Synthesis and biological evaluation of α- and γ-carboxamide derivatives of 10-CF3CO-DDACTHF

Article abstract of DOI:10.1016/j.bmc.2004.11.050

Structurally-related, but non-polyglutamylatable, derivatives of 10-CF ₃CO-DDACTHF (1), which incorporate l-glutamine (2) and l-isoglutamine (3) in place of l-glutamate, were prepared and evaluated as inhibitors of recombinant human (rh) GAR Tf

Full text of DOI:10.1016/j.bmc.2004.11.050

Bioorganic & Medicinal Chemistry 13 (2005) 3587–3592
Synthesis and biological evaluation of a- and
c-carboxamide derivatives of 10-CF3CO-DDACTHF
Youhoon Chong,a,c Inkyu Hwang,a,c Ali Tavassoli,d Yan Zhang,b,c Ian A. Wilson,b,c
Stephen J. Benkovicd and Dale L. Bogera,c,
*
aDepartment of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
bDepartment of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
cThe Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, CA 92037, USA
dDepartment of Chemistry, Pennsylvania State University, University Park, PA 16802, USA
Received 13 September 2004; revised 24 November 2004; accepted 24 November 2004
Available online 2 April 2005
Abstract—Structurally-related, but non-polyglutamylatable, derivatives of 10-CF3CO-DDACTHF (1), which incorporate L-glutam-
ine (2) and L-isoglutamine (3) in place of L-glutamate, were prepared and evaluated as inhibitors of recombinant human (rh) GAR
Tfase. While the L-glutamate a-carboxamide derivative 3 was much less effective as a rhGAR Tfase inhibitor (Ki = 4.8 lM) and inac-
tive in cellular functional assays, the c-carboxamide derivative 2 was found to be a potent and selective rhGAR Tfase inhibitor
(Ki = 0.056 lM) being only 4-fold less potent than 1 (Ki = 0.015 lM). Moreover, 2 was effective in cellular functional assays exhib-
iting purine sensitive cytotoxic activity (IC50 = 300 nM, CCRF-CEM) only 20-fold less potent than 1 (IC50 = 16 nM), consistent
with inhibition of de novo purine biosynthesis via selective inhibition of GAR Tfase. Like 1, 2 is transported into the cell by the
reduced folate carrier. Unlike 1, the functional activity of 2 is not dependent upon FPGS polyglutamylation.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
amylated.6–10 For example, recent studies suggest that
the polyglutamate metabolites of DDATHF are the
principal species responsible for cell growth inhibition
and that DDATHF itself may be a minor contributor
to the cellular cytotoxic activity.7 The pentaglutamate
of DDATHF has been reported to be about 11 or 100
times more potent against human8 and mouse7 glycin-
amide ribonucleotide transformylase (GAR Tfase),
respectively, than DDATHF. In addition, reduced
polyglutamylation by down-regulation of FPGS has
been described as a mechanism of inherent and acquired
resistance to classical antifolates,10 and deficient poly-
glutamylation renders cell lines resistant to both short
and long term drug exposure.11 Indeed, antifolate poly-
glutamylation and FPGS activity have been shown to be
important determinants for both methotrexate cytotox-
icity in vitro12 and outcome in clinical studies.12 None-
theless, the long term and enhanced intracellular
accumulation of antifolates that results from their poly-
glutamylation contributes to their cumulative toxicity.
