Synthesis and biological evaluation of N-{4-[5-(2,4-diamino-6-oxo-1,6- dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl}-L-glutamic acid as a potential inhibitor of GAR Tfase and the de novo purine biosynthetic pathway

Article abstract of DOI:10.1016/j.bmc.2004.11.049

The synthesis and evaluation of N-{4-[5-(2,4-diamino-6-oxo-1,6- dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl}-l-glutamic acid (2) as an inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide rib

Full text of DOI:10.1016/j.bmc.2004.11.049

Bioorganic & Medicinal Chemistry 13 (2005) 3593–3599
Synthesis and biological evaluation of
N-{4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-
trifluoroacetyl)pentyl]benzoyl}-L-glutamic acid as a potential
inhibitor of GAR Tfase and the de novo purine biosynthetic pathway
Heng Cheng,a,b Inkyu Hwang,a,b Youhoon Chong,a,b Ali Tavassoli,c Michael E. Webb,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 27 August 2004; revised 24 November 2004; accepted 24 November 2004
Available online 28 March 2005
Abstract—The synthesis and evaluation of N-{4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pent-
yl]benzoyl}-L-glutamic acid (2) as an inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carb-
oxamide ribonucleotide transformylase (AICAR Tfase) are reported. The inhibitor 2 was prepared in a convergent synthesis
involving C-alkylation of methyl 4-(4,4,4-trifluoro-3-dimethylhydrazonobutyl)benzoate with 1-chloro-3-iodopropane followed by
construction of the pyrimidinone ring. Compound 2 was found to be an effective inhibitor of recombinant human GAR Tfase
(Ki = 0.50 lM), whereas it was inactive (Ki > 100 lM) against E. coli GAR Tfase as well as recombinant human AICAR Tfase.
Compound 2 exhibited modest, purine-sensitive growth inhibitory activity against the CCRF-CEM cell line (IC50 = 6.0 lM).
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
DNA and RNA, inhibition of enzymes in the purine
biosynthetic pathway has been found to be an effective
approach for antineoplastic intervention. Notably, the
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,
Fig. 1).1 Since purines are required components of
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 pur-
ine biosynthesis and GAR Tfase as viable targets for
antineoplastic intervention.2–4
Herein, we report the synthesis and evaluation of N-{4-
[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-
(2,2,2-trifluoroacetyl)pentyl]benzoyl}-L-glutamic
(2), an analogue of the potent and selective GAR Tfase
inhibitor 10-CF3CO-DDACTHF (1) (Fig. 2).
acid
2. Inhibitor design
*
In previous studies, we have disclosed studies that
examined folate-based inhibitors which incorporate
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.2004.11.049

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H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3593–3599
GAR Tfase inhibitors (3, Ki = 0.014 lM against rhGAR
O
O
Tfase; 1, Ki = 0.015 lM against rhGAR Tfase).8–11 Both
were found to be effectively transported into the cell by
the reduced folate carrier and polyglutamated by FPGS,
which contributes to their cytotoxic activity by enhanc-
ing their intracellular accumulation (3, CCRF-CEM
IC50 = 60 nM; 1, CCRF-CEM IC50 = 16 nM).8–11 Nota-
bly, 10-CF3CO-DDACTHF (1) is the most potent and
selective GAR Tfase disclosed to date, being >10-fold
more potent than Lometrexol, and, unlike 3, is suitably
stable for in vivo evaluation.10
−2O3PO
−2O3PO
NHCHO
NH2
HN
HN
GAR Tfase
O
O
HO OH
NH2
HO OH
GAR
H
N
N
O
O
NH
N
H
O
H
−2O3PO
N
N
HN
H
N
O
H
OH
COOH
A preceding analogue of 1, compound 4 which contains
an additional carbon in the chain linking the trifluoro-
methyl ketone and the diaminopyrimidinone, was
recently reported.9 The analogue 4 with this one carbon
extension in the flexible spacer exhibited a >1000-fold
decrease in activity against both rhGAR Tfase
(Ki = 22 lM) and CCRF-CEM cell growth inhibition
(IC50 = 50 lM). Herein, we describe the synthesis and
evaluation of N-{4-[5-(2,4-diamino-6-oxo-1,6-dihydro-
pyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)-pentyl]benzoyl}-
L-glutamic acid (2, Fig. 2), an analogue of 1 which
contains an additional carbon between C10 and the ben-
zoyl glutamate.
