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

Article abstract of DOI:10.1016/j.bmc.2004.12.004

The synthesis and evaluation of 10-methanesulfonyl-DDACTHF (1), 10-methanesulfonyl-5-DACTHF (2), and 10-methylthio-DDACTHF (3) as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide t

Full text of DOI:10.1016/j.bmc.2004.12.004

Bioorganic & Medicinal Chemistry 13 (2005) 3577–3585
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
Heng Cheng,a,b Youhoon Chong,a,b Inkyu Hwang,a,b Ali Tavassoli,c Yan Zhang,b,d
Ian A. Wilson,b,d Stephen J. Benkovicc and Dale L. Bogera,b,
*
aDepartment of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
bThe Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
cDepartment of Chemistry, Pennsylvania State University, University Park, PA 16802, USA
dDepartment of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 30 September 2004; revised 2 December 2004; accepted 2 December 2004
Available online 31 March 2005
Abstract—The synthesis and evaluation of 10-methanesulfonyl-DDACTHF (1), 10-methanesulfonyl-5-DACTHF (2), and 10-meth-
ylthio-DDACTHF (3) as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carb-
oxamide ribonucleotide transformylase (AICAR Tfase) are reported. The compounds 10-methanesulfonyl-DDACTHF (1,
Ki = 0.23 lM), 10-methanesulfonyl-5-DACTHF (2, Ki = 0.58 lM), and 10-methylthio-DDACTHF (3, Ki = 0.25 lM) were found
to be selective and potent inhibitors of recombinant human GAR Tfase. Of these, 3 exhibited exceptionally potent, purine sensitive
growth inhibition activity (3, IC50 = 100 nM) against the CCRF–CEM cell line being 3-fold more potent than Lometrexol and 30-
fold more potent than the parent, unsubstituted DDACTHF, whereas 1 and 2 exhibited more modest growth inhibition activity (1,
IC50 = 1.0 lM and 2, IC50 = 2.0 lM).
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Fig. 1).1 Since purines are critical components of DNA
and RNA, inhibition of enzymes in the purine biosyn-
thetic pathway had been envisioned as an effective ap-
proach for antineoplastic intervention. The disclosure
that (6R)-5,10-dideazatetrahydrofolate (Lometrexol,
(6R)-DDATHF, Fig. 2) is an efficacious antitumor
agent that acts as an effective inhibitor of GAR Tfase
(Ki = 0.1 lM) established inhibition of purine biosynthe-
sis and GAR Tfase as viable targets for antineoplastic
intervention.2–4 Herein, we report the synthesis and
evaluation of 10-methanesulfonyl-DDACTHF (1), 10-
methanesulfonyl-5-DACTHF (2), and 10-methylthio-
DDACTHF (3) as novel folate-based inhibitors of
GAR Tfase, Figure 2.
Glycinamide ribonucleotide transformylase (GAR
Tfase) and aminoimidazole carboxamide ribonucleotide
transformylase (AICAR Tfase) are folate-dependent
enzymes central to the de novo purine biosynthetic path-
way. GAR Tfase utilizes the cofactor (6R)-N10-formyl-
tetrahydrofolate to transfer a formyl group to the
primary amine of its substrate, glycinamide ribonucleo-
tide (GAR, Fig. 1). This one carbon transfer incorpo-
rates the C-8 carbon of the purines and is the first of
two formyl transfer reactions. The second formyl trans-
fer reaction is catalyzed by the enzyme AICAR Tfase
which also employs (6R)-N10-formyltetrahydrofolate to
transfer a formyl group to the C-5 amine of its substrate,
aminoimidazole carboxamide ribonucleotide (AICAR,
2. Inhibitor design
*
In previous studies, we described folate-based inhibitors
of GAR Tfase that incorporated electrophilic functional
Corresponding author. Tel.: +1 858 784 7522; fax: +1 858 784
7550; e-mail: boger@scripps.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.004

3578
H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3577–3585
GAR Tfase inhibitors. X-ray and NMR studies of the
O
O
inhibitor–enzyme complexes revealed that both inhibi-
tors bound GAR Tfase as their gem-diols.10,11 The for-
mation of the gem-diol mimics the formyl transfer
reaction tetrahedral intermediate and provides strong
stabilizing hydrogen bond interactions between the
inhibitor and active site catalytic residues of the protein.
−2O3PO
−2O3PO
NHCHO
NH2
HN
HN
O
GAR Tfase
O
HO OH
NH2
HO OH
GAR
H
N
N
O
O
NH
N
H
O
Both 10-formyl-DDACTHF (4) and 10-CF3CO-
DDACTHF (5) were shown to be selective and potent
GAR Tfase inhibitors (4, Ki = 0.014 lM against rhGAR
Tfase; 5, Ki = 0.015 lM against rhGAR Tfase).8–11 They
were both found to be effectively transported into the
cell by the reduced folate carrier and polyglutamated
by FPGS, which contributes to their cytotoxic activity
by enhancing intracellular accumulation (4, CCRF–
CEM IC50 = 60 nM; 5, CCRF–CEM IC50 = 16 nM).8–11
10-CF3CO-DDACTHF (5) proved to be 10-fold more
potent against tumor cell proliferation than Lometrexol
and suitable for in vivo evaluation.10
H
−2O3PO
N
N
HN
H
N
O
H
OH
COOH
HO OH
N
COOH
N
CONH2
NH2
CONH2
NHCHO
−2O3PO
−2O3PO
N
O
N
O
AICAR Tfase
HO OH
HO OH
AICAR
Beyond the intrinsic importance of these two inhibitors
themselves, their discovery led to the recognition that
such a tetrahedral intermediate mimic provides a unique
design feature that conveys selectivity for GAR Tfase
inhibition over all other folate-dependent enzymes that
do not enlist a formyl transfer reaction. Herein we re-
port the extension of these observations to the prepara-
tion and examination of the tetrahedral transition state
inhibitors 10-methanesulfonyl-DDACTHF (1) and 10-
methanesulfonyl-5-DACTHF (2), which were antici-
pated to be a nice complement to the corresponding
aldehyde and ketones (Fig. 2). Since one potential limi-
tation of the carbonyl-based inhibitors is their in vivo
reduction, the sulfone 1 and sulfonamide 2 may well
prove to be more stable and efficacious in vivo, albeit
not as intrinsically potent against GAR Tfase in vitro.
We also describe the synthesis and evaluation of 10-
methylthio-DDACTHF (3), a comparison analogue of
1 where the methanesulfonyl group was replaced by a
methylthio group.
Figure 1.
H
H2N
N
O
O
NH2
NH
N
N
NH2
NH
O
Me
S
H
N
H
N
O
O
COOH
COOH
O
10-CH SO -DDACTHF (1)
COOH
DDATHF
H2N
COOH
NH2
3
2
H2N
N
O
O
NH2
N
NH
NH
Me
Me
S
N
O
O
S
H
N
H
N
O
COOH
COOH
COOH
O
10-CH SO -5-DACTHF (2)
10-CH3S-DDACTHF (3) COOH
3
2
3. Chemistry
H2N
N
O
O
NH2
NH
H2N
N
O
O
NH2
NH
The synthesis of 10-methanesulfonyl-DDACTHF (1)
was accomplished through the alkylation of methyl
4-(methanesulfonylmethyl)benzoate12 with 1,3-dibromo-
propane, as presented in Scheme 1. This was
accomplished upon NaH deprotonation of methyl 4-
(methanesulfonylmethyl)benzoate (DMF, 0 ꢁC, 25 min)
and subsequent treatment with excess 1,3-dibromopro-
pane (10 equiv, DMF, 25 ꢁC, 2.5 h, 35%) to give 7.
