10-Formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-formyl-DDACTHF): a potent cytotoxic agent acting by selective inhibition of human GAR Tfase and the de novo purine biosynthetic pathway.

Article abstract of DOI:10.1016/S0968-0896(02)00102-5

The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. Aldehyde 3, the corresponding gamma- and alpha-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR Tfase and AICAR Tfase. The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC(50) for 3=60nM) that exceeded their enzyme inhibition potency [K(i) (3)=6 and 1 microM for Escherichia coli GAR and human AICAR Tfase, respectively]. Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from GAR versus AICAR Tfase inhibition. The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGS(-)) establishing that their potent activity requires both reduced folate carrier transport and polyglutamation. Unexpectedly, the pentaglutamates displayed surprisingly similar K(i)'s versus E. coli GAR Tfase and only modestly enhanced K(i)'s versus human AICAR Tfase. On the surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca. 100-fold higher intracellular concentrations). However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed they and the corresponding gamma-pentaglutamates were unexpectedly much more potent against the human versus E. coli enzyme (K(i) for 3, 14nM against rhGAR Tfase versus 6 microM against E. coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.

Full text of DOI:10.1016/S0968-0896(02)00102-5

Bioorganic & Medicinal Chemistry 10 (2002) 2739–2749
10-Formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic Acid
(10-Formyl-DDACTHF):
A Potent Cytotoxic Agent Acting by Selective Inhibition of
Human GAR Tfase and the De Novo
Purine Biosynthetic Pathway
Thomas H. Marsilje,a Marc A. Labroli,a Michael P. Hedrick,a Qing Jin,a
Joel Desharnais,a Stephen J. Baker,c Lata T. Gooljarsingh,c Joseph Ramcharan,c
Ali Tavassoli,c Yan Zhang,b Ian A. Wilson,b G. Peter Beardsley,d
Stephen J. Benkovicc and Dale L. Bogera,*
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
cDepartment of Chemistry, Pennsylvania State University, University Park, PA16802, USA
dDepartment of Pediatrics and Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA
Received 7 December 2001; accepted 11 February 2002
Abstract—The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR
Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. Aldehyde 3, the corresponding
g- and a-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR
Tfase and AICAR Tfase. The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC50 for 3=60 nM) that
exceeded their enzyme inhibition potency [Ki (3)=6 and 1 mM for Escherichia coli GAR and human AICAR Tfase, respectively].
Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective
purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from
GAR versus AICAR Tfase inhibition. The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell
lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGSꢀ) establishing
that their potent activity requires both reduced folate carrier transport and polyglutamation. Unexpectedly, the pentaglutamates
displayed surprisingly similar Ki’s versus E. coli GAR Tfase and only modestly enhanced Ki’s versus human AICAR Tfase. On the
surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential
intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca. 100-fold higher intra-
cellular concentrations). However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed
they and the corresponding g-pentaglutamates were unexpectedly much more potent against the human versus E. coli enzyme (Ki for
3, 14 nM against rhGAR Tfase versus 6 mM against E. coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.
# 2002 Elsevier Science Ltd. All rights reserved.
Glycinamide ribonucleotide transformylase (GAR
Tfase) is an enzyme central to de novo purine
biosynthesis.1ꢀ12 Since purines are crucial components
of DNA and RNA, inhibition of enzymes in the purine
biosynthetic pathway has been proposed to be an effec-
tive approach for antineoplastic intervention.13 The
disclosure
that
(6R)-5,10-dideazatetrahydrofolate
[Lometrexol, (6R)-DDATHF] is an efficacious anti-
tumor agent that acts as an effective inhibitor of
GAR Tfase (Ki=0.1 mM) established inhibition of
purine biosynthesis and GAR Tfase as viable targets
for antineoplastic intervention.14ꢀ23 GAR Tfase uses
*Corresponding author. Tel.: +1-858-784-7522; fax: +1-858-784-
7550; e-mail: boger@scripps.edu
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00102-5

2740
T. H. Marsilje et al. / Bioorg. Med. Chem. 10 (2002) 2739–2749
Figure 2.
and 10-formyl-5,8,10-trideazafolate (10-formyl-TDAF),
the most potent of the inhibitors examined to date,
revealed that the aldehyde inhibitor (Ki=260 nM) binds
in the active site as its hydrate mimicking the tetrahedral
intermediate involved in formyl transfer.32 Thus, no
enzyme-assembled imine adduct with the substrate
b-GAR or covalent adduct with nucleophiles of the
GAR Tfase active site residues were observed, and the
potent inhibitory activity could be attributed to the
H-bonding interactions of the inhibitor aldehyde hydrate
with the catalytically important residues of the enzyme
active site. Despite these efforts, none of the potent
GAR Tfase inhibitors in this series, including 10-formyl-
TDAF, exhibited cytotoxic activity consistent with their
level of enzyme inhibition potency; observations that
could be attributed in part to their instability and in-
effective transport by the reduced folate carrier.28
Figure 1.
Numerous reports have described acyclic analogues of
(6R)-5,10-dideazatetrahydrofolate [4, Lometrexol or
(6R)-DDATHF, Fig. 2].33ꢀ42 Several of these analogues,
including the acyclic derivative 5 (Fig. 2, X=CH2) of
DDATHF,33 have been shown to retain the potent
cytotoxic and enzyme inhibitory properties of 4. Addi-
tionally, several analogues of 4 with substituents at C-10
(e.g., 10-methyl and 10-hydroxymethyl) exhibit equiva-
lent or increased biological activity relative to 4.36 Con-
sequently, we were interested in establishing the
properties of 3, an acyclic analogue of DDATHF bear-
ing a non-transferable C-10 formyl group.
(6R)-10-formyl-5,6,7,8-tetrahydrofolate (1) to transfer a
formyl group to the primary amine of its substrate, gly-
cinamide ribonucleotide (2a, GAR; Fig. 1). This one
carbon transfer constitutes the incorporation of the C-8
carbon of the purines and is the first of two formyl
transfer reactions. The second formyl transfer reaction
is catalyzed by aminoimidazole carboxamide ribonu-
cleotide transformylase (AICAR Tfase) which also
employs 1 to transfer a formyl group to the C-5 amine
of its substrate, aminoimidazole carboxamide ribonu-
cleotide (2b, AICAR; Fig. 1).1,24ꢀ27 Herein, we detail the
preparation and evaluation of 10-formyl-DDACTHF
(3) in our continued efforts to identify potent inhibitors
of GAR Tfase and AICAR Tfase.28
Chemistry
The synthesis of 10-formyl-DDACTHF (3) was accom-
plished in a convergent manner through alkylation43 of
the known N,N-dimethylhydrazone 628 with 1,3-dibro-
mopropane. LDA deprotonation of 6 (THF, ꢀ78 ꢁC,
30 min) and subsequent treatment with excess 1,3-
dibromopropane (10 equiv, HMPA, ꢀ78 ꢁC, 2 h, 52%)
provided the key intermediate 7 (Scheme 1). The pre-
formed sodium salt of ethyl cyanoacetate (NaH, DMF,
0 ꢁC, 30 min) was alkylated with 7 (DMF, 25 ꢁC, 2.5 h,
49%) providing 8. Cyclization with the free base of gua-
nidine (1.1 equiv, CH3OH, 25 ꢁC, 12 h, 52%) under basic
conditions gave the desired pyrimidine 9. Treatment of
9 with LiOH (3.0 equiv, 3:1 CH3OH–H2O, 25 ꢁC, 12 h,
88%) cleanly provided the carboxylic acid 10 which was
coupled with di-tert-butyl l-glutamate hydrochloride
(EDCI, NaHCO3, DMF, 25ꢁC, 12h) to provide 11. Sub-
sequent hydrolysis of the dimethylhydrazone was
Inhibitor Design
In previous studies, we examined aldehyde containing
folate-based inhibitors incapable of transferring the
formyl group.28 Thus, replacement of N10 with a
carbon atom prevents the transfer of the formyl
group from the cofactor analogue providing unique
opportunities for enzyme inhibition. This could entail
either competitive inhibition of the enzymes through
gem-diol binding of the aldehyde mimicking the for-
myl transfer tetrahedral intermediate or covalent trap
of the substrate at the active site to provide enzyme-
assembled tight binding inhibitors of GAR or AICAR
Tfase.28ꢀ32 Co-crystallization of GAR Tfase, b-GAR

T. H. Marsilje et al. / Bioorg. Med. Chem. 10 (2002) 2739–2749
2741
Scheme 2.