HO OH
N
COOH
N
CONH2
NH2
CONH2
NHCHO
−2O3PO
−2O3PO
N
O
N
O
AICAR Tfase
HO OH
AICAR
HO OH
Figure 1.
H
N
N
NH2
H2N
(
N
O
O
NH2
3. Chemistry
NH
NH
)
n
O
O
F3C
The synthesis of 2 was accomplished in a convergent
manner as presented in Scheme 1.8–11 3-(4-Carbometh-
oxyphenyl)propanic acid12 was treated with oxalyl chlo-
ride in CH2Cl2 to afford the corresponding acid
chloride. Without further purification, the acid chloride
was converted to trifluoromethyl ketone 6 by reaction
with trifluoroacetic anhydride (pyridine, CH2Cl2, 0 ꢁC,
20 min) followed by an aqueous quench (70%).13 Reac-
tion of 6 with N,N-dimethylhydrazine (glacial AcOH,
anhydrous EtOH, 25 ꢁC, 24 h, 64%) provided the key
N,N-dimethylhydrazone 7. Sodium bis(trimethylsil-
yl)amide deprotonation of 7 (THF, À78 ꢁC) and subse-
quent treatment with excess 1-chloro-3-iodopropane
(10 equiv, THF, 25 ꢁC, 1 h) provided the monoalkyl-
ation product 8, inseparable from 7. The use of 1,3-di-
bromopropane in place of 1-chloro-3-iodopropane
gave only elimination versus alkylation product. a-
Alkylation of the dimethylhydrazone versus alkylation
at the acidic benzylic position was confirmed by 1H
COSY NMR of 8 and related intermediates. The pre-
formed sodium salt of ethyl cyanoacetate (NaH,
DMF, 0 ꢁC, 30 min) was alkylated with 8 (DMF,
50 ꢁC, 9 h, 19% for two steps from 7) to give 9, and
treatment with free base guanidine (1.2 equiv,
CH3OH–DMF, 75 ꢁC, overnight, 58%) under basic con-
ditions gave the desired pyrimidinone 10.
H
N
H
N
COOH
COOH
COOH
O
n = 1
DDATHF
10-CF3CO-DDACTHF (1)
COOH
NH2
n = 2, (4)
H2N
N
O
H2N
N
O
O
NH2
NH
NH
F3C
O
H
CH2
H
N
H
N
COOH
COOH
O
COOH
(2)
(3)
O
COOH
Figure 2.
electrophilic functional groups that could potentially
interact either with active site nucleophiles or the
GAR/AICAR substrate amines.5–8 The most significant
of these were the folate-based inhibitors 10-formyl-
DDACTHF (3)8 and 10-CF3CO-DDACTHF (1)9,10
(Fig. 2), bearing a nontransferable formyl or trifluoro-
acetyl group, which both proved to be potent GAR
Tfase inhibitors. X-ray and NMR studies of the inhibi-
tor–enzyme complexes revealed that both inhibitors
bound as their gem-diols.10,11 The formation of the
gem-diol mimics the formyl transfer reaction tetrahedral
intermediate and provides strong hydrogen bond inter-
actions between the inhibitor and protein.
