10-(2-Benzoxazolcarbonyl)-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid: A potential inhibitor of GAR transformylase and AICAR transformylase

Article abstract of DOI:10.1016/S0968-0896(03)00457-7

The design and synthesis of 10-(2-benzoxazolcarbonyl)-DDACTHF (1) as an inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. Ketone 1 and the corresponding alcohol 13 were evaluated for inhibition of GAR Tfase and AICAR Tfase and the former was found to be a potent inhibitor of recombinant human (rh) GAR Tfase (K_i=600 nM).

Full text of DOI:10.1016/S0968-0896(03)00457-7

Bioorganic & Medicinal Chemistry 11 (2003) 4503–4509
10-(2-Benzoxazolcarbonyl)-5,10-dideaza-acyclic-5,6,7,8-
tetrahydrofolic Acid: A Potential Inhibitor of GAR
Transformylase and AICAR Transformylase
Thomas H. Marsilje,a,c Michael P. Hedrick,a,c Joel Desharnais,a,c Kevin Capps,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, CA92037, USA
bDepartment of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA92037, USA
cThe Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA92037, USA
dDepartment of Chemistry, Pennsylvania State University, University Park, PA16802, USA
Received 10 October 2002; accepted 22 April 2003
Abstract—The design and synthesis of 10-(2-benzoxazolcarbonyl)-DDACTHF (1) as an inhibitor of glycinamide ribonucleotide
transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. Ketone 1 and the
corresponding alcohol 13 were evaluated for inhibition of GAR Tfase and AICAR Tfase and the former was found to be a potent
inhibitor of recombinant human (rh) GAR Tfase (Ki=600 nM).
# 2003 Elsevier Ltd. All rights reserved.
Inhibitor design
Glycinamide ribonucleotide transformylase (GAR
Tfase) and aminoimidazole carboxamide ribonucleotide
transformylase (AICAR Tfase) are folate-dependent
The use of a-keto heterocycles as electrophilic, tight-
binding reversible enzyme inhibitors was first disclosed
by Edwards et al. in 1992.4 Since then, a number of
enzymes central to the de novo purine biosynthetic
pathway. GAR Tfase utilizes the cofactor (6R)-N10-for-
myltetrahydrofolate (Fig. 1) to transfer a formyl group
potent enzyme inhibitors have been disclosed based
upon analogous design principles,5 including our own
to the primary amine of its substrate, glycinamide ribo-
work in the development of fatty acid amide hydrolase
inhibitors.6 In previous studies, we examined folate-
nucleotide (GAR, Fig. 1). This one carbon transfer
incorporates the C-8 carbon of the purines and is the
based inhibitors which incorporated electrophilic func-
first of two formyl transfer reactions. The second formyl
tional groups that could potentially interact either with
transfer reaction is catalyzed by the enzyme AICAR
active site nucleophiles or the GAR/AICAR substrate
Tfase which also employs (6R)-N10-formyltetra-
hydrofolate to transfer a formyl group to the C-5 amine
of its substrate, aminoimidazole carboxamide ribonu-
cleotide (AICAR, Fig. 1).1 The discovery that (6R)-
amines.7 It was envisioned that the properly positioned
electrophilic carbonyl of an a-keto heterocycle could
potentially form an imine or a tetrahedral adduct with
these same potential nucleophiles or serve to stabilize
gem diol formation of the electrophilic carbonyl and
promote active site binding mimicking the tetrahedral
intermediate of the formyl transfer reactions. This latter
effect was observed with folate-based inhibitors bearing
a nontransferable formyl group and has provided
potent and efficacious GAR Tfase inhibitors.7À9 In
addition to the electrophilic carbonyl, an appropriately
positioned nitrogen atom within the heterocyclic ring
might form additional hydrogen bonds with an active
site residue (e.g., protonated His-108), thereby further
stabilizing the inhibitor complex.