Scheme 3.
In efforts to establish the origin of the potent cytotoxic
activity of 3 and related compounds, the corresponding
g- and a-pentaglutamates of 3 and 15 were also pre-
pared. Whereas only g-polyglutamates have been found
in eurkaryotes, bacteria including Escherichia coli pro-
duce folate conjugates which contain two g and sub-
sequent
a glutamate linkages [i.e., pAB(g-Glu)2-
(a-Glu)n].46 To establish the importance of the nature of
the linkage, both the g- and a-pentaglutamates were
prepared. The carboxylic acid 10 was coupled with the
known free amine of the tert-butyl ester protected
g-pentaglutamate 1844 (EDCI, NaHCO3, DMF, 25 ꢁC,
12 h, 31%) to provide 19 (Scheme 4) as well as with the
known free amine of the tert-butyl ester protected
a-pentaglutamate 2345 (EDCI, NaHCO3, DMF, 25 ꢁC,
48 h, 22%) to provide 24 (Scheme 5). Subsequent
hydrolysis of the dimethylhydrazone in 19 was accom-
plished to generate the sensitive aldehyde 20 by treat-
ment with CuCl2 (5.0 equiv, 0 ꢁC, 1.5 h, 48%) in THF–
H2O buffered to pH 7. Subsequent deprotection of the
tert-butyl esters was accomplished by treatment with
trifluoroacetic acid (1:4 v/v TFA/CHCl3, 12 h, 100%) to
provide the 10-formyl-DDACTHF g-pentaglutamate
21. In a similar manner, hydrolysis of the dimethyl-
hydrazone in 24 was accomplished to generate the cor-
responding sensitive aldehyde by treatment with CuCl2
(5.0 equiv, 0 ꢁC, 1 h) in THF–H2O buffered to pH 7.
Subsequent deprotection of the tert-butyl esters was
accomplished by treatment with trifluoroacetic acid (1:4
v/v TFA/CHCl3, 12 h, 22% from 24) to provide the 10-
formyl-DDACTHF a-pentaglutamate 25. Similarly, the
N,N-dimethylhydrazones 19 and 24 were converted to
22 and 26, respectively, by acid-catalyzed deprotection
of the di-tert-butyl esters (1:4 v/v TFA/CHCl3, 12 h,
100%) for direct comparison (Schemes 4 and 5).
Scheme 1.
accomplished to provide the sensitive aldehyde 12 by
treatment with CuCl2 (5.0 equiv, 0 ꢁC, 1 h, 39%) in
THF–H2O buffered to pH 7. In addition to obtaining
aldehyde 12, the oxidative deformylation product 13
(21–44%, Scheme 2) was also obtained. Deprotection of
12 was accomplished by treatment with trifluoroacetic
acid (1:5 v/v TFA/CHCl3, 12 h, 89%) to provide
10-formyl-DDACTHF (3).
Acid-catalyzed deprotection of 13 by treatment with
trifluoroacetic acid (10 equiv, CHCl3, 12 h, 83%) pro-
vided 14 (Scheme 2).
In addition, the stable N,N-dimethylhydrazone 11 was
also converted to 15 by acid-catalyzed deprotection of
the di-tert-butyl esters by treatment with trifluoroacetic
acid (1:4 v/v TFA/CHCl3, 12 h, quantitative) (Scheme
1). For comparative purposes, the aldehyde 12 was
reduced to the alcohol 16 with NaBH4 (3.0 equiv,
CH3OH, 4 h, 88%) followed by deprotection of 16 with
trifluoroacetic acid (1:10 v/v TFA/CHCl3, 12 h, 98%) to
provide the known alcohol 1736 (Scheme 3).

2742
T. H. Marsilje et al. / Bioorg. Med. Chem. 10 (2002) 2739–2749
Scheme 5.
Scheme 4.
Table 1. GAR Tfase, AICAR Tfase, and DHFR inhibition (Ki, mM)a
GAR Tfase, AICAR Tfase, and DHFR inhibition
Compd
Ki GAR Tfase
Ki AICAR Tfase
Ki DHFR
Compounds 3, 9–12, 14, 15, 17, 21, 22, 25, and 26 were
tested initially for inhibition of E. coli GAR Tfase,
human AICAR Tfase, and E. coli DHFR, and the
results are presented in Table 1. With the exception of
11, all compounds demonstrate inhibition of GAR
Tfase within one order of magnitude Ki range
(1.9–48 mM). Compounds 3 and 12 were also found to
be effective inhibitors of AICAR Tfase with identical Ki
values of 1 mM, surprisingly comparable to the Ki’s
observed with GAR Tfase. This lack of potentiation by
the glutamate (3 vs 12) suggests that the enzyme inhibi-
tion properties are being dominated by the presence of
the aldehyde in 3 and 12 and we will return to this
unusual observation when we discuss human GAR
Tfase. A more conventional glutamate potentiation of
the dimethylhydrazone 15 (vs 11) was observed.
Although the GAR Tfase inhibition by 15 proved com-
parable to that of the aldehyde 3 (Ki=6 mM), 15 was
30-fold less effective than 3 against AICAR Tfase. Also
representative of this importance of the aldehyde in 3,
the corresponding alcohol 17 and the norketone 14 were
significantly less active against GAR Tfase and inactive
against AICAR Tfase. Similar observations have been
made with related series of aldehyde-based inhibitors
(CHO>HC=NNMe2>CH2OH>C¼O)28 with the
exception that the inhibition of GAR Tfase has gen-
erally been greater than AICAR Tfase. While this was
observed with 14, 15, and 17, the aldehyde 3 was found
to be slightly more potent against AICAR Tfase.
9
17
48
>100
5
>100
>100
>100
1
>100
>100
>100
>100
10
11
12
3
14
15
17
21
22
25
26
6
24
6
16
2.7
1.9
16
23
0.1
1
>100
28
>100
0.26
0.20
16
>100
>100
>100
>100
25
62
>200
>200
ndb
7.1
ndb
Lometrexol
aE. coli GAR Tfase, human AICAR Tfase, and E. coli DHFR.
bnd, not done.
higher binding affinity for E. coli GAR Tfase as com-
pared to the monoglutamate inhibitors. A similar mod-
est 4-fold increase in potency was observed with the
aldehyde g-pentaglutamate 21 versus 3 against AICAR
Tfase, whereas the dimethylhydrazone 22 exhibited a
much more substantial 140-fold increase relative to 15.
Moreover, both g-pentaglutamate derivatives exhibit an
ca. 10ꢂ higher binding affinity for AICAR Tfase than
GAR Tfase.
