Conformationally restricted analogues designed for selective inhibition of GAR Tfase versus thymidylate synthase or dihydrofolate reductase

Article abstract of DOI:10.1016/S0968-0896(00)00051-1

The synthesis and evaluation of a series of conformationally restricted analogues of 10-formyl-tetrahydrofolate as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) or aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00051-1

Bioorganic & Medicinal Chemistry 8 (2000) 1075±1086
Conformationally Restricted Analogues Designed for Selective
Inhibition of GAR Tfase Versus Thymidylate Synthase or
Dihydrofolate Reductase
Dale L. Boger, a,* Marc A. Labroli, a Thomas H. Marsilje, a Qing Jin, a Michael P. Hedrick, a
Stephen J. Baker, b Jae Hoon Shim b and Stephen J. Benkovic b
aDepartment of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
bDepartment of Chemistry, Pennsylvania State University, University Park, PA 16802, USA
Received 29 September 1999; accepted 10 January 2000
AbstractÐThe synthesis and evaluation of a series of conformationally restricted analogues of 10-formyl-tetrahydrofolate as
potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) or aminoimidazole carboxamide transformylase
(AICAR Tfase) are reported. # 2000 Elsevier Science Ltd. All rights reserved.
Inhibitor design
Glycinamide ribonucleotide transformylase (GAR
Tfase) is an enzyme central to de novo purine
biosynthesis.1 12 Since purines play a crucial role as
required components of DNA and RNA, inhibition of
enzymes in the purine biosynthetic pathway has been
pursued as an e€ective approach for antineoplastic
intervention.13 The disclosure that (6R)-5,10-dideazate-
trahydrofolate (Lometrexol, DDATHF) is an eca-
cious antitumor agent that acts as an e€ective inhibitor
of GAR Tfase (Ki=0.1 mM) established inhibition of
purine biosynthesis and GAR Tfase as viable targets for
antineoplastic intervention.14 23 GAR Tfase uses (6R)-
10-formyl-tetrahydrofolate (1) to transfer a formyl
group to the primary amine of its substrate, glycinamide
ribonucleotide (2a, GAR; Fig. 1). This one carbon
transfer constitutes the incorporation of the C-8 carbon
of the purines and is the ®rst of two formyl transfer
reactions. The second formyl transfer reaction is cata-
lyzed by the enzyme aminoimidazole carboxamide
ribonucleotide transformylase (AICAR Tfase) which
also employs (6R)-10-formyl-tetrahydrofolate (1) to
transfer a formyl group to the C-5 amine of its sub-
strate, aminoimidazole carboxamide ribonucleotide (2b,
AICAR; Fig. 1).1,24 27
The X-ray crystal structure28 of 5-DATHF (5-deazate-
trahydrofolic acid) complexed with GAR Tfase indicated
that it adopts a bound L-shaped conformation with an
axial C6±C9 bond that is perpendicular to the bicyclic
ring with an extended or trans (169ꢀ) versus gauche (50 to
69ꢀ) C5±C6±C9±N10 dihedral angle characteristic of
bound TS or DHFR cofactor and inhibitors (Fig. 2).29
An identical conformation was observed with the GAR
Tfase bound inhibitor 1476U8929 and the analogous
dihedral angles of the two bound molecules in the unit
cell were found to be 166 and 174ꢀ. Thus, a folate inhi-
bitor designed to lock this trans conformation has the
potential for speci®city against GAR Tfase.28
Based on these observations, two classes of con-
formationally restricted inhibitors that embody these
characteristics were designed for examination. These
classes of conformationally restricted inhibitors are
represented by 3 and 4 incorporating an acyclic folate
core (Fig. 3). Mimics of the more stable trans versus cis
amide29b are constrained to the GAR Tfase bound trans
orientation (180ꢀ) by their incorporation into an aro-
matic pyrrole or pyridine ring where the basic nitrogen
may serve to accept an active site H-bond (protonation)
from the active site His-108.29c The atoms corresponding
to the X5±C6±C9±X10 dihedral angle are locked in the
key trans orientation (180ꢀ) by virtue of incorporation
into the pyrrole or pyridine ring.
*Corresponding author. Tel.: +1-858-784-7522; fax: +1-858-784-
7550; e-mail: boger@scripps.edu
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00051-1

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D. L. Boger et al. / Bioorg. Med. Chem. 8 (2000) 1075±1086
observed at d 5.08 and 5.28 for 7 and its isomer,
respectively, in agreement with those observed for N-
benzyl 4- and 5-hydroxymethylimidazole (d 5.13 and
5.30, respectively).30 The hydroxyl group of 7 was
cleanly converted to the chloride 8 on warming with
thionyl chloride (10.0 equiv, CH2Cl2, 40 ꢀC, 3 h, 89%).
Alkylation of the potassium salt of ethyl cyanoacetate
(9) with 8 (K2CO3, DMF, 70 ꢀC, 3 h, 76%) a€orded 10.
Cyclization with the free base of guanidine (1.0 equiv,
EtOH, 25 ꢀC, 3 h, 62%) gave the desired pyrimidine 11.
Treatment of 11 with LiOH (5.0 equiv, 3:1 MeOH±
H2O, 60 ꢀC, 84%) cleanly provided the carboxylic acid
12 which was coupled with di-tert-butyl-l-glutamate
hydrochloride (13, EDCI, NaHCO3, DMF, 54%) to
provide 14. Deprotection of 14 was accomplished by
treatment with tri¯uoroacetic acid (10.0 equiv, CH2Cl2,
82%) to provide the desired conformationally restricted
analogue 3a.
The synthesis of 4a was accomplished in a similar man-
ner (Scheme 2). The preparation of 15 was accomplished
in three steps starting from commercially available 5-
bromonicotinic acid according to published procedure.31
Palladium(0)-catalyzed Suzuki coupling of 15 with com-
mercially available methyl 5-bromobenzoate 16 (diboron
pinacol ester, KOAc, DMF, 80 ꢀC, 2 h, 54%) with in situ
generation of the boronic ester provided the desired
diphenyl derivative 17.32 The preferential mixed versus
homo coupling of the reactants was controlled by ®rst
forming the boronic ester derived from 16 in situ, prior
to the addition of 15. Deprotection of 17 was accom-
plished by treatment with Bu4NF (3.0 equiv, THF,
90%) and was followed by conversion of the alcohol 18
to the chloride 19 (5.0 equiv SOC12, CH2Cl2, 3 h, 92%).
Alkylation of the potassium salt of ethyl cyanoacetate
(9) with 19 (K2CO3, DMF, 70 ꢀC, 3 h, 79%) a€orded 20.
Cyclization with the free base of guanidine (1.0 equiv,
EtOH, 45 ꢀC, 12 h, 61%) gave the desired pyrimidine 21.