5,10-dideazatetrahydrofolate
[Lometrexol,
(6R)-
DDATHF, Fig. 2] achieves its potent anticancer activity
by selective GAR Tfase inhibition established GAR
Tfase and the purine de novo biosynthetic pathway as
viable targets for antineoplastic intervention.2,3 Herein,
we report the design, synthesis and evaluation of a novel
folate analogue, 10-(2-benzoxazolcarbonyl)-DDACTHF
(1, Fig. 2).
*Corresponding author. Tel.: +1-858-784-7522; fax: +1-858-784-
7550; e-mail:
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00457-7

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T. H. Marsilje et al. / Bioorg. Med. Chem. 11 (2003) 4503–4509
Recently, we reported the synthesis and biological eval-
uation of 10-formyl-DDACTHF 2.8 This analogue was
shown to be a potent GAR Tfase inhibitor (Ki=0.014
mM against rhGAR Tfase) and an effective cytotoxic
agent (CCRF–CEM IC50=60 nM).8 Nonetheless, a
facile oxidative decarbonylation of the formyl group
conveyed a chemical instability to 2 precluding con-
sideration for in vivo use. Consequently, we have
examined a number of alternatives5,7 to the incor-
poration of a formyl group. In the preceding article we
detailed the preparation of an extensive series of sim-
plified folate analogues bearing an electrophilic a-keto-
heterocycle in place of the formyl group in structures
which lack the benzoylglutamate subunit.5 Herein, we
report the synthesis and examination of 1, 10-(2-ben-
zoxazolcarbonyl)-DDACTHF which bears the addi-
tional benzoylglutamate subunit and may be
representative of the entire series. In addition to active
site interactions of the benzoxazole, the electrophilic
carbonyl of the benzoxazolcarbonyl moiety in the
DDACTHF scaffold could potentially interact with the
active site of the enzyme either via formation of a
covalent bond with the substrate GAR amine or by
formation of hydrogen bonds (with its gem diol) with
active site residues. Because of these possibilities, it
was envisioned that 1 may exhibit interesting phar-
macological properties in comparison to the formyl
derivative 2.8,9
Chemistry
of 10-(2-benzoxazolcarbonyl)-
The
synthesis
DDACTHF (1) was accomplished in a convergent
manner as shown in Schemes 1 and 2. Known aldehyde
310 was converted to the corresponding cyanohydrin
(KCN, THF/H2O, 25 ꢀC, 20 h). Following aqueous
workup, the unstable crude cyanohydrin was converted
into the corresponding ethyl imidate (HCl, anhydrous
EtOH/CHCl3, 25 ꢀC, 12 h). Following concentration in
vacuo, the unstable crude imidate was converted into
benzoxazole
4 (2-aminophenol, anhydrous EtOH,
60 ꢀC, 5 h).11 Compound 4 was oxidized (Dess–Martin
Figure 2.
Figure 1.
Scheme 1.

T. H. Marsilje et al. / Bioorg. Med. Chem. 11 (2003) 4503–4509
4505
periodinane, anhydrous CH2Cl2, 0–25 ꢀC, 2 h, 92%)
generating ketone 5. The H NMR (CDCl3) of 5 clearly
LiOH (2.1 equiv, 3:1 CH3OH–H2O, 25 ꢀC, 24 h) cleanly
provided the carboxylic acid 9 which was coupled with
di-tert-butyl l-glutamate hydrochloride (EDCI,
NaHCO3, DMF, 25 ꢀC, 24 h, 25%) to provide 10. Acid-
catalyzed deprotection of 10 (1:4 v/v TFA/CHCl3, 24 h,
89%) provided 11. Hydrolysis of the dimethylhydrazone
by treatment with CuCl2 (5.0 equiv, 0–25 ꢀC, 7 h, 64%)
in THF–H2O buffered to pH 7,12 provided ketone 12.