Since it has been shown previously that there are two
folylpolyglutamate synthetase activities (both a and g)
in E. coli,46 the a-pentaglutamate derivatives 25 and 26
were also synthesized and evaluated. The aldehyde
a-pentaglutamate 25 was 3–16ꢂ less potent than the
aldehyde monoglutamate 3 against GAR Tfase and
AICAR Tfase, respectively. Likewise, the dimethyl-
hydrazone a-pentaglutamate 26 was 4ꢂ less potent than
the hydrazone monoglutamate 15 against GAR Tfase,
whereas it was 4ꢂ more potent against AICAR Tfase
Interestingly, the aldehyde g-pentaglutamate 21 and
dimethylhydrazone g-pentaglutamate 22 did not exhibit
as large an increase in affinity for GAR Tfase as expected.
Both g-pentaglutamate derivatives only exhibit a 2–3ꢂ

T. H. Marsilje et al. / Bioorg. Med. Chem. 10 (2002) 2739–2749
Table 3. In vitro cytotoxic activity in the presence of AICAR
2743
Table 2. In vitro cytotoxic activity
Compd
CCRF-CEM (IC50, mM)
Compd
CCRF-CEM (IC50, mM)
(+) T, (+) Ha (ꢀ) T, (+) H (+) T, (ꢀ) H (ꢀ) T, (ꢀ) H
(ꢀ)T, (ꢀ) H, (+) T, (ꢀ) H, (ꢀ) T, (+) H, (ꢀ) T, (ꢀ) H,
(+) A
(ꢀ) Aa
(ꢀ) A
(ꢀ) A
9
225
>250
50
>250
>250
50
80
>250
50
90
>250
40
10
11
12
3
14
15
17
0.07
0.10
0.03
0.03
0.15
0.06
0.20
0.04
0.04
0.20
>150
>200
>200
>200
>200
>150
>200
>200
>200
>200
50
50
40
50
3
14
15
17
21
22
25
26
150
80
170
80
>200
160
>100
>100
60
>100
>250
0.06
0.07
Lometrexol
0.20
0.04
0.04
0.10
0.03
0.03
>200
>200
>100
>100
80
aT, thymidine; H, hypoxanthine; A, AICAR monophosphate.
>100
>100
>100
>100
Table 4. In vitro cytotoxic activity
9
7
0.20
7
6
0.15
>100
>250
(+) T, (+) Ha
(ꢀ) T, (+) H
(+) T, (ꢀ) H
(ꢀ) T, (ꢀ)H
Lometrexol
Compd
aT, thymidine; H, hypoxanthine.
CCRF-CEM/MTX (IC50, mM)
3
14
15
17
130
>200
nd
>200
nd
>200
140
nd
>200
>100
>200
>100
>200
>100
>200
>100
>200
>200
nd
>200
and both were 1–2 orders of magnitude less potent than
the corresponding g-pentaglutamate 22. Thus, the
g-pentaglutamates were notably more potent than the
a-pentaglutamates. However, with the exception of 22
versus AICAR Tfase, the g-pentaglutamates were not
significantly more potent enzyme inhibitors than the
corresponding monoglutamates. While interesting, this
behavior toward E. coli GAR Tfase proved not to be
consistent with the functional potency of the com-
pounds and we will return to this point later.
Lometrexol
CCRF-CEM/FPGSꢀ (IC50, mM)
3
14
15
17
>100
nd
nd
nd
nd
nd
nd
>100
>100
>100
55
>100
>100
25
nd
nd
nd
nd
Lometrexol
>100
>100
aT, thymidine; H, hypoxanthine.
15, or 17, the reversal of the cytotoxicity with hypox-
anthine (100 mM) resulted in a ꢃ103–104 change in the
IC50 value indicating that the activity was being observed
through selective inhibition of purine biosynthesis.
None of the compounds tested for inhibition of DHFR
exhibited activity, establishing a selectivity for GAR
Tfase and AICAR Tfase versus DHFR.
The aldehyde pentaglutamate derivatives 21 and 25 and
dimethylhydrazone pentaglutamate derivatives 22 and
26 exhibited little or no cytotoxic activity presumably
due to difficulty in traversing the cellular membrane.
Cytotoxic activity
Compounds 3, 9–12, 14, 15, 17, 21, 22, 25, and 26 were
examined for cytotoxic activity both in the presence (+)
and absence (ꢀ) of added hypoxanthine against the
CCRF-CEM cell line (Table 2). The cytotoxic activity
of the precursor agents (9–12) was relatively nonpotent
and uniform against the CCRF-CEM cell line regard-
less of whether the assay was conducted in the presence
or absence of a media purine (hypoxanthine) or pyr-
imidine (thymidine). In contrast, aldehyde 3, like
Lometrexol, exhibits no activity against the CCRF-
CEM cell line cultured in media supplemented with a
purine. However, both Lometrexol and aldehyde 3
exhibit potent cytotoxic activity (IC50=0.15 and 0.06–
0.07 mM, respectively) when purines are absent in the
media. This sensitivity to the presence of purines, but
not pyrimidines, indicates that the activity of aldehyde 3
is derived from its inhibition of enzymes in the de novo
purine biosynthetic pathway.
AICAR rescue experiments were performed using 3, 14,
15, and 17 in order to further elucidate the source of
their cytotoxic activity (Table 3). In each case, the
reversal or rescue of the cytotoxicity with hypoxanthine
(100 mM) or AICAR monophosphate (100 mM) resulted
in a ꢃ103–104 increase in the IC50 value. This indicates
that the activity is being observed through selective
inhibition of purine biosynthesis prior to the AICAR
Tfase enzymatic step, presumably through inhibition of
GAR Tfase. This selective sensitivity to GAR Tfase is
the expected behavior of the inhibitors 14, 15, and 17,
whereas the aldehyde 3 and the corresponding g-penta-
glutamates 21 (from 3) and 22 (from 15) would be
expected to be more effective or at least as effective at
acting on AICAR Tfase.
The extent to which the potent cytotoxic activity of 3,
14, 15, and 17 was dependent on reduced folate trans-
port across the cellular membrane was established by
assaying against a mutant CCRF-CEM cell line (CEM/
MTX) (Table 4). This cell line has been shown to have
an impaired reduced folate carrier.47 All the potent
inhibitors including 3 and 15 lost cytotoxic activity
against this mutant CCRF-CEM/MTX cell line
(IC50>100 mM), indicating that reduced folate carrier
transport is essential for their biological activity.
Significantly, the degradation product 14, obtained by
oxidative deformylation of 3, proved to be only slightly less
potent or roughly equivalent in potency (IC50=0.10 mM)
to 3 and it also exhibited the selective purine rescue.
Even more significantly, both the stable N,N-dimethyl-
hydrazone 15 and the alcohol 17 exhibited a purine
sensitive cytotoxic potency (IC50=0.03 and 0.03 mM,
respectively) that was at least as great or exceeded that
of the aldehyde 3 or ketone 14. In each case with 3, 14,

2744
T. H. Marsilje et al. / Bioorg. Med. Chem. 10 (2002) 2739–2749
Table 5. E. coli and rhGAR Tfase inhibition (Ki, mM)
(Ki=170 nM), 100-fold more potent than the corre-
sponding alcohol 17 (Ki=1.7 mM), and 1000-fold more
potent than the degradation ketone 14 (Ki=13 mM).
The g-pentaglutamates of 3 and 15 (21 and 22) were
roughly 3–4ꢂ more potent than the corresponding
a-pentaglutamates 25 and 26. Whereas the g-pentaglu-
tamate of aldehyde 3 did not enhance its remarkable
potency against rhGAR Tfase (3 vs 21), the g-penta-
glutamate of the hydrazone 15 did increase the potency
5-fold (15 vs 22). Most notably, this potency of the g-
pentaglutamates against rhGAR Tfase is comparable to
the cytotoxic potencies observed with 3 and 15 and
consistent with the studies indicating that their target is
GAR Tfase and not AICAR Tfase.