The preformed sodium salt of ethyl cyanoacetate
(NaH, DMF, 0 ꢁC, 30 min) was alkylated with 7
(DMF, 25 ꢁC, 2 h, 70%) to give 8, and its subsequent
treatment with the free base of guanidine (1.2 equiv,
CH3OH–DMF, 75 ꢁC, overnight, 58%) under basic con-
ditions gave the desired pyrimidinone 9. Treatment of 9
with LiOH (3 equiv, CH3OH–H2O 3:1, 25 ꢁC, over-
night, 100%) cleanly provided the carboxylic acid 10,
which was coupled with di-tert-butyl-L-glutamate
hydrochloride (1.5 equiv, EDCI, NaHCO3, DMF,
F3C
H
H
N
H
N
COOH
COOH
COOH
COOH
O
O
10-formyl-DDACTHF (4)
Figure 2.
10-CF CO-DDACTHF (5)
3
groups that could potentially interact either with
active site nucleophiles or the GAR/AICAR substrate
amines.5–8 The most significant of these were 10-
formyl-DDACTHF (4)8 and 10-CF3CO-DDACTHF
(5)9,10 (Fig. 2), bearing a nontransferable formyl or tri-
fluoroacetyl group, which both proved to be potent

H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3577–3585
3579
1,3-dibromopropane
H
N
Me
1,3-dibromopropane
NaH
Me
S
S
S
NaH
S
S
S
O
O
O
O
CO2Me
CO2Me
anhyd DMF
0−25 °C, 35%
anhyd DMF
0−25 °C, 76%
CO2Me
CO2Me
6
Br
COOEt
_
Br
Na+
COOEt
CN
_
Na+
Me
CN
Me
N
O
O
O
anhyd DMF, 25 °C
70%
O
anhyd DMF, 25 °C
73%
7
12
CN
CN
Guanidine
NaOCH3, CH3OH−DMF
COOEt
Guanidine
NaOCH3, CH3OH−DMF
COOEt
Me
Me
N
O
75 °C, 58%
O
O
O
75 °C, 58%
CO2Me
8
CO2Me
NH2
NH
13
H2N
H2N
N
O
NH2
NH
N
LiOH, CH3OH/H2O
100%
LiOH, CH3OH/H2O
100%
Me
S
S
Me
N
O
S
S
O
O
COOtBu
O
O
CO2Me
H2N
COOtBu
9
H2N
CO2Me
14
H2N
H2N
N
NH2
NH
N
NH2
NH
COOtBu
COOtBu
Me
O
EDCI, anhyd DMF
25 °C, 45%
EDCI, anhyd DMF
25 °C, 45%
Me
O
N
O
O
O
O
H2N
N
O
O
NH2
CO2H
CO2H
H2N
N
O
O
NH2
NH
10
15
NH
Me
S
Me
S
N
O
H
N
O
O
H
N
COOR
COOR
O
COOR
COOR
11, R = tBu
1, R = H
TFA
16, R = tBu
2, R = H
100%
TFA
100%
Scheme 1.
Scheme 2.
25 ꢁC, overnight, 45%) to provide 11 as a 1:1 mixture of
the inseparable diastereomers. Deprotection of 11 was
accomplished by treatment with trifluoroacetic acid
(TFA/CHCl3, 25 ꢁC, overnight, 100%) to provide 10-
methanesulfonyl-DDACTHF (1).
accomplished by treatment with trifluoroacetic acid
(TFA/CHCl3, 25 ꢁC, overnight, 100%) to provide 10-
methanesulfonyl-5-DACTHF (2).
The synthesis of 10-methylthio-DDACTHF (3) is pre-
sented in Scheme 3. This was accomplished by sodium
bis(trimethylsilyl)amide deprotonation of methyl 4-
(methylthiomethyl)benzoate12 (THF, À78 ꢁC) and sub-
sequent treatment with excess 1-chloro-3-iodopropane
(10 equiv, THF, 25 ꢁC, 45 min, 31%) to give 17. The
use of NaH for deprotonation failed to provide the de-
sired product 17 and the use of excess 1,3-dibromopro-
pane or 1,3-diiodopropane to alkylate the sodium
bis(trimethylsilyl)amide-derived anion provided compet-
itive elimination products. The preformed sodium salt of
ethyl cyanoacetate (NaH, DMF, 0 ꢁC, 30 min) was
alkylated with 17 (DMF, 50 ꢁC, 9 h, 56%) to give 18,
which was treated with the free base of guanidine
(1.2 equiv, CH3OH–DMF, 75 ꢁC, overnight, 57%)
under basic conditions to afford the desired pyrimidi-
none 19. Treatment of 19 with LiOH (3 equiv,
CH3OH–H2O 3:1, 25 ꢁC, overnight, 100%) cleanly pro-
vided the carboxylic acid 20, which was coupled with
di-tert-butyl-L-glutamate hydrochloride (1.5 equiv,
EDCI, NaHCO3, DMF, 25 ꢁC, overnight, 44%) to
The synthesis of 10-methanesulfonyl-5-DACTHF (2)
was accomplished through the alkylation of methyl
4-(methanesulfonylamino)benzoate13 with 1,3-dibromo-
propane, as presented in Scheme 2.4c This was
accomplished upon NaH deprotonation of methyl 4-
(methanesulfonylamino)benzoate (DMF, 0 ꢁC, 25 min)
and subsequent treatment with excess 1,3-dibromopro-
pane (10 equiv, DMF, 25 ꢁC, 2.5 h, 76%) to give 12.
The preformed sodium salt of ethyl cyanoacetate
(NaH, DMF, 0 ꢁC, 30 min) was alkylated with 12
(DMF, 25 ꢁC, 2 h, 73%) to give 13, and its treatment
with the free base of guanidine (1.2 equiv, CH3OH–
DMF, 75 ꢁC, overnight, 58%) under basic conditions
gave the desired pyrimidinone 14. Treatment of 14 with
LiOH (3 equiv, CH3OH–H2O 3:1, 25 ꢁC, overnight,
100%) cleanly provided the carboxylic acid 15, which
was coupled with di-tert-butyl-L-glutamate hydrochlo-
ride (1.5 equiv, EDCI, NaHCO3, DMF, 25 ꢁC, over-
night, 45%) to provide 16. Deprotection of 16 was

3580
H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3577–3585
Table 1. GAR and AICAR Tfase inhibition (Ki, lM)
1-chloro-3-iodopropane
NaN(SiMe3)2
Me
Me
S
S
Compound
E. coli GAR
Tfasea
rhGAR
Tfaseb
rhAICAR
Tfasec
anhyd THF
−78 to 25 °C, 31%
CO2Me
CO2Me
1
>100
>100
>100
6d
0.23
0.58
>100
>100
>100
1d
65e
NDf
1e
Br
COOEt
_
2
Na+
3
0.25
CN
4
0.014d
0.015e
1.7
1.9e
5
anhyd DMF, 50 °C
56%
5
DDACTHF
Lometrexol
17
18
0.1e
0.06g
CN
a E. coli GAR Tfase.
