhGAR
Tfase. Of all the inhibitors, only 10R-3 and 10S-3 exhibited
potent activity in this cell-based assay (IC50 ) 80 and 50 nM,
respectively), being only 3-5 times less potent than 2, the most
potent inhibitor of GAR Tfase disclosed to date. While both
C10-diastereomers displayed this potent cytotoxic activity, the
10S-diastereomer proved to be slightly more active than the 10R-
L-glutamate hydrochloride and deprotected using trifluoroacetic
acid to provide 10S-8.
Finally, 9, which incorporates two C10 sulfur atoms and, in a
sense, simultaneously incorporates both C10-diastereomers of 3,
was judged as an attractive additional derivative to examine that
avoids the presence of two C10-diastereomers. Synthesis of
inhibitor 9 started with alkylation of 12 by using ethyl cyanoacetate
to give 29 (Scheme 5). Intermediate 29 was converted to 9 by
using the same route detailed for the previous inhibitors, starting
with formation of pyrimidine 30 and saponification to give 31.
Compound 31 was coupled with di-tert-butyl L-glutamate and
deprotected by using trifluoroacetic acid to provide 9.
Results and Discussion
Each diastereomer of 3, 7, 8, and 9 was examined for
inhibition of rhGAR Tfase and aminoimidazole carboxamide
ribonucleotide transformylase (rhAICAR Tfase), and the results
are summarized in Table 1. Remarkably, both diastereomers of
the thiomethyl derivative 3 exhibited potent activity against
rhGAR Tfase with 10S-3 being slightly more potent than 10R-3
(Ki ) 180 nM vs Ki ) 210 nM, respectively). This nearly
indistinguishable activity of the two C10-diastereomers of 3 is
only consistent with both possessing near equivalent capabilities
for binding at the GAR Tfase active site and suggests that the
flexible linker region joining the pyrimidine and phenyl ring
conformationally adjusts to allow a comparable positioning of
the thiomethyl substituent in the formyl transfer pocket of the

5444 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
DeMartino et al.
Table 3. In Vitro Cytotoxic Activity in Mutant Cell Lines
IC50, µM [(-)T, (-)H]
bearing a C10-thiomethyl substituent were surprisingly potent
and efficacious. Presumably this reflects the conformational
flexibility of the linking region between the pyrimidine and
phenyl group, which contains the C10-substituent that allows a
comparable positioning of the thiomethyl group in the formyl
transfer pocket of the GAR Tfase active site. In addition to being
substantially more potent than the corresponding C10-methoxy
derivatives highlighting the unique properties embodied in 3,
the two diastereomers of 3 proved especially efficacious in
inhibiting cell growth (CCRF-CEM IC50 ) 80 and 50 nM for
10R-3 and 10S-3, respectively) that could be rescued by added
purine (but not pyrimidine) and that relies on intracellular FPGS
polyglutamation but not intracellular transport by the reduced
folate carrier. Similarly, both diastereomers of 3 were found to
be potent and essentially indistinguishable inhibitors of rhGAR
Tfase (Ki ) 210 and 180 nM for 10R-3 and 10S-3, respectively),
albeit with potencies that are slightly lower than their actual
cell growth inhibitory activity. This latter observation suggests
that the required intracellular polyglutamation observed with 3
functionally enhances its potency in the cell-based assays.
compound
CCRF-CEM CCRF-CEM/FPGS- CCRF-CEM/MTX
10R-3
10S-3
10R-7
10S-7
10R-8
10S-8
9
DDACTHF
2
lometrexol
0.09
0.06
5.50
0.60
0.60
0.80
>100
2.7
5.5
5.0
>10
>10
6.0
>10
nd
>10
>10
>10
0.07
0.06
4.4
0.6
0.6
0.9
nd
>100
0.06
0.20
nd
nd (>100)a
a Ref 18.
diastereomer, and both display a potency that slightly exceeds
that observed on the isolated enzyme. Compounds 10R-7, 10S-
7, 10R-8, and 10S-8 were all approximately 10 times less potent
than the thiomethyl inhibitors. All still showed modest activity,
and there was little distinction between the individual C10
diastereomers. Inhibitor 9, on the other hand, showed no activity
in this cell-based assay. An especially interesting set of
comparisons are the activities of the two diastereomers of 3
and the related inhibitors with DDACTHF lacking a C10
substituent. These comparisons indicate that each of the sub-
stituents actually enhance, or at least maintain, the GAR Tfase
inhibition of DDACTHF and enhance and maintain its corre-
sponding cell growth inhibitory activity. Thus, the differences
observed among one another and relative to DDACTHF may
be attributed to relative binding enhancements provided by the
key C10 substituents and not to destabilizing interactions they
introduce at the enzyme active site. Finally, in the presence of
thymidine, a pyrimidine, all inhibitors retained their activity,
whereas the inhibitors were inactive in the presence of added
hypoxanthine. This indicates that these compounds, like 2,
inhibit cell growth by selectively inhibiting an enzyme within
the purine biosynthetic (not pyrimidine) pathway consistent with
their potent inhibition of GAR Tfase.
All seven inhibitors were subsequently examined in mutant
CCRF-CEM cell lines that lack folypolyglutamate synthetase
(CCRF-CEM/FPGS-) or the reduced folate carrier (CCRF-
CEM/MTX) (Table 3). Like the past inhibitors of GAR Tfase
that we have examined, all inhibitors including 3 lost activity
against the cell line that lacks FPGS (ca. 100-fold for 3),
indicating that they benefit from intracellular polyglutamation.
This observation suggests that the potent activity of both
diastereomers of 3 in the cell growth assays, which exceed their
inhibition of GAR Tfase roughly 3-fold, may be attributed to
their FPGS polyglutamation and the resulting intracellular
accumulation or enhanced target affinity. In contrast, none of
the inhibitors disclosed herein exhibited altered activity against
the cell line lacking the reduce folate carrier, indicating its
transport of the inhibitors is not essential to their activity. Thus,
cell lines and tumors that derive their resistance to antifolates
through down-regulation of the reduced folate carrier would be
expected to remain sensitive to the two diastereomers of 3.
Experimental Section
Characterization of the compounds with the alternative C10-
stereochemistry is provided in the Supporting Information. Our use
of “concentrated” in the following experimental refers to evapora-
tion to dryness on a rotary evaporator.
Methyl 4-(1,3-Dithian-2-yl)benzoate (11). A solution of methyl
4-formylbenzoate (14.10 g, 85.9 mmol) in anhydrous CH2Cl2 (282
mL) was treated with 1,3-propanedithiol (9.48 mL, 94.5 mmol, 1.1
equiv), and the mixture was stirred for 1 h at room temperature.
The solution was cooled to 0 °C, and boron trifluoride diethyl
etherate was added (11.9 mL, 94.5 mmol, 1.1 equiv). The reaction
mixture was stirred and allowed to warm to room temperature
overnight. The mixture was diluted with CH2Cl2 and quenched with
the addition of saturated aqueous NaHCO3. The organic layer was
washed with H2O and saturated aqueous NaCl, dried over Na2SO4,
and concentrated (evaporated to dryness on a rotary evaporator).