Compound
Ki E. coli GAR Tfase
Ki rhGAR Tfase
3 R=CHO
6
24
6
16
2.7
1.9
16
23
0.1
0.014
13
0.17
1.7
0.013
0.032
0.034
0.12
nd
14 R=O¼
15 R=CH¼NNMe2
17 R=CH2OH
21(gGlu5-3)
To our knowledge, this striking difference in the beha-
vior of the E. coli versus human GAR Tfase toward the
inhibitors represents the first such demonstration of an
unexpectedly selective inhibition of the human enzyme.
In total, there are 20 enzyme residues that constitute the
core of the folate binding site and 12 enlist their side
chains to stabilize folate binding. These 12 are identical
in the human and E. coli enzymes except for one con-
servative Leu-143 (E. coli) versus Val-143 (human) sub-
stitution. This has led to the expectation that little
inhibitor distinction between the E. coli and human
enzymes might be observed. The results with 3, which is
over 400-fold more potent against the human versus
E. coli enzyme, indicate that this need not be the case.
22 (gGlu5-15)
25 (aGlu5-3)
26 (aGlu5-15)
Lometrexol
Finally, the extent to which the potent cytotoxic com-
pounds were dependent upon polyglutamation was
established by assaying against a mutant CCRF-CEM
cell line (CEM/FPGSꢀ) that lacks folylpolyglutamate
synthase (FPGS)48 (Table 4). All the potent inhibitors
including 3 and 15 lost cytotoxic activity against this cell
line indicating that inhibitor polyglutamation is essen-
tial for their biological activity.
Human GAR Tfase inhibition
Conclusions
In the preceding studies, the four key inhibitors 3, 14,
15, and 17 exhibited exceptionally potent cytotoxic
activity (e.g., 3 IC50=60 nM) that proved sensitive to
media purines and AICAR, that required reduced folate
carrier transport into the cells, and that required poly-
glutamation. This level of functional potency surpassed
the enzyme inhibition activity approximately 100-fold
(Ki=6 mM for 3 vs E. coli GAR Tfase) suggesting that
the cytotoxic potency enhancement might rest with
intracellular accumulation of the inhibitors. However,
human AICAR Tfase was found to be more potently
inhibited by the g-pentaglutamates inconsistent with
GAR Tfase being the target suggesting that intracellular
accumulation by transport and polyglutamation might
be only part of the answer. Consequently, we examined
the inhibitors against recombinant human GAR Tfase
(rhGAR Tfase) and found a remarkable and unprece-
dented sensitivity to the aldehyde inhibitor 3.
A series of compounds were synthesized and evaluated
as potential inhibitors of GAR Tfase and AICAR
Tfase. Four compounds (3, 14, 15, and 17) were identi-
fied as having potent biological activity (IC50 values
ꢄ0.20 mM) in the absence of media purines, indicating
selective cytotoxicity through the inhibition of the
purine de novo biosynthetic pathway. Purine and
AICAR rescue experiments indicate that they exhibit
their potent cytotoxic activity specifically through
intracellular GAR Tfase inhibition even though none of
the compounds examined demonstrated sub-micro-
molar in vitro inhibition of E. coli GAR Tfase or
human AICAR Tfase.
Subsequent assays were performed in order to deter-
mine if polyglutamation and/or reduced folate carrier
transport were responsible for the significant increase in
cellular biological activity compared to in vitro enzy-
matic activity. The lack of cytotoxic activity of agents
(3, 14, 15, and 17) against CCRF-CEM cells with
impaired reduced folate active transport (CCRF-CEM/
MTX) indicates that these agents require the reduced
folate carrier for biological activity and their inactivity
against CCRF-CEM/FPGSꢀ lacking folylpolygluta-
mate synthase establishes that their polyglutamation is
also required for activity. The g-pentaglutamate deriva-
tives 21 and 22 demonstrated only marginal enhanced
binding affinity for E. coli GAR Tfase, and a more sig-
nificant 4ꢂ (21) and 140ꢂ (22) enhanced binding affinity
for human AICAR Tfase resulting in inhibitors with a
The results of the examination of 3, 14, 15, and 17 as
well as the corresponding a- and g-pentaglutamates of 3
and 15 (21, 22, 25 and 26) against recombinant human
GAR Tfase are summarized in Table 5 alongside the
results against E. coli GAR Tfase. Remarkably, the
inhibitors displayed trends against rhGAR Tfase that
might be expected of the original design. For the
monoglutamates, the aldehyde 3 proved to be an
exceptionally potent inhibitor of rhGAR Tfase
(Ki=14 nM) and it was approximately 10-fold more
potent than the corresponding dimethylhydrazone 15

T. H. Marsilje et al. / Bioorg. Med. Chem. 10 (2002) 2739–2749
2745
10ꢂ higher affinity for human AICAR Tfase over E.
coli GAR Tfase in vitro. These observations on the
pentaglutamates, while interesting, were inconsistent
with GAR Tfase as a primary site of action. Subsequent
examination of the inhibitors against human GAR
Tfase revealed that they and the corresponding g-penta-
glutamates were unexpectedly much more potent
against the human versus E. coli enzyme which also
contributes to their exceptional cytotoxic potency.
Methyl 4-[5-(2,4-diamino-6(1H)-pyrimidinon-5-yl)-1-(di-
methylhydrazono)-pent-2-yl]benzoate (9). A solution of
Na metal (0.035 g, 1.54 mmol, 2.2 equiv) in CH3OH
(0.87 mL) at 25 ꢁC was treated with guanidine hydro-
chloride (0.073 g, 0.769 mmol, 1.1 equiv). The solution
was stirred at 25 ꢁC for 30 min and then treated with a
solution of 8 (0.261 mg, 0.698 mmol) in CH3OH
(0.87 mL). The solution was stirred at 25 ꢁC for 12 h.
The excess NaOCH3 was neutralized by the addition of
HOAc (0.045 mL). Chromatography (SiO2, 3ꢂ15 cm,
10% CH3OH–CHCl3) provided 9 (0.140 g, 52%) as a
white solid: 1H NMR (CD3OD, 250 MHz) d 7.94
(d, J=8.4 Hz, 2H), 7.36 (d, J=8.4 Hz, 2H), 6.81 (d,
J=7.1 Hz, 1H), 3.88 (s, 3H), 3.59 (dd, J=14.7, 7.4 Hz,
1H), 2.70 (s, 6H), 2.30 (t, J=7.7 Hz, 2H), 1.94–1.78 (m,
2H), 1.55–1.29 (m, 2H); MALDIFTMS (DHB) m/z
409.1963 (M+Na+, C19H26N6O3 requires 409.1964).
Experimental
Methyl 4-[1-dimethylhydrazono-5-bromopent-2-yl]benzoate
(7).
A
solution of diisopropylamine (1.82 mL,
13.0 mmol, 1.5 equiv) in THF (13 mL) cooled to 0 ꢁC
was treated with n-BuLi (2.5 M in hexanes, 4.50 mL,
11.2 mmol, 1.3 equiv) and stirred at 0 ꢁC for 30 min and
at ꢀ78 ꢁC for 20 min. A solution of 6 (1.91 g, 8.65 mmol,
1.0 equiv) in THF (3.9 mL) was added dropwise and the
resulting solution was stirred at ꢀ78 ꢁC for 30 min. A
solution of 1,3-dibromopropane (8.79 mL, 86.5 mmol,
10.0 equiv) in HMPA (5.5 mL) was added and the mix-
ture was stirred at ꢀ78 ꢁC for 2 h. The reaction mixture
was quenched by the dropwise addition of saturated
aqueous NH4Cl (10 mL) and allowed to warm to 25 ꢁC.