Hydrolysis of the ester 21 with LiOH (5.0 equiv, 3:1
MeOH±H2O, 25 ꢀC, 88%), followed by glutamate cou-
pling of the resulting carboxylic acid 22 (EDCI,
NaHCO3, DMF, 45%) provided 23. Deprotection of 23
was accomplished using tri¯uoroacetic acid (10.0 equiv,
CH2Cl2, 89%) to provide the desired conformationally
restricted analogue 4a.
Syntheses of conformationally restricted analogues 3b and
Figure 1.
4b (XˆCH2, n=1)
The synthesis of 3b was accomplished in a manner
similar to the synthesis of 3a (Scheme 3). Commercially
available tryptamine dihydrochloride was treated with
NaNO2 in 6 N HCl to provide the corresponding chlo-
ride 24.33 N-alkylation of 4-(chloroethyl)imidazole
hydrochloride (24) with methyl (4-bromomethyl)benzo-
ate (6, K2CO3, DMF, 70 ꢀC, 1 h, 80%) provided the
desired alkylated imidazole 25. In addition to being the
Syntheses of conformationally restricted analogues 3a and
4a (X=CH2, n=0)
The synthesis of 3a is depicted in Scheme 1. Starting from
commercially available reagents, N-alkylation of 4-
(hydroxymethyl)imidazole hydrochloride (5) with
methyl (4-bromomethyl)benzoate (6, K2CO3, DMF,
70 ꢀC, 1 h, 49%) provided the desired alkylated imidazole
7 in a 1.6:1 ratio of N1-aryl-4-(hydroxymethyl)imidazole
and N1-aryl-5-(hydroxymethyl)imidazole. In addition to
being the predicted major product, 7 exhibited 1H NMR
chemical shifts for the benzylic protons consistent with
this assignment. The N-benzyl methlyene protons were
1
expected major product, 25 exhibited H NMR chemi-
cal shifts for the N-benzyl protons at d 5.12 (versus d
5.30) consistent with this assignment. Alkylation of the
potassium salt of ethyl cyanoacetate (9) with 23
(K2CO3, DMF, 70 ꢀC, 3 h, 56%) a€orded 26 and its
cyclization with the free base of guanidine (1.0 equiv,

D. L. Boger et al. / Bioorg. Med. Chem. 8 (2000) 1075±1086
1077
Figure 2. Left: structure overlay of tetrahydrofolate bound to dihydrofolate reductase (taken from an X-ray structure) and 5,10-dideazatetrahy-
drofolate bound to GAR Tfase (taken from an X-ray structure) illustrating the gauche (50±69ꢀ) versus the extended (trans, 166±174ꢀ) bound con-
formations that may be used to distinguish conformationally restricted folate analogues.29 Right: comparison of the relative stability of the trans and
cis amide conformations of 10-formyl-tetrahydrofolate.
Figure 3. Models of 3 (brown, top; n=0, XˆCH2) and 4 (brown, bottom; n=0, X=CH2) bound in the GAR Tfase active site and structural
overlays with the structure of 5,10-dideazatetrahydrofolate (yellow) taken from the X-ray crystal structure bound to GAR Tfase..28
EtOH, 25 ꢀC, 3 h, 62%) gave the desired pyrimidine 27.
Hydrolysis of the ester 27 with LiOH±H2O (5.0 equiv,
3:1 MeOH±H2O, 60 ꢀC, 81%), followed by glutamate
coupling with the resulting carboxylic acid 28 (EDCI,
NaHCO3, DMF, 61%) provided 29. Deprotection of 29
was accomplished using tri¯uoroacetic acid (10.0 equiv,
CH2Cl2, 81%) to provide 3b.
diphenyl derivative 31. Deprotection of 31 (2.0 equiv
Bu4NF, THF, 91%), followed by chlorination of the
terminal hydroxyl group of 32 on warming with thionyl
chloride (10.0 equiv, CH2Cl2, 3 h, 87%) provided 33.
Alkylation of the potassium salt of ethyl cyanoacetate
(9) with 33 (K2CO3, DMF, 70 ꢀC, 3 h, 63%) a€orded 34
and cyclization of 34 with the free base of guanidine (1.0
equiv, EtOH, 25 ꢀC, 3 h, 56%) gave the desired pyr-
imidine 35. Hydrolysis of the ester 35 with LiOH±H2O
(5.0 equiv, 3:1 MeOH:H2O, 60 ꢀC, 91%), followed by
glutamate coupling with the resulting carboxylic acid 36
(EDCI, NaHCO3, DMF, 48%) provided 37. Deprotec-
tion of 37 (10.0 equiv TFA, CH2Cl2, 83%) provided the
desired conformationally restricted analogue 4b.
The synthesis of 4b was accomplished in a manner similar
to 3b (Scheme 4). Palladium(0)-catalyzed Suzuki coupling
of 30, derived in three steps from commercially available
5-bromo-3-pyridine acetic acid,31 with commercially
available methyl 5-bromobenzoate (16, diboron pinacol
ester, KOAc, DMF, 80 ꢀC, 2 h, 63%) provided the desired

1078
D. L. Boger et al. / Bioorg. Med. Chem. 8 (2000) 1075±1086
Scheme 3.
Scheme 1.
Syntheses of conformationally restricted analogues 3c and
4c (X=S)
The syntheses of conformationally restricted analogues
3c and 4c were accomplished by an alternative pathway.
The syntheses of 3c and 4c were envisioned to proceed
via a base-catalyzed coupling of a thiol to 5-bromo-2,6-
diamino-4(3H)-pyrimidinone (40) using diisopropy-
lethylamine in dimethylformamide.34 The syntheses of
the thiols 39 and 45 were accomplished by displacement
of the chlorides 8 and 19 with potassium thioacetate
(acetone, 50 ꢀC, 2 h) followed by acid hydrolysis of the
thiol esters 38 and 44 to provide the corresponding
thiols 39 and 45, respectively (Schemes 5 and 6). Several
attempts to couple the thiol in the presence of base
(iPr2NEt, Et3N) led exclusively to the dimerized thiol.
Thus, coupling was accomplished without the use of
base (DMF, 85 ꢀC, 3 h) to provide the pyrimidines 42
and 46. Hydrolysis, coupling and deprotection to pro-
vide 3c, 4c was accomplished in a manner similar to that
previously reported for 3a,b and 4a,b.
GAR Tfase inhibition
The agents 3 and 4 and their respective precursors were
tested for inhibition of GAR Tfase and AICAR Tfase
and the results are presented in Table 1. Compounds
3a±3c, 4a and 4c all demonstrate similar binding to
GAR Tfase, with a Ki range of 11±24 mM. Compound
4b inhibited GAR Tfase with a Ki of 61 mM. The inter-
mediates in the preparation of 4b, compounds 36 and
37, showed the best GAR Tfase inhibition with Ki's of
Scheme 2.