Column chromatography (SiO2, 25-50% EtOAc/hexanes gradient)
1
yielded 11 (21.0 g, 96%) as a white solid: mp 132-134 °C. H
NMR (500 MHz, CDCl3) δ 8.02 (d, 2H, J ) 8.3 Hz), 7.55 (d, 2H,
J ) 8.3 Hz), 5.22 (s, 1H), 3.92 (s, 3H), 3.10-3.04 (m, 2H),
2.96-2.92 (m, 2H), 2.22-2.19 (m, 1H), 1.99-1.95 (m, 1H). 13C
NMR (125 MHz, CDCl3) δ 166.8, 144.2, 130.3 (2C), 130.2, 128.1
(2C), 52.4, 51.3, 32.1 (2C), 25.2. IR (film) νmax 2903, 1707, 1272,
1108 cm-1. ESI-TOF m/z 255.0507 (M + H+, C12H14O2S2 requires
255.0508).
Methyl
4-(2-(3-Chloropropyl)-1,3-dithian-2-yl)benzoate
(12). A solution of 11 (20.96 g, 82.4 mmol) in THF (629 mL)
cooled to -78 °C was treated with NaHMDS (2 M in THF, 51.5
mL, 103 mmol, 1.25 equiv) dropwise. The solution was stirred for
30 min, followed by the addition of 1-chloro-3-iodopropane (44.3
mL, 412 mmol, 5.0 equiv). The reaction mixture was allowed to
warm to room temperature overnight and was quenched with the
addition of saturated aqueous NH4Cl. The mixture was diluted with
EtOAc and subsequently washed with saturated aqueous NaCl, dried
over Na2SO4, and concentrated. Column chromatography (SiO2,
0-10% EtOAc/hexanes gradient) yielded 12 (21.73 g, 80%) as a
1
white solid: mp 75-77 °C. H NMR (500 MHz, CDCl3) δ 8.05
(d, 2H, J ) 8.7 Hz), 8.00 (d, 2H, J ) 8.7 Hz), 3.92 (s, 3H), 3.40
(t, 2H, J ) 6.4 Hz), 2.75-2.60 (m, 4H), 2.17-2.14 (m, 2H),
1.96-1.93 (m, 2H), 1.75-1.70 (m, 2H). 13C NMR (125 MHz,
CDCl3) δ 166.8, 147.2, 130.0 (2C), 129.2, 129.0 (2C), 58.1, 52.3,
44.7, 42.3, 27.8 (2C), 27.3, 25.0. IR (film) νmax 2944, 1723, 1277
cm-1. ESI-TOF m/z 331.0587 (M + H+, C15H19ClO2S2 requires
331.0588).
Methyl 4-(4-Chlorobutanoyl)benzoate (13). A solution of 12
(10.79 g, 32.6 mmol) in 9:1 MeCN/H2O (163 mL) was treated with
bis(trifluoro)acetoxyiodobenzene (21.0 g, 48.9 mmol, 1.5 equiv)
Conclusions
Examination of the importance of the C10 stereochemistry
of DDACTHF-based inhibitors was addressed with the asym-
metric synthesis of the two diastereomers of 10-methylthio-
DDACTHF (3) and a series of key analogues incapable of in
situ racemization of the C10 center. Remarkably, the activities
of the C10-substituted DDACTHF derivatives proved to be
essentially independent of the C10 stereochemistry and those

Inhibitors of Glycinamide Ribonucleotide Transformylase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5445
and allowed to stir for 30 min. The reaction mixture was quenched
with the addition of saturated aqueous NaHCO3 and diluted with
EtOAc. The mixture was extracted with H2O (2×) and saturated
aqueous NaCl, dried over Na2SO4, and concentrated. Column
chromatography (SiO2, 20% EtOAc/hexanes) provided 13 (6.37 g,
81%) as a white solid: mp 45-46 °C. 1H NMR (500 MHz, CDCl3)
δ 8.15 (d, 2H, J ) 8.3 Hz), 8.04 (d, 2H, J ) 8.2 Hz), 3.96 (s, 3H),
3.70 (t, 2H, J ) 6.3 Hz), 3.22 (t, 2H, J ) 6.9 Hz), 2.28-2.22 (m,
2H). 13C NMR (125 MHz, CDCl3) δ 198.6, 166.3, 140.0, 134.1,
130.0 (2C), 128.0 (2C), 52.6, 44.6, 35.8, 26.7. IR (film) νmax 2944,
1723, 1682, 1272, 1108 cm-1. ESI-TOF m/z 241.0624 (M + H+,
C12H13ClO3 requires 241.0626).
1.29/1.28 (two t, 3H, J ) 7.0 Hz). 13C NMR (125 MHz, CDCl3)
δ 194.4, 166.8, 165.9, 146.6, 130.2 (2C), 129.5, 127.7 (2C), 116.4,
63.0, 52.3, 47.2, 37.4, 35.1, 30.6, 29.4, 24.7, 14.1. IR (film) νmax
3733, 2974, 1733, 1641, 1256, 1108 cm-1. ESI-TOF m/z 400.1191
(M + Na+, C19H23NO5S requires 400.1189).
Methyl 4-((1R)-5-Cyano-6-ethoxy-1-mercapto-6-oxohexyl)-
benzoate (R-17). A solution of R-16 (2.01 g, 5.3 mmol) in EtOH
(26.6 mL) was treated with K2CO3 (3.68 g, 26.6 mmol, 5.0 equiv)
and stirred for 12 h. The reaction mixture was diluted with EtOAc
and washed with H2O and saturated aqueous NaCl, dried over
Na2SO4, and concentrated. Column chromatography (SiO2, 30%
EtOAc/hexanes) yielded the unstable free thiol R-17 (291 mg, 16%)
as a colorless oil. 1H NMR (600 MHz, CDCl3) δ 8.00 (d, 2H, J )
7.8 Hz), 7.38 (d, 2H, J ) 8.2 Hz), 4.26-4.21 (m, 2H), 4.01-3.98
(m, 1H), 3.91 (s, 3H), 3.47-3.43 (m, 1H), 1.99-1.92 (m, 4H),
1.96 (d, 1H, J ) 5.6 Hz), 1.66-1.58 (m, 1H), 1.49-1.40 (m, 1H),
1.30-1.26 (m, 3H). 13C NMR (125 MHz, CDCl3) δ 166.7, 165.9,
149.4, 130.3 (2C), 129.4, 126.9 (2C), 116.4, 63.0, 52.2, 43.4, 38.6,
37.4, 29.4, 25.2, 14.1. IR (film) νmax 2933, 1718, 1436, 1271, 1184,
1102 cm-1. ESI-TOF m/z 336.1267 (M + H+, C17H21NO4S requires
336.1264).