The reaction mixture was diluted with EtOAc (200 mL)
and washed successively with H2O (2ꢂ50 mL) and
saturated aqueous NaCl (50 mL). The organic layer was
dried (Na2SO4), filtered, and concentrated under
reduced pressure. Chromatography (SiO2, 5ꢂ15 cm,
70% hexanes–EtOAc) provided 7 (1.55 g, 52%) as a
yellow oil: 1H NMR (CDCl3, 250 MHz) d 7.96
(d, J=8.8 Hz, 2H), 7.28 (d, J=8.4 Hz, 2H), 6.65 (d,
J=5.8 Hz, 1H), 3.89 (s, 3H), 3.58–3.49 (m, 1H), 3.38 (t,
J=6.2 Hz, 2H), 2.74 (s, 6H), 2.08–1.78 (m, 4H); MAL-
DIFTMS (DHB) m/z 341.0856 (M+H+, C15H21BrN2O2
requires 341.0859).
4-[5-(2,4-Diamino-6(1H)-pyrimidinon-5-yl)-1-(dimethyl-
hydrazono)pent-2-yl]benzoic acid (10). A solution of 9
(0.063 g, 0.163 mmol) in 3:1 CH3OH–H2O (1.63 mL)
was treated with LiOH–H2O (0.021 g, 0.489 mmol, 3.0
equiv) and the mixture was stirred at 25 ꢁC for 12 h. The
mixture was diluted with H2O (10 mL) and the aqueous
layer was washed with EtOAc (3ꢂ3 mL). The aqueous
layer was acidified to pH=4 by the addition of 1 M
aqueous HCl. The solution was concentrated under
reduced pressure and the residue was treated with tolu-
ene (3ꢂ5 mL) to remove traces of H2O to provide 10
(0.053 g, 88%): 1H NMR (CD3OD, 400 MHz) d 7.90 (d,
J=8.4 Hz, 2H), 7.28 (d, J=8.1 Hz, 2H), 6.83
(d, J=7.3 Hz, 1H), 3.51 (dd, J=15.0, 7.3 Hz, 1H), 2.31
(t, J=7.3 Hz, 2H), 1.92–1.82 (m, 2H), 1.52–1.28 (m,
2H); MALDIFTMS (DHB) m/z 395.1824 (M+Na+,
C18H24N6O3 requires 395.1824).
Di-tert-butyl N-{4-[5-(2,4-diamino-6(1H)-pyrimidinon-
5-yl)-1-(dimethylhydrazono)pent-2-yl]benzoyl}-L-glutamate
(11). A solution of 10 (0.029 g, 0.078 mmol) and di-tert-
butyl l-glutamate hydrochloride (0.034 g, 0.117 mmol,
1.5 equiv) in DMF (0.31 mL) was treated with NaHCO3
(0.020 g, 0.234 mmol, 3.0 equiv) followed by EDCI
(0.045 g, 0.234 mmol, 3.0 equiv). The reaction mixture
was stirred at 25 ꢁC for 12 h before the solvent was
removed under reduced pressure. The residue was dis-
solved in CHCl3 (5 mL) and extracted with saturated
aqueous NaHCO3 (2 mL). The organic layer was dried
(Na2SO4), filtered, and concentrated under reduced
pressure. PCTLC (SiO2, 2 mm plate, 10% CH3OH–
Methyl
4-[6-carboethoxy-6-cyano-1-(dimethylhydrazo-
no)hex-2-yl]benzoate (8). A suspension of NaH (60%
dispersion, 0.211 g, 5.28 mmol, 1.2 equiv) in anhydrous
DMF (25 mL) at 0 ꢁC was treated dropwise with ethyl
cyanoacetate (0.61 mL, 5.7 mmol, 1.3 equiv). The solu-
tion was stirred at 0 ꢁC for 30 min, forming the sodium
salt as a clear solution. This solution was treated with a
solution of 7 (1.50 g, 4.40 mmol) in anhydrous DMF
(10 mL). The resulting reaction mixture was stirred at
25 ꢁC for 2.5 h. The reaction mixture was diluted with
EtOAc (200 mL) and washed successively with H2O
(3ꢂ50 mL) and saturated aqueous NaCl (50 mL). The
organic layer was dried (Na2SO4), filtered, and con-
centrated under reduced pressure. Chromatography
(SiO2, 4ꢂ15 cm, 60% hexanes–EtOAc) provided 8
(0.798 g, 49%) as a yellow oil: 1H NMR (CDCl3,
250 MHz) d 7.97 (d, J=8.0 Hz, 2H), 7.28 (d, J=8.1 Hz,
2H), 6.61–6.55 (m, 1H), 4.23 (q, J=7.0 Hz, 2H), 3.89 (s,
3H), 3.58–3.42 (m, 2H), 2.74 (s, 6H), 2.01–1.70 (m, 4H),
1.61–1.42 (m, 2H), 1.26 (t, J=7.1 Hz, 3H); MAL-
DIFTMS (DHB) m/z 374.2066 (M+H+, C20H27N3O4
requires 374.2074).
1
CHCl3) provided 11 (0.017 g, 36%) as a white solid: H
NMR (CD3OD, 250 MHz) d 7.78 (d, J=8.4 Hz, 2H),
7.27 (d, J=8.4 Hz, 2H), 6.82 (d, J=7.1 Hz, 1H), 4.51–
4.45 (m, 1H), 3.53 (dd, J=14.4, 7.0 Hz, 1H), 2.71 (s,
6H), 2.42–2.12 (m, 5H), 2.08–1.89 (m, 5H), 1.48 (s, 9H),
1.43 (s, 9H); MALDIFTMS (DHB) m/z 614.3678
(M+H+, C31H47N7O6 requires 614.3666).
Di-tert-butyl N-{4-[4-(2,4-diamino-6(1H)-pyrimidinon-
5-yl)-1-formyl-but-2-yl]benzoyl}-L-glutamate (12).
solution of 11 (30 mg, 0.049 mmol) in THF (0.9 mL) and
A
pH 7 aqueous phosphate buffer (0.02 mL) cooled to 0 ꢁC
was treated with a solution of CuCl2 (33 mg,
0.244 mmol, 5.0 equiv) in H2O (0.3 mL). The solution

2746
T. H. Marsilje et al. / Bioorg. Med. Chem. 10 (2002) 2739–2749
was stirred at 0 ꢁC for 1 h before it was quenched by the
dropwise addition of a pH 8 saturated aqueous NH4Cl–
NH4OH solution (20 mL). The solution was extracted
with CHCl3 (3ꢂ20 mL), purged with N2, dried
(Na2SO4), filtered, and concentrated under reduced
pressure. PCTLC (SiO2, 1 mm plate, 20% CH3OH–
CHCl3) provided 12 (11 mg, 39%; typically 21–44%) as
a white solid: 1H NMR (DMSO-d6, 400 MHz) d 9.69 (s,
1H), 9.65 (s, 1H), 8.56 (d, J=7.3 Hz, 1H), 7.84 (d,
J=8.1 Hz, 2H), 7.33 (d, J=8.1 Hz, 2H), 5.86 (br s, 2H),
5.60 (br s, 2H), 4.34–4.30 (m, 1H), 3.78–3.70 (m, 1H),
2.33 (t, J=6.6 Hz, 2H), 2.17 (t, J=7.0 Hz, 2H), 2.04–
2.01 (m, 3H), 1.93–1.88 (m, 2H), 1.69–1.62 (m, 1H), 1.41
(s, 9H), 1.39 (s, 9H); MALDIFTMS (DHB) m/z
594.2904 (M+Na+, C29H41N5O7 requires 594.2904).