Guanidine
NaOCH3, CH3OH−DMF
COOEt
b Recombinant human GAR Tfase.
Me
Me
S
S
c Recombinant human AICAR Tfase.
75 °C, 57%
d Ref. 8.
e Ref. 9.
CO2Me
H2N
N
NH2
NH
f Not determined.
g Ref. 14.
LiOH, CH3OH/H2O
100%
O
fer tetrahedral intermediate, but that it does so less
effectively than the corresponding trifluoromethyl
ketone (bound as gem-diol). Substitution of the C-10
carbon of compound 1 by a nitrogen results in a 2-fold
decrease in its potency against rhGAR Tfase (2,
Ki = 0.58 lM). Compound 3 bearing a C-10 methylthio
group was a surprisingly potent inhibitor of rhGAR
Tfase (3, Ki = 0.25 lM) being essentially equipotent with
1 bearing the 10-methanesulfonyl group. While inhibitor
3 does not contain an apparent mimic of the formyl
transfer reaction intermediate, it does incorporate a po-
tential hydrogen bond acceptor and presents a soft
hydrophobic substituent for active site binding, both
of which presumably contribute to its 7-fold increase
in Ki relative to the unsubstituted, parent DDACTHF.
Compounds 1, 2, and 3 were inactive against rhAICAR
Tfase (Ki > 100 lM).
CO2Me
H2N
COOtBu
19
H2N
N
NH2
NH
COOtBu
Me
O
EDCI, anhyd DMF
25 °C, 44%
S
CO2H
H2N
N
O
O
NH2
20
NH
Me
S
H
N
COOR
COOR
21, R = tBu
3, R = H
TFA
100%
Scheme 3.
provide 21 as a 1:1 mixture of the inseparable diastereo-
mers. Deprotection of 21 was accomplished by treatment
with trifluoroacetic acid (TFA/CHCl3, 25 ꢁC, overnight,
100%) to provide 10-methylthio-DDACTHF (3).
5. Cytotoxic activity
Compounds 1, 2, and 3 were examined for cytotoxic
activity both in the presence (+) and absence (À) of
added hypoxanthine (purine) and thymidine (pyrimi-
dine) against the CCRF–CEM cell line (Table 2). Each
exhibited potent cytotoxic activity against the CCRF–
CEM cell line when purines (hypoxanthine) were absent
4. GAR Tfase and AICAR Tfase inhibition
Compounds 1, 2, and 3 were tested for inhibition of
E. coli GAR Tfase, recombinant human GAR Tfase,
and recombinant human AICAR Tfase, and the results
are presented in Table 1. All of the compounds exhibited
selective and potent inhibition of recombinant human
GAR Tfase, while all three failed to inhibit E. coli
GAR Tfase (Ki > 100 lM). The analogous selective inhi-
bition of human versus E. coli GAR Tfase was similarly
observed previously with 4 and 5, but contrasts the near
equipotent and less active inhibition of the two enzymes
by the parent unsubstituted DDACTHF. Compound 1
(Ki = 0.23 lM) was 7-fold more potent than the parent
unsubstituted DDACTHF, 4-fold less potent than the
reported activity of Lometrexol, and only 15-fold less
potent than 5, the most potent rhGAR Tfase inhibitor
disclosed to date.10 From this one can infer that C-10
sulfone of 1 increases enzyme active site binding consis-
tent with its predicted ability to mimic the formyl trans-
in the cell culture media and were inactive (IC50 >
100 lM) in the presence of media purines, but this activ-
ity was insensitive to the presence of thymidine. This
sensitivity to the presence of purines, but not pyrimi-
dines (thymidine), indicate that the cytotoxic activity
of 1–3 is derived from their inhibition of an enzyme in
the de novo purine biosynthetic pathway consistent with
inhibition of GAR Tfase. Sulfone 1 and sulfonamide 2
were found to be two to three times more potent than
the parent unsubstituted DDACTHF and 3- or 7-fold,
respectively, less potent than Lometrexol. Both exhib-
ited IC50 values roughly 4-fold higher than their enzy-
matic (GAR Tfase) KiÕs. In contrast to design
expectations, inhibitor 3 proved to be exceptionally
potent exhibiting a CCRF–CEM IC50 of 100 nM. This
proved to be 2- to 3-fold more potent than Lometrexol,
30- to 40-fold more potent than DDACTHF, only 6-
fold less potent than the most potent and selective

H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3577–3585
Table 2. In vitro cyctotoxic activity (IC50, lM)
3581
Tfase inhibitor (Ki = 0.25 lM) and exhibited exception-
ally potent, purine sensitive cytotoxic activity (CCRF–
CEM IC50 = 100 nM). This functional cellular activity
of 3 exceeded its in vitro enzymatic activity and was
shown to benefit from both FPGS polyglutamation
and reduced folate carrier transport into the cell. As
such, its properties indicate it merits in vivo examination
alongside related recently disclosed inhibitors.
Compound
CCRF–CEM
(À)T, (À)Ha
1.0
2.0
(+)T, (À)H
0.8
2.0
(À)T, (+)H
>100
1
2
>100
>100
170b
>100c
>100
>100
3
0.1
0.08
0.06b
0.017c
4.0
4
0.07b
0.016c
3.0
5
DDACTHF
Lometrexol
0.3
2.0
7. Experimental
Compounda
CCRF–CEM
CCRF–CEM/
MTX
CCRF–CEM/
FPGSÀ
7.1. Methyl 4-(4-bromo-1-methanesulfonylbutyl)-
benzoate (7)
1
1.0
2.0
20
20
>100
>100
50
>100b
>100c
2
3
0.1
2.5
A suspension of NaH (60% dispersion, 0.225 g,
4
0.07b
0.016c
3.0
>200b
>100c
ND (>100)c
ND (>100)c
5.62 mmol, 1.2 equiv) in freshly distilled DMF (3 mL)
was treated with a solution of methyl 4-(methanesulfo-
nylmethyl)benzoate (1.07 g, 4.69 mmol) in freshly dis-
tilled DMF (20 mL) at 0 ꢁC. After the solution was
stirred at 0 ꢁC for 25 min, 1,3-dibromopropane
(4.78 mL, 46.9 mmol, 10 equiv) was added to the reac-
tion mixture. The cooling bath was removed and the
reaction mixture was stirred at 25 ꢁC for 2.5 h before
being quenched by the addition of saturated aqueous
NH4Cl (50 mL). The resulting aqueous solution was ex-
tracted with EtOAc (5 · 20 mL). The combined organic
layers were washed with saturated aqueous NaCl
(50 mL), dried (Na2SO4), filtered, and concentrated.
Column chromatography (SiO2, 25–50% EtOAc–
hexanes) afforded 7 (0.57 g, 35%) as a colorless oil:
1H NMR (CDCl3, 400 MHz): d 8.04 (d, J = 8.2 Hz,
2H), 7.52 (d, J = 8.2 Hz, 2H), 4.10 (dd, J = 4.1, 11.1 Hz,
1H), 3.89 (s, 3H), 3.38–3.28 (m, 2H), 2.63 (s, 3H), 2.61–
2.51 (m, 1H), 2.31–2.21 (m, 1H), 1.89–1.81 (m, 1H),
1.78–1.65 (m, 1H); 13C NMR (CDCl3, 100 MHz): d
166.3, 137.5, 131.1, 130.4, 129.4, 68.7, 52.4, 38.8, 32.3,
29.6, 26.1; IR (neat) mmax 2954, 1718, 1605, 1436, 1282,
1128, 1113 cmÀ1; MALDI–FTMS (DHB) m/z 370.9929
(M+Na+, C13H17BrO4S requires 370.9923).