Methyl ester hydrolysis and cleavage of the hydrazone
was accomplished by treatment of 10 with LiOH
(3 equiv, CH3OH–H2O 3:1, 25 ꢁC, overnight) followed
by acidification with aqueous 1 N HCl to pH 1
(30 min, 100%) to provide 11, which was coupled with
di-tert-butyl L-glutamate hydrochloride (1.5 equiv,
EDCI, NaHCO3, DMF, 25 ꢁC, overnight, 44%) to pro-
Both 10-formyl-DDACTHF (3) and 10-CF3CO-
DDACTHF (1) were shown to be selective and potent

H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3593–3599
3595
HOOC
LiOH, CH3OH/H2O, 100%
(1) (COCl)2, CH2Cl2
10
Me2N
CO2Me
CO2Me
(2) TFAA, pyridine
N
t
H2N
CO2 Bu
0−25 °C, 70%
O
F3C
t
CO2 Bu
H2NNMe2, AcOH, EtOH
25 °C, 64%
F3C
CO2H
O
EDCI, anhyd DMF
25 °C, 46%
13
14
6
7
HN
Me2N
N
1-chloro-3-iodopropane
NaN(SiMe3)2
NH2
N
N
H2N
F3C
Me2N
(1) TFA, CHCl3
Me2N
THF, −78 to 25 °C
2
CO2Me
CO2Me
(2) H2O, pH 1
100%
COOEt
Na
N
F3C
O
H
N
t
CN
CO2 Bu
F3C
anhyd DMF, 50 °C, 9 h
O
HN
19% from 7
8
9
t
CO2 Bu
Cl
NH2
N
H2N
Scheme 2.
Me2N
F3C
N
Guanidine
NaOCH3, CH3OH−DMF
75 °C, 58%
CO2Me
46%) to provide 14 as a 1:1 mixture of the inseparable
diastereomers. Deprotection of 14 was accomplished
by treatment with trifluoroacetic acid (TFA–CHCl3,
25 ꢁC, overnight) followed by acidification to pH 1
(30 min, 100%) to provide 2.
NC
COOEt
Me2N
N
(1) LiOH, CH3OH/H2O
(2) pH 1, 30 min, 100%
F3C
CO2Me
O
4. GAR Tfase and AICAR Tfase inhibition
10
11
HN
H2N
COOtBu
Compound 2 was tested for inhibition of E. coli GAR
Tfase, recombinant human GAR Tfase, and recombi-
nant human AICAR Tfase, and the results are presented
in Table 1. Compound 2 exhibited selective and potent
inhibition of recombinant human GAR Tfase
(Ki = 0.50 lM), whereas it was inactive against E. coli
GAR Tfase (Ki > 100 lM). This unusual selective inhibi-
tion of human GAR Tfase was also observed with 1 and
3. Compound 2 was roughly 10-fold less potent than
Lometrexol, and 30-fold less potent than 1 against
rhGAR Tfase. Compound 2 was inactive against rhAI-
CAR Tfase (Ki > 100 lM).
NH2
O
N
H2N
COOtBu
F3C
EDCI, anhyd DMF
25 °C, 44%
CO2H
O
O
HN
H2N
NH2
N
F3C
H
N
COOR
O
O
HN
H2N
COOR
NH2
N
12, R = tBu
2, R = H
TFA
5. Cell growth inhibition
100%
Scheme 1.
Compounds 2, as well as 10–14, were examined for cell
growth inhibition both in the presence (+) and absence
(À) of added hypoxanthine against the CCRF-CEM cell
line (Table 2). Compound 2 exhibited modest inhibitory
activity against the CCRF-CEM cell line (2,
IC50 = 6.0 lM) when purines (hypoxanthine) were
vide 12 as a 1:1 mixture of the inseparable diastereo-
mers. Deprotection of 12 was accomplished by treat-
ment with trifluoroacetic acid (TFA–CHCl3, 25 ꢁC,
overnight, 100%) to provide N-{4-[5-(2,4-diamino-6-
oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)-
pentyl]benzoyl}-L-glutamic acid (2).