D. L. Boger et al. / Bioorg. Med. Chem. 8 (2000) 1075±1086
1079
10 and 8 mM, respectively. Compounds 3b, 3c, 4b, and
4c did not inhibit AICAR Tfase and with the exception
of 4a, all were less active against AICAR versus GAR
Tfase demonstrating a selectivity for GAR Tfase over
AICAR Tfase. However, these results would appear to
demonstrate that the conformationally restricted inhi-
bitors do not have a positive e€ect on binding. The
compounds are several orders of magnitude less potent
than DDATHF, but bind to GAR Tfase with anities
roughly equivalent to a large series of TDAF inhibi-
tors.23 Given this weak GAR Tfase inhibition, the
compounds were not examined as inhibitors of TS or
DHFR.
Cytotoxic activity
The agents 3 and 4 and their precursors were examined
for cytotoxic activity both in the presence (+) and
Scheme 6.
Table 1. GAR and AICAR Tfase inhibition (Ki, mM)
Scheme 4.
Compound
Ki GAR Tfase
Ki AICAR Tfase
3a
4a
3b
4b
3c
4c
11
20
24
61
17
18
150
11
nia
ni
ni
ni
11
12
14
21
22
23
27
28
29
35
36
37
41
42
43
46
47
48
70
ni
ni
ni
ni
120
50
ni
ni
50
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
35
40
52
24
28
10
8
22
37
16
40
58
54
Lometrexol
0.1
Not tested
Scheme 5.
ani, no inhibition.

1080
D. L. Boger et al. / Bioorg. Med. Chem. 8 (2000) 1075±1086
absence ( ) of added hypoxanthine and/or thymidine
against the CCRF-CEM cell line in purine and pyr-
imidine free medium (Table 2). Consistent with their
weak enzyme inhibition properties, the agents exhibited
no or only modest cytotoxic activity. None exhibited a
sensitivity to the medium absence of either purines or
pyrimidines indicating that the modest activity is not
due to inhibition of either purine or pyrimidine bio-
synthesis.
Methyl 4-[(4-chloromethyl-1H-imidazol-1-yl)methyl]-
benzoate (8). A stirred solution of 7 (0.50 g, 2.03 mmol)
and thionyl chloride (1.48 mL, 20.3 mmol, 10.0 equiv) in
CH2Cl2 (20 mL) was warmed at 40ꢀC for 3 h. After cool-
ing to room temperature, the excess thionyl choride was
quenched by the addition of saturated aqueous NaHCO3
(50 mL), dried (Na2SO4), and concentrated under reduced
pressure. Chromatography (SiO2, 4Â15cm, 10±20%
MeOH±CHCl3 gradient elution) a€orded 8 (0.48 g, 89%)
1
as a white solid: H NMR (CDCl3, 400 MHz) d 8.02 (d,
J=8.4 Hz, 2H), 7.52 (s, 1H), 7.21 (d, J=8.1 Hz, 2H),
6.92 (s, 1H), 5.13 (s, 2H), 4.57 (s, 2H), 3.91 (s, 3H);
FABHRMS (NBA/NaI) m/z 265.0749 (M+H+,
C13H13ClN2O2 requires 265.0744).
Experimental
Methyl 4-[(4-hydroxymethyl-1H-imidazol-1-yl)methyl]-
benzoate (7). A stirred solution of 4-(hydroxymethyl)
imidazole (5, 1.0 g, 7.4 mmol), methyl 4-(bromomethyl)
benzoate (6, 2.04 g, 8.9 mmol, 1.2 equiv), and K2CO3
(3.08 g, 22.3 mmol, 3.0 equiv) in DMF (37 mL) was
warmed at 100 ꢀC for 3 h. The solution was diluted with
H2O (150mL) and extracted with EtOAc (3Â25mL). The
organic layer was dried (Na2SO4), and concentrated under
reduced pressure. Chromatography (SiO2, 5Â15cm, 10±
40% MeOH±CHCl3 gradient elution) a€orded 7 (0.90 g,
49%) as a white solid: 1H NMR (CDCl3, 250 MHz) d 7.96
(d, J=8.3 Hz, 2H), 7.46 (s, 1H), 7.16 (d, J=8.2 Hz, 2H),
6.81 (s, 1H), 5.08 (s, 2H), 4.67 (br s, 1H), 4.53 (s, 2H), 3.86
(s, 3H); FABHRMS (NBA/NaI) m/z 247.1079 (M+H+,
C13H14N2O3 requires 247.1083). The undesired regioi-
somer methyl 4-[(5-hydroxymethyl-1H-imidazol-1-yl)
methyl]benzoate was obtained as a higher Rf compound
Methyl 4-[4-(3-carboethoxy-3-cyanoprop-1-yl)-1H-imida-
zol-1-yl]methyl]benzoate (10). A stirred solution of 8
(0.40 g, 1.51 mmol), ethyl cyanoacetate 9 (0.32 mL,
3.02 mmol, 2.0 equiv), and K2CO3 (1.04 g, 7.55 mmol,
5.0 equiv) in DMF (15 mL) was warmed at 70 ꢀC for 3 h.
The solution was cooled to 25 ꢀC and diluted with H2O
(75 mL). The aqueous layer was extracted with EtOAc
(4Â20 mL), dried (Na2SO4), and concentrated under
reduced pressure. Chromatography (SiO2, 4Â15cm, 10±
20% MeOH±CHCl3 gradient elution) a€orded 10 (0.39 g,
76%) as a white solid: 1H NMR (CDCl3, 250 MHz) d 8.00
(d, J=8.3 Hz, 2H), 7.48 (s, 1H), 7.18 (d, J=8.3 Hz, 2H),
6.83 (s, 1H), 5.13 (s, 2H), 4.23 (dd, J=14.3, 7.1 Hz, 2H),
3.96 (dd, J=8.7, 5.9 Hz, 1H), 3.90 (s, 3H), 3.22 (dd,
J=14.5, 5.8 Hz, 1H), 3.11 (dd, J=14.5, 8.7 Hz, 2H), 1.27
(t, J=7.2 Hz, 3H); FABHRMS (NBA/NaI) m/z 328.1288
(M+H+, C17H17N3O4 requires 328.1297).
1
in 24% yield: H NMR (CDCl3, 250 MHz) d 7.95 (d,
J=8.3 Hz, 2H), 7.45 (s, 1H), 7.14 (d, J=8.2 Hz, 2H),
6.92 (s, 1H), 5.28 (s, 2H), 4.46 (s, 2H), 3.88 (s, 3H).