5
DDACTHF
Lometrexol
ND (>100)c
30 (>100)c
0.3
a (À)T, thymidine; (À)H, hypoxanthine.
b Ref. 8.
c Ref. 9.
GAR Tfase inhibitor disclosed to date (10-CF3CO-
DDACTHF, IC50 = 16 nM), and 2- to 3-fold more po-
tent than its in vitro GAR Tfase Ki. Like Lometrexol
and inhibitors 48 and 59, assay of 1–3 against CCRF–
CEM cell lines deficient in the reduced folate carrier
(CCRF–CEM/MTX)15 or FPGS (CCRF–CEM/
FPGSÀ)16 revealed that each lacked or lost activity indi-
cating that each benefits from the reduced folate carrier
transport, each is a substrate for FPGS, and that poly-
glutamation by FPGS contributes to their cellular activ-
ity. This may not only increase the intracellular
accumulation of the inhibitors, but the corresponding
polyglutamates may additionally exhibit an increased
GAR Tfase binding affinity, both of which may account
for the cellular potency of 3 which exceeds that of
its inherent enzyme inhibitory potency. Finally,
compounds 9–11, 14–16, and 19–21 were inactive
(IC50 > 100 lM) in the CCRF–CEM assay in the pres-
ence or absence of media purines or pyrimidines.
7.2. Methyl 4-(5-cyano-5-ethoxycarbonyl-1-methane-
sulfonylpentyl)benzoate (8)
A suspension of NaH (60% dispersion, 78.7 mg,
1.97 mmol, 1.2 equiv) in freshly distilled DMF
(1.5 mL) was treated with ethyl cyanoacetate
(0.227 mL, 2.13 mmol, 1.3 equiv) at 0 ꢁC. The reaction
mixture was stirred at 0 ꢁC for 30 min, forming the so-
dium salt as a clear solution. A solution of 7 (0.57 g,
1.64 mmol) in freshly distilled DMF (1.5 mL) was added
at 0 ꢁC and the reaction mixture was stirred at 25 ꢁC for
2 h before being quenched by the addition of saturated
aqueous NH4Cl (30 mL). The resulting aqueous solu-
tion was extracted with EtOAc (5 · 10 mL). The com-
bined organic layers were washed with saturated
aqueous NaCl (30 mL), dried (Na2SO4), filtered, and
concentrated. Column chromatography (SiO2, 33–67%
EtOAc–hexanes) afforded 8 (0.44 g, 70%) as a colorless
6. Conclusions
10-Methanesulfonyl-DDACTHF (1) and 10-methane-
sulfonyl-5-DACTHF (2) were prepared as sulfone
and sulfonamide mimics of the GAR Tfase-catalyzed
formyl transfer reaction tetrahedral intermediate. Both
proved to be effective GAR Tfase inhibitors (Ki = 0.23
and 0.58 lM) being 5- to 10-fold more potent than the
parent, unsubstituted DDACTHF. Both exhibited pur-
ine sensitive cytotoxic activity at concentrations roughly
4-fold higher than their in vitro enzymatic activity con-
sistent with functional inhibition GAR Tfase and de
novo purine biosynthesis, and both benefit from trans-
port into the cell by the reduced folate carrier and FPGS
polyglutamation. More interestingly, 10-methylthio-
DDACTHF (3), prepared for direct comparison along-
side 1 and 2, proved to be a surprisingly potent GAR
oil: 1H NMR (CDCl3, 500 MHz):
d
8.08 (d,
J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 4.21 (q,
J = 7.4 Hz, 2H), 4.08 (dd, J = 4.1, 11.4 Hz, 1H), 3.92
(s, 3H), 3.46–3.42 (m, 1H), 2.61 (s, 3H), 2.48–2.42

3582
H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3577–3585
(m, 1H), 2.20–2.12 (m, 1H), 2.01–1.91 (m, 2H), 1.52–
1.47 (m, 1H), 1.41–1.37 (m, 1H), 1.24 (t, J = 7.4 Hz,
3H); 13C NMR (CDCl3, 125 MHz): d 166.7, 166.0,
138.0, 131.7, 130.9, 129.7, 116.4, 69.7, 63.4, 52.8, 39.2,
37.5, 29.5, 26.9, 24.4, 14.4; IR (neat) mmax 2933, 1738,
1718, 1436, 1282, 1190, 1133, 1108, 1021 cmÀ1; MAL-
DI–FTMS (DHB) m/z 404.1142 (M+Na+, C18H23NO6S
requires 404.1138).
CHCl3 (10 mL). The resulting solution was washed with
saturated aqueous NaHCO3 (2 · 10 mL), dried
(Na2SO4), filtered, and concentrated. Column chroma-
tography (SiO2, 5–17% MeOH–CHCl3) afforded 11
1
(78 mg, 45%) as a light yellow solid: H NMR (CDCl3,
400 MHz):
d 7.81 (d, J = 7.6 Hz, 2H), 7.48 (d,
J = 7.6 Hz, 2H), 4.67–4.63 (m, 1H), 4.42–4.34 (m,
0.5H), 4.22 (t, 0.5H, J = 5.9 Hz), 2.63 (s, 3H), 2.48–
2.36 (m, 4H), 2.29–2.19 (m, 2H), 2.13–2.03 (m, 2H),
1.49 (s, 9H), 1.42 (s, 9H), 1.39–1.28 (m, 2H); IR (neat)
mmax 3342, 2910, 1731, 1637, 1618, 1453, 1362, 1286,
7.3. Methyl 4-[4-(2,4-diamino-6-oxo-1,6-dihydropyrimi-
din-5-yl)-1-methanesulfonylbutyl]benzoate (9)
1147 cmÀ1
; MALDI–FTMS (DHB) m/z 622.2912
A solution of NaOMe prepared by dissolving sodium
(40.5 mg, 1.76 mmol, 2.1 equiv) in anhydrous MeOH
(1.06 mL) was treated with guanidine hydrochloride
(96.2 mg, 1.01 mmol, 1.2 equiv) at ambient temperature.
After the reaction mixture was stirred at ambient tem-
perature for 30 min, compound 8 (0.32 g, 0.84 mmol)
in freshly distilled DMF (1.06 mL) was added. The reac-
tion mixture was allowed to warm to 75 ꢁC and stirred
for 16 h. The reaction was quenched by the addition
of acetic acid (53.3 lL, 0.92 mmol, 1.1 equiv), and col-
umn chromatography (SiO2, 6–13% MeOH–CHCl3)
afforded 9 (0.19 g, 58%) as a light yellow solid: 1H
NMR (CD3OD, 500 MHz): d 7.81 (d, J = 8.1 Hz, 2H),
7.36 (d, J = 8.1 Hz, 2H), 4.29 (dd, J = 3.3, 11.4 Hz,
1H), 3.68 (s, 3H), 2.54 (s, 3H), 2.15–2.00 (m, 3H),
1.95–1.91 (m, 1H), 1.15–1.09 (m, 2H); 13C NMR
(CD3OD, 100 MHz): d 168.2, 165.3, 164.5, 155.1,
139.9, 131.9, 131.5, 131.0, 89.2, 70.0, 52.9, 39.2, 28.1,
24.2, 22.8; IR (neat) mmax 2911, 2351, 1712, 1644, 1610,
1432, 1281, 1118 cmÀ1; MALDI–FTMS (DHB) m/z
417.1184 (M+Na+, C17H22N4O5S requires 417.1203).