Thus, lack of polyglutamylation is potentially a thera-
peutic asset, especially in the treatment of antifolate-
resistant tumors whose resistance is derived from a
Folylpolyglutamate synthetase (FPGS) plays a critical
role in endogenous folate metabolism and retention
and contributes to the cellular pharmacology of classical
antifolates.1 When folate or antifolates (as their a-
monoglutamate) enter mammalian cells, they are con-
verted by folylpolyglutamate synthetase (FPGS) to
poly-c-glutamates, containing between 2 and 9 addi-
tional c-glutamate residues. Intracellularly formed c-oli-
goglutamyl conjugates of reduced folates generally bind
better than the parent monoglutamates to their cognate
enzymes and are better retained in the cell.2 For the
same reasons, the potency of antifolates may be corre-
lated with their state of polyglutamylation.3 Recognition
of the significance of polyglutamylation for the thera-
peutic efficacy of antifolates has led to drug discovery
efforts to identify agents that are both inhibitors of fo-
late-dependent enzymes4,5 and are efficiently polyglut-
*
Corresponding author. Tel.: +1 858 784 7522; fax: +1 858 784
7550; e-mail: boger@scripps.edu
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.11.050

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Y. Chong et al. / Bioorg. Med. Chem. 13 (2005) 3587–3592
reduced capacity for polyglutamylation. In such instan-
ces, dose escalation should be tolerated better with non-
polyglutamylatable compounds than with classical
antifolates.
amylation, the investigation of structurally-related, but
non-polyglutamylatable, derivatives of 1 were pursued
which would also provide valuable insight into the role
of polyglutamylation on the activity of 1. Herein, we re-
port the synthesis and evaluation of two derivatives of
the 10-CF3CO-DDACTHF (1) that incorporate L-glut-
amine (2) and L-isoglutamine (3) in place of L-glutamate
which preclude their ability to serve as substrates for
FPGS. To our knowledge, the analogous monoamides
of methotrexate are the only antifolates reported in
which the L-glutamate side chain has been derivatized
as the corresponding carboxamides.16
In previous studies,13 we reported the design, synthesis,
and biological evaluation of 10-trifluoroacetyl-
DDACTHF (10-CF3CO-DDACTHF, 1, Fig. 1), which
was shown to be a selective and exceptionally potent
GAR Tfase inhibitor (Ki = 0.015 lM, rhGAR Tfase)
and an effective cytotoxic agent (CCRF-CEM cell line,
IC50 = 16 nM). Thus, gem-diol binding of the electro-
philic carbonyl of 1, that mimics the tetrahedral inter-
mediate of the formyl transfer reaction, conveys an
approximate 100-fold enhancement in binding affinity
for GAR Tfase accounting for its exquisite selectivity
and exceptional potency over other folate-dependent en-
zymes. As such, 1 constitutes the most potent GAR
Tfase inhibitor disclosed to date. Like other folate-based
inhibitors incorporating the DDACTHF scaffold,14 this
inhibitor was not only an effective enzyme inhibitor, but
it is also transported into the cell by the reduced folate
carrier and polyglutamylated by FPGS. However, the
polyglutamates of 1 exhibited only a modestly enhanced
Ki against GAR Tfase (1- to 3-fold) implying that the
FPGS and polyglutamylation requirement for observ-
ation of the potent, purine sensitive cytotoxic activity
of 1 is the result of enhanced intracellular accumul-
ation (retention) and not enhanced enzyme inhibitory
potency.15
2. Chemistry
The syntheses of a- and c-carboxamide derivatives of
10-CF3CO-DDACTHF (2 and 3) were accomplished
in a divergent manner as shown in Schemes 1 and 2.