Deprotection of 12 was accomplished by treatment with
trifluoroacetic acid (1:4 v/v TFA/CHCl3, 25 ꢀC, 12 h,
100%) to provide 1 (Scheme 2).
1
shows a peak corresponding to the benzylic methylene
at d 3.92, as well as the absence of a peak corresponding
to an enol methine, indicating that this compound exists
in the keto, rather than enol, form. Reaction of ketone 5
with N,N-dimethylhydrazine (glacial AcOH, anhydrous
EtOH, 25 ꢀC, 12 h, 87%) provided the key N,N-dime-
thylhydrazone 6 (Scheme 1). NaH deprotonation of 6
(DMF, 0 ꢀC, 15 min) and subsequent treatment with
excess 1,3-dibromopropane (10 equiv, DMF, 25 ꢀC, 2 h,
39%) provided the mono-alkylation product 7. The pre-
formed sodium salt of ethyl cyanoacetate (NaH, DMF,
0 ꢀC, 30 min) was alkylated with 7 (DMF, 25 ꢀC, 2 h).
Following aqueous workup, the crude alkylation inter-
mediate was used without further purification. Cycli-
zation with the free base of guanidine (1.2 equiv,
CH3OH, 25 ꢀC, 1 h, 32% from 7) under basic conditions
gave the desired pyrimidinone 8. Treatment of 8 with
For comparative purposes, ketone 12 was reduced with
NaBH4 (2.0 equiv, CH3OH, À20 ꢀC, 30 min) followed by
acid-catalyzed deprotection (4 N HCl–dioxane, 0–25 ꢀC,
3 h, 50% from 12) to provide alcohol 13 (Scheme 3).
GAR Tfase and AICAR Tfase Inhibition
Compounds 1, 11 and 13 were tested for inhibition of
GAR Tfase and AICAR Tfase and the results are pre-
sented in Table 1. The 10-(2-benzoxazolcarbonyl)-
DDACTHF folate-based analogue (1) was the only
derivative to show moderate activity against Escherichia
coli GAR Tfase (Ki=11 mM). The importance of the
keto functionality in the binding affinity of 1 is apparent
in the comparison of the analogous dimethylhydrazone
(11) and hydroxy (13) derivatives which were 10-fold
less potent (Ki=100 mM) and inactive (Ki>100 mM),
respectively. Interestingly, of the three compounds
examined for inhibition of rhAICAR Tfase, only the
dimethylhydrazone 11 showed activity (Ki=30 mM)
whereas 1 and 13 were inactive (Ki>100 mM). When
tested against rhGAR Tfase, compound 1 showed an
18-fold increase in its potency (11 mM against E. coli vs
0.6 mM against rhuman GAR Tfase) now displaying a
useful and potent level of activity. Although compound
11 was still inactive in this assay, the alcohol derivative
13 showed significant activity against rhGAR Tfase
(Ki=1.8 mM), while it was inactive against E. coli GAR
Tfase. Consistent with the anticipated importance of the
electrophilic carbonyl, this alcohol 13 was 3-fold less
potent than the corresponding ketone 1 against rhGAR
Scheme 2.
Scheme 3.

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T. H. Marsilje et al. / Bioorg. Med. Chem. 11 (2003) 4503–4509
Table 1. GAR and AICAR Tfase inhibition (Ki, mM)
Experimental
Methyl 4-(2-benzoxazol-2-yl-2-hydroxyethyl)benzoate (4).
Known aldehyde 310 (1.000 g, 5.612 mmol) was dis-
solved in a mixture of THF (25 mL) and H2O (30 mL).
Potassium cyanide (1.462 g, 22.45 mmol, 4.0 equiv) was
added and the resulting solution was stirred at 25 ꢀC for
20 h. The reaction mixture was partitioned between
H2O (100 mL) and EtOAc (400 mL). The organic layer
was washed with H2O (2Â100 mL), saturated aqueous
NaHCO3 (1Â100 mL), and saturated aqueous NaCl
(1Â100 mL) followed by concentration under reduced
pressure. Due to instability, the crude cyanohydrin
product was used without further purification. The
crude cyanohydrin was dissolved in CHCl3 (12 mL).