(M+H+, C29H43N5O8S requires 622.2900).
7.6. N-{4-[4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-
yl)-1-methanesulfonylbutyl]benzoyl}-L-glutamic acid (1)
A solution of 11 (3.8 mg, 6.11 lmol) in CHCl3 (0.2 mL)
was treated with trifluoroacetic acid (1 mL) at 0 ꢁC. The
reaction solution was allowed to warm to 25 ꢁC, and
stirred overnight. The solution was concentrated, and
triturated with Et2O (3 · 1 mL) to give 1–CF3COOH
(3.8 mg, 100%) as a white solid: 1H NMR (D2O,
500 MHz):
d 7.63 (d, J = 7.7 Hz, 2H), 7.32 (d,
J = 7.7 Hz, 2H), 4.44–4.36 (m, 1H), 4.23–4.19 (m, 1H),
2.49 (s, 3H), 2.26–2.20 (m, 2H), 2.13–2.03 (m, 4H),
1.91–1.82 (m, 2H), 1.16–1.05 (m, 2H); MALDI–FTMS
(DHB) m/z 510.1637 (M+H+, C21H27N5O8S requires
510.1648).
7.7. Methyl 4-[(3-bromopropyl)methanesulfonyl-
amino]benzoate (12)
A
suspension of NaH (60% dispersion, 0.175 g,
7.4. 4-[4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-
1-methanesulfonylbutyl]benzoic acid (10)
4.36 mmol, 1 equiv) in freshly distilled DMF (3 mL)
was treated with a solution of methyl 4-(methanesulfo-
nylamino)benzoate (1 g, 4.36 mmol) in freshly distilled
DMF (20 mL) at 0 ꢁC. After the solution was stirred
at 0 ꢁC for 25 min, 1,3-dibromopropane (4.47 mL,
43.6 mmol, 10 equiv) was added to the reaction mixture.
The cooling bath was removed and the reaction mixture
was stirred at 25 ꢁC for 2.5 h before being quenched by
the addition of saturated aqueous NH4Cl (50 mL). The
resulting aqueous solution was extracted with EtOAc
(5 · 20 mL). The combined organic layers were washed
with saturated aqueous NaCl (50 mL), dried (Na2SO4),
filtered, and concentrated. Chromatography (SiO2, 25–
50% EtOAc–hexanes) afforded 12 (1.16 g, 76%) as a col-
orless oil: 1H NMR (CDCl3, 500 MHz): d 8.07 (d,
J = 8.8 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H), 3.91 (s, 3H),
3.88 (t, J = 7.0 Hz, 2H), 3.40 (t, J = 6.2 Hz, 2H), 2.89
(s, 3H), 2.08–2.02 (m, 2H); 13C NMR (CDCl3,
100 MHz): d 166.5, 143.7, 131.4, 130.0, 127.5, 52.8,
49.3, 37.6, 32.1, 30.1; IR (neat) mmax 2952, 1719, 1602,
1499, 1431, 1341, 1283, 1152, 1111 cmÀ1; MALDI–
FTMS (DHB) m/z 371.9885 (M+Na+, C12H16BrNO4S
requires 371.9876).
A solution of 9 (0.117 g, 0.30 mmol) in MeOH (4.1 mL)
was treated with LiOH monohydrate (37.4 mg,
0.89 mmol) in water (1.3 mL), and the reaction solution
was stirred at ambient temperature for 24 h. The reac-
tion solution was diluted with water (10 mL), washed
with EtOAc (2 · 10 mL), acidified to pH 4 by addition
of 1 N aqueous HCl, and concentrated. Removal of
traces of water by treatment of the residue with benzene
(3 · 5 mL) provided 10 (0.113 g, 100%) as a white solid:
1H NMR (D2O, 400 MHz): d 7.92 (d, J = 8.2 Hz, 2H),
7.44 (d, J = 8.2 Hz, 2H), 4.48 (dd, J = 3.2, 11.4 Hz,
1H), 2.84 (s, 3H), 2.26–2.21 (m, 1H), 2.19–2.14 (m,
2H), 2.03–1.98 (m, 1H), 1.37–1.30 (m, 1H), 1.20–1.14
(m, 1H); MALDI–FTMS (DHB) m/z 381.1220
(M+H+, C16H20N4O5S requires 381.1227).
7.5. Di-tert-butyl-N-{4-[4-(2,4-diamino-6-oxo-1,6-dihydro-
pyrimidin-5-yl)-1-methanesulfonylbutyl]benzoyl}-L-gluta-
mate (11)
A solution of 10 (0.108 g, 0.28 mmol), di-tert-butyl-L-
glutamate hydrochloride (0.126 g, 0.43 mmol, 1.5 equiv)
and NaHCO3 (71.6 mg, 0.85 mmol, 3 equiv) in DMF
(2.7 mL) was treated with EDCI (0.163 g, 0.85 mmol,
3 equiv) at 0 ꢁC. The reaction mixture was stirred at
ambient temperature overnight before the addition of
7.8. Methyl 4-[(4-cyano-4-ethoxycarbonylbutyl)methane-
sulfonylamino]benzoate (13)
A suspension of NaH (60% dispersion, 59.0 mg,
1.47 mmol, 1.2 equiv) in freshly distilled DMF

H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3577–3585
3583
(1.2 mL) was treated with ethyl cyanoacetate (0.17 mL,
1.6 mmol, 1.3 equiv) at 0 ꢁC. The reaction mixture was
stirred at 0 ꢁC for 30 min, forming the sodium salt as
a clear solution. A solution of 12 (0.43 g, 1.23 mmol)
in freshly distilled DMF (1.2 mL) was added at 0 ꢁC
and the reaction mixture was stirred at 25 ꢁC for 2 h be-
fore being quenched by the addition of saturated aque-
ous NH4Cl (30 mL). The resulting aqueous solution
was extracted with EtOAc (5 · 10 mL). The combined
organic layers were washed with saturated aqueous
NaCl (30 mL), dried (Na2SO4), filtered, and concen-
trated. Flash chromatography (SiO2, 33–67% EtOAc–
hexanes) afforded 13 (0.34 g, 73%) as a colorless oil:
1H NMR (CDCl3, 400 MHz): d 8.00 (d, J = 7.9 Hz,
2H), 7.38 (d, J = 7.9 Hz, 2H), 4.13 (q, J = 6.8 Hz, 2H),
3.84 (s, 3H), 3.74 (t, J = 5.6 Hz, 2H), 3.53 (t, J =
6.5 Hz, 1H), 2.82 (s, 3H), 2.00–1.91 (m, 2H), 1.62–
1.57 (m, 2H), 1.19 (t, J = 7.0 Hz, 3H); 13C
NMR (CDCl3, 100 MHz): d 166.0, 165.7, 142.8,
130.9, 129.6, 127.8, 116.3, 62.8, 52.3, 48.9, 37.0, 36.7,
26.3, 25.2, 13.9; IR (neat) mmax 2943, 1742, 1719,
1602, 1337, 1278, 1152 cmÀ1; MALDI–FTMS (DHB)
m/z 405.1095 (M+Na+, C17H22N2O6S requires
405.1091).