N,N-Dimethylhydrazone 413 was alkylated to give the
corresponding chloropropyl adduct 5 (NaHMDS, 3-
chloro-1-iodopropane, THF, À78 °C, 63%). This alkyl-
ation procedure proved to be an improvement over that
initially reported for the preparation of 1 enlisting 1,3-
dibromopropane.13 Although comparable conversions
were observed, the desired monoalkylation was more
dependably controlled with use of 3-chloro-1-iodopro-
pane and the conditions detailed herein. The preformed
sodium salt of ethyl cyanoacetate (NaH, DMF, 0 °C,
30 min) was alkylated with 5 (DMF, 60 °C, 8 h) to give
6 (82%), and its treatment with the free base of guan-
idine under basic conditions gave the desired pyrimid-
inone 7 (63%) as previously detailed.13 Treatment of 7
with LiOH (3 equiv, 3:1 CH3OH/H2O, 25 °C, 48 h) fol-
lowed by acidification to pH 4 with 1 N aqueous HCl
Thus, in conjunction with our interest in establishing the
potential benefits of an antifolate incapable of polyglut-
H
N
N
O
O
NH2
NH
H2N
N
O
O
NH2
NH
F3C
NaHMDS
Me2NN
I(CH2)3Cl, THF
4
5
CO2Me
H
N
H
N
63%
CO2H
CO2H
CO2H
Cl
NaH
F3C
CNCH2CO2Et
82%
CO2H
DDATHF
DDACTHF
Me2NN
CO2Me
H2N
N
O
O
NH2
NH
CN
CO2Et
guanidine
F3C
F3C
NaOMe, MeOH
63%
H
N
CO2H
O
Me2NN
CO2Me
NH2
NH
6
7
H2N
N
10-CF3CO-DDACTHF (1)
CO2H
H2N
LiOH
H2N
N
O
O
NH2
NH
N
O
O
NH2
NH
F3C
O
100%
Me2NN
CO2Me
H2N
F3C
F3C
N
NH2
H
N
H
N
CO2H
CONH2
CO2H
O
O
NH
8
F3C
O
CONH2
O
CO2H
2
3
Figure 1.
Scheme 1.

Y. Chong et al. / Bioorg. Med. Chem. 13 (2005) 3587–3592
3589
Table 1. GAR and AICAR Tfase inhibition (Ki, lM)
H2N
N
O
NH2
NH
Compd
E. coli GAR Tfasea rhGAR Tfaseb rhAICAR Tfasec
1. ClCO2Et, Et3N
2. Gln-OH
1
1.9
>100
0.015
0.056
4.8
>100
>100
>100
nde
F3C
2
H
N
3
>100
5
O
O
CO2H
CONH2
DDACTHF
Lometrexol
1.7
0.06d
0.1
nde
2
O
N
H2N
NH2
8
a E. coli GAR Tfase.
b Recombinant human GAR Tfase.
c Recombinant human AICAR Tfase.
d Ref. 17.
NH
F3C
O
O
1. ClCO2Et, Et3N
2. iso-Gln-OH
H
N
e Not determined.
CONH2
CO2H
3
Scheme 2.
320-fold decreased affinity (Ki = 4.8 lM). Thus, 2 exhib-
its the selective inhibition of human versus E. coli GAR
Tfase analogous to 1 and is only 4-fold less potent than
1. Moreover, 2 is roughly equipotent with lometrexol
and 30-fold more potent than DDACTHF itself against
rhGAR Tfase. This latter comparison indicates that the
gem diol binding of the trifluoroacetyl group increases
the enzyme binding affinity significantly (30-fold) consis-
tent with expectations. The roughly 100-fold difference
in inhibition observed between the c-carboxamide (2)
and a-carboxamide (3) derivatives clearly defines the
role of the two glutamate carboxylates where the a-car-
boxylic acid contributes significantly to the binding
affinity, whereas the c-carboxylic acid does not.
cleanly hydrolyzed both the methyl ester and the di-
methylhydrazone, providing the common intermediate
813 (100%). In order to arrive at the target molecules,
L-glutamine and L-isoglutamine17 were coupled, under
basic conditions (aq NaOH), with the mixed anhydride
formed by the treatment of 8 with ethyl chloroformate
(ClCO2Et, Et3N, DMF, À10 °C).18 Acidification of the
reaction mixture to pH 2 with 6 N HCl provided the
HCl salts of 2 and 3 (Scheme 2).
3. GAR Tfase and AICAR Tfase inhibition
This result is consistent with the X-ray structure of com-
pound 1 bound with human GAR Tfase13 which defined
a critical salt bridge between the glutamate a-carboxyl-
ate and Arg64 while the glutamate c-carboxylate ex-
tends into solvent without any specific interactions
with enzyme residues. None of the compounds tested
showed inhibitory activity against rhAICAR Tfase.