Methyl 4-((1R)-5-Cyano-6-ethoxy-1-(methylthio)-6-oxohexyl)-
benzoate (R-18). Free thiol R-17 (291 mg, 0.87 mmol) dissolved
in MeOH (8.68 mL) was treated with MeI (542 µL, 8.68 mmol, 10
equiv) and NaHCO3 (80 mg, 0.97 mmol, 1.1 equiv), and the mixture
was stirred for 3 h at room temperature. The reaction mixture was
diluted with EtOAc and washed with H2O and saturated aqueous
NaCl, dried over Na2SO4, and concentrated. Column chromatog-
raphy (SiO2, 30% EtOAc/hexanes) provided R-18 (238 mg, 79%)
as a colorless oil. 1H NMR (600 MHz, CDCl3) δ 8.01 (d, 2H, J )
7.3 Hz), 7.37 (d, 2H, J ) 7.8 Hz), 4.26-4.20 (m, 2H), 3.91 (s,
3H), 3.69 (t, 1H, J ) 7.4 Hz), 3.46-3.41 (m, 1H), 1.99-1.87 (m,
3H), 1.85 (s, 3H), 1.64-1.56 (m, 1H), 1.50-1.45 (m, 1H),
1.31-1.26 (m, 3H). 13C NMR (150 MHz, CDCl3) δ 166.9, 166.0,
147.4, 130.1 (2C), 129.3, 127.9 (2C), 116.4, 63.0, 52.2, 50.9, 37.5,
35.1, 29.6, 25.1, 14.4, 14.1. IR (film) νmax 3358, 2923, 1718, 1431,
1267, 1097, 1010 cm-1. ESI-TOF m/z 372.1252 (M + Na+,
C18H23NO4S requires 372.1240).
Methyl (S)-4-(4-Chloro-1-hydroxybutyl)benzoate (S-14). A
solution of R-Me-CBS (1 M in toluene, 1.09 mL, 1.09 mmol, 0.1
equiv) in anhydrous THF (10.1 mL) cooled to 0 °C was treated
with borane dimethylsulfide complex (2 M in THF, 6.80 mL, 13.6
mmol, 1.25 equiv) and stirred for 15 min at 0 °C. A solution of 13
(2.62 g, 10.9 mmol) in THF (54.4 mL) was added dropwise to the
reaction mixture and stirred for 1 h, allowing the reaction mixture
to warm to room temperature. The reaction was quenched with the
addition of MeOH and allowed to stir for 30 min at room
temperature. The mixture was diluted with EtOAc and washed with
1 N HCl (3×), H2O (2×), dried over Na2SO4, and concentrated.
Column chromatography (SiO2, 40% EtOAc/hexanes) afforded S-14
1
(2.55 g, 97%) as a clear oil. H NMR (500 MHz, CDCl3) δ 8.04
(d, 2H, J ) 8.2 Hz), 7.44 (d, 2H, J ) 8.4 Hz), 4.81 (t, 1H, J ) 5.9
Hz), 3.92 (s, 3H), 3.60-3.53 (m, 2H), 1.97-1.82 (m, 4H). 13C
NMR (125 MHz, CDCl3) δ 167.0, 149.6, 130.0 (2C), 129.5, 125.8
(2C), 73.4, 52.2, 45.0, 36.3, 28.8. IR (film) νmax 2923, 1748, 1277
cm-1. ESI-TOF m/z 243.0781 (M + H+, C12H15ClO3 requires
243.0782); [R]D -30 (c 1.0, CHCl3). Enantiomeric excess was
determined (99% ee) using a Chiralcel AD-H analytical column
(250 mm × 4.6 mm, 3% i-PrOH/hexane, 1 mL/min, R ) 1.07).
Methyl 4-((1S)-5-Cyano-6-ethoxy-1-hydroxy-6-oxohexyl)-
benzoate (S-15). A suspension of NaH (60% dispersion, 1.11 g,
27.7 mmol, 3.0 equiv) in anhydrous DMF (27.7 mL) was treated
with ethyl cyanoacetate (2.96 mL, 27.7 mmol, 3.0 equiv) at 0 °C.
The reaction mixture was stirred for 30 min while allowing the
mixture to warm to room temperature, forming the sodium salt as
a clear solution. A solution of S-14 (2.24 g, 9.24 mmol) in DMF
(18.5 mL) was added at room temperature, and the reaction mixture
was stirred at 60 °C for 12 h. The reaction mixture was cooled to
room temperature and quenched with the addition of saturated
aqueous NH4Cl. The mixture was concentrated and then diluted
with EtOAc. The organic layer was washed with H2O and saturated
aqueous NaCl, dried over Na2SO4, and concentrated. Column
chromatography (SiO2, 50% EtOAc/hexanes) provided S-15 (1.76
Methyl (R)-4-(4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-
5-yl)-1-(methylthio)butyl)benzoate (R-19). A solution of NaOMe
(60 mg, 1.12 mmol, 3.0 equiv) in anhydrous MeOH (3 mL) was
treated with guanidine hydrochloride (107 mg, 1.12 mmol, 3.0
equiv) at room temperature. After the reaction mixture was stirred
for 30 min, a solution of R-18 (130 mg, 0.372 mmol) in MeOH
(2.10 mL) was added. The reaction mixture was warmed at reflux
for 16 h before being quenched with acetic acid after cooling to
room temperature. After concentration, column chromatography
(SiO2, 10% MeOH/CH2Cl2) gave R-19 (39 mg, 29%) as a white
1
g, 60%) as a yellow oil. H NMR (500 MHz, CDCl3) δ 8.04 (d,
1
2H, J ) 8.2 Hz), 7.43 (d, 2H, J ) 8.2 Hz), 4.78 (t, 1H, J ) 6.0
Hz), 4.26/4.25 (two q, 2H, J ) 7.1 Hz), 3.92 (s, 3H), 3.49/3.48
(two t, 1H, J ) 6.9 Hz), 2.00-1.97 (m, 2H), 1.85-1.65 (m, 4H),
1.31/1.30 (two t, 3H, J ) 7.1 Hz). 13C NMR (125 MHz, CDCl3)
δ 167.0, 166.1, 149.6, 130.0 (2C), 129.6, 125.8 (2C), 116.5, 73.6,
63.0, 52.2, 38.1, 37.6, 29.8, 23.2, 14.1. IR (film) νmax 3467, 2923,
1744, 1713, 1282, 1195, 1113 cm-1. ESI-TOF m/z 342.1309 (M
+ Na+, C17H21NO5 requires 342.1312).
solid: mp 180-185 °C. H NMR (500 MHz, CD3OD) δ 7.96 (d,
2H, J ) 8.4 Hz), 7.43 (d, 2H, J ) 8.4 Hz), 3.89 (s, 3H), 3.81 (t,
1H, J ) 6.7 Hz), 2.29 (t, 2H, J ) 7.2 Hz), 1.97-1.86 (m, 2H),
1.84 (s, 3H), 1.53-1.47 (m, 1H), 1.42-1.34 (m, 1H). 13C NMR
(125 MHz, CD3OD) δ 168.4, 165.2, 164.1, 154.8, 150.2, 130.6
(2C), 129.8, 129.2 (2C), 89.8, 52.6, 52.2, 36.3, 27.2, 23.1, 14.2. IR
(film) νmax 3353, 2933, 1713, 1610, 1431, 1282, 1108 cm-1. ESI-
TOF m/z 363.1489 (M + H+, C17H22N4O3S requires 363.1485);
[R]D +70 (c 0.1, MeOH).