Table 1. GAR and AICAR Tfase inhibition (Ki, lM)
Compound E. coli GAR Tfasea rhGAR Tfaseb rhAICAR Tfasec
The synthesis of 2 was accomplished by an alternative
route as presented in Scheme 2 that also provided access
to additional intermediates that still contain the N,N-
dimethylhydrazone. Treatment of 10 with lithium
hydroxide (3 equiv, CH3OH–H2O 3:1, 25 ꢁC, overnight,
100%) provided the carboxylic acid 13, which was cou-
pled with di-tert-butyl L-glutamate hydrochloride
(1.5 equiv, EDCI, NaHCO3, DMF, 25 ꢁC, overnight,
2
>100
1.9
0.1
0.50
0.015
0.06d
>100
>100
1
1
Lometrexol
a E. coli GAR Tfase.
b Recombinant human GAR Tfase.
c Recombinant human AICAR Tfase.
d Ref. 14.

3596
H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3593–3599
Table 2. In vitro cytotoxic activity, CCRF-CEM (IC50, lM)
between the trifluoromethyl ketone and the benzoylglut-
amate, was examined and found to be an effective GAR
Tfase inhibitor, albeit 30-fold less potent than 1. It was
established to possess modest, purine-sensitive cell
growth inhibitory activity against cultured cells consis-
tent with functional inhibition of GAR Tfase and, like
1, was found to benefit from reduced folate carrier trans-
port into the cell. Unlike 1, the functional activity of 2
was not dependent on FPGS (i.e., not a substrate for
FPGS) suggesting that the 75–400-fold loss in cellular
activity with 2 versus 1 results from both its reduced
enzymatic inhibition and less effective intracellular
retention attributable to polyglutamation. Regardless
of the origin of the effects, the comparison of 2 (and
4)9 with 1 highlights the unique combination of proper-
ties embodied in 1, which provides an unusually potent
and effective antineoplastic candidate.
Compound
(À)T (À)Ha
6
60
(+)T (À)H
10
(À)T (+)H
2
10
>100
>100
>100
>100
>100
>100
>100
>100
60
>100
20
11
12
>100
20
13
14
>100
30
0.08 (0.016)b
0.3
>100
20
1
Lometrexol
0.08 (0.016)b
0.2
a T, thymidine; H, hypoxanthine.
b Ref. 10.
Table 3. In vitro cytotoxic activity (IC50, lM)a
Compd CCRF-CEM CCRF-CEM/MTX CCRF-CEM/FPGSÀ
>100
2
6
6
a (À)T, thymidine; (À) H, hypoxanthine.
7. Experimental
absent in the media. This sensitivity to the presence of
purines, but not pyrimidines (thymidine), indicates that
the cell growth inhibitory activity of 2 is derived from its
inhibition of enzymes (i.e., GAR Tfase) in the de novo
purine biosynthetic pathway. Compound 2 was 20-fold
less potent than Lometrexol, and >75-fold less potent
than 1 against the CCRF-CEM cell line. In addition,
compounds 10, 12, and 14 exhibited weak cell growth
inhibitory activity against the CCRF-CEM cell line
(10, IC50 = 60 lM; 12, IC50 = 20 lM; 14, IC50 = 30 lM),
7.1. Methyl 4-(4,4,4-trifluoro-3-oxobutyl)benzoate (6)
A solution of 3-(4-carbomethoxyphenyl)propionic acid
(9.81 g, 47.1 mmol) in anhydrous CH2Cl2 (167 mL)
was treated with oxalyl chloride (12.4 mL, 0.141 mol,
3.0 equiv), and the resulting solution was stirred at
25 ꢁC for 2 h. After evaporation of the solvent and ex-
cess oxalyl chloride, a solution of the resulting residue
and trifluoroacetic anhydride (40.7 mL, 0.394 mol,
6.1 equiv) in CH2Cl2 (294 mL) was treated with pyridine
(31.9 mL, 0.394 mol, 8.4 equiv) at 0 ꢁC for 20 min.
Water (72 mL) was added cautiously, and the reaction
mixture was stirred at 25 ꢁC for 1 h. The reaction mix-
ture was poured into water (300 mL), and extracted with
CH2Cl2 (3 · 150 mL). The combined organic layers were
washed with saturated aqueous NaCl (250 mL), dried
(Na2SO4), filtered, and concentrated. Column chroma-
while compounds 11 and 13 were inactive (IC50
>
100 lM) highlighting the importance of the trifluoroace-
tyl and glutamate subunits of 2 for effective cell growth
inhibitory activity.