(DHB) m/z 382.1167 (M+H+, C15H19N5O5S requires
382.1180).
7.11. Di-tert-butyl-N-{4-[[3-(2,4-diamino-6-oxo-1,6-di-
hydropyrimidin-5-yl)propyl]methanesulfonylamino]benz-
oyl}-L-glutamate (16)
A solution of 15 (96.4 mg, 0.25 mmol), di-tert-butyl-L-
glutamate hydrochloride (0.112 g, 0.38 mmol, 1.5 equiv)
and NaHCO3 (63.8 mg, 0.76 mmol, 3 equiv) in DMF
(2.4 mL) was treated with EDCI (0.146 g, 0.76 mmol,
3 equiv) at 0 ꢁC. The reaction mixture was stirred at
ambient temperature overnight before the addition
of CHCl3 (10 mL). The resulting solution was washed
with saturated aqueous NaHCO3 (2 · 10 mL), dried
(Na2SO4), filtered, and concentrated. PCTLC (SiO2,
1 mm plate, 14% MeOH–CHCl3) afforded 16 (70.8 mg,
45%) as a light yellow solid: 1H NMR (CDCl3,
400 MHz):
d 7.63 (d, J = 7.9 Hz, 2H), 7.27 (d,
J = 7.9 Hz, 2H), 4.25 (dd, J = 4.7, 8.8 Hz, 1H), 3.52 (t,
J = 7.6 Hz, 2H), 2.71 (s, 3H), 2.15 (t, J = 7.0 Hz,
2H), 2.07 (t, J = 6.5 Hz, 2H), 1.97–1.92 (m, 0.5H),
1.82–1.74 (m, 1H), 1.56–1.52 (m, 0.5H), 1.39–1.34
(m, 2H), 1.24 (s, 9H), 1.19 (s, 9H); IR (neat) mmax
3342, 3173, 2983, 2930, 1727, 1616, 1600, 1489,
1447, 1368, 1331, 1146 cmÀ1; MALDI–FTMS (DHB)
m/z 645.2662 (M+Na+, C28H42N6O8S requires
645.2677).
7.9. Methyl 4-[[3-(2,4-diamino-6-oxo-1,6-dihydropyrimi-
din-5-yl)propyl]methanesulfonylamino]benzoate (14)
A solution of NaOMe prepared by dissolving sodium
(43.1 mg, 1.87 mmol, 2.1 equiv) in anhydrous MeOH
(1.13 mL) was treated with guanidine hydrochloride
(102 mg, 1.07 mmol, 1.2 equiv) at ambient temperature.
After the reaction mixture was stirred at ambient tem-
perature for 30 min, 13 (0.341 g, 0.892 mmol) in freshly
distilled DMF (1.13 mL) was added. The reaction mix-
ture was allowed to warm to 75 ꢁC and stirred for
16 h. The reaction was quenched by the addition of ace-
tic acid (56.7 lL, 0.98 mmol, 1.1 equiv), and column
chromatography (SiO2, 8–25% MeOH–CHCl3) afforded
14 (0.204 g, 58%) as a light yellow solid: 1H NMR
(CD3OD, 500 MHz): d 7.95 (d, J = 8.5 Hz, 2H), 7.51
(d, J = 8.5 Hz, 2H), 3.84 (s, 3H), 3.66 (t, J = 7.4 Hz,
2H), 3.01 (s, 3H), 2.12 (t, J = 7.3 Hz, 2H), 1.40–1.34
(m, 2H); IR (neat) mmax 2920, 1716, 1706, 1652, 1590,
1431, 1320, 1278 cmÀ1; MALDI–FTMS (DHB) m/z
396.1330 (M+H+, C16H21N5O5S requires 396.1336).
7.12. N-{4-[[3-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-
5-yl)propyl]methanesulfonylamino]benzoyl}-L-glutamic
acid (2)
A solution of 16 (10 mg, 16.1 lmol) in CHCl3 (0.2 mL)
was treated with trifluoroacetic acid (1 mL) at 0 ꢁC.
The reaction solution was allowed to warm to 25 ꢁC,
and stirred overnight. The solution was concentrated,
and triturated with Et2O (3 · 1 mL) to give 2–
1
CF3COOH (10.0 mg, 100%) as a white solid: H NMR
(D2O, 400 MHz): d 7.67 (d, J = 8.2 Hz, 2H), 7.34 (d,
J = 8.2 Hz, 2H), 4.50–4.45 (m, 1H), 3.56 (t, J = 7.6 Hz,
2H), 2.69 (s, 3H), 2.40 (t, J = 6.7 Hz, 2H), 2.19–2.13
(m, 1H), 1.99–1.95 (m, 1H), 1.88–1.82 (m, 1H), 1.50–
1.47 (m, 2H), 0.98–0.94 (m, 1H); MALDI–FTMS
(DHB) m/z 511.1594 (M+H+, C20H26N6O8S requires
511.1606).
7.10. 4-[[3-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-
yl)propyl]methanesulfonylamino]benzoic acid (15)
7.13. Methyl 4-(4-chloro-1-methylthiobutyl)benzoate (17)
After a solution of sodium bis(trimethylsilyl)amide
(1.0 M in THF, 1.07 mL, 1.07 mmol, 1.05 equiv) was
treated with a solution of methyl 4-(methylthiom-
ethyl)benzoate (0.2 g, 1.02 mmol) in freshly distilled
THF (3 mL) at À78 ꢁC, 1-chloro-3-iodopropane
(1.1 mL, 10.2 mmol, 10 equiv) was added quickly to
the reaction mixture. The cooling bath was removed,
and the reaction mixture was stirred at 25 ꢁC for
45 min before being quenched by addition of saturated
aqueous NH4Cl (10 mL). The resulting aqueous solu-
tion was extracted with EtOAc (4 · 5 mL). The com-
bined organic layers were washed with saturated
aqueous NaCl (15 mL), dried (Na2SO4), filtered, and
concentrated. Column chromatography (SiO2, 5–17%
A solution of 14 (0.1 g, 0.25 mmol) in MeOH (3.5 mL)
was treated with LiOH monohydrate (31.9 mg,
0.76 mmol) in water (1.14 mL), and the reaction solu-
tion was stirred at ambient temperature for 24 h. The
reaction solution was diluted with water (10 mL),
washed with EtOAc (2 · 10 mL), acidified to pH 4 by
addition of 1 N aqueous HCl, and concentrated.