Methyl 4-((1R)-1-(Acetylthio)-5-cyano-6-ethoxy-6-oxohexyl)-
benzoate (R-16). A cooled (0 °C) solution of triphenylphosphine
(358 mg, 1.36 mmol, 2.0 equiv) in THF (3.4 mL) was treated with
DIAD (270 µL, 1.36 mmol, 2.0 equiv). The reaction mixture was
stirred at 0 °C for 30 min before a solution of S-15 (218 mg, 0.68
mmol) and thiolacetic acid (121 uL, 1.36 mmol, 2.0 equiv) in THF
(1.7 mL) was added dropwise. The mixture was stirred for 1 h,
while allowing it to warm to room temperature. The reaction
mixture was diluted with EtOAc, washed with saturated aqueous
NaHCO3 (3×), dried over Na2SO4, and concentrated. Column
chromatography (SiO2, 30% EtOAc/hexanes) yielded R-16 (246
mg, 95%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ 8.00 (d,
2H, J ) 8.3 Hz), 7.36 (d, 2H, J ) 8.2 Hz), 4.60 (t, 1H, J ) 7.7
Hz), 4.25/4.24 (two q, 2H, J ) 7.1 Hz), 3.91 (s, 3H), 3.47-3.43
(m, 1H), 2.31 (s, 3H), 2.02-1.94 (m, 4H), 1.54-1.43 (m, 2H),
(R)-4-(4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-
(methylthio)butyl)benzoic Acid (R-20). A solution of R-19 (39
mg, 0.11 mmol) in MeOH (1.5 mL) was treated with LiOH
monohydrate (23 mg, 0.54 mmol, 5 equiv) in water (840 µL), and
the reaction mixture was stirred at room temperature for 24 h. The
reaction mixture was diluted with H2O, washed with EtOAc,
acidified to pH 4 by the addition of aqueous 1 N HCl, and
concentrated. The resulting white solid, R-20 (38 mg, 100%), was
used directly in the next step: mp 210 °C (dec). 1H NMR (500
MHz, CD3OD) δ 7.97 (d, 2H, J ) 8.3 Hz), 7.43 (d, 2H, J ) 8.4
Hz), 3.81 (t, 1H, J ) 6.8 Hz), 2.35-2.31 (m, 2H), 1.97-1.87 (m,
2H), 1.85 (s, 3H), 1.55-1.48 (m, 1H), 1.43-1.36 (m, 1H). 13C
NMR (125 MHz, CD3OD) δ 169.6, 164.3, 153.4, 152.6, 149.8,
130.8 (2C), 130.5, 129.1 (2C), 89.6, 52.0, 36.2, 26.9, 22.5, 14.3.

5446 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
DeMartino et al.
IR (film) νmax 3356, 1569, 1405, 1241 cm-1. ESI-TOF m/z 349.1336
(M + H+, C16H20N4O3S requires 349.1329); [R]D +30 (c 0.1,
MeOH).
) 7.3 Hz), 2.36 (t, 2H, J ) 7.4 Hz), 1.78-1.73 (m, 2H), 1.61-1.55
(m, 1H), 1.50-1.44 (m, 1H). 13C NMR (125 MHz, CD3OD) δ
169.8, 165.2, 163.5, 158.4, 152.2, 130.8 (2C), 130.5, 127.0 (2C),
89.9, 74.6, 39.4, 25.2, 22.8. IR (film) νmax 3374, 2923, 1636, 1390
cm-1. ESI-TOF m/z 319.1405 (M + H+, C15H18N4O4 requires
319.1401); [R]D -12 (c 0.1, MeOH).
Di-tert-butyl (S)-2-(4-((R)-4-(2,4-Diamino-6-oxo-1,6-dihydro-
pyrimidin-5-yl)-1-(methylthio)butyl)benzamido)pentanedioate
(10R-21). A solution of R-20 (38 mg, 0.11 mmol), di-tert-butyl
L-glutamate hydrochloride (48 mg, 0.16 mmol, 1.5 equiv) and
NaHCO3 (27 mg, 0.32 mmol, 3.0 equiv) in DMF (1.08 mL) was
treated with EDCI (62 mg, 0.32 mmol, 3.0 equiv). The reaction
mixture was stirred for 24 h at room temperature before the addition
of CHCl3. The resulting solution was washed with saturated aqueous
NaHCO3 (2×), dried over Na2SO4, and concentrated. Column
chromatography (SiO2, 10% MeOH/CH2Cl2) provided the protected
glutamate intermediate 10R-21 (27 mg, 43%) as a white solid: mp
Dimethyl
(S)-2-(4-((S)-4-(2,4-Diamino-6-oxo-1,6-dihydro-
pyrimidin-5-yl)-1-hydroxybutyl)benzamido)pentanedioate (10S-
24). A solution of S-23 (29 mg, 0.090 mmol), dimethyl L-glutamate
hydrochloride (29 mg, 0.14 mmol, 1.5 equiv), and NaHCO3 (23
mg, 0.27 mmol, 3.0 equiv) in DMF (0.90 mL) was treated with
EDCI (52 mg, 0.27 mmol, 3.0 equiv). The reaction mixture was
stirred for 24 h at room temperature before being concentrated.
Column chromatography (SiO2, 20% MeOH/CH2Cl2) provided 10S-
24 (31 mg, 72%) as a white solid: mp 105-110 °C. 1H NMR (600
MHz, CD3OD) δ 7.82 (d, 2H, J ) 8.4 Hz), 7.46 (d, 2H, J ) 7.8
Hz), 4.73 (t, 1H, J ) 6.0 Hz), 4.65-4.63 (m, 1H), 3.75 (s, 3H),
3.65 (s, 3H), 2.50 (t, 2H, J ) 7.2 Hz), 2.35-2.27 (m, 3H),
2.14-2.09 (m, 1H), 1.79-1.74 (m, 2H), 1.57-1.54 (m, 1H),
1.46-1.43 (m, 1H). 13C NMR (150 MHz, CD3OD) δ 174.9, 173.7,
170.3, 165.2, 164.3, 154.8, 151.2, 133.5, 128.5 (2C), 127.1 (2C),
90.0, 74.7, 60.4, 53.7, 52.8, 52.2, 39.7, 31.3, 27.4, 25.5, 23.3. IR
(film) νmax 3364, 1723, 1631, 1441 cm-1. ESI-TOF m/z 476.2142
(M + H+, C22H29N5O7 requires 476.2140); [R]D -18 (c 0.1,
MeOH).
1
108-112 °C. H NMR (500 MHz, CD3OD) δ 7.80 (d, 2H, J )
8.3 Hz), 7.43 (d, 2H, J ) 8.3 Hz), 4.52-4.49 (m, 1H), 3.81 (t, 1H,
J ) 6.8 Hz), 2.40 (t, 2H, J ) 7.4 Hz), 2.30 (t, 2H, J ) 7.6 Hz),
2.24-2.17 (m, 1H), 2.06-1.99 (m, 1H), 1.97-1.86 (m, 2H), 1.84
(s, 3H), 1.53-1.50 (m, 1H), 1.49 (s, 9H), 1.44 (s, 9H), 1.41-1.36
(m, 1H). 13C NMR (125 MHz, CD3OD) δ 173.9, 172.7, 170.2,
165.1, 164.2, 154.8, 148.6, 133.7, 129.2 (2C), 128.6 (2C), 89.8,
83.0, 81.9, 54.5, 52.1, 36.4, 32.9, 28.3 (6C), 27.6, 27.3, 23.1, 14.2.