solution of 11 (7.5 mg, 0.0122 mmol) in CHCl3
(0.20 mL) cooled to 0 ꢁC was treated with trifluoroacetic
acid (0.05 mL). The solution was stirred at 0 ꢁC for 2 h
and 25 ꢁC for 12 h. The reaction was concentrated under
reduced pressure. The product was triturated with Et2O
(1ꢂ1 mL) and dried in vacuo to give 15-CF3CO2H
1
(7.5 mg, 100%) as a white solid: H NMR (CD3OD,
400 MHz) d 7.83 (d, J=8.2 Hz, 2H), 7.37 (d, J=8.2 Hz,
2H), 4.68–4.55 (m, 1H), 3.68–3.61 (m, 1H), 2.87 (s, 6H),
2.85 (t, J=7.4 Hz, 2H) 2.49 (t, J=7.7 Hz, 2H), 2.40–
2.30 (m, 2H), 2.26–1.90 (m, 4H); MALDIFTMS (DHB)
m/z 524.2248 (M+Na+, C23H31N7O6 requires
524.2233).
Di-tert-butyl N-{4-[5-(2,4-diamino-6(1H)-pyrimidinon-5-
A
yl)-1-hydroxypent-2-yl]benzoyl}-L-glutamate (16).
Di-tert-butyl N-{4-[4-(2,4-diamino-6(1H)-pyrimidinon-5-
yl)-1-oxo-but-1-yl]benzoyl}-L-glutamate (13). Obtained
as the higher Rf spot from the reaction that provided 12.
PCTLC (SiO2, 1 mm plate, 20% CH3OH–CHCl3) pro-
vided 13 (12 mg, 44%) as a white solid: 1H NMR
(DMSO-d6, 400 MHz) d 9.75 (s, 1H), 8.79 (d, J=7.6 Hz,
1H), 8.02 (d, J=8.5 Hz, 2H), 7.96 (d, J=8.5 Hz, 2H),
5.92 (br s, 2H), 5.76 (br s, 1H), 4.37–4.31 (m, 1H), 3.02
(t, J=7.3 Hz, 2H), 2.35 (t, J=7.0 Hz, 2H), 2.23 (t,
J=7.0 Hz, 2H), 2.07–2.00 (m, 1H), 1.97–1.88 (m, 1H),
1.73–1.59 (m, 2H), 1.41 (s, 9H), 1.39 (s, 9H); MAL-
DIFTMS (DHB) m/z 580.2730 (M+Na+, C28H39N5O7
requires 580.2747).
solution of 12 (6.1 mg, 0.0107 mmol) in CH3OH
(0.11 mL) at 0 ꢁC was treated with NaBH4 (1.2 mg,
0.032 mmol, 3.0 equiv). The solution was stirred at 0 ꢁC
for 2 h and 25 ꢁC for 2 h before the solvent was removed
under reduced pressure. The residue was diluted with
CHCl3 (2 mL) and washed successively with saturated
aqueous NH4Cl (1 mL) and saturated aqueous NaCl
(1 mL), dried (Na2SO4), filtered, and concentrated
under reduced pressure. PCTLC (SiO2, 1 mm plate, 8%
CH3OH–CHCl3) provided 16 (5.4 mg, 88%) as a white
1
solid: H NMR (DMSO-d6, 400 MHz) d 9.68 (s, 1H),
8.50 (d, J=7.6 Hz, 1H), 7.75 (d, J=7.9 Hz, 2H), 7.26 (d,
J=8.2 Hz, 2H), 5.86 (br s, 2H), 5.57 (br s, 2H), 4.58 (d,
J=5.0 Hz, 1H), 4.34–4.28 (m, 1H), 3.04–2.95 (m, 2H),
2.71–2.66 (m, 1H), 2.32 (t, J=7.4 Hz, 2H), 2.10 (t,
J=7.0 Hz, 2H), 2.04–1.97 (m, 1H), 1.95–1.87 (m, 1H),
1.76–1.69 (m, 2H), 1.40 (s, 9H), 1.38 (s, 9H); 1.17–1.09
(m, 2H); MALDIFTMS (DHB) m/z 574.3263 (M+H+,
C29H43N5O7 requires 574.3241).
N-{4-[4-(2,4-Diamino-6(1H)-pyrimidinon-5-yl)-1-formyl-
but-2-yl]benzoyl}-L-glutamic acid (3). A solution of 12
(2.9 mg, 0.0051 mmol) in CHCl3 (0.20 mL) cooled to
0 ꢁC was treated with trifluoroacetic acid (0.04 mL). The
solution was stirred at 0 ꢁC for 2 h and 25 ꢁC for 12 h.
Et2O (1 mL) was added and a precipitate formed. The
precipitate was triturated with Et2O (3ꢂ1 mL) and dried
in vacuo to give 3-CF3CO2H (2.6 mg, 89%) as a tan
solid: 1H NMR (CD3OD, 400 MHz) d 7.91 (d,
J=8.4 Hz, 2H), 7.52 (d, J=8.4 Hz, 2H), 4.70–4.59 (m,
2H), 4.45–4.38 (m, 1H), 2.51–2.43 (m, 2H), 2.38–2.25
(m, 2H), 2.19–1.95 (m, 3H), 1.94–1.79 (m, 1H); MAL-
DIFTMS (DHB) m/z 482.1652 (M+Na+, C21H25N5O7
requires 482.1670).
N-{4-[5-(2,4-Diamino-6(1H)-pyrimidinon-5-yl)-1-hydroxy-
pent-2-yl]benzoyl}-L-glutamic acid (17). A solution of 16
(4.2 mg, 0.0073 mmol) in CHCl3 (0.20 mL) cooled to
0 ꢁC was treated with trifluoroacetic acid (0.02 mL). The
solution was stirred at 0 ꢁC for 2 h and 25 ꢁC for 12 h.
Et2O (1 mL) was added and a precipitate formed. The
precipitate was triturated with Et2O (3ꢂ1 mL) and dried
in vacuo to give 17-CF3CO2H (4.1 mg, 98%) as a white
solid: 1H NMR (DMSO-d6, 400 MHz) d 9.72 (br s, 1H),
8.59 (d, J=7.9 Hz, 1H), 7.80 (d, J=8.2 Hz, 2H), 7.36 (d,
J=8.2 Hz, 2H), 6.04 (br s, 2H), 5.67 (br s, 1H), 4.56 (d,
J=6.2 Hz, 1H), 4.41–4.35 (m, 1H), 3.16–3.10 (m, 1H),
3.05–2.97 (m, 1H), 2.76 (t, J=7.3 Hz, 2H), 2.14–2.06
(m, 3H), 1.96–1.89 (m, 1H), 1.75–1.58 (m, 2H), 1.23–
1.13 (m, 2H); MALDIFTMS (DHB) m/z 484.1824
(M+Na+, C21H27N5O7 requires 484.1808).
N-{4-[4-(2,4-Diamino-6(1H)-pyrimidinon-5-yl)-1-oxo-but-
1-yl]benzoyl}-L-glutamic acid (14). A solution of 13
(3.3 mg, 0.0059 mmol) in CHCl3 (0.12 mL) cooled to
0 ꢁC was treated with trifluoroacetic acid (0.01 mL). The
solution was stirred at 0 ꢁC for 2 h and 25 ꢁC for 12 h.