Separately, anhydrous absolute EtOH (5.08 mL, 87.6
mmol, 25 equiv) was dissolved in CHCl3 (9 mL) and
cooled to 0 ꢀC. Acetyl chloride (5.23 mL, 73.58 mmol,
21 equiv) was added dropwise. Following this addition,
the cyanohydrin starting material in CHCl3 solution
was added dropwise and the resulting reaction mixture
was allowed to warm to 25 ꢀC. After stirring for 12 h,
the reaction mixture was concentrated under reduced
pressure. Due to instability, the crude imidate product
was used without further purification. The crude imi-
date was dissolved in anhydrous absolute EtOH (15
mL). 2-Aminophenol (0.344 mg, 3.15 mmol, 0.90 equiv)
was added and the resulting solution was warmed at
60 ꢀC for 5 h. The reaction mixture was cooled to 25 ꢀC
and diluted with EtOAc (400 mL). The organic layer
was washed with saturated aqueous NaHCO3 (1Â100
mL), 1 N aqueous HCl (3Â100 mL), and saturated
aqueous NaCl (1Â100 mL) followed by concentration
under reduced pressure. Chromatography (SiO2, 1:1
EtOAc/hexanes) afforded 4 (0.250 g, 15% from 3) as an
Compd
E. coli GAR Tfasea rhGAR Tfaseb rhAICAR Tfasec
1
11
11
100
>100
0.6
>100
1.8
>100
30
>100
nde
13
Lometrexol
0.1
0.06d
aE. coli GAR Tfase.
bRecombinant human GAR Tfase.
cRecombinant human AICAR Tfase.
dRef 13.
end, not determined.
Table 2. In vitro cytotoxic activity
Compd
CCRF–CEM (IC50, mM)
(+) T, (+) Ha(À) T, (+) H (À) T, (À) H
1
11
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.52
>100
>100
>100
0.23
13
Lometrexol
aT, thymidine; H, hypoxanthine.
Tfase. Although not easily anticipated, this preferential
ca. 20-fold inhibition of rhGAR Tfase versus E. coli
GAR Tfase and the relative potency of the series
(1>13>11) is analogous to observations first made
with 2 (10-formyl-DDACTHF).8
Cytotoxic Activity
Compounds 1, 11 and 13 were examined for cytotoxic
activity both in the presence (+) and absence (À) of
added hypoxanthine against the CCRF–CEM cell line
(Table 2). 10-(2-benzoxazolcarbonyl)-DDACTHF (1)
and the related compounds (11 and 13) were inactive
against the CCRF–CEM cell line in vitro. Thus,
although compound 1 potently inhibits GAR Tfase in
vitro (Ki=600 nM), it does not display functional cyto-
toxic activity, perhaps due to ineffective cellular pene-
tration or polyglutamation by FPGS.
1
orange solid: H NMR (CDCl3, 250 MHz) d 7.93 (d,
J=8.4 Hz, 2H), 7.69–7.61 (m, 1H), 7.58–7.50 (m, 1H),
7.41–7.28 (m, 4H), 5.21 (dd, J=4.8, 7.7 Hz, 1H), 3.88 (s,
3H), 3.49–3.28 (m, 2H); MALDIFTMS (DHB) m/z
298.1065 (M+H+, C17H15NO4 requires 298.1074).
Methyl 4-(2-benzoxazol-2-yl-2-oxoethyl)benzoate (5).