IR (film) νmax 2913, 1733, 1631, 1149 cm-1. ESI-TOF m/z 590.2999
(M + H+, C29H43N5O6S requires 590.3007); [R]D +36 (c 0.1,
MeOH).
(S)-2-(4-((R)-4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-
yl)-1-(methylthio)butyl)benzamido)pentanedioic Acid (10R-3).
A solution of 10R-21 (21 mg, 0.035 mmol) in CHCl3 (1.26 mL)
was treated with trifluoroacetic acid (1 mL) at 0 °C. The reaction
mixture was allowed to warm to room temperature and stirred
overnight. The solution was concentrated and triturated with Et2O
to give 10R-3 (20 mg, 100%) as a white solid: mp 190 °C (dec).
1H NMR (500 MHz, CD3OD) δ 7.82 (d, 2H, J ) 8.4 Hz), 7.42 (d,
2H, J ) 8.3 Hz), 4.66-4.63 (m, 1H), 3.81 (t, 1H, J ) 6.6 Hz),
2.49 (t, 2H, J ) 7.5 Hz), 2.35-2.28 (m, 3H), 2.15-2.07 (m, 1H),
1.98-1.91 (m, 1H), 1.88-1.82 (m, 1H), 1.84 (s, 3H), 1.54-1.47
(m, 1H), 1.42-1.33 (m, 1H). 13C NMR (125 MHz, CD3OD) δ
176.6, 175.1, 170.2, 164.6, 154.8, 153.0, 148.4, 133.8, 129.2, 128.6,
89.6, 53.8, 52.0, 36.3, 31.5, 27.6, 26.9, 22.6, 14.3. IR (film) νmax
2964, 1653, 1544, 1210 cm-1. ESI-TOF m/z 478.1752 (M + H+,
C21H27N5O6S requires 478.1755); [R]D +32 (c 0.1, MeOH). Agilent
1100 LC/MS: reverse phase (2-40% acetonitrile/water/0.1% formic
acid, flow rate 0.75 mL/min); tR, 12.0 min; purity, 99.2%.
Methyl (S)-4-(4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-
5-yl)-1-hydroxybutyl)benzoate (S-22). A solution of NaOMe (270
mg, 5.01 mmol, 3.0 equiv) in anhydrous MeOH (13.8 mL) was
treated with guanidine hydrochloride (478 mg, 5.01 mmol, 3.0
equiv) at room temperature. After the reaction mixture was stirred
for 30 min, a solution of S-15 (533 mg, 1.67 mmol) in MeOH (9.4
mL) was added. The reaction mixture was warmed at reflux for
16 h and quenched with acetic acid after cooling to room
temperature. After concentration, column chromatography (SiO2,
10% MeOH/CH2Cl2) gave S-22 (336 mg, 61%) as a white solid:
(S)-2-(4-((S)-4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-
yl)-1-hydroxybutyl)benzamido)pentanedioic Acid (10S-7). A
solution of 10S-24 (26 mg, 0.055 mmol) in MeOH (0.55 mL) was
treated with aqueous 1 N NaOH (0.22 mL, 4 equiv). The reaction
mixture was stirred for 18 h and then acidified with the addition of
Dowex 50X8-200. The solution was filtered and concentrated to
1
give 10S-7 (21 mg, 87%) as a white solid: mp 200 °C (dec). H
NMR (500 MHz, CD3OD) δ 7.82 (d, 2H, J ) 8.3 Hz), 7.45 (d,
2H, J ) 8.3 Hz), 4.72 (t, 1H, J ) 6.1 Hz), 4.64-4.61 (m, 1H),
2.40-2.24 (m, 5H), 2.17-2.08 (m, 1H), 1.79-1.74 (m, 2H),
1.59-1.54 (m, 1H), 1.49-1.42 (m, 1H). 13C NMR (150 MHz,
CD3OD) δ 176.7, 175.1, 174.3, 170.3, 163.7, 163.6, 151.0, 133.8,
128.5 (2C), 127.1 (2C), 89.9, 74.6, 53.8, 39.4, 35.4, 31.0, 25.2,
23.0. IR (film) νmax 3333, 1600, 1451, 1405, 1318, 1108 cm-1
.
ESI-TOF m/z 448.1836 (M + H+, C20H25N5O7 requires 448.1827);
[R]D +4 (c 0.1, MeOH). Agilent 1100 LC/MS: reverse phase
(2-40% acetonitrile/water/0.1% formic acid, flow rate 0.75 mL/
min); tR, 9.9 min; purity, 99.5%.
Methyl 4-((1S)-5-Cyano-6-ethoxy-1-methoxy-6-oxohexyl)-
benzoate (S-25). A solution of S-15 (246 mg, 0.77 mmol), 49%
aqueous fluoroboric acid (97 µL, 1.54 mmol, 2 equiv) in CH2Cl2
(3.08 mL) was cooled to 0 °C. TMSCHN2 (2 M in hexanes, 0.385
mL, 0.77 mmol, 1 equiv) was added to the mixture, and stirring
was continued at 0 °C. Three further portions of TMSCHN2 (0.193
mL (0.5 equiv), 0.097 mL (0.25 equiv), and 0.097 mL (0.25 equiv))
were added dropwise at intervals of 20 min. The reaction mixture
was stirred a further 30 min, poured into H2O, and extracted with
EtOAc. The organic layer was washed with H2O, dried over
Na2SO4, filtered, and concentrated. Column chromatography (SiO2,
30% EtOAc/hexanes) gave S-25 (110 mg, 43%) as a colorless oil.
1H NMR (400 MHz, CDCl3) δ 8.04 (d, 2H, J ) 8.2 Hz), 7.36 (d,
2H, J ) 8.1 Hz), 4.28-4.22 (m, 2H), 4.18-4.15 (m, 1H), 3.92 (s,
3H), 3.49-3.45 (m, 1H), 3.22 (s, 1.5H), 3.21 (s, 1.5H), 1.98-1.93
(m, 2H), 1.82-1.78 (m, 1H), 1.71-1.63 (m, 2H), 1.54-1.48 (m,
1H), 1.32-1.28 (m, 3H). 13C NMR (125 MHz, CDCl3) δ 166.9,
166.0, 147.3, 129.9 (2C), 129.7, 126.5 (2C), 116.5, 83.1, 62.8, 57.0,
52.1, 37.6, 37.2, 29.7, 23.2, 14.0. IR (film) νmax 3405, 1708, 1272,
1103 cm-1. ESI-TOF m/z 356.1465 (M + Na+, C18H23NO5 requires
356.1468).
1
mp 120 °C (dec). H NMR (500 MHz, CD3OD) δ 7.97 (d, 2H, J
) 8.4 Hz), 7.46 (d, 2H, J ) 8.2 Hz), 4.73 (t, 1H, J ) 7.4 Hz), 3.89
(s, 3H), 2.35-2.32 (m, 2H), 1.77-1.74 (m, 2H), 1.61-1.52 (m,
1H), 1.50-1.42 (m, 1H). 13C NMR (125 MHz, CD3OD) δ 168.0,
164.7, 163.7, 152.1, 152.0, 130.0 (2C), 129.9, 126.6 (2C), 89.5,
74.1, 52.0, 39.1, 24.9, 22.7. IR (film) νmax 3333, 2912, 1718, 1615,
1436, 1287, 1102 cm-1. ESI-TOF m/z 333.1562 (M + H+,
C16H20N4O4 requires 333.1557); [R]D -8 (c 0.1, MeOH).