Compound 2 was also examined for cell growth inhibi-
tory activity in CCRF-CEM cell lines that lack the re-
duced folate carrier (CCRF-CEM/MTX) and folyl
polyglutamate synthase (CCRF-CEM/FPGSÀ). Its inac-
tivity against CCRF-CEM/MTX, like that of 1, indi-
cates that 2 is actively transported into the cell by the
reduced folate carrier, Table 3. Unlike 1, which is also
inactive against CCRF-CEM/FPGSÀ, the cytotoxic
activity of 2 is unaltered with this cell line indicating that
polyglutamation of 2 does not contribute to its cellular
activity and likely that it is not an effective FPGS sub-
strate. This may contribute to the relative potency differ-
ences between 1 and 2 in the cellular functional assays
where polyglutamation of 1, but not 2, increases intra-
cellular retention and concentration. In contrast and
as expected, the activity of 10–14 was unaffected in both
the CCRF-CEM/MTX and CCRF-CEM/FPGS-cell
lines indicating that their modest, purine-sensitive cell
growth inhibitory activity is not dependent on either re-
duced folate carrier transport or FPGS polyglutamation
(data not shown).
tography (SiO2, 25% EtOAc–hexanes) afforded
(8.57 g, 70%) as a yellow oil: 1H NMR (CDCl3,
400 MHz) 7.92 (d, J = 8.2 Hz, 2H), 7.21 (d,
6
d
J = 8.2 Hz, 2H), 3.85 (s, 3H), 3.01 (t, J = 4.7 Hz, 4H);
IR (neat) mmax 3415, 2941, 1767, 1713, 1611, 1435,
1286, 1178, 1110 cmÀ1; ESITOF-HRMS m/z 259.0589
(MÀH+, C12H11F3O3 requires 259.0587).
7.2. Methyl 4-(4,4,4-trifluoro-3-dimethylhydrazonobutyl)-
benzoate (7)
A solution of 6 (4.95 g, 19.0 mmol), N,N-dimethylhydr-
azine (3.28 mL, 43.2 mmol, 2.27 equiv) in anhydrous
EtOH (79 mL) was treated with acetic acid (1.11 mL,
19.4 mmol, 1.02 equiv), and the reaction solution was
stirred at 25 ꢁC overnight. The reaction solution was
concentrated under reduced pressure, and column chro-
matography (SiO2, 8–25% EtOAc–hexanes) afforded 7
(3.67 g, 64%) as a yellow oil: 1H NMR (CDCl3,
400 MHz)
d 7.98 (d, J = 8.2 Hz, 2H), 7.28 (d,
J = 8.2 Hz, 2H), 3.91 (s, 3H), 2.96–2.92 (m, 2H), 2.77
(s, 6H), 2.79–2.75 (m, 2H); 13C NMR (CDCl3,
100 MHz) d 166.9, 145.6, 130.0, 128.7, 128.2, 121.4 (q,
J = 275 Hz), 51.9, 47.1, 31.6, 28.8; IR (neat) mmax 2941,
2860, 1719, 1604, 1428, 1354, 1279, 1185, 1103,
6. Conclusions
Compound 2, an analogue of the potent antineoplastic
compound 1, which contains an additional methylene

H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3593–3599
3597
1022 cmÀ1; MALDI-FTHRMS (DHB) m/z 303.1320
MeOH (1.58 mL) was treated with guanidine hydrochlo-
ride (0.143 g, 1.5 mmol, 1.2 equiv) at ambient tempera-
ture. After the reaction mixture was stirred at ambient
temperature for 30 min, 9 (0.57 g, 1.25 mmol) in freshly
distilled DMF (1.58 mL) was added. The reaction mix-
ture was warmed to 75 ꢁC and stirred for 16 h. The reac-
tion mixture was cooled and quenched by addition of
acetic acid (79.7 lL, 1.5 mmol, 1.1 equiv). Column chro-
matography (SiO2, 8–25% MeOH–CHCl3) afforded 10
(0.32 g, 58%) as a light yellow solid: 1H NMR (CD3OD,
400 MHz) d 7.67 (d, J = 8.2 Hz, 2H), 7.09 (d, J = 8.2 Hz,
2H), 3.63 (s, 3H), 3.56–3.52 (m, 1H), 2.75 and 2.66 (AB
in ABX system, J = 7.0, 13.8 Hz, 2H), 2.12 (s, 6H), 2.10–
1.97 (m, 2H), 1.48–1.37 (m, 2H), 1.24–1.16 (m, 2H); IR
(neat) mmax 3330, 3180, 2941, 1709, 1604, 1492, 1440,
(M+H+, C14H17F3N2O2 requires 303.1315).