Ethyl 4-[4-[(2,4-diamino-6(1H)-pyrimidinon-5-yl)methyl]-
1H - imidazol - 1 - yl]methyl]benzoate (11). A solution of
NaOEt (0.051 g, 0.75 mmol, 1.0 equiv) in absolute EtOH
(1.3 mL) was treated successively with a solution of 10
(0.20 g, 0.76 mmol) in EtOH (2.5 mL) and a solution of
guanidine hydrochloride (0.072 g, 0.75 mmol, 1.0 equiv)
and NaOEt (0.051 g, 0.75 mmol, 1.0 equiv) in EtOH
(2.5 mL). The solution was stirred at 25 ꢀC for 3 h. The
solution was acidi®ed to pH=1 with the addition of
tri¯uoracetic acid followed by concentration under
reduced pressure. Chromatography (SiO2, 2Â15 cm, 10±
80% MeOH±CHCl3 gradient elution) a€orded 11
(0.165 g, 62%) as a white solid: 1H NMR (CD3OD,
250 MHz) d 7.99±7.96 (m, 3H), 7.32 (d, J=8.2 Hz, 2H),
7.10 (s, 1H), 5.29 (s, 2H), 4.34 (dd, J=14.3, 7.1 Hz, 2H),
3.16 (d, J=9.1 Hz, 1H), 3.08 (d, J=9.3 Hz, 1H), 1.36 (t,
J=7.1 Hz, 3H); FABHRMS (NBA/NaI) m/z 369.1675
(M+H+, C18H20N6O3 requires 369.1675).
Table 2. In vitro cytotoxic activity
CCRF-CEM (IC50, mM)
Compd
(+) T, (+)Ha
(
)T, (+)Ha (+)T, ( )Ha
(
)T, ( )Ha
3a
4a
3b
4b
3c
4c
60
60
80
50
35
40
120
65
>200
150
50
70
90
40
65
70
130
60
>200
>200
>200
60
>200
140
11
12
14
21
22
23
27
28
29
35
36
37
41
42
43
46
47
48
>250
>250
100
>250
>250
100
>250
>250
100
>250
>250
100
90
90
140
170
>250
40
90
200
55
90
>250
50
>250
>250
>250
0.9
>250
40
80
>250
100
90
>250
30
145
>250
175
>250
30
70
>250
75
90
>250
45
>250
>250
>250
>250
40
130
>250
60
80
225
35
>250
>250
>250
0.5
4-[4-[(2,4-Diamino-6(1H)-pyrimidinon-5-yl)methyl]-1H-
imidazol-1-yl]methyl]benzoic acid (12). A solution of 11
(0.010g, 0.027 mmol), LiOH±H2O (0.0057 g, 0.135 mmol,
5.0 equiv) in 3:1 MeOH±H2O (0.27 mL) was warmed at
60 ꢀC for 5 h. The mixture was cooled to 25 ꢀC and
diluted with H2O (10 mL) and the aqueous layer was
washed with EtOAc (3Â3 mL) and acidi®ed to pH=1
with the addition of 1 M aqueous HCl. The solution
was concentrated under reduced pressure and the resi-
due was azeotropically treated with toluene (3Â5 mL) to
remove traces of glacial acetic acid and H2O, dried
1.1
200
75
1.5
>250
>250
>250
60
>250
>250
Lometrexol
>250
>250
0.70
1.1
aT, thymidine, H, hypoxanthine.

D. L. Boger et al. / Bioorg. Med. Chem. 8 (2000) 1075±1086
1081
(Na2SO4), and concentrated under reduced pressure to
provide 12 (0.008 g, 84%). The compound was used with-
out further puri®cation: 1H NMR (DMSO-d6, 400 MHz)
d 7.81 (d, J=7.8 Hz, 2H), 7.61 (s, 1H), 7.14 (d, J=8.2 Hz,
2H), 6.63 (s, 1H), 6.47 (br s, 2H), 5.89 (br s, 2H), 5.05 (s,
2H), 3.16 (s, 3H); FABHRMS (NBA/NaI) m/z 363.1167
(M+Na+, C16H16N6O3 requires 363.1182).
(br s, 1H), 8.59 (br s, 1H), 8.14 (d, J=8.4 Hz, 2H), 7.89 (s,
1H), 7.65 (d, J=8.4 Hz, 2H), 4.83 (s, 2H), 3.94 (s, 3H),
0.93 (s, 9H), 0.13 (s, 6H); MALDIFTMS (DHB) m/z
358.1832 (M+H+, C20H27NO3Si requires 358.1838).
Methyl 4-[5-hydroxymethyl-3-pyridinyl]benzoate (18). A
solution of 17 (0.32 g, 0.89 mmol) in THF (10 mL) at
25 ꢀC was treated with Bu4NF (2.67 mL, 2.67 mmol, 3.0
equiv) as a 1 M solution in THF. The solution was
stirred at 25 ꢀC for 3 h and quenched by the addition of
saturated aqueous NH4Cl (50 mL). The product was
extracted into EtOAc (3Â10 mL), dried (Na2SO4), ®l-
tered, and concentrated under reduced pressure. Chro-
matography (SiO2, 4Â15 cm, 8% MeOH±CHCl3)
di-tert-Butyl N-{4-[4-[(2,4-diamino-6(1H)-pyrimidinon-5-
yl)methyl]-1H-imidazol-1-yl]methyl]benzoyl}-L-glutamate
(14). A solution of 12 (9.2 mg, 0.027 mmol) and di-tert-
butyl l-glutamate hydrochloride (13, 11.2 mg,
0.041 mmol, 1.5 equiv) in DMF (2.7 mL) was treated
succesively with NaHCO3 (6.8 mg, 0.081 mmol, 3.0
equiv) and EDCI (15.5 mg, 0.081 mmol, 3.0 equiv). The
solution was stirred at 25ꢀC for 12h and concentrated
under reduced pressure. The residue was dissolved in
CHCl3 (5 mL) and extracted with saturated aqueous
NaHCO3 (2 mL). The organic layer was dried (Na2SO4),
®ltered, and concentrated under reduced pressure.
Chromatography (SiO2, 1Â15 cm, 10±20% MeOH±
CHCl3 gradient elution) a€orded 14 (8.5 mg, 54%) as a
white solid: 1H NMR (CDCl3, 400 MHz) d 7.69 (d,
J=7.6 Hz, 2H), 7.54 (br s, 1H), 7.34 (s, 1H), 7.04 (d,
J=7.6 Hz, 2H), 6.67 (s, 1H), 5.76 (br s, 2H), 5.66 (br s,
1H), 4.93 (s, 2H), 4.62 (dd, J=12.7, 8.1 Hz, 2H), 3.54 (s,
2H), 2.45±2.30 (m, 2H), 2.20±2.17 (m, 1H), 2.09±2.01 (m,
1H), 1.46 (s, 9H), 1.39 (s, 1H); FABHRMS (NBA/NaI)
m/z 714.2040 (M+Cs+, C29H39N7O6 requires 714.2016).
1
provided 18 (0.20 g, 90%) as a white solid: H NMR
(CDCl3, 400 MHz) d 8.80 (s, 1H), 8.62 (s, 1H), 8.15 (d,
J=8.1 Hz, 2H), 7.97 (s, 1H), 7.67 (d, J=8.4 Hz, 2H),
4.84 (s, 2H), 3.95 (s, 3H); MALDIFTMS (DHB) m/z
244.0972 (M+H+, C14H13NO3 requires 244.0974).