(S)-4-(4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-
hydroxybutyl)benzoic Acid (S-23). A solution of S-22 (36 mg,
0.11 mmol) in MeOH (1.5 mL) was treated with LiOH monohydrate
(23 mg, 0.54 mmol, 5 equiv) in water (840 µL), and the reaction
mixture was stirred at room temperature for 24 h. The reaction
mixture was diluted with H2O, washed with EtOAc, acidified to
pH 4 by the addition of aqueous 1 N HCl, and concentrated. The
resulting white solid, S-23 (35 mg, 100%), was used directly in
Methyl (S)-4-(4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-
5-yl)-1-methoxybutyl)benzoate (S-26). A solution of NaOMe (44
mg, 0.81 mmol, 3.0 equiv) in anhydrous MeOH (2.2 mL) was
treated with guanidine hydrochloride (77 mg, 0.81 mmol, 3.0 equiv)
at room temperature. After the reaction mixture was stirred for 30
min, a solution of S-25 (90 mg, 0.27 mmol) in MeOH (1.53 mL)
1
the next step: mp 220 °C (dec). H NMR (500 MHz, CD3OD) δ
7.98 (d, 2H, J ) 8.3 Hz), 7.45 (d, 2H, J ) 8.4 Hz), 4.73 (t, 1H, J

Inhibitors of Glycinamide Ribonucleotide Transformylase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5447
was added. The reaction mixture was warmed at reflux for 16 h
and quenched with acetic acid after cooling to room temperature.
After concentration, column chromatography (SiO2, 10% MeOH/
CH2Cl2) gave S-26 (27 mg, 29%) as a white solid: mp 160-164
°C. 1H NMR (600 MHz, CD3OD) δ 8.00 (d, 2H, J ) 8.4 Hz), 7.41
(d, 2H, J ) 7.8 Hz), 4.28-4.25 (m, 1H), 3.90 (s, 3H), 3.20 (s,
3H), 2.30 (t, 2H, J ) 7.5 Hz), 1.84-1.78 (m, 1H), 1.68-1.62 (m,
1H), 1.56-1.52 (m, 1H), 1.43-1.36 (m, 1H). 13C NMR (150 MHz,
CD3OD) δ 168.4, 165.1, 164.0, 154.8, 149.3, 130.7 (2C), 130.4,
127.9 (2C), 89.8, 84.4, 57.1, 52.6, 38.5, 25.4, 23.2. IR (film) νmax
3354, 2933, 1610, 1431, 1282 cm-1. ESI-TOF m/z 347.1729 (M
+ H+, C17H22N4O4 requires 347.1714); [R]D -30 (c 0.1, MeOH).
(S)-4-(4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-
yl)-1-methoxybutyl)benzoic Acid (S-27). The intermediate pyri-
midine S-26 (30 mg, 0.086 mmol) in MeOH (1.2 mL) was treated
with LiOH monohydrate (18 mg, 0.43 mmol, 5 equiv) in water
(675 µL), and the reaction mixture was stirred at room temperature
for 24 h. The reaction mixture was diluted with H2O, washed with
EtOAc, acidified to pH 4 by the addition of 1 N aqueous HCl, and
concentrated. The resulting white solid, S-27 (29 mg, 100%), was
used directly in the next step: mp 175 °C (dec). 1H NMR (600
MHz, CD3OD) δ 7.95 (d, 2H, J ) 8.2 Hz), 7.32 (d, 2H, J ) 8.2
Hz), 4.24 (t, 1H, J ) 5.8 Hz) 3.19 (s, 3H), 2.30 (t, 2H, J ) 7.5
Hz), 1.84-1.78 (m, 1H), 1.70-1.63 (m, 1H), 1.56-1.52 (m, 1H),
1.42-1.36 (m, 1H). 13C NMR (150 MHz, CD3OD) δ 173.2, 165.1,
164.0, 154.9, 147.3, 135.4, 130.6 (2C), 127.4 (2C), 90.0, 85.2, 57.0,
38.5, 25.5, 23.3. IR (film) νmax 3385, 2923, 1626, 1395, 1246, 1087
cm-1. ESI-TOF m/z 333.1563 (M + H+, C16H20N4O4 requires
333.1557); [R]D -20 (c 0.1, MeOH).
Di-tert-butyl (S)-2-(4-((S)-4-(2,4-Diamino-6-oxo-1,6-dihydro-
pyrimidin-5-yl)-1-methoxybutyl)benzamido)pentanedioate (10S-
28). A solution of S-27 (29 mg, 0.086 mmol), di-tert-butyl
L-glutamate hydrochloride (38 mg, 0.13 mmol, 1.5 equiv), and
NaHCO3 (22 mg, 0.26 mmol, 3.0 equiv) in DMF (0.86 mL) was
treated with EDCI (50 mg, 0.26 mmol, 3.0 equiv). The reaction
mixture was stirred for 24 h at room temperature before the addition
of CHCl3. The resulting solution was washed with saturated aqueous
NaHCO3 (2×), dried over Na2SO4, and concentrated. Column
chromatography (SiO2, 10% MeOH/CH2Cl2) provided 10S-28 (14
mg, 28%) as a white solid: mp 120-125 °C. 1H NMR (600 MHz,
CD3OD) δ 7.83 (d, 2H, J ) 7.8 Hz), 7.40 (d, 2H, J ) 7.8 Hz),
4.51-4.49 (m, 1H), 4.27 (t, 1H, J ) 7.4 Hz), 3.21 (s, 3H), 2.40 (t,
2H, J ) 7.2 Hz), 2.30 (t, 2H, J ) 7.2 Hz), 2.24-2.18 (m, 1H),
2.05-1.99 (m, 1H), 1.85-1.79 (m, 1H), 1.70-1.64 (m, 1H),
1.56-1.52 (m, 1H), 1.49 (s, 9H), 1.44 (s, 9H), 1.41-1.37 (m, 1H).
13C NMR (150 MHz, CD3OD) δ 173.9, 172.6, 170.3, 165.2, 164.3,
154.8, 148.1, 134.4, 128.6 (2C), 127.9 (2C), 90.0, 85.0, 83.0, 81.9,
57.0, 54.5, 38.5, 32.9, 28.3 (6C), 27.5, 25.4, 23.3. IR (film) νmax
3344, 2923, 1723, 1626, 1446, 1369, 1149 cm-1. ESI-TOF m/z
574.3234 (M + H+, C29H43N5O7 requires 574.3235); [R]D -48 (c
0.1, MeOH).
Methyl 4-(2-(4-Cyano-5-ethoxy-5-oxopentyl)-1,3-dithian-2-
yl)benzoate (29). A suspension of NaH (60% dispersion, 9.19 g,
230 mmol, 3.5 equiv) in anhydrous DMF (202 mL) was treated
with ethyl cyanoacetate (24.5 mL, 230 mmol, 3.5 equiv) at 0 °C.