7.3. Methyl 4-[5-chloro-2-(2,2,2-trifluoro-1-dimethylhydr-
azonoethyl)pentyl]benzoate (8)
A solution of sodium bis(trimethylsilyl)amide (1.0 M in
THF, 1.88 mL, 1.88 mmol, 1.05 equiv) was treated with
a solution of 7 (0.54 g, 1.79 mmol) in freshly distilled
THF (8.1 mL) at À78 ꢁC, and 1-chloro-3-iodopropane
(1.92 mL, 17.9 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 1 h be-
fore being quenched by addition of saturated aqueous
NH4Cl (100 mL). The resulting aqueous solution was
extracted with EtOAc (5 · 30 mL). The combined or-
ganic layers were washed with saturated aqueous NaCl
(60 mL), dried (Na2SO4), filtered, and concentrated.
Column chromatography (SiO2, 7–25% EtOAc–hex-
anes) afforded 8 mixed with 7 as a yellow oil, which
was used directly in the next reaction. For 8: MALDI-
1328, 1275, 1178, 1126, 1014, 753 cmÀ1
; MAL-
DIÀFTHRMS (DHB) m/z 469.2155 (M+H+,
C21H27F3N6O3 requires 469.2169).
7.6. 4-[5-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-
2-(2,2,2-trifluoroacetyl)pentyl]benzoic acid (11)
FTHRMS
C17H22ClF3N2O2 requires 379.1395).
(DHB)
m/z
379.1398
(M+H+,
A solution of 10 (30 mg, 64.0 lmol) in MeOH (0.87 mL)
was treated with LiOH monohydrate (8.1 mg,
0.192 mmol, 3 equiv) in water (0.30 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 1 by addition of
1 N aqueous HCl, stirred for 30 min, and concentrated.
Removal of traces of water by treatment of the residue
with benzene (3 · 5 mL) provided 11 (26.4 mg, 100%) as
a white solid: 1H NMR (D2O, 400 MHz) d 7.68 (d,
J = 8.2 Hz, 2H), 7.13 (d, J = 8.2 Hz, 2H), 2.98–2.93 (m,
1H), 2.37 (t, J = 12.3 Hz, 1H), 2.04–2.00 (m, 1H), 1.90–
1.84 (m, 2H), 1.36–1.26 (m, 2H), 1.08–1.02 (m, 1H),
0.87–0.79 (m, 1H); ESITOF-HRMS m/z 413.1436
(M+H+, C18H19F3N4O4 requires 413.1431).
7.4. Methyl 4-[6-cyano-6-ethoxycarbonyl-2-(2,2,2-triflu-
oro-1-dimethylhydrazonoethyl)hexyl]benzoate (9)
A suspension of sodium hydride (60% dispersion,
85.9 mg, 2.15 mmol, 1.2 equiv) in freshly distilled
DMF (1.69 mL) was treated with ethyl cyanoacetate
(0.248 mL, 2.33 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 8 (8,
0.68 g, 1.79 mmol, mixed with 7) in freshly distilled
DMF (1.69 mL) was added at 0 ꢁC. The reaction mix-
ture was allowed to warm to 50 ꢁC, and stirred for 9 h
before being quenched by addition of saturated aqueous
NH4Cl (50 mL). The resulting aqueous solution was ex-
tracted with EtOAc (5 · 15 mL). The combined organic
layers were washed with saturated aqueous NaCl
(50 mL), dried (Na2SO4), filtered, and concentrated.