Removal of traces of water by treatment of the
residue with benzene (3 · 5 mL) provided 15 (96.4 mg,
100%) as a white solid: 1H NMR (D2O, 400 MHz):
d 7.68 (d, J = 7.0 Hz, 2H), 7.18 (d, J = 7.0 Hz, 2H),
3.47 (t, J = 7.4 Hz, 2H), 2.84 (s, 3H), 2.00 (t,
J = 7.3 Hz, 2H), 1.41–1.36 (m, 2H); MALDI–FTMS

3584
H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3577–3585
EtOAc–hexanes) afforded 17 (86 mg, 31%) as a colorless
oil: H NMR (CDCl3, 500 MHz): d 8.00 (d, J = 8.1 Hz,
7.16. 4-[4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-
yl)-1-methylthiobutyl]benzoic acid (20)
1
2H), 7.36 (d, J = 8.1 Hz, 2H), 3.91 (s, 3H), 3.71 (t,
J = 7.7 Hz, 1H), 3.51 (t, J = 6.6 Hz, 2H), 2.10–1.96 (m,
2H), 1.86 (s, 3H), 1.89–1.83 (m, 1H), 1.75–1.69 (m,
1H); 13C NMR (CDCl3, 100 MHz): d 166.8, 147.4,
129.9, 129.1, 127.8, 52.1, 50.5, 44.5, 32.9, 30.5, 14.2;
IR (neat) mmax 2955, 1719, 1430, 1280, 1189,
A solution of 19 (29.6 mg, 81.7 lmol) in MeOH
(1.13 mL) was treated with LiOH monohydrate
(10.3 mg, 0.245 mmol, 3 equiv) in water (0.38 mL), and
the reaction solution was stirred at ambient temperature
for 24 h. The reaction solution was diluted with water
(10 mL), washed with EtOAc (2 · 10 mL), acidified to
pH 4 by addition of 1 N aqueous HCl, and concen-
trated. Removal of traces of water by treatment of
the residue with benzene (3 · 5 mL) provided 20
(28.4 mg, 100%) as a white solid: 1H NMR (D2O,
1111 cmÀ1
;
ESI–MS (NBA) m/z 295.1 (M+Na+,
C13H17ClO2S requires 295.1).
7.14. Methyl 4-(5-cyano-5-ethoxycarbonyl-1-methylthio-
pentyl)benzoate (18)
500 MHz):
d 7.70 (d, J = 8.1 Hz, 2H), 7.21 (d,
A suspension of NaH (60% dispersion, 73.9 mg,
1.85 mmol, 1.2 equiv) in freshly distilled DMF
(1.5 mL) was treated with ethyl cyanoacetate
(0.214 mL, 2.01 mmol, 1.3 equiv) at 0 ꢁC. The reaction
mixture was stirred at 0 ꢁC for 30 min, forming the so-
dium salt as a clear solution. A solution of 17 (0.42 g,
1.54 mmol) in freshly distilled DMF (1.5 mL) was added
at 0 ꢁC and the reaction mixture was allowed to warm to
50 ꢁC, and stirred for 9 h before being quenched by
addition of saturated aqueous NH4Cl (30 mL). The
resulting aqueous solution was extracted with EtOAc
(5 · 5 mL). The combined organic layers were
washed with saturated aqueous NaCl (20 mL), dried
(Na2SO4), filtered, and concentrated. Column chromato-
graphy (SiO2, 6–33% EtOAc–hexanes) afforded 18
(0.30 g, 56%) as a colorless oil: 1H NMR (CDCl3,
J = 8.1 Hz, 2H), 3.68 (dd, J = 6.3, 9.2 Hz, 1H), 2.12 (t,
J = 7.0 Hz, 2H), 1.94–1.83 (m, 2H), 1.77 (s, 3H), 1.38–
1.32 (m, 1H), 1.24–1.21 (m, 1H); MALDI–FTMS
(DHB) m/z 349.1334 (M+H+, C16H20N4O3S requires
349.1329).
7.17. Di-tert-butyl-N-{4-[4-(2,4-diamino-6-oxo-1,6-di-
hydropyrimidin-5-yl)-1-methylthiobutyl]benzoyl}-L-
glutamate (21)
A solution of 20 (25.5 mg, 73.4 lmol), di-tert-butyl-L-
glutamate
hydrochloride
(32.6 mg,
0.11 mmol,
1.5 equiv) and NaHCO3 (18.5 mg, 0.22 mmol, 3 equiv)
in DMF (0.71 mL) was treated with EDCI (42.2 mg,
0.22 mmol, 3 equiv) at 0 ꢁC. The reaction mixture
was stirred at ambient temperature overnight before
the addition of CHCl3 (10 mL). The resulting solution
was washed with saturated aqueous NaHCO3
(2 · 10 mL), dried (Na2SO4), filtered, and concentrated.
PCTLC (SiO2, 1 mm plate, 14% MeOH–CHCl3)
afforded 21 (19.0 mg, 44%) as a light yellow solid:
1H NMR (CDCl3, 500 MHz): d 7.73 (d, J = 7.0 Hz,
2H), 7.33 (d, J = 7.0 Hz, 2H), 4.70–4.66 (m, 1H), 3.74
(t, J = 6.6 Hz, 1H), 2.46–2.40 (m, 1H), 2.37–2.30
(m, 1H), 2.27–2.20 (m, 2H), 2.08–2.03 (m, 1H),
1.96–1.90 (m, 1H), 1.87–1.83 (m, 1H), 1.81 (s, 3H),
1.77–1.69 (m, 1H), 1.50 (s, 9H), 1.41 (s, 9H), 1.26–
1.24 (m, 2H); IR (neat) mmax 3329, 2922, 1722,
500 MHz):
d 8.00 (d, J = 7.9 Hz, 2H), 7.36 (d,
J = 7.9 Hz, 2H), 4.23 (q, J = 7.4 Hz, 2H), 3.92 (s, 3H),
3.70 (t, J = 7.0 Hz, 1H), 3.43 (dd, J = 7.4, 14.1 Hz,
1H), 2.02–1.91 (m, 4H), 1.85 (s, 3H), 1.65–1.45 (m,
2H), 1.28 (t, J = 7.3 Hz, 3H); IR (neat) mmax 2919,
1725, 1705, 1611, 1430, 1274, 1177, 1105 cmÀ1; MALDI
–FTMS (DHB) m/z 372.1243 (M+Na+, C18H23NO4S
requires 372.1240).
7.15. Methyl 4-[4-(2,4-diamino-6-oxo-1,6-dihydropyrimi-
din-5-yl)-1-methylthiobutyl]benzoate (19)
A solution of NaOMe prepared by dissolving sodium
(23.8 mg, 1.03 mmol, 2.1 equiv) in anhydrous MeOH
(0.62 mL) was treated with guanidine hydrochloride
(56.4 mg, 0.59 mmol, 1.2 equiv) at ambient temperature.
After the reaction mixture was stirred at ambient
temperature for 30 min, 18 (0.172 g, 0.49 mmol) in
freshly distilled DMF (0.62 mL) was added. The reac-
tion mixture was allowed to warm to 75 ꢁC and stirred
for 16 h. The reaction was quenched by addition of ace-
tic acid (31.3 lL, 0.54 mmol, 1.1 equiv), and column
chromatography (SiO2, 8–25% MeOH–CHCl3) afforded
1624, 1601, 1360, 1142 cmÀ1
;
MALDI–FTMS
(DHB) m/z 590.3000 (M+H+, C29H43N5O6S requires
590.3007).