Compounds 2 and 3 were tested for inhibition of GAR
Tfase and aminoimidazole carboxamide ribonucleotide
transformylase (AICAR Tfase) and the results are pre-
sented in Table 1. Unlike 10-CF3CO-DDACTHF (1)13
which showed moderate inhibitory activity against
E. coli GAR Tfase (Ki = 1.9 lM), the carboxamide
derivatives 2 and 3 were not active against the
bacterial enzyme. However, c-carboxamido-10-CF3-
CO-DDACTHF (2) exhibited potent inhibitory activity
(Ki = 56 nM) against recombinant human GAR Tfase
comparable with that observed with 1 (Ki = 15 nM),13
whereas the a-carboxamide derivative 3 exhibited a
4. Cytotoxic activity
Compounds 2 and 3 were examined for cytotoxic activ-
ity both in the presence (+) and absence (À) of added
Table 2. In vitro cytotoxic activity
Compound
CCRF-CEM (IC50, lM)
(+) T, (+) Ha
(À) T, (+) H
(+) T, (À) H
0.017
0.4
(À) T, (À) H
0.016
0.3
1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2
3
>100
3.6
0.2
>100
2.7
0.2
DDACTHF
Lometrexol
Compound
IC50, lM [(À)T, (À)H]
CCRF-CEM
CCRF-CEM/FPGSÀ
CCRF-CEM/MTX
1
0.016
0.3
>100
0.5
>100
40
2
3
>100
3
0.2
>100
ndc (>100b)
25.0 (>100b)
ndc
DDACTHF
Lometrexol
ndc (>100b)
ndc (>100b)
a T = Thymidine, H = Hypoxanthine.
b Ref. 15.
c Not determined.

3590
Y. Chong et al. / Bioorg. Med. Chem. 13 (2005) 3587–3592
hypoxanthine (purine) or thymidine (pyrimidine)
against the CCRF-CEM cell line (Table 2). Like 1, the
c-carboxamide derivative 2 exhibits potent cytotoxic
activity (IC50 = 0.3 lM) against the CCRF-CEM cell
line when purines (hypoxanthine) are absent in the med-
ium, but was inactive (IC50 > 100 lM) in the presence of
medium purines. This sensitivity to the presence of pur-
ines, but not pyrimidines (thymidine), indicates that the
cytotoxic activity of 2, like that of 1, is derived from
inhibition of an enzyme in the de novo purine biosyn-
thetic pathway consistent with its potent and selective
inhibition of rhGAR Tfase. In addition and in spite of
the moderate differences in Ki (4-fold, Table 1), 2 is
20-fold less cytotoxic than 1 in this assay. This would
imply that, along with the decreased affinity of 2 for
the target enzyme, a decrease in intracellular accumul-
ation of 2 derived from a lack of polyglutamylation may
reduce the effective intracellular concentration of 2 and
thereby reduce its cytotoxic activity. Importantly, assay
of 1 and 2 against a FPGS-deficient CCRF-CEM cell
line (CCRF-CEM/FPGSÀ, Table 2) performed to estab-
lish the extent to which the cytotoxic activity of 1 and 2
was dependent on the FPGS-polyglutamylation indi-
cates that 2 acts independent of the FPGS level. Unlike
1 and lometrexol which lost or lacked activity against
this cell line, compound 2 remains equipotent against
the FPGS-deficient cell line. In addition, the assay of 2
against a CCRF-CEM cell line with an impaired re-
duced folate carrier (CCRF-CEM/MTX) revealed that
2 is roughly 100-fold less active and that its cellular func-
tional activity, like that of 1, may benefit from reduced
folate carrier transport into the cell. Thus, conversion
of the glutamate c-carboxylic acid of 1 to a carboxamide
(2) precludes FPGS polyglutamylation, but does not ap-
pear to impact transport into the cell.
potent than 1 (IC50 = 16 nM), consistent with inhibition
of de novo purine biosynthesis via selective inhibition of
GAR Tfase. Like 1, 2 is transported into the cell by the
reduced folate carrier. Unlike 1, the functional activity
of 2 is not dependent upon FPGS polyglutamylation.