Dess–Martin periodane (1.028 g, 2.42 mmol, 3.0 equiv)
was added to a stirred solution of 4 (0.240 g, 0.808
mmol) in anhydrous CH2Cl2 (13 mL) at 25 ꢀC. The
solution was stirred at 25 ꢀC for 2 h. The reaction was
diluted with Et2O (50 mL) and quenched by the addi-
tion of 1:1 (v/v) aqueous Na2S2O3/NaHCO3 (50 mL)
and stirred for 10 min. The resulting mixture was parti-
tioned between Et2O (400 mL) and H2O (100 mL). The
organic layer was washed with H2O (2Â100 mL) and
saturated aqueous NaCl (1Â100 mL) followed by con-
centration under reduced pressure affording 5 (0.219 g,
Conclusions
10-(2-Benzoxazolcarbonyl)-DDACTHF (1), and the
related dimethylhydrazone 11 and the hydroxy deriva-
tive 13 have been synthesized and evaluated as potential
inhibitors of GAR Tfase and AICAR Tfase. Compound
1 was the most active derivative in the series exhibiting
potent rhGAR Tfase inhibition (Ki=600 nM) being 3-
fold more potent than the corresponding alcohol
(Ki=1.8 mM), and >150-fold more potent than the
corresponding N,N-dimethylhydrazone. Thus, the pres-
ence of a benzoxazolcarbonyl group on the DDACTHF
framework produced folate analogues with potent
enzyme inhibiting activity, but did not provide com-
pounds that exhibited functional cytotoxic activity. The
latter we suggest may be due to ineffective transport
into the cell by the reduced folate carrier and/or lack of
polyglutamation by FPGS.
1
92%) as a yellow oil: H NMR (CDCl3, 250 MHz) d
8.10–7.39 (m, 8H), 3.92 (s, 2H), 3.89 (s, 3H); MAL-
DIFTMS (DHB) m/z 296.0917 (M+H+, C17H13NO4
requires 296.0917).
Methyl 4-(2-benzoxazol-2-yl-2-dimethylhydrazonoethyl)-
benzoate (6). Compound 5 (0.320 g, 1.08 mmol) was
dissolved in anhydrous EtOH (20 mL). N,N-Dimethyl-
hydrazine (0.42 mL, 5.42 mmol, 5.0 equiv) was added
followed by glacial acetic acid (0.06 mL, 1.08 mmol, 1.0

T. H. Marsilje et al. / Bioorg. Med. Chem. 11 (2003) 4503–4509
4507
equiv) and the resulting solution was stirred at 25 ꢀC for
12 h. The reaction mixture was concentrated under
reduced pressure. Chromatography (SiO2, 1:2 EtOAc/
hexanes) afforded 6 (0.318 g, 87%) as a red oil: 1H
NMR (CDCl3, 250 MHz) d 7.92 (d, J=8.4 Hz, 2H),
7.70–7.65 (m, 1H), 7.58–7.52 (m, 1H), 7.39–7.28 (m,
4H), 4.47 (s, 2H), 3.86 (s, 3H), 2.91 (s, 6H); MAL-
DIFTMS (DHB) m/z 338.1486 (M+H+, C19H19N3O3
requires 338.1499).
(CD3OD, 250 MHz) d 7.91–7.80 (m, 2H), 7.71–7.50 (m,
3H), 7.39–7.30 (m, 3H), 4.20 (t, J=7.6 Hz, 1H), 3.85 (s,
1H), 3.83 (s, 2H), 2.79 (s, 4H), 2.70 (s, 2H), 2.42–2.22
(m, 1H), 2.35 (t, J=7.4 Hz, 2H), 2.01–1.84 (m, 1H),
1.62–1.42 (m, 2H); MALDIFTMS (DHB) m/z 504.2349
(M+H+, C26H29N7O4 requires 504.2354).
4-[1-(Benzoxazol-2-yl-dimethylhydrazonomethyl)-4-(2,4-
diamino-6-oxo-1,6-dihydropyrimidin-5-yl)butyl]benzoic
acid (9). A solution of 8 (0.100 g, 0.199 mmol) in 3:1
.