Column chromatography (SiO2, 8–33% EtOAc–hex-
anes) afforded 9 (0.57 g, 19% from 7) as a yellow oil:
1H NMR (CDCl3, 400 MHz) d 7.97 (d, J = 7.9 Hz,
2H), 7.27 (d, J = 7.9 Hz, 2H), 4.24 (q, J = 7.0 Hz, 2H),
3.91 (s, 3H), 3.82–3.74 (m, 1H), 3.46–3.40 (m, 1H),
2.98–2.90 (m, 2H), 2.43 (s, 3H), 2.42 (s, 3H), 1.97–1.87
(m, 1H), 1.75–1.69 (m, 1H), 1.68–1.55 (m, 3H), 1.49–
1.43 (m, 1H), 1.30 (t, J = 7.0 Hz, 3H); 13C NMR
(CDCl3, 100 MHz) d 166.8, 165.8, 163.0, 144.2, 129.8,
129.0, 128.6, 121.1 (q, J = 278 Hz), 116.3, 113.1, 62.9,
62.8, 52.0, 47.5, 40.2, 38.1, 37.3, 30.4, 29.5, 25.1, 24.9,
24.7, 13.9; IR (neat) mmax 2941, 2868, 1745, 1715, 1678,
1611, 1434, 1282, 1178, 1105, 1014 cmÀ1; MALDI-
FTHRMS (DHB) m/z 456.2107 (M+H+, C22H28F3N3O4
requires 456.2105).
7.7. Di-tert-butyl N-{4-[5-(2,4-diamino-6-oxo-1,6-dihy-
dropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]ben-
zoyl}-L-glutamate (12)
A solution of 11 (26.4 mg, 64.0 lmol), di-tert-butyl L-
glutamate hydrochloride (28.4 mg, 0.096 mmol,
1.5 equiv) and NaHCO3 (16.1 mg, 0.19 mmol, 3 equiv)
in DMF (0.62 mL) was treated with EDCI (36.8 mg,
0.19 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 12
(18.4 mg, 44%) as a light yellow solid: 1H NMR
(CD3OD, 400 MHz) d 7.50 (d, J = 7.6 Hz, 1H), 7.44
(d, J = 7.6 Hz, 1H), 7.02 (d, J = 7.6 Hz, 2H), 4.28–4.22
(m, 1H), 3.41–3.38 (m, 0.5H), 3.19–3.15 (m, 0.5H),
2.94–2.88 (m, 0.5H), 2.81–2.75 (m, 0.5H), 2.68–2.61
(m, 0.5H), 2.50–2.48 (m, 0.5H), 2.17–2.01 (m, 2H),
1.99–1.78 (m, 2H), 1.48–1.36 (m, 2H), 1.24 (s, 9H),
1.19 (s, 9H), 1.08–1.04 (m, 2H), 0.72–0.60 (m, 2H); IR
(neat) mmax 3340, 3215, 2921, 1724, 1616, 1446, 1355,
1151 cmÀ1; ESITOF-HRMS m/z 654.3109 (M+H+,
C31H42F3N5O7 requires 654.3109).
7.5. Methyl 4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrim-
idin-5-yl)-2-(2,2,2-trifluoro-1-dimethylhydrazono-
ethyl)pentyl]benzoate (10)
A solution of sodium methoxide prepared by dissolving
sodium (60.4 mg, 2.63 mmol, 2.1 equiv) in anhydrous

3598
H. Cheng et al. / Bioorg. Med. Chem. 13 (2005) 3593–3599
7.8. N-{4-[5-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-
yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl}-L-glutamic
acid (2)
7.11. N-{4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-
yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl}-L-glutamic
acid (2)
A solution of 12 (10 mg, 15.3 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-
A solution of 14 (10 mg, 14.4 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 solvent was evaporated, and
water (0.5 mL) was added. The resulting solution
(pH = 1) was stirred at 25 ꢁC for 30 min, before the sol-
vent was evaporated to give 2-CF3COOH (9.4 mg,
100%) as a white solid identical to the sample described
previously.