7.18. N-{4-[4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-
5-yl)-1-methylthiobutyl]benzoyl}-L-glutamic acid (3)
A solution of 21 (3.3 mg, 5.60 lmol) in CHCl3 (0.2 mL)
was treated with trifluoroacetic acid (1 mL) at 0 ꢁC. The
reaction solution was allowed to warm to 25 ꢁC, and
stirred overnight. The solution was concentrated,
and triturated with Et2O (3 · 1 mL) to give 3–
CF3COOH (3.3 mg, 100%) as a white solid: 1H
NMR (D2O, 500 MHz): d 7.59 (d, J = 6.2 Hz, 2H),
7.26 (d, J = 6.2 Hz, 2H), 4.47–4.44 (m, 1H), 3.68–
3.65 (m, 1 H), 2.41 (t, J = 6.2 Hz, 2H), 2.19–2.15 (m,
1H), 2.08 (t, J = 7.4 Hz, 2H), 2.00–1.97 (m, 1H), 1.83–
1.79 (m, 1H), 1.72 (s, 3H), 1.69–1.65 (m, 1H), 1.38–
1.32 (m, 1H), 1.19–1.11 (m, 1H); MALDI–FTMS
(DHB) m/z 478.1746 (M+H+, C21H27N5O6S requires
478.1755).
19 (0.102 g, 57%) as
a
light yellow solid: 1H
NMR (CD3OD, 500 MHz): d 7.95 (d, J = 8.1 Hz, 2H),
7.33 (d, J = 8.1 Hz, 2H), 3.88 (s, 3H), 3.72
(t, J = 7.4 Hz, 1H), 2.23–2.19 (m, 2H), 1.91–1.81 (m,
2H), 1.80 (s, 3H), 1.51–1.45 (m, 1H), 1.42–1.36 (m,
1H); 13C NMR (CDCl3, 100 MHz): d 166.9, 164.4,
161.9, 153.3, 148.0, 129.8, 128.8, 127.9, 89.6, 52.1,
50.9, 36.5, 35.3, 25.9, 14.2; IR (neat) mmax 2909, 1707,
1632, 1601, 1428, 1277 cmÀ1; MALDI–FTMS (DHB)
m/z 363.1490 (M+H+, C17H22 N4O3S requires 363.1485).

H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3577–3585
3585
L. S.; Worzalla, J. F.; Rinzel, S. M.; Grindey, G. B.;
Harrington, P. M.; Taylor, E. C. J. Med. Chem. 1992, 35,
1109; (f) Shih, C.; Habeck, L. L.; Mendelsohn, L. G.;
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135.
7.19. GAR and AICAR Tfase assay
The Ki values for the folate analogues were measured as
previously described.7 For the GAR Tfase inhibition as-
say, each compound was dissolved in DMSO and then
diluted in assay buffer and the concentration of DMSO
did not affect enzyme activity. Thus, all assays were con-
ducted by mixing 10 lM of 10-formyl-5,8-dideazafolic
acid (fDDF), 20 lM of inhibitor in total volume of
1 mL buffer (0.1 M HEPES, pH 7.5) at 26 ꢁC, and the
reaction initiated by the addition of 76 nM E. coli or
rhGAR Tfase. The assay monitors the deformylation
of fDDF (De = 18.9 mMÀ1 cmÀ1 at 295 nm) resulting
from the transfer of the formyl group to GAR. If the
inhibitor was found to be active, a series of 1/vi versus
1/[GAR] at different, fixed concentrations of I (e.g., 1,
2, 4, 8, 12, 16, 20, 32 lM) were generated in order to
determine Ki using the Michaelis–Menton equation for
competitive inhibition. AICAR Tfase inhibition studies
was conducted in the absence of 5 lM b-mercapto-
ethanol and screened with 10 nM enzyme, 25 lM
inhibitor and 22.5 lM of cofactor. The results for the
inhibition assays are summarized in Table 1.
5. Marsilje, T. H.; Hedrick, M. P.; Desharnais, J.; Tavassoli,
A.; Ramcharan, J.; Zhang, Y.; Wilson, I. A.; Benkovic, S.
J.; Boger, D. L. Bioorg. Med. Chem. 2003, 11, 4487.
6. Greasley, S. E.; Marsilje, T. H.; Cai, H.; Baker, S.;
Benkovic, S. J.; Boger, D. L.; Wilson, I. A. Biochemistry
2001, 40, 13538.
7. Boger, D. L.; Haynes, N.-E.; Kitos, P. A.; Warren, M. S.;
Ramcharan, J.; Marolewski, A. E.; Benkovic, S. J. Bioorg.
Med. Chem. 1997, 5, 1817; Boger, D. L.; Haynes, N.-E.;
Warren, M. S.; Gooljarsingh, L. T.; Ramcharan, J.; Kitos,
P. A.; Marolewski, A. E.; Benkovic, S. J. Bioorg. Med.
Chem. 1997, 5, 1831; Boger, D. L.; Haynes, N.-E.;
Warren, M. S.; Ramcharan, J.; Kitos, P. A.; Benkovic,
S. J. Bioorg. Med. Chem. 1997, 5, 1839; Boger, D. L.;
Haynes, N.-E.; Warren, M. S.; Ramcharan, J.; Marolew-
ski, A. E.; Kitos, P. A.; Benkovic, S. J. Bioorg. Med.
Chem. 1997, 5, 1847; Boger, D. L.; Haynes, N.-E.;
Warren, M. S.; Ramcharan, J.; Kitos, P. A.; Benkovic,
S. J. Bioorg. Med. Chem. 1997, 5, 1853; Boger, D. L.;
Kochanny, M. J.; Cai, H.; Wyatt, D.; Kitos, P. A.;
Warren, M. S.; Ramcharan, L. T.; Gooljarsingh, L. T.;
Benkovic, S. J. Bioorg. Med. Chem. 1998, 6, 643; Boger, D.
L.; Labroli, M. A.; Marsilje, T. H.; Jin, Q.; Hedrick, M.
P.; Baker, S. J.; Shim, J. H.; Benkovic, S. J. Bioorg. Med.
Chem. 2000, 8, 1075; Boger, D. L.; Marsilje, T. H.; Castro,
R. A.; Hedrick, M. P.; Jin, Q.; Baker, S. J.; Shim, J. H.;
Benkovic, S. J. Bioorg. Med. Chem. Lett. 2000, 10, 1471;
Marsilje, T. H.; Hedrick, M. P.; Desharnais, J.; Capps, K.;
Tavassoli, A.; Zhang, Y.; Wilson, I. A.; Benkovic, S. J.;
Boger, D. L. Bioorg. Med. Chem. 2003, 11, 4503.
8. Marsilje, T. H.; Labroli, M. A.; Hedrick, M. P.; Jin, Q.;
Desharnais, J.; Baker, S. J.; Gooljarsingh, L. T.; Ramch-
aran, J.; Tavassoli, A.; Zhang, Y.; Wilson, I. A.; Beards-
ley, G. P.; Benkovic, S. J.; Boger, D. L. Bioorg. Med.
Chem. 2002, 10, 2739.
7.20. Cytotoxic assay
The cytotoxic activity of the compounds was measured
using the CCRF–CEM human leukemia cell lines as de-
scribed previously (72 h assay).10
Acknowledgements
We gratefully acknowledge the financial support of the
National Institute of Health (CA 63536, to D.L.B.,
I.A.W., and S.J.B.), and the Skaggs Institute for Chem-
ical Biology. Y.Z. is a Skaggs Fellow.
9. Desharnais, J.; Hwang, I.; Zhang, Y.; Tavassoli, A.;
Baboval, J.; Benkovic, S. J.; Wilson, I. A.; Boger, D. L.
Bioorg. Med. Chem. 2003, 11, 4511.
References and notes
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