Methyl 4-[5-chloromethyl-3-pyridinyl]benzoate (19). A
stirred solution of 18 (0.197 g, 0.810 mmol) and thionyl
chloride (0.29 mL, 4.1 mmol, 5.0 equiv) in CH2Cl2
(8.2 mL) was warmed at 50 ꢀC for 3 h. After cooling to
room temperature, the excess thionyl chloride was
quenched by the addition of saturated aqueous
NaHCO3 (25 mL), dried (Na2SO4), and concentrated
under reduced pressure. Chromatography (SiO2,
4Â15 cm, 50% hexanes±EtOAc) a€orded 19 (0.198 g,
1
92%) as a white solid: H NMR (CDCl3, 400 MHz) d
N-{4-[4-[(2,4-Diamino-6(1H)-pyrimidinon-5-yl)methyl]-
1H-imidazol-1-yl]methyl]benzoyl}-L-glutamic acid (3a).
A solution of 14 (5.0 mg, 0.009 mmol) in CH2Cl2
(0.1 mL) cooled to 0 ꢀC was treated with tri¯uoracetic
acid (0.03 mL). The solution was stirred at 0 ꢀC for 2 h
and warmed to 25 ꢀC and stirred for 12 h. Et2O (1 mL)
was added to the reaction mixture and a white pre-
cipitate formed. The precipitate was triturated with
Et2O (3Â1 mL) and dried in vacuo to give 3a±CF3CO2H
8.84 (s, 1H), 8.65 (s, 1H), 8.16 (d, J=7.9 Hz, 2H), 7.99
(s, 1H), 7.67 (d, J=7.9 Hz, 2H), 4.68 (s, 2H), 3.96 (s,
3H); MALDIFTMS (DHB) m/z 262.0644 (M+H+,
C14H12ClNO2 requires 244.0635).
Methyl 4-[5-[3-carboethoxy-3-cyanoprop-1-yl]-3-pyridi-
nyl]benzoate (20). A stirred solution of 19 (0.21 g,
0.81 mmol), ethyl cyanoacetate (9, 0.26 mL, 2.43 mmol,
3.0 equiv), and K2CO3 (0.45 g, 3.24 mmol, 4.0 equiv) in
DMF (8.1 mL) was warmed at 70 ꢀC for 3 h. The solution
was cooled to 25 ꢀC and diluted with H2O (50 mL). The
aqueous layer was extracted with EtOAc (3Â20 mL),
dried (Na2SO4) and, concentrated under reduced pres-
sure. Chromatography (SiO2, 3Â15 cm, 25% hexanes±
1
(3.4 mg, 82%) as a white solid: H NMR (DMSO-d6,
400 MHz) d 9.10 (s, 1H), 8.65 (d, J=7.8 Hz, 1H), 7.90 (d,
J=7.8 Hz, 2H), 7.48 (d, J=8.1 Hz, 2H), 7.31 (s, 1H),
7.06 (br s, 2H), 6.49 (br s, 2H), 5.40 (s, 2H), 4.42±4.36 (m,
1H), 2.36±2.33 (m, 2H), 2.11±2.07 (m, 1H), 1.98±1.91 (m,
1H); MALDIFTMS (DHB) m/z 470.1791 (M+H+,
C21H23N7O6 requires 470.1788).
1
EtOAc) a€orded 20 (0.22 g, 79%): H NMR (CDCl3,
250 MHz) d 8.82 (br s, 1H), 8.56 (br s, 1H), 8.14 (d,
J=8.5 Hz, 2H), 7.89 (s, 1H), 7.65 (d, J=8.6 Hz, 2H),
4.27 (q, J=14.3, 7.2 Hz, 2H), 3.95 (s, 3H), 3.82 (dd,
J=7.6, 6.0 Hz, 1H), 3.36±3.32 (m, 2H), 1.27 (t,
J=7.2 Hz, 3H); MALDIFTMS (DHB) m/z 339.1336
(M+H+, C19H18N2O2 requires 339.1345).
Methyl 4-[5-[[(1,1-dimethylethyl)silyl]oxy]methyl]-3-pyri-
dinyl]benzoate (17). A solution of methyl 4-bromobenzo-
ate (16, 0.81g, 3.77mmol, 1.0 equiv), diboron pinacol
ester (1.05 g, 4.17mmol, 1.1 equiv), KOAc (0.93 g,
11.3 mmol, 3.0 equiv), and PdCl2(dppf) (0.09 g, 3 mol%)
in DMF (22.5 mL) was warmed at 80ꢀC for 2 h. The
solution was cooled to 25ꢀC and was treated successively
with 15 (2.19 g, 7.55mmol, 2.0 equiv), PdCl2(dppf) (0.09g,
3 mol%), and 2 M aqueous Na2CO3 (9.40 mL, 5.0 equiv).
The solution was heated at 80 ꢀC for 12h, cooled to
25 ꢀC and diluted with Et2O (100 mL). The organic layer
was washed with H2O (50 mL) and saturated aqueous
NaCl (50 mL), dried (Na2SO4), ®ltered, and concentrated
under reduced pressure. Chromatography (SiO2,
5Â15cm, 15% hexanes±EtOAc) a€orded 17 (0.72 g,
54%) as a white solid: 1H NMR (CDCl3, 400 MHz) d 8.77
Methyl 4-[5-[(2,4-diamino-6(1H)-pyrimidinon-5-yl)me-
thyl] - 3 - pyridinyl]benzoate (21). A solution of NaOEt
(5.1 mg, 0.74 mmol, 1.0 equiv) in absolute EtOH
(0.13 mL) was treated successively with a solution of 20
(0.025 g, 0.074 mmol) in EtOH (0.26 mL) and a solution
of guanidine hydrochloride (7.8 mg, 0.074 mmol, 1.0
equiv) and NaOEt (5.1 mg, 0.74 mmol 1.0 equiv) in
EtOH (0.13 mL). The solution was warmed at 45 ꢀC for
12 h. The solution was acidi®ed with the addition of
glacial acetic acid (pH=1) and concentrated under
reduced pressure. Chromatography (SiO2, 1Â15 cm,

1082
D. L. Boger et al. / Bioorg. Med. Chem. 8 (2000) 1075±1086
1
15% MeOH±CH2Cl2) a€orded 21 (0.016 g, 61%): H
NMR (DMSO-d6, 500 MHz) d 9.88 (s, 1H), 8.69 (s, 1H),
8.53 (s, 1H), 8.07 (d, J=8.4 Hz, 2H), 7.99 (s, 1H), 7.80
(d, J=8.4 Hz, 2H), 6.01 (s, 2H), 5.93 (s, 2H), 3.88 (s,
3H), 3.60 (s, 2H); MALDIFTMS (DHB) m/z 352.1427
(M+H+, C18H17N5O3 requires 352.1410).
the procedure described for 7. Chromatography (SiO2,
5Â15cm, 10% MeOH±EtOAc) a€orded 25 (0.81 g, 44%)
1
as a white solid: H NMR (CDCl3, 400 MHz) d 8.00 (d,
J=8.4 Hz, 2H), 7.48 (s, 1H), 7.13 (d, J=8.4 Hz, 2H), 6.73
(s, 1H), 5.12 (s, 2H), 3.91 (s, 3H), 3.76 (t, J=7.1 Hz, 2H),
3.00 (t, J=7.1 Hz, 2H); FABHRMS (NBA/NaI) m/z
279.0904 (M+H+, C14H15ClN2O2 requires 279.0900).