Methyl 4-[1-(benzoxazol-2-yl-2-dimethylhydrazonome-
thyl)-4-bromobutyl]benzoate (7). NaH (60% dispersion,
0.013 g, 0.313 mmol, 1.1 equiv) was added to a stirred
solution of 6 (0.096 g, 0.285 mmol) in anhydrous DMF
(1.5 mL) at 0 ꢀC, and the solution was stirred at 0 ꢀC for
15 min. 1,3-Dibromopropane (0.29 mL, 2.85 mmol, 10.0
equiv) was added to the reaction mixture and the cool-
ing bath was removed. The solution was stirred at 25 ꢀC
for 2 h. The reaction was quenched by the addition of
saturated aqueous NH4Cl (1 mL). The resulting mixture
was partitioned between EtOAc (100 mL) and H2O (50
mL). The organic layer was washed with H2O (2Â50
mL) and saturated aqueous NaCl (1Â50 mL) followed
by concentration under reduced pressure. Chromato-
graphy (SiO2, 1:2 EtOAc/hexanes) afforded 7 (0.051 g,
CH3OH–H2O (4.0 mL) was treated with LiOH H2O
(0.018 g, 0.417 mmol, 2.1 equiv) and the mixture was
stirred at 25 ꢀC for 24 h. The mixture was diluted with
H2O (15 mL) and the aqueous layer was washed with
EtOAc (3Â5 mL). The aqueous layer was acidified to
pH=4 by the addition of 1 N aqueous HCl. The solu-
tion was concentrated under reduced pressure and the
residue was treated with toluene (3Â10 mL) to remove
traces of H2O to provide crude 9 (0.090 g) which was
1
used without further purification: H NMR (CD3OD,
250 MHz) d 7.87–7.23 (m, 8H), 4.16 (t, J=7.6 Hz, 1H),
2.76 (s, 4H), 2.69 (s, 2H), 2.45–2.22 (m, 2H), 2.34 (t,
J=7.4 Hz, 2H), 1.62–1.40 (m, 2H); MALDIFTMS
(DHB) m/z 490.2205 (M+H+, C25H27N7O4 requires
490.2197).
1
39%) as a yellow oil: H NMR (CDCl3, 250 MHz) d
7.98–7.90 (m, 2H), 7.75–7.70 (m, 1H), 7.60–7.52 (m,
1H), 7.48–7.29 (m, 4H), 4.22 (t, J=7.2 Hz, 1H), 3.89 (s,
1H), 3.85 (s, 2H), 3.41 (t, J=6.4 Hz, 2H), 3.03 (s, 1H),
2.90 (s, 3.5H), 2.70 (s, 1.5H), 2.38–2.23 (m, 1H), 2.09–
1.79 (m, 3H); MALDIFTMS (DHB) m/z 458.1082
(M+H+, C22H24BrN3O3 requires 458.1074).
Di-tert-butyl N-{4-[1-(benzoxazol-2-yl-dimethylhydrazo-
nomethyl)-4-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-
yl)butyl]benzoyl}-L-glutamate (10). A solution of 9
(0.100 g, 0.204 mmol) and di-tert-butyl l-glutamate
hydrochloride (0.091 g, 0.307 mmol, 1.5 equiv) in DMF
(1.0 mL) was treated with NaHCO3 (0.077 g, 0.920
mmol, 4.5 equiv) followed by EDCI(0.118 g, 0.613
mmol, 3.0 equiv). The reaction mixture was stirred at
25 ꢀC for 24 h before the solvent was removed under
reduced pressure. The resulting residue was suspended
in CHCl3 (50 mL) and washed with saturated aqueous
NaHCO3 (3Â5 mL). The organic layer was dried
(Na2SO4), filtered, and concentrated under reduced
pressure. Chromatography (SiO2, 10:1 CHCl3/CH3OH)
Methyl 4-[1-(benzoxazol-2-yl-2-dimethylhydrazonome-
thyl)-4-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)bu-
tyl]benzoate (8). A suspension of NaH (60% dispersion,
0.786 g, 19.7 mmol, 15 equiv) in anhydrous DMF (7.5
mL) at 0 ꢀC was treated dropwise with ethyl cyanoace-
tate (2.09 mL, 19.7 mmol, 15 equiv). The solution was
stirred at 0 ꢀC for 30 min, forming the sodium salt as a
clear solution. This was treated with a solution of 7
(0.600 g, 1.31 mmol) in anhydrous DMF (7.5 mL). The
resulting reaction mixture was stirred at 25 ꢀC for 2 h.