Et2O (1 mL) was added and a precipitate formed. The
precipitate was triturated with Et2O (3ꢂ1 mL) and dried
in vacuo to give 14-CF3CO2H (2.7 mg, 83%) as a white
solid: 1H NMR (DMSO-d6, 400 MHz) d 8.81 (d,
J=7.6 Hz, 1H), 8.01 (d, J=8.8 Hz, 2H), 7.98 (d,
J=8.8 Hz, 2H), 4.44–4.37 (m, 1H), 3.05 (t, J=7.0 Hz,
2H), 2.36 (t, J=7.4 Hz, 2H), 2.30 (t, J=7.4 Hz, 2H),
2.13–2.06 (m, 1H), 2.00–1.87 (m, 2H), 1.72–1.66 (m,
1H); MALDIFTMS (DHB) m/z 446.1674 (M+H+,
C20H23N5O7 requires 446.1676).
N-{4-[5-(2,4-Diamino-6(1H)-pyrimidinon-5-yl)-1-(dimethyl-
hydrazono)pent-2-yl]benzoyl}-L-ꢀ-glutamyl-L-ꢀ-glutamyl-
L-ꢀ-glutamyl-L-ꢀ-glutamyl-L-ꢀ-glutamic acid hexa-tert-
butyl ester (19). A solution of 10 (90 mg, 0.24 mmol)
and 1844 (242 mg, 0.24 mmol, 1.0 equiv) in anhydrous
DMF (1.0 mL) was treated with NaHCO3 (61 mg,
0.73 mmol, 3.0 equiv) followed by EDCI (139 mg,
0.73 mmol, 3.0 equiv) and stirred at 25 ꢁC for 12 h. The
reaction mixture was diluted with EtOAc (50 mL) and
washed with saturated aqueous NaHCO3 (2ꢂ20 mL)
N-{4-[5-(2,4-Diamino-6(1H)-pyrimidinon-5-yl)-1-(dimethyl-
hydrazono)pent-2-yl]benzoyl}-L-glutamic acid (15). A

T. H. Marsilje et al. / Bioorg. Med. Chem. 10 (2002) 2739–2749
2747
followed by saturated aqueous NaCl (20 mL). The
organic layer was dried (Na2SO4), filtered, and con-
centrated under reduced pressure. Chromatography
(SiO2, 3ꢂ15 cm, 10% CH3OH–CHCl3) provided 19
(102 mg, 31%) as a white solid: 1H NMR (CD3OD,
400 MHz) d 7.83 (d, J=8.2 Hz, 2H), 7.36 (d, J=8.2,
2H), 6.82 (d, J=7.0 Hz, 1H), 4.58–4.49 (m, 1H), 4.40–
4.25 (m, 4H), 3.57–3.51 (m, 1H), 2.70 (s, 6H), 2.45–1.72
(m, 26H), 1.51–1.41 (m, 54H); MALDIFTMS (DHB)
m/z 1354.7844 (M+H+, C67H107N11O18 requires
1354.7868).
N-{4-[5-(2,4-Diamino-6(1H)-pyrimidinon-5-yl)-1-(dimethyl-
hydrazono)pent-2-yl]benzoyl}-L-ꢁ-glutamyl-L-ꢁ-glutamyl-
L-ꢁ-glutamyl-L-ꢁ-glutamyl-L-ꢁ-glutamic acid hexa-tert-
butyl ester (24). A solution of 10 (7.5 mg, 0.020 mmol)
and 2345 (20.2 mg, 0.020 mmol, 1.0 equiv) in DMF
(0.1 mL) was treated with NaHCO3 (5.1 mg,
0.060 mmol, 3.0 equiv) followed by EDCI (11.6 mg,
0.060 mmol, 3.0 equiv) and stirred at 25 ꢁC for 48 h. The
reaction was diluted with EtOAc (20 mL) and washed
with saturated aqueous NaHCO3 (5 mL). The organic
layer was dried (Na2SO4), filtered, and concentrated
under reduced pressure. PCTLC (SiO2, 2 mm plate,
10% CH3OH–CHCl3) provided 24 (6.0 mg, 22%) as a
N-{4-[4-(2,4-Diamino-6(1H)-pyrimidinon-5-yl)-1-formyl-
but-2-yl]benzoyl}-L-ꢀ-glutamyl-L-ꢀ-glutamyl-L-ꢀ-gluta-
myl-L-ꢀ-glutamyl-L-ꢀ-glutamic acid hexa-tert-butyl ester
(20). A solution of 19 (41 mg, 0.030 mmol) in THF
1
white solid: H NMR (CD3OD, 400 MHz) d 7.86 (d,
J=8.5 Hz, 2H), 7.36 (d, J=8.2 Hz, 2H), 6.80 (d,
J=4.4 Hz, 1H), 4.49–4.30 (m, 5H), 4.08–4.00 (m, 1H),
2.70 (s, 6H), 2.50–2.31 (m, 12H), 2.21–1.83 (m, 14H),
1.49–1.38 (m, 54H); MALDIFTMS (DHB) m/z
(0.43 mL) and pH
7 aqueous phosphate buffer
(0.09 mL) cooled to 0 ꢁC was treated with a solution of
CuCl2 (20.4 mg, 0.15 mmol, 5.0 equiv) in H2O
(0.15 mL). The solution was stirred at 0 ꢁC for 1.5 h
before it was quenched by the dropwise addition of a
pH 8 saturated aqueous NH4Cl–NH4OH solution
(5 mL). The solution was extracted with CHCl3
(3ꢂ10 mL), purged with N2, dried (Na2SO4), filtered,
and concentrated under reduced pressure. PCTLC
(SiO2, 1 mm plate, 8% CH3OH–CHCl3) provided 20
(19 mg, 48%) as a white solid: 1H NMR (CD3OD,
400 MHz) d 7.79 (d, J=7.6 Hz, 2H), 7.36 (d, J=7.9 Hz,
2H), 4.63–4.51 (m, 2H), 4.38–4.30 (m, 5H), 2.49–1.70
(m, 26H), 1.49–1.41 (m, 54H); MALDIFTMS (DHB)
m/z 1312.7312 (M+H+, C65H101N9O19 requires
1312.7286).
1376.7730
1376.7687).
(M+Na+,
C67H107N11O18
requires
N-{4-[5-(2,4-Diamino-6(1H)-pyrimidinon-5-yl)-1-(dimethyl-
hydrazono)pent-2-yl]benzoyl}-L-ꢁ-glutamyl-L-ꢁ-glutamyl-
L-ꢁ-glutamyl-L-ꢁ-glutamyl-L-ꢁ-glutamic acid (25). A
solution of 24 (19 mg, 0.014 mmol) in THF (0.2 mL) and
pH 7 aqueous phosphate buffer (0.04 mL) cooled to 0 ꢁC
was treated with
a solution of CuCl2 (9.4 mg,
0.070 mmol, 5.0 equiv) in H2O (0.07 mL). The solution
was stirred at 0 ꢁC for 1 h before it was quenched by the
dropwise addition of a pH 8 saturated aqueous NH4Cl–
NH4OH solution (5 mL). The solution was extracted
with CHCl3 (3ꢂ5 mL), purged with N2, dried (Na2SO4),
filtered, and concentrated under reduced pressure.