Thus, not only is 2 roughly equipotent with lometrexol
and only slightly less active than 1, but its functional
activity is independent of FPGS levels and polyglutam-
ylation providing a superb candidate for in vivo exami-
nation alongside 1. Such studies are in progress and will
be detailed in due course.
6. Experimental
6.1. Methyl 4-{4-chloro-1-[1-(dimethylhydrazono)-2,2,2-
trifluoroethyl]butyl}-benzoate (5)
NaHMDS (2 M solution in THF, 0.17 mL, 0.35 mmol)
was slowly added to a stirred solution of 413 (100 mg,
0.35 mmol) in anhydrous THF (5 mL) at À78 °C. After
stirring the reaction mixture for 15 min at À78 °C, 1-
chloro-3-iodopropane (0.38 mL, 3.46 mmol) was added.
The reaction mixture was slowly warmed to 23 °C and
stirred for 10 h, before being quenched with the addition
of saturated aqueous NH4Cl. The aqueous layer was ex-
tracted three times with EtOAc, and the combined or-
ganic layers were washed with saturated aqueous NaCl,
dried (MgSO4), filtered, and concentrated. Column chro-
matography (SiO2, 8% EtOAc–hexane) provided 80 mg
(0.22 mmol, 63%) of a 2.8:1 mixture (cis/trans hydra-
1
zone) of 5 as a yellow oil: H NMR (CDCl3, 400 MHz)
d 7.99 (d, J = 8.2 Hz, 2H), 7.98 (d, J = 8.2 Hz, 0.7H),
7.38 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 8.5 Hz, 0.7H),
4.79 (t, J = 7.6 Hz, 1H), 3.78 (t, J = 7.0 Hz, 0.3H), 3.59
(td, J = 6.4, 2.6 Hz, 2H), 3.49 (t, J = 6.4 Hz, 0.7H),
2.77 (s, 2.1H), 2.61 (s, 6H), 2.43–2.33 (m, 1H), 2.22–
2.07 (m, 1.3H), 1.93–1.74 (m, 2.7H), 1.66–1.56 (m,
0.3H); MALDIFTHRMS (DHB) m/z 364.1242
(M+H+, C16H20ClF3N2O2 requires 364.1238).
In contrast, the a-carboxamide derivative 3 was inactive
against the CCRF-CEM cell line (Table 2). Thus, the L-
glutamate a-carboxylate is critical and its derivatization
to a carboxamide results in both low affinity for the tar-
get enzyme (Ki = 4.8 lM) and a loss in activity in cellu-
lar functional assays.
6.2. Methyl 4-{5-cyano-1-[1-(dimethylhydrazono)-2,2,2-
trifluoroethyl]-5-(ethoxycarbonyl)pentyl}benzoate (6)
5. Conclusions
A suspension of NaH (158 mg, 3.95 mmol) in anhydrous
DMF (5 mL) at 0 °C was treated with ethyl cyanoace-
tate (0.42 mL, 3.95 mL) dropwise. The reaction mixture
was stirred at 0 °C for 30 min, forming the sodium salt
as a clear solution. This anion was treated with a
solution of 5 (80 mg, 0.22 mmol) in anhydrous DMF
(5 mL). The reaction mixture was stirred at 25 °C for
8 h and then warmed at 60 °C for 8 h before it was
quenched by the addition of saturated aqueous NH4Cl.