The reaction mixture was stirred for 30 min while allowing the
mixture to warm to room temperature, forming the sodium salt as
a clear solution. A solution of 12 (21.7 g, 65.7 mmol) in DMF
(122 mL) was added at room temperature, and the reaction mixture
was stirred at 60 °C for 12 h. The reaction mixture was cooled to
room temperature and quenched with the addition of saturated
aqueous NH4Cl. The mixture was concentrated and then diluted
with EtOAc. The organic layer was washed with H2O and saturated
aqueous NaCl, dried over Na2SO4, and concentrated. Column
chromatography (SiO2, 15-50% EtOAc/hexanes gradient) followed
by vacuum distillation provided 29 (14.06 g, 53%) as a white solid:
1
mp 54-56 °C. H NMR (500 MHz, CDCl3) δ 8.06 (d, 2H, J )
8.6 Hz), 8.00 (d, 2H, J ) 8.6 Hz), 4.21 (q, 2H, J ) 7.1 Hz), 3.93
(s, 3H), 3.37 (t, 1H, J ) 7.6 Hz), 2.70-2.63 (m, 4H), 2.03 (t, 2H,
J ) 8.3 Hz), 1.97-1.93 (m, 2H), 1.84-1.81 (m, 2H), 1.57-1.42
(m, 2H), 1.26 (t, 3H, J ) 7.2 Hz). 13C NMR (125 MHz, CDCl3) δ
166.8, 165.9, 130.1 (2C), 129.2, 129.0 (2C), 116.3, 62.9, 58.4, 52.3,
44.2, 37.4, 29.8, 27.8 (2C), 25.0, 21.6, 14.1. IR (film) 2924, 2360,
1719, 1276, 1105, 1017 cm-1. ESI-FTMS m/z 408.1292 (M + H+,
C20H25NO4S2 requires 408.1298).
Methyl 4-(2-(3-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-
yl)propyl)-1,3-dithian-2-yl)benzoate (30). A solution of NaOMe
(3.26 g, 60.4 mmol, 3.5 equiv) in anhydrous MeOH (166 mL) was
treated with guanidine hydrochloride (5.77 g, 60.4 mmol, 3.5 equiv)
at room temperature. After the reaction mixture was stirred for 30
min, a solution of 29 (7.03 g, 17.25 mmol) in MeOH (97.5 mL)
was added. The reaction mixture was warmed at reflux for 16 h
and quenched with acetic acid after cooling to room temperature.
After concentration, column chromatography (SiO2, 10% MeOH/
CH2Cl2) gave 30 (6.04 g, 83%) as a white solid: mp dec 150 °C.
1H NMR (500 MHz, CD3OD) δ 8.00 (s, 4H), 3.91 (s, 3H),
2.74-2.59 (m, 4H), 2.19 (t, 2H, J ) 7.2 Hz), 2.10-2.05 (m, 2H),
1.94-1.88 (m, 2H), 1.42-1.36 (m, 2H). 13C NMR (125 MHz,
DMSO) δ 165.9, 162.3, 161.4, 152.9, 147.7, 129.3 (2C), 128.7 (2C),
128.1, 86.8, 58.1, 52.1, 43.2, 27.0 (2C), 24.6, 22.9, 21.9. IR (film)
νmax 3361, 1711, 1646, 1435, 1282, 1111 cm-1. ESI-FTMS m/z
421.1364 (M + H+, C19H24N4O3S2 requires 421.1363).
4-(2-(3-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-
propyl)-1,3-dithian-2-yl)benzoic Acid (31). A solution of 30 (2.01
g, 4.78 mmol) in MeOH (36 mL) was treated with LiOH
monohydrate (1.00 g, 23.9 mmol, 5 equiv) in water (12 mL), and
the reaction was stirred at room temperature for 24 h. The reaction
mixture was diluted with H2O, washed with Et2O, acidified to pH
4 by the addition of aqueous 1 N HCl, and concentrated. The
resulting white solid, 31 (1.48 g, 76%), was used directly in the
1
next step. H NMR (500 MHz, CD3OD) δ 8.04 (d, 4H, J ) 16.6
Hz), 2.72-2.62 (m, 4H), 2.22 (t, 2H, J ) 7.3 Hz), 2.07-2.04 (m,
2H), 1.95-1.89 (m, 2H), 1.41-1.38 (m, 2H). 13C NMR (125 MHz,
DMSO) δ 167.8, 167.1, 151.0, 151.4, 147.2, 129.7 (2C), 129.5,
128.6 (2C), 87.2, 58.0, 42.9, 27.2 (2C), 24.7, 22.6, 21.3.
(S)-2-(4-((S)-4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-
yl)-1-methoxybutyl)benzamido)pentanedioic Acid (10S-8). The
protected glutamate 10S-28 (8 mg, 0.015 mmol) in CHCl3 (0.52
mL) was treated with trifluoroacetic acid (0.5 mL) at 0 °C. The
reaction mixture was allowed to warm to room temperature and
stirred overnight. The solution was concentrated and triturated with
Et2O to give 10S-8 (6 mg, 91%) as a white solid: mp 130 °C (dec).
1H NMR (600 MHz, CD3OD) δ 7.84 (d, 2H, J ) 8.4 Hz), 7.40 (d,
2H, J ) 8.4 Hz), 4.65-4.63 (m, 1H), 4.26 (t, 1H, J ) 7.2 Hz),
3.20 (s, 3H), 2.50-2.47 (m, 2H), 2.33-2.30 (m, 3H), 2.14-2.08
(m, 1H), 1.85-1.79 (m, 1H), 1.70-1.64 (m, 1H), 1.58-1.51 (m,
1H), 1.44-1.37 (m, 1H). 13C NMR (150 MHz, CD3OD) δ 176.7,
175.1, 170.2, 165.0, 159.3, 154.0, 147.9, 134.4, 128.6 (2C), 127.9
(2C), 90.0, 85.0, 57.0, 53.8, 38.3, 31.6, 27.6, 25.1, 23.0. IR (film)
Di-tert-butyl (S)-2-(4-(2-(3-(2,4-Diamino-6-oxo-1,6-dihydropyrim-
idin-5-yl)propyl)-1,3-dithian-2-yl)benzamido)pentanedioate (32).
Compound 31 (1.48 g, 3.65 mmol), di-tert-butyl L-glutamate
hydrochloride (1.62 g, 5.48 mmol, 1.5 equiv), and NaHCO3 (675
mg, 8.03 mmol, 2.2 equiv) in DMF (32 mL) were treated with
EDCI (1.40 g, 7.30 mmol, 2.0 equiv). The reaction mixture was
stirred for 24 h at room temperature before the addition of CHCl3.