The reaction was quenched by the addition of saturated
aqueous NH4Cl (5 mL). The reaction was diluted with
EtOAc (200 mL) and washed successively with H2O
(3Â50 mL) and saturated aqueous NaCl (50 mL). The
organic layer was dried (Na2SO4), filtered, and con-
centrated under reduced pressure. Sodium metal (1.108
g, 48.20 mmol, 2.3 equiv) was added to anhydrous
CH3OH (15 mL) and stirred at 25 ꢀC for 10 min to
generate NaOCH3. Guanidine–HCl (2.40 g, 25.2 mmol,
1.2 equiv) was added to this solution and stirred at
25 ꢀC for 30 min. Separately, the crude alkylation pro-
duct was dissolved in anhydrous CH3OH (20 mL) and
the resulting solution quickly added to the stirring
reaction mixture. The solution was stirred at 25 ꢀC for 1
h. The reaction mixture was applied directly to a SiO2
plug. After complete air evaporation of the reaction
solvent, impurities were removed by washing with 5:1
hexanes/EtOAc. The product was subsequently eluted
by washing the SiO2 with 10:1 CHCl3/CH3OH to afford
1
afforded 10 (0.038 g, 25%) as a yellow solid: H NMR
(CD3OD, 250 MHz) d 7.87–7.31 (m, 8H), 4.49–4.42 (m,
1H), 4.20 (t, J=7.6 Hz, 1H), 2.79 (s, 4H), 2.71 (s, 2H),
2.50–2.12 (m, 4H), 2.35 (t, J=7.4 Hz, 2H), 2.06–1.90
(m, 2H), 1.62–1.40 (m, 2H), 1.45 (s, 9H), 1.38 (s, 9H);
MALDIFTMS (DHB) m/z 731.3901 (M+H+,
C38H50N8O7 requires 731.3875).
N-{4-[1-(Benzoxazol-2-yl-dimethylhydrazonomethyl)-4-
(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)butyl]ben-
zoyl}-L-glutamic acid (11). A solution of 10 (0.005 g,
0.007 mmol) in CHCl3 (1.0 mL) cooled to 0 ꢀC was
treated with trifluoroacetic acid (0.25 mL). The solution
was allowed to warm and stirred at 25 ꢀC for 24 h. The
reaction was concentrated under reduced pressure. Et2O
(1 mL) was added and a precipitate formed. The pre-
cipitate was triturated with Et2O (3Â1 mL) and dried in
vacuo to give 11–CF3CO2H (0.004 mg, 89%) as a gray
1
solid: H NMR (CD3OD, 400 MHz) d 7.86 (d, J=8.5
Hz, 1H), 7.70 (d, J=7.9 Hz, 1H), 7.57 (d, J=7.6 Hz,
1H), 7.42–7.35 (m, 4H), 4.62–4.51 (m, 1H), 4.31 (t,
J=7.5 Hz, 1H), 2.91 (s, 6H), 2.46–2.20 (m, 4H), 2.37 (t,
J=7.2 Hz, 2H), 2.09–1.90 (m, 2H), 1.58–1.40 (m, 2H);
1
8 (0.210 g, 32% from 7) as a yellow solid: H NMR

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T. H. Marsilje et al. / Bioorg. Med. Chem. 11 (2003) 4503–4509
MALDIFTMS (DHB) m/z 619.2596 (M+H+,
C30H34N8O7 requires 619.2623).