The reaction mixture was diluted with EtOAc and
washed with water and saturated aqueous NaCl. The
organic layer was dried (MgSO4), filtered, and
concentrated under reduced pressure. The excess ethyl
cyanoacetate was distilled off, and the residual product
was purified by column chromatography (SiO2, 12%
EtOAc–hexanes) to give 80 mg (0.18 mmol, 82%) of a
2.4:1 mixture (cis/trans hydrazone) of 6 as a yellow oil:
1H NMR (CDCl3, 400 MHz) d 7.99 (d, J = 8.2 Hz,
2H), 7.98 (d, J = 7.6 Hz, 0.8H), 7.36 (d, J = 8.0 Hz,
To address antifolate resistance derived from reduced
polyglutamylation and to avoid the cumulative toxicity
of polyglutamylatable antifolates, efforts have been di-
rected at the development of novel antifolates that are
designed to act irrespective of the level of FPGS expres-
sion in the cell.19–22 For this purpose and in this study,
non-polyglutamylatable c-carboxamide (2) and a-car-
boxamide (3) derivatives of 10-CF3CO-DDACTHF
were prepared. Whereas the L-glutamate a-carboxamide
derivative 3 was much less effective as a rhGAR Tfase
inhibitor (Ki = 4.8 lM) and inactive in cellular func-
tional assays, the c-carboxamide derivative 2 was found
to be a potent and selective rhGAR Tfase inhibitor
(Ki = 0.056 lM) being only 4-fold less potent than 1
(Ki = 0.015 lM). Moreover, 2 was effective in cellular
functional assays exhibiting purine sensitive cytotoxic
activity (IC50 = 300 nM, CCRF-CEM) only 20-fold less

Y. Chong et al. / Bioorg. Med. Chem. 13 (2005) 3587–3592
3591
2H), 7.28 (d, J = 7.0 Hz, 0.8H), 4.78 (t, J = 7.6 Hz, 1H),
4.26 (t, J = 7.2 Hz, 0.4H), 3.76 (t, J = 7.4 Hz, 0.4H), 3.53
(td, J = 7.3, 2.6 Hz, 1H), 2.78 (s, 1.2H), 2.77 (s, 1.2H),
2.61 (s, 3H), 2.60 (s, 3H), 2.26–2.20 (m, 1H), 2.07–1.91
(m, 4.6H), 1.70–1.49 (m, 2.4H), 1.44–1.30 (m, 0.4H);
MALDIFTHRMS (DHB) m/z 464.1768 (M+Na+,
C21H26F3N3O4Na requires 464.1773).
tration under reduced pressure, the residue was purified
by preparative TLC (reverse phase, 20% CH3OH–H2O)
followed by trituation with ether to give 21 mg
1
(0.04 mmol, 35%) of 2 as an off-white solid: H NMR
(CDCl3, 400 MHz) d 7.85 (d, J = 8.2 Hz, 0.6H), 7.74
(d, J = 8.2 Hz, 1.6H), 7.39 (d, J = 7.6 Hz, 1.6H), 7.36
(d, J = 7.9 Hz, 0.6H), 4.59–4.48 (m, 2.2H), 3.10 (t,
J = 5.2 Hz, 2.2H), 2.38 (t, J = 7.9 Hz, 2.2H), 2.42–2.20
(m, 2.2H), 2.30 (t, J = 8.2H, 1.6H), 2.22 (t, J = 8.2 Hz,
0.6H), 2.12–1.94 (m, 3.3H), 1.91–1.73 (m, 1.1H);
MALDIFTHRMS (DHB) m/z 527.1872 (M+H+,
C22H25F3N6O6 requires 527.186).