The resulting solution was washed with saturated aqueous NaHCO3
(2×), dried over Na2SO4, and concentrated. Column chromatog-
raphy (SiO2, 10% MeOH/CH2Cl2) provided 32 (1.76 g, 75%) as a
white solid: mp 120-124 °C. 1H NMR (500 MHz, CD3OD) δ 8.00
(d, 2H, J ) 8.5 Hz), 7.84 (d, 2H, J ) 8.5 Hz), 4.53-4.50 (m, 1H),
2.72-2.62 (m, 4H), 2.41 (t, 2H, J ) 6.7 Hz), 2.37-2.30 (m, 2H),
2.19 (t, 2H, J ) 7.5 Hz), 2.10-2.06 (m, 2H), 1.97-1.91 (m, 2H),
1.49 (s, 9H), 1.45 (s, 9H), 1.42-1.36 (m, 2H). 13C NMR (125 MHz,
DMSO) δ 171.5, 171.0, 166.4, 161.4, 157.1, 153.0, 145.5, 132.5,
128.2 (2C), 127.6 (2C), 86.8, 80.6, 79.7, 58.2, 52.5, 43.3, 31.4,
ν
max 3354, 2933, 1636, 1426, 1205 cm-1. ESI-TOF m/z 462.1997
(M + H+, C21H27N5O7 requires 462.1983); [R]D -22 (c 0.1,
MeOH). Agilent 1100 LC/MS: reverse phase (2-40% acetonitrile/
water/0.1% formic acid, flow rate 0.75 mL/min); tR, 10.9 min;
purity, 97.1%.

5448 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
DeMartino et al.
synthesis. Mol. Pharmacol. 1997, 52, 903–911.
30.9, 27.7 (3C), 27.6 (3C), 27.0, 25.9, 23.0, 22.0. IR (film) νmax
3349, 2961, 1728, 1617, 1440, 1364, 1183 cm-1. ESI-FTMS m/z
648.2890 (M + H+, C31H45N5O6S2 requires 648.2884).
(7) Jackson, R. C.; Harkrader, R. J. The contributions of de novo and
salvage pathways of nucleotide biosynthesis in normal and malignant
cells. In In: Nucleosides and Cancer Treatment. (Eds.).; Tattersall,
M. H. N., Fox, R. M., Eds.; Academic Press: Sydney, 1981, pp 18-
31.
(8) Marsilje, T. H.; Labroli, M. A.; Hedrick, M. P.; Jin, Q.; Desharnais,
J.; Baker, S. J.; Gooljarsingh, L. T.; Ramcharan, J.; Tavassoli, A.;
Zheng, Y.; Wilson, I. A.; Beardsley, G. P.; Benkovic, S. J.; Boger,
D. L. 10-Formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-
formyl-DDACTHF): A potent cytotoxic agent acting by selective
inhibition of human GAR Tfase and the de novo purine biosynthetic
pathway. Bioorg. Med. Chem. 2002, 10, 2739–2749.
(9) Zhang, Y.; Desharnais, J.; Marsilje, T. H.; Li, C.; Hedrick, M. P.;
Gooljarsingh, L. T.; Tavassoli, A.; Benkovic, S. J.; Olson, A. J.; Boger,
D. L.; Wilson, I. A. Rational design, synthesis, evaluation, and crystal
structure of a potent inhibitor of human GAR Tfase: 10-trifluoroacetyl-
5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid. Biochemistry 2003,
42, 6043–6056.
(10) Greasley, S. E.; Yamashita, M. M.; Cai, H.; Benkovic, S. J.; Boger,
D. L.; Wilson, I. A. New insights into inhibitor design from the crystal
structure and NMR studies of Escherichia coli GAR transformylase
in complex with ꢀ-GAR and 10-formyl-5,8,10-trideazafolic acid.
Biochemistry 1999, 38, 16783–16793.
(S)-2-(4-(2-(3-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-
yl)propyl)-1,3-dithian-2-yl)benzamido)pentanedioic Acid (9). A
solution of 32 (54 mg, 0.083 mmol) in CHCl3 (833 µL) was treated
with trifluoroacetic acid (1 mL) at 0 °C. The reaction mixture was
allowed to warm to room temperature and stirred overnight. The
solution was concentrated and triturated with Et2O to give 9 (41
1
mg, 91%) as a white solid: mp dec 125-130 °C. H NMR (500
MHz, CD3OD) δ 7.98 (d, 2H, J ) 8.6 Hz), 7.84 (d, 2H, J ) 8.5
Hz), 4.68-4.65 (m, 1H), 2.72-2.62 (m, 4H), 2.51-2.48 (m, 2H),
2.43-2.39 (m, 2H), 2.19 (t, 2H, J ) 7.1 Hz), 2.15-2.11 (m, 2H),
1.94-1.98 (m, 2H), 1.40-1.37 (m, 2H). 13C NMR (125 MHz,
CD3OD) δ 176.9, 175.8, 170.0, 164.9, 154.0 (2C), 147.7, 133.7,
130.2 (2C), 128.5 (2C), 87.9, 59.5, 53.9, 44.8, 31.1, 28.6 (2C), 27.6,
26.9, 23.2, 22.7. IR (film) νmax 3380, 2961, 1718, 1634 cm-1. ESI-
FTMS m/z 536.1625 (M + H+, requires C23H29N5O6S2 536.1632).
Agilent 1100 LC/MS: reverse phase (2-40% acetonitrile/water/
0.1% formic acid, flow rate 0.75 mL/min); tR, 13.7 min; purity,
99.5%.
(11) Cheng, H.; Chong, Y.; Hwang, I.; Tavassoli, A.; Zhang, Y.; Wilson,
I. A.; Benkovic, S. J.; Boger, D. L. Design, synthesis, and biological
evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-
5-DACTHF, and 10-methylthio-DDACTHF as potent inhibitors of
GAR Tfase and the de novo purine biosynthetic pathway. Bioorg. Med.
Chem. 2005, 13, 3593–3599.
(12) Mendelsohn, L. G.; Shih, C.; Schultz, R. M.; Worzalla, J. F.
Biochemistry and pharmacology of glycinamide ribonucleotide formyl-
transferase inhibitors: LY309887 and lometrexol. InVest. New Drugs
1996, 14, 287–294.
(13) Boritzki, T. J.; Bartlett, C. A.; Zhang, C.; Howland, E. F.; Margosiak,
S. A.; Palmer, C. L.; Romines, W. H.; Jackson, R. C. AG2034: a
novel inhibitor of glycinamide ribonucleotide formyltransferase. InVest.
New Drugs 1996, 14, 295–303.
GAR and AICAR Tfase Assay. Enzyme assays for rhGAR and
rhAICAR were performed as described previously.8,9 Kinetics of
the enzyme reactions were monitored for 2 min after initiation of
the reaction. Ki’s of the inhibitors were calculated using Dixon plots.
Cytotoxic Activity. The cytotoxic activity of the compounds was
measured using the CCRF-CEM human leukemia cell lines as
described previously.8,9,19
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health and the Skaggs
Institute for Chemical Biology.
Supporting Information Available: Full details and character-
ization for the alternative C10-diastereomers are provided. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(14) Kisliuk, R. L. Deaza analogs of folic acid as antitumor agents. Curr.
Pharm. Des. 2003, 9, 2615–2625.
(15) Corey, E. J.; Bakshi, R. K.; Shibata, S.; Chen, C. -P.; Singh, V. K. A
stable and easily prepared catalyst for the enantioselective reduction
of ketones: applications to multistep syntheses. J. Am. Chem. Soc.
1987, 109, 7925–7926.
(16) Corey, E. J.; Helal, C. J. Reduction of carbonyl compounds with chiral
oxazaborolidine catalysts: a new paradigm for enantioselective catalysis
and a powerful new synthetic method. Angew. Chem., Int. Ed. 1998,
37, 1986–2012.
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JM800555H