1
CF3COOH (10.0 mg, 100%) as a white solid: H NMR
(D2O, 500 MHz) d 7.54 (d, J = 8.1 Hz, 1H), 7.52 (d,
J = 8.1 Hz, 1H), 7.19 (d, J = 8.1 Hz, 2H), 4.51–4.48
(m, 1H), 3.02–2.99 (m, 1H), 2.46–2.41 (m, 3H), 2.23–
2.18 (m, 1H), 2.06–1.99 (m, 2H), 1.93–1.89 (m, 2H),
1.35–1.27 (m, 2H), 1.10–1.04 (m, 1H), 0.88–0.81 (m,
7.12. GAR and AICAR Tfase assay
1H);
C23H26F3N5O7 requires 542.1857).
ESITOF-HRMS
m/z
542.1858
(M+H+,
The Ki values for the folate analogues were measured as
previously described.7 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 rhGAR 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
generated in order to determine Ki using the Michae-
lis-Menton equation for competitive inhibition. AICAR
Tfase inhibition studies was conducted in the absence of
5 lM b-mercaptoethanol 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.
7.9. 4-[5-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-
2-(2,2,2-trifluoro-1-dimethylhydrazonoethyl)pentyl]ben-
zoic acid (13)
A solution of 10 (30 mg, 64.0 lmol) in MeOH (0.87 mL)
was treated with LiOH monohydrate (8.1 mg,
0.192 mmol, 3 equiv) in water (0.30 mL), and the reac-
tion 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 13 (29.1 mg,
1
100%) as a white solid: H NMR (D2O, 500 MHz) d
7.61 (d, J = 8.1 Hz, 2H), 7.10 (d, J = 8.1 Hz, 2H),
3.45–3.40 (m, 1H), 2.77 (d, J = 7.8 Hz, 2H), 2.11 (s,
6H), 2.07 (t, J = 6.6 Hz, 2H), 1.49–1.43 (m, 2H), 1.25–
1.20 (m, 2H); ESITOF-HRMS m/z 455.2017 (M+H+,
C20H25F3N6O3 requires 455.2013).
7.10. Di-tert-butyl N-{4-[5-(2,4-diamino-6-oxo-1,6-dihy-
dropyrimidin-5-yl)-2-(2,2,2-trifluoro-1-dimethylhydrazono-
ethyl)pentyl]benzoyl}-L-glutamate (14)
7.13. Cytotoxic assay
The cytotoxic activity of the compounds was measured
using the CCRF-CEM human leukemia cell lines as de-
scribed previously.10
A solution of 13 (29.1 mg, 64.0 lmol), di-tert-butyl L-
glutamate
hydrochloride
(28.4 mg,
0.096 mmol,
1.5 equiv) and NaHCO3 (16.1 mg, 0.19 mmol, 3 equiv)
in DMF (0.62 mL) was treated with EDCI (36.8 mg,
0.19 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 14
(20.5 mg, 46%) as a light yellow solid: 1H NMR
(CD3OD, 500 MHz) d 7.53 (d, J = 8.1 Hz, 2H), 7.09
(d, J = 8.1 Hz, 2H), 4.26–4.24 (m, 1H), 3.58–3.54 (m,
1H), 2.76 and 2.68 (AB in ABX system, J = 7.8,
13.6 Hz, 2H), 2.16 (s, 6H), 2.11–2.06 (m, 1H), 2.04–
1.94 (m, 2H), 1.82–1.76 (m, 1H), 1.51–1.40 (m, 2H),
1.25 (s, 9H), 1.21 (s, 9H), 1.13–1.10 (m, 2H), 0.74–0.68
(m, 2H); IR (neat) mmax 3333, 3186, 2915, 2847, 2349,
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.
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