4-[5-[(2,4-Diamino-6(1H)-pyrimidinon-5-yl)methyl]-3-pyr-
idinyl]benzoic Acid (22). A solution of 21 (0.036 g,
0.087 mmol) in 3:1 CH3OH:H2O (0.87 mL) was treated
with LiOH±H2O (0.011 g, 0.261 mmol, 3.0 equiv) and
the mixture was stirred at 25 ꢀC for 12 h. The mixture was
diluted with H2O (5 mL) and the aqueous layer was
washed with EtOAc (3Â2 mL) and acidi®ed to pH=1
with the addition of 1 M aqueous HCl. The solution was
concentrated under reduced pressure and the residue was
azeotropically treated with toluene (3Â5 mL) to remove
Methyl 4-[4-(3-carboethoxy-3-cyanobut-1-yl)-1H-imida-
zol-1-yl]ethyl]benzoate (26). Obtained from 25 (0.191 g,
0.683 mmol) using the procedure described for 10.
Chromatography (SiO2, 3Â15 cm, 100% EtOAc) a€or-
ded 26 (0.14 g, 56%) as a white solid: 1H NMR (CDCl3,
400 MHz) d 8.00 (d, J=6.1 Hz, 2H), 7.52 (s, 1H), 7.19 (d,
J=6.1 Hz, 2H), 6.69 (s, 1H), 5.09 (s, 2H), 4.21 (dd,
J=14.3, 7.1 Hz, 2H), 3.88 (s, 3H), 3.56 (dd, J=8.9,
6.0 Hz, 1H), 2.81±2.71 (m, 2H), 2.41±2.34 (m, 1H), 2.26±
2.19 (m, 1H), 1.26 (t, J=14.0 Hz, 3H); FABHRMS
(NBA/NaI) m/z 356.1619 (M+H+, C19H21N3O4
requires 356.1610).
1
traces of H2O to provide 22 (0.053 g, 88%): H NMR
(DMSO-d6, 400 MHz) d 9.04 (s, 1H), 8.71 (s, 1H), 8.62 (s,
1H), 8.44 (s, 2H), 8.08 (d, J=8.5 Hz, 2H), 7.94 (d,
J=8.2 Hz, 2H), 7.46 (br s, 2H), 3.49 (s, 2H); MAL-
DIFTMS (DHB) m/z 338.1240 (M+H+, C17H15N5O3
requires 338.1240).
Ethyl 4-[4-[(2,4-diamino-6(1H)-pyrimidinon-5-yl)ethyl]-
1H-imidazol-1-yl]methyl]benzoate (27). Obtained from
26 (0.042 g, 0.117 mmol) using the procedure described
for 11. Chromatography (SiO2, 1Â15 cm, 10±80%
MeOH±CHCl3 gradient elution) a€orded 27 (0.165 g,
62%) as a tan solid: 1H NMR (DMF-d7, 400 MHz) d 9.99
(br s, 1H), 8.00 (d, J=8.1 Hz, 2H), 7.74 (br s, 1H), 7.43 (d,
J=8.1 Hz, 2H), 6.95 (s, 1H), 6.21 (br s, 1H), 5.73 (br s,
1H), 5.34 (s, 2H), 4.35 (dd, J=14.2, 7.3 Hz, 2H), 2.61 (m,
4H), 1.35 (t, J=7.0 Hz, 3H); FABHRMS (NBA/NaI) m/z
383.1829 (M+H+, C19H22N6O3 requires 383.1832).
di-tert-Butyl N-{4-[5-[(2,4-diamino-6(1H)-pyrimidinon-5-
yl)methyl]-3-pyridinyl]benzoyl}-L-glutamate (23). A solu-
tion of 22 (0.026 g, 0.076 mmol) and 13 (0.033 g,
0.114 mmol, 1.5 equiv) in DMF (0.76 mL) was treated
with NaHCO3 (0.019g, 0.228 mmol, 3.0 equiv) followed
by EDCI (0.044g, 0.228 mmol, 3.0 equiv). The reaction
mixture was stirred at 25ꢀC for 12h 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), ®ltered, and concentrated under reduced pres-
sure. Chromatography (SiO2, 1Â15cm, 10% MeOH±
4-[4-[(2,4-Diamino-6(1H)-pyrimidinon-5-yl)ethyl]-1H-imi-
dazol-1-yl]methyl]benzoic acid (28). Obtained from 27
(0.107 g, 0.300 mmol) using the procedure described for
12. The solution was concentrated under reduced pres-
sure and the residue was treated with toluene (3Â5 mL)
to remove traces of H2O to provide 28 (0.083g, 81%). The
compound was used without further puri®cation: 1H
NMR (DMF-d7, 400 MHz) d 9.22 (s, 1H), 7.97 (d,
J=8.1 Hz, 2H), 7.55 (s, 1H), 7.45 (d, J=8.1 Hz, 2H), 5.48
(s, 2H), 2.66±2.62 (m, 2H), 2.57±2.50 (m, 2H); FABHRMS
(NBA/NaI) m/z 355.1512 (M+H+, C17H18N6O3 requires
355.1519).
1
CHCl3) provided 23 (0.019 g, 45%) as a white solid: H
NMR (CD3OD, 400 MHz) d 8.88 (s, 1H), 8.31 (s, 1H),
7.98 (d, J=8.3 Hz, 2H), 7.85 (s, 1H), 7.73 (d, J=8.3 Hz,
2H), 6.83 (s, 1H), 5.13 (s, 2H), 4.54±4.49 (m, 1H), 3.73 (s,
1H), 3.58 (s, 1H), 2.42 (t, J=7.2 Hz, 2H), 2.26±2.17 (m,
1H), 2.08±2.01 (m, 1H), 1.46 (s, 9H), 1.41 (s, 9H); MAL-
DIFTMS (DHB) m/z 601.2739 (M+Na+, C30H38N6O6
requires 601.2751).