PCTLC (SiO2, 1 mm plate, 8% CH3OH–CHCl3)
removed baseline impurities. The isolated product was
dissolved in CHCl3 (1.00 mL), cooled to 0 ꢁC and trea-
ted with trifluoroacetic acid (0.25 mL). The solution was
stirred at 0 ꢁC for 2 h and 25 ꢁC for 12 h. The solution
was concentrated under reduced pressure. The solid
residue was triturated with Et2O (3ꢂ5 mL) and dried in
vacuo to give 25-CF3CO2H (3.4 mg, 22% over two steps
from 24) as a tan solid: 1H NMR (CD3OD, 400 MHz) d
7.84 (d, J=8.5 Hz, 2H), 7.48 (d, J=8.5 Hz, 2H), 4.54–
4.32 (m, 5H), 2.78–2.70 (m, 2H), 2.55–2.36 (m, 14H),
2.28–2.10 (m, 5H), 2.09–1.92 (m, 5H); MALDIFTMS
(DHB) m/z 976.3517 (M+H+, C41H53N9O19 requires
976.3530).
N-{4-[5-(2,4-Diamino-6(1H)-pyrimidinon-5-yl)-1-(dimethyl-
hydrazono)pent-2-yl]benzoyl}-L-ꢀ-glutamyl-L-ꢀ-glutamyl-
L-ꢀ-glutamyl-L-ꢀ-glutamyl-L-ꢀ-glutamic acid (22). A
solution of 19 (25 mg, 0.019 mmol) in CHCl3 (1.00 mL)
cooled to 0 ꢁC was treated with trifluoroacetic acid
(0.25 mL). The solution was stirred at 0 ꢁC for 2 h and
25 ꢁC for 12 h. The solution was concentrated under
reduced pressure. The solid residue was triturated with
Et2O (3ꢂ5 mL) and dried in vacuo to give 22-CF3CO2H
(21 mg, 100%) as a tan solid: 1H NMR (CD3OD,
400 MHz) d 7.85 (d, J=7.4 Hz, 2H), 7.41 (bs, 1H), 7.36
(d, J=8.2 Hz, 2H), 4.68–4.60 (m, 1H), 4.48–4.39 (m,
5H), 2.88 (s, 6H), 2.51–1.82 (m, 26H); MALDIFTMS
(DHB) m/z 1018.4102 (M+H+, C43H59N11O18 requires
1018.4112).
N-{4-[5-(2,4-Diamino-6(1H)-pyrimidinon-5-yl)-1-formyl-
but-2-yl]benzoyl}-L-ꢁ-glutamyl-L-ꢁ-glutamyl-L-ꢁ-gluta-
myl-L-ꢁ-glutamyl-L-ꢁ-glutamic acid (26). A solution of
24 (5.4 mg, 0.0040 mmol) in CHCl3 (1.00 mL) cooled to
0 ꢁC was treated with trifluoroacetic acid (0.25 mL). The
solution was stirred at 0 ꢁC for 2 h and 25 ꢁC for 12 h.
The solution was concentrated under reduced pressure.
The solid residue was triturated with Et2O (3ꢂ5 mL)
and dried in vacuo to give 26-CF3CO2H (4.5 mg, 100%)
N-{4-[5-(2,4-Diamino-6(1H)-pyrimidinon-5-yl)-1-formyl-
but-2-yl]benzoyl}-L-ꢀ-glutamyl-L-ꢀ-glutamyl-L-ꢀ-glutamyl-
L-ꢀ-glutamyl-L-ꢀ-glutamic acid (21). A solution of 20
(16 mg, 0.012 mmol) in CHCl3 (1.00 mL) cooled to 0 ꢁC
was treated with trifluoroacetic acid (0.25 mL). The
solution was stirred at 0 ꢁC for 2 h and 25 ꢁC for 12 h.
The solution was concentrated under reduced pressure.
The solid residue was triturated with Et2O (3ꢂ5 mL)
and dried in vacuo to give 21-CF3CO2H (13 mg, 100%)
1
as a tan solid: H NMR (CD3OD, 400 MHz) d 7.85 (d,
1
as a tan solid: H NMR (CD3OD, 400 MHz) d 7.86 (d,
J=8.5 Hz, 2H), 7.35 (d, J=8.5 Hz, 2H), 7.18–7.14 (m,
1H), 4.52–4.33 (m, 5H), 4.09–4.02 (m, 1H), 2.79 (s, 6H),
2.52–2.38 (m, 16H), 2.23–2.08 (m, 5H), 2.05–1.92 (m,
5H); MALDIFTMS (DHB) m/z 1018.4157 (M+H+,
C43H59N11O18 requires 1018.4112).
J=8.2 Hz, 2H), 7.50 (d, J=8.5 Hz, 2H), 4.69–4.61 (m,
2H), 4.48–4.40 (m, 5H), 2.52–1.83 (m, 26H); MAL-
DIFTMS (DHB) m/z 976.3570 (M+H+, C41H53N9O19
requires 976.3530).

2748
T. H. Marsilje et al. / Bioorg. Med. Chem. 10 (2002) 2739–2749
GAR Tfase, AICAR Tfase, and DHFR inhibition
16. Taylor, E. C.; Wong, G. S. K.; Fletcher, S. R.; Harring-
ton, P. J.; Beardsley, G. P.; Shih, C. J. In Chemistry and Biol-
ogy of Pteridines; Cooper, B. A., Whitehead, V. M., Eds.;
Walter de Gruyter: Berlin, 1986; p 61.
17. Beardsley, G. P.; Moroson, B. A.; Taylor, E. C.; Moran,
R. G. J. Biol. Chem. 1989, 264, 328.
18. Moran, R. G.; Baldwin, S. W.; Taylor, E. C.; Shih, C.
J. Biol. Chem. 1989, 264, 21047.
19. Taylor, E. C.; Wong, G. S. K. J. Org. Chem. 1989, 54,
3618. Taylor, E. C.; Harrington, P. M.; Warner, J. C. Hetero-
cycles 1988, 27, 1925. Taylor, E. C.; Harrington, P. M. J. Org.
Chem. 1990, 55, 3222.
GAR and AICAR Tfase inhibition studies were con-
ducted as previously detailed28 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 cofac-
tor. The DHFR inhibition study was conducted as pre-
viously detailed49 with 10 nM enzyme, 30 mM H2F,
100 mM NADPH and 30 mM inhibitor.
20. Barnett, C. J.; Wilson, T. M.; Wendel, S. R.; Winning-
ham, M. J.; Deeter, J. B. J. Org. Chem. 1994, 59, 7038.
21. Taylor, E. C. J. Heterocycl. Chem. 1990, 27, 1.
Acknowledgements
22. Baldwin, S. W.; Tse, A.; Gossett, L. S.; Taylor, E. C.;
Rosowsky, A.; Shih, C.; Moran, R. G. Biochemistry 1991, 30,
1997.
We gratefully acknowledge the financial support of the
National Institute of Heath (CA 63536) and The Skaggs
Institute for Chemical Biology. We gratefully thank Dr.
Gerrit Jansen (University Hospital Utrecht, The Neth-
erlands) for kindly supplying the CCRF-CEM/MTX
cell line for the cytotoxicity studies.
23. For related analogues and studies, see: Caperelli, C. A.
J. Med. Chem. 1987, 30, 1254. Taylor, E. C.; Hamby, J. M.;
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33, 561. Taylor, E. C.; Gillespie, P.; Patel, M. J. Org. Chem.
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Tetrahedron 1992, 48, 19. Bigham, E. C.; Hodson, S. J.; Mal-
lory, W. R.; Wilson, D.; Duch, D. S.; Smith, G. K.; Ferone, R.
J. Med. Chem. 1992, 35, 1399. Taylor, E. C.; Kuhnt, D.; Shih,
C.; Rinzel, S. M.; Grindey, G. B.; Barredo, J.; Jannatipour,
M.; Moran, R. G. J. Med. Chem. 1992, 35, 4450. Piper, J. R.;
Johnson, C. A.; Otter, G. M.; Sirotnak, F. M. J. Med. Chem.
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