Cytotoxicity, GAR Tfase and AICAR Tfase inhibition.
Cytotoxicity, rhGAR14 and rhAICAR15 Tfase inhibi-
tion studies were conducted as previously detailed8 with
the exception that the AICAR Tfase inhibition was
conducted in the absence of 5 mM b-mercaptoethanol
and screened with 10 nM enzyme, 25 mM inhibitor and
22.5 mM of cofactor.
Di-tert-butyl N-{4-[1-(benzoxazol-2-carbonyl)-4-(2,4-dia-
mino-6-oxo-1,6-dihydropyrimidin-5-yl)butyl]benzoyl}-L-
glutamate (12). A solution of 10 (0.030 g, 0.041 mmol)
in THF (0.9 mL) and pH 7 aqueous phosphate buffer
(0.2 mL) cooled to 0 ꢀC was treated with a solution of
CuCl2 (0.028 g, 0.205 mmol, 5.0 equiv) in H2O (0.3 mL).
The solution was stirred at 0 ꢀC for 1 h and then at
25 ꢀC for 6 h before it was quenched by the dropwise
addition of a pH 8 saturated aqueous NH4Cl-NH4OH
solution (20 mL). The product was extracted with
CHCl3 (2Â50 mL), purged with N2, dried (Na2SO4),
filtered, and concentrated under reduced pressure.
Chromatography (SiO2, 10:1 CHCl3/CH3OH) afforded
Acknowledgements
We gratefully acknowledge the financial support of the
National Institutes of Health (CA 63536, to D.L.B.,
I.A.W., and S.J.B.), the Skaggs Institute for Chemical
Biology, and a Skaggs predoctoral fellowship (Y.Z.,
K.C.).
1
12 (0.018 g, 64%) as a white solid: H NMR (CD3OD,
250 MHz) d 7.95–7.42 (m, 8H), 5.10 (d, J=7.2 Hz, 1H),
4.49–4.40 (m, 1H), 2.40–1.90 (m, 6H), 2.35 (t, J=7.0
Hz, 2H), 1.51–1.31 (m, 2H), 1.45 (s, 9H), 1.38 (s, 9H);
MALDIFTMS (DHB) m/z 711.3109 (M+Na+,
C36H44N6O8 requires 711.3113).
References and Notes
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J=7.4 Hz, 1H), 4.62–4.52 (m, 1H), 2.44 (t, J=7.2 Hz,
2H), 2.37 (t, J=7.4 Hz, 2H), 2.32–2.20 (m, 2H), 2.15–
1.90 (m, 2H), 1.55–1.35 (m, 2H); MALDIFTMS (DHB)
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glutamic acid (13). A solution of 12 (0.010 g, 0.015
mmol) in anhydrous CH3OH (0.5 mL) at À20 ꢀC was
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reaction mixture was diluted with EtOAc (5 mL) and
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25 ꢀC for 3 h. The reaction was purged with N2 and then
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1H NMR (CD3OD, 400 MHz) d 7.74 (d, J=7.9 Hz,
2H), 7.70–7.59 (m, 2H), 7.40–7.32 (m, 4H), 5.12 (d,
J=6.5 Hz, 1H), 4.65–4.58 (m, 1H), 2.46 (t, J=7.1 Hz,
2H), 2.38–2.25 (m, 1H), 2.23 (t, J=7.2 Hz, 2H), 2.12–
2.00 (m, 1H), 1.89–1.80 (m, 1H), 1.70–1.57 (m, 1H),
1.39–1.21 (m, 2H); MALDIFTMS (DHB) m/z 579.2211
(M+H+, C28H30N6O8 requires 579.2198).

T. H. Marsilje et al. / Bioorg. Med. Chem. 11 (2003) 4503–4509
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