6.3. Methyl 4-{4-(2,4-diamino-6-oxo-1,6-dihydropyrim-
idin-5-yl)-1-[1-(dimethylhydrazono)-2,2,2-trifluoro-
ethyl]butyl}benzoate (7)
A stirred solution of NaOMe (367 mg, 6.80 mmol) in
MeOH (6 mL) was treated with guanidineÆHCl
(650 mg, 6.80 mmol) and the mixture was stirred for
30 min at 23 °C. A solution of 6 (1.0 g, 2.27 mmol) in
MeOH (4 mL) was added and the mixture was stirred
at reflux for 24 h. After concentration under reduced
pressure, the residual product was purified by column
chromatography (SiO2, 5–10% MeOH–CH2Cl2) to give
650 mg (1.43 mmol, 63%) of 7 as a colorless syrup iden-
tical in all respects with that previously described:13 1H
NMR (CDCl3, 400 MHz) d 7.95 (d, J = 6.8 Hz, 2H),
7.42 (d, J = 6.6 Hz, 2H), 4.79 (t, J = 6.2 Hz, 1H), 3.88
(s, 3H), 2.62 (s, 6H), 2.48–2.34 (m, 2H), 2.31–2.27 (m,
1H), 2.02–1.95 (m, 1H), 1.59–1.56 (m, 1H), 1.52–1.46
(m, 1H); MALDIFTHRMS (DHB) m/z 455.2001
(M+H+, C20H25F3N6O3 requires 455.2013).
6.6. 4-Carbamoyl-4-{4-[4-(2,4-diamino-6-oxo-1,6-dihy-
dropyrimidin-5-yl)-1-(2,2,2-trifluoroacetyl)butyl]benzo-
ylamino}butyric acid (3)
A sample of 8 (33 mg, 0.084 mmol) and Et3N (0.013 mL,
0.093 mmol) were dissolved in anhydrous DMF (5 mL)
and cooled to À10 °C under a N2 atmosphere. ClCO2Et
(0.0088 mL, 0.093 mmol) was added and, after 20 min, a
solution of L-isoglutamine (25 mg, 0.17 mmol) in 0.3 N
aqueous NaOH (0.7 mL, 0.20 mmol) was added. The
solution was allowed to warm to 23 °C and stirred for
25 min at 23 °C. The DMF was removed in vacuo and
the residue was dissolved in MeOH, cooled to 0 °C,
and acidified with dilute aqueous HCl. After concentra-
tion under reduced pressure, the residue was purified by
preparative TLC (reverse phase, 20% CH3OH–H2O)
followed by trituation with ether to give 13 mg
6.4. 4-[4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-
1-(2,2,2-trifluoro-acetyl)butyl]benzoic acid (8)
1
(0.025 mmol, 30%) of 3 as an off-white solid: H NMR
(CDCl3, 400 MHz) d 8.00 (d, J = 6.4 Hz, 0.6H), 7.90
(d, J = 6.4 Hz, 1.6H), 7.40 (d, J = 7.2 Hz, 1.6H), 7.36
(d, J = 6.4 Hz, 0.6H), 4.94–4.83 (m, 2.2H), 3.21 (t, J =
5.2 Hz, 0.6H), 3.20 (t, J = 5.2 Hz, 1.6H), 2.31 (t,
J = 5.6 Hz, 2.2H), 2.25–2.20 (m, 2.2H), 2.24 (t, J =
5.6 Hz, 1.6H), 2.23 (t, J = 5.6 Hz, 0.6H), 2.05–1.98
(m, 3.3H), 1.89–1.80 (m, 1.1H); MALDIFTHRMS
(DHB) m/z 527.1844 (M+H+, C22H25F3N6O6 requires
527.186).
A solution of 7 (111 mg, 0.24 mmol) in a 2:1 mixture of
MeOH (2 mL) and H2O (1 mL) was treated with LiOH-
H2O (31 mg, 7.33 mmol) and stirred for 24 h at 23 °C.