N-{4-[5-[(2,4-Diamino-6(1H)-pyrimidinon-5-yl)methyl]-3-
pyridinyl]benzoyl}-L-glutamic acid (4a). A solution of 23
(3.2 mg, 0.0055 mmol) in CH2Cl2 (0.20 mL) cooled to
0 ꢀC was treated with tri¯uoroacetic 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 to the reaction mixture and a
white precipitate formed. The precipitate was triturated
with Et2O (3Â1 mL) and dried in vacuo to give 4a±
CF3CO2H (2.1 mg, 89%) as a white solid: 1H NMR
(CD3OD, 400 MHz) d 8.86 (s, 1H), 8.42 (s, 1H), 8.02 (s,
1H), 8.01 (d, J=8.1 Hz, 2H), 7.75 (d, J=8.3 Hz, 2H),
6.83 (s, 1H), 5.13 (s, 2H), 4.71±4.65 (m, 1H), 3.82 (s,
1H), 3.70 (s, 1H), 2.53±2.46 (m, 2H), 2.32±2.28 (m, 1H),
2.20±2.12 (m, 1H); MALDIFTMS (DHB) m/z 467.1679
(M+Na+, C22H22N6O6 requires 467.1683).
di-tert-Butyl N-{4-[4-[(2,4-diamino-6(1H)-pyrimidinon-5-
yl)ethyl]-1H-imidazol-1-yl]methyl]benzoyl}-L-glutamate
(29). Obtained from 28 (0.052 g, 0.152 mmol) using the
procedure described for 14. Chromatography (SiO2,
1Â15 cm, 50% hexanes±EtOAc) a€orded 29 (0.055 g,
1
61%) as a white solid: H NMR (CDCl3, 400 MHz) d
7.78 (d, J=7.3 Hz, 2H), 7.48 (s, 1H), 7.44 (s, 1H), 7.08 (d,
J=7.6 Hz, 2H), 6.55 (s, 1H), 5.04 (br s, 2H), 5.04 (s, 2H),
4.65±4.60 (m, 1H), 2.65±2.60 (m, 4H), 2.43±2.30 (m, 2H),
2.24±2.18 (m, 1H), 2.09±1.99 (m, 1H), 1.46 (s, 9H), 1.40
(s, 9H); FABHRMS (NBA/NaI) m/z 596.3214 (M+H+,
C30H41N7O6 requires 596.3197).
N-{4-[4-[(2,4-Diamino-6(1H)-pyrimidinon-5-yl)ethyl]-1H
-imidazol-1-yl]methyl]benzoyl}-L-glutamic acid (3b).
Obtained from 29 (5.1 mg, 0.009 mmol) using the pro-
cedure described for 3a. The precipitate was triturated
Methyl 4-[(4-chloromethyl-1H-imidazol-1-yl)ethyl]benzo-
ate (25). Obtained from 24 (1.10 g, 6.58 mmol) using

D. L. Boger et al. / Bioorg. Med. Chem. 8 (2000) 1075±1086
1083
with Et2O (3Â1 mL) and dried in vacuo to give 3b±
CF3CO2H (3.6 mg, 81%) as a white solid: 1H NMR
(DMSO-d6, 400 MHz) d 9.24 (s, 1H), 7.78 (d, J=7.3 Hz,
2H), 7.48 (s, 1H), 7.44 (s, 1H), 7.08 (d, J=7.6 Hz, 2H),
6.55 (s, 1H), 5.04 (br s, 2H), 5.04 (s, 2H), 4.65±4.60 (m,
1H), 2.65±2.60 (m, 4H), 2.43±2.30 (m, 2H), 2.24±2.18
(m, 1H), 2.09±1.99 (m, 1H); FABHRMS (NBA/NaI) m/
z 484.1928 (M+H+, C22H25N7O6 requires 484.1945).
4-[5-[(2,4-Diamino-6(1H)-pyrimidinon-5-yl)ethyl]-3-pyri-
dinyl]benzoic acid (36). Obtained from 35 (0.020 g,
0.055 mmol) using the procedure described for 22. The
solution was concentrated under reduced pressure and
the residue was treated with toluene (3Â5 mL) to remove
1
traces of H2O to provide 36 (0.017 g, 91%): H NMR
(DMSO-d6, 500 MHz) 9.05 (s, 1H), 8.76 (s, 1H), 8.66 (s,
1H), 8.32 (br s, 2H), 8.08 (d, J=8.0Hz, 2H), 7.99 (d,
J=8.4Hz, 2H), 7.29 (br s, 2H), 2.86 (t, J=7.0Hz, 2H),
2.64 (t, J=7.4 Hz, 2H); MALDIFTMS (DHB) m/z
352.1424 (M+H+, C18H17N5O3 requires 352.1410).
Methyl 4-[5-[[(1,1-dimethylethyl)silyl]oxy]ethyl]-3-pyridi-
nyl]benzoate (31). Obtained from 30 (1.62 g, 5.12 mmol)
using the procedure described for 17. Chromatography
(SiO2, 5Â15 cm, 25% hexanes±EtOAc) a€orded 31
(0.598 g, 63%) as a white solid: 1H NMR (CDCl3,
500 MHz) d 8.72 (d, J=1.8 Hz, 1H), 8.49 (d, J=1.8 Hz,
1H), 8.13 (d, J=8.4 Hz, 2H), 7.77 (t, J=1.8 Hz, 1H),
7.64 (d, J=8.5 Hz, 2H), 3.94 (s, 3H), 3.87 (t, J=6.6 Hz,
2H), 2.88 (t, J=6.3 Hz, 2H), 0.84 (s, 9H), 0.03 (s, 6H);
MALDIFTMS (DHB) m/z 372.1998 (M+H+,
C21H29NO3Si requires 372.1995).
di-tert-Butyl N-{4-[5-[(2,4-diamino-6(1H)-pyrimidino-5-
yl) ethyl] - 3 - pyridinyl]benzoyl} - L - glutamate (37).
Obtained from 36 (0.010 g, 0.028 mmol) using the pro-
cedure described for 23. Chromatography (SiO2,
1Â15 cm, 20% MeOH±CHCl3 gradient elution) a€orded
1
37 (0.008 g, 48%) as a white solid: H NMR (CDCl3,
500 MHz) d 8.60 (s, 1H), 8.41 (s, 1H), 7.84 (d, J=8.0 Hz,
2H), 7.69 (s, 1H), 7.45 (d, J=6.6 Hz, 1H), 5.47 (br s, 2H),
4.71±4.65 (m, 3H), 2.81 (s, 2H), 2.60 (s, 2H), 2.48±2.42
(m, 1H), 2.40±2.34 (m, 1H), 2.27±2.20 (m, 1H), 2.12±2.05
(m, 1H), 1.49 (s, 9H), 1.42 (s, 9H); MALDIFTMS
(DHB) m/z 593.3089 (M+H+, C31H40N6O6 requires
593.3087).
Methyl 4-[5-hydroxyethyl-3-pyridinyl]benzoate (32).
Obtained from 31 (0.579 g, 1.56 mmol) using the proce-
dure described for 18. Chromatography (SiO2, 4Â15 cm,
25% hexanes±EtOAc) a€orded 32 (0.365 g, 91%) as a
white solid: 1H NMR (CDCl3, 400 MHz) d 8.57 (s, 1H),
8.41 (s, 1H), 8.05 (d, J=8.1 Hz, 2H), 7.76 (s, 1H), 7.55
(d, J=8.4 Hz, 2H), 3.91 (s, 3H), 2.90 (t, J=5.0 Hz, 2H),
2.84±2.81 (m, 2H); MALDIFTMS (DHB) m/z 258.1121
(M+H+, C15H15NO3 requires 258.1130).