After diluting the reaction mixture with water, the aque-
ous layer was washed with EtOAc two times and treated
with 1 N aqueous HCl to adjust the pH to 4. After con-
centration under reduced pressure, the residue was trea-
ted with CH3CN to remove traces of water to provide 8
(97 mg, 0.24 mmol, 100%), identical in all respects with
material disclosed previously,13 which was used for the
next step without further purification: 1H NMR
(CDCl3, 400 MHz) d 7.89 (d, J = 8.0 Hz, 2H), 7.30 (d,
J = 8.0 Hz, 2H), 4.37 (t, J = 7.2 Hz, 1H), 2.25 (t,
J = 7.4 Hz, 2H), 2.19–1.77 (m, 2H), 1.44–1.25 (m, 2H);
MALDIFTHRMS (DHB) m/z 399.1275 (M+H+,
C17H17F3N4O4 requires 399.1275).
6.7. GAR and AICAR Tfase assay
The Ki values for the folate analogs were measured as
previously described.13 For the GAR Tfase inhibition
assay, each compound was dissolved in DMSO and then
diluted in assay buffer using a concentration of DMSO
that did not affect enzyme activity. Thus, all assays were
conducted by mixing 10 lM of fDDF, 20 lM of inhibi-
tor in total volume of 1 mL buffer (0.1 M HEPES,
pH 7.5) at 26 °C, and the reaction initiated by the addi-
tion of 76 nM E. coli or rh GAR Tfase. The assay mon-
itors 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 concen-
trations of I (e.g., 1, 2, 4, 8, 12, 16, 20, 32 lM) were gen-
erated in order to determine Ki using the Michaelis–
Menten equation for competitive inhibition. AICAR
Tfase inhibition studies was conducted in the absence
of 5 lM b-mercaptoethanol and screened with 10 nM en-
zyme, 25 lM inhibitor and 22.5 lM of cofactor. The re-
sults of the inhibition assays are summarized in Table 1.
6.5. 4-Carbamoyl-2-{4-[4-(2,4-diamino-6-oxo-1,6-dihy-
dropyrimidin-5-yl)-1-(2,2,2-trifluoroacetyl)butyl]benzo-
ylamino}butyric acid (2)
A sample of 8 (45 mg, 0.11 mmol) and Et3N (0.017 mL,
0.12 mmol) were dissolved in anhydrous DMF (5 mL)
and cooled to À10 °C under a N2 atmosphere. ClCO2Et
(0.012 mL, 0.12 mmol) was added and, after 20 min, a
solution of L-glutamine (33 mg, 0.23 mmol) in 0.3 N
aqueous NaOH (0.88 mL, 0.27 mmol) was added. The
solution was allowed to warm to 23 °C and stirred for
25 min at 23 °C. The DMF was removed in vacuo and
the residue was dissolved in MeOH, cooled to 0 °C,
and acidified with diluted aqueous HCl. After concen-

3592
Y. Chong et al. / Bioorg. Med. Chem. 13 (2005) 3587–3592
R.; Brown, M.; Gibson, W.; Aherne, G. W.; Hardcastle,
A.; Boyle, F. T. J. Cancer 1995, 71, 914.
6.8. Cytotoxic assay
11. Takemura, Y.; Kobayashi, H.; Gibson, W.; Kimbell, R.;
Miyachi, H.; Jackman, A. L. Int. J. Cancer 1996, 66, 29.
12. Whitehead, V. M.; Rosenblatt, D. S.; Vuchich, M.-J.;
Shuster, J. J.; Witte, A.; Beaulieu, D. Blood 1990, 76,
44.
The cytotoxic activity of the compounds was measured
using the CCRF-CEM human leukemia cell lines as de-
scribed previously.13–15
13. 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. Biochemistry
2003, 42, 6043.
14. Bigham, E. C.; Hodson, S. J.; Mallory, W. R.; Wilson, D.;
Duch, D. S.; Smith, G. K.; Ferone, R. J. Med. Chem.
1992, 35, 1399; 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. Bioorg.
Med. Chem. 2002, 10, 2739.
Acknowledgements
We gratefully acknowledge the financial support of the
National Institutes of Health (CA 63536, to DLB, SJB
and IAW), and the Skaggs Institute for Chemical
Biology.
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