N-{4-[5-[(2,4-Diamino-6(1H)-pyrimidinon-5-yl)ethyl]-3-
pyridinyl]benzoyl}-L-glutamic acid (4b). Obtained from
37 (4.1 mg, 0.007 mmol) using the procedure described
1
for 4a to provide 4b (2.8 mg, 83%) as a white solid: H
NMR (DMSO-d6, 400 MHz) d 8.90 (s, 1H), 8.75 (d,
J=7.6 Hz, 2H), 8.56 (s, 1H), 8.32 (s, 1H), 8.04 (d,
J=8.5 Hz, 2H), 7.89 (d, J=8.2 Hz, 2H), 7.51 (br s, 1H),
6.77 (br s, 2H), 4.45±4.41 (m, 1H), 2.78 (t, J=6.8 Hz, 2H),
2.60 (t, J=6.8 Hz, 2H), 2.38 (t, J=7.3 Hz, 2H), 2.16±2.08
(m, 1H), 2.02±1.92 (m, 1H), FABHRMS (NBA/NaI) m/z
481.1825 (M+H+, C23H24N6O6 requires 481.1836).
Methyl 4 - [5 - chloroethyl - 3 - pyridinyl]benzoate (33).
Obtained from 32 (0.150 g, 0.581 mmol) using the proce-
dure described for 19. Chromatography (SiO2, 3Â15 cm,
40% hexanes±EtOAc) a€orded 33 (0.14 g, 87%) as a
white solid: 1H NMR (CDCl3, 500 MHz) d 8.78 (s, 1H),
8.53 (s, 1H), 8.15 (d, J=8.4 Hz, 2H), 7.79 (s, 1H), 7.66 (d,
J=8.4 Hz, 2H), 3.95 (s, 3H), 3.79 (t, J=7.0 Hz, 2H), 3.17
(t, J=7.0 Hz, 2H); MALDIFTMS (DHB) m/z 276.0785
(M+H+, C15H14ClNO2 requires 276.0791).
Methyl 4-[(4-(acetylthio)methyl-1H-imidazol-1-yl)me-
thyl]benzoate (39). A solution of 8 (1.31 g, 4.91 mmol)
and potassium thioacetate (1.68 g, 14.7 mmol, 3.0 equiv)
in acetone (20 mL) was warmed at re¯ux for 30 min. The
solution was cooled to 25 ꢀC, diluted with H2O (100 mL)
and extracted with EtOAc (3Â20mL). The EtOAc was
dried (Na2SO4) and concentrated under reduced pressure.
Chromatography (SiO2, 5Â15 cm, 100% EtOAc) a€orded
39 (1.11 g, 74%) as a white solid: 1H NMR (CDCl3,
400 MHz) d 7.99 (d, J=8.4Hz, 2H), 7.44 (s, 1H), 7.17 (d,
J=8.1Hz, 2H), 6.81 (s, 1H), 5.08 (s, 2H), 4.05 (s, 2H), 3.90
(s, 3H), 2.31 (s, 3H); MALDIFTMS (DHB) m/z 305.0975
(M+H+, C15H16N2O3S requires 305.0960).
Methyl 4-[5-[3-carboethoxy-3-cyanobut-1-yl]-3-pyridinyl]-
benzoate (34). Obtained from 33 (0.161 g, 0.583 mmol)
using the procedure described for 20. Chromatography
(SiO2, 3Â15 cm, 50% EtOAc) a€orded 34 (0.13 g, 63%)
1
as a white solid: H NMR (CDCl3, 400 MHz) d 8.74 (s,
1H), 8.50 (s, 1H), 8.11 (d, J=8.1 Hz, 2H), 7.74 (s, 1H), 7.62
(d, J=8.1 Hz, 2H), 4.22 (dd, J=14.3, 7.2 Hz, 2H), 3.92 (s,
3H), 3.54 (t, J=7.0 Hz, 1H), 2.30±2.87 (m, 2H), 2.34±2.29
(m, 2H), 1.29 (t, J=7.0 Hz, 3H); FABHRMS (NBA/NaI)
m/z 353.1508 (M+H+, C20H20N2O4 requires 353.1501).
Methyl 4-[5-[(2,4-diamino-6(1H)-pyrimidinon-5-yl)ethyl]-
3 - pyridinyl]benzoate (35). Obtained from 34 (0.149 g,
0.423 mmol) using the procedure described for 21. Chro-
matography (SiO2, 3Â15cm, 10±30% MeOH±CHCl3 gra-
dient elution) a€orded 35 (0.086 g, 56%) as a white solid:
1H NMR (DMSO-d6, 500 MHz) d 9.80 (br s, 1H), 8.74 (d,
J=1.8 Hz, 1H), 8.45 (d, J=1.8 Hz, 1H), 8.07 (d, J=8.4 Hz,
2H), 8.03 (t, J=1.8 Hz, 1H), 7.88 (d, J=8.4 Hz, 2H), 5.92
(s, 2H), 5.81 (s, 2H), 3.89 (s, 3H), 2.70 (t, J=7.0 Hz, 2H),
2.53 (t, J=7.0 Hz, 2H); MALDIFTMS (DHB) m/z
366.1576 (M+H+, C19H19N5O3 requires 366.1566).
Methyl 4-[(4-mercaptomethyl-1H-imidazol-1-yl)methyl]-
benzoate (40). A solution of 39 (0.050 g, 0.164 mmol) in 1
N HCl±EtOH (0.75 mL) was warmed at 40ꢀC for 2 h. The
solution was cooled to 25ꢀC and the solvent was removed
under reduced pressure and the residue was dissolved in
H2O (10 mL). The aqueous layer was extracted with
CH2Cl2 (3Â3 mL). The organic layer was dried (Na2SO4)
and concentrated under reduced pressure. Chromato-
graphy (SiO2, 1Â15 cm, 16% MeOH±CHCl3) a€orded
1
40 (0.024 g, 56%) as a white solid: H NMR (CDCl3,
500 MHz) d 8.00 (d, J=8.1 Hz, 2H), 7.58 (s, 1H), 7.20

1084
D. L. Boger et al. / Bioorg. Med. Chem. 8 (2000) 1075±1086
(d, J=8.1 Hz, 2H), 6.78 (s, 1H), 5.12 (s, 2H), 3.90 (s,
3H), 3.69 (s, 2H); FABHRMS (NBA/NaI) m/z 263.0853
(M+H+, C13H14N2O2S requires 263.0854).
Methyl 4-[5-mercaptomethyl-3-pyridinyl]benzoate (45).
Obtained from 44 (0.040g, 0.133 mmol) using the proce-
dure described for 40. Chromatography (SiO2, 1Â15cm,
40% hexanes±EtOAc) a€orded 45 (0.032 g, 93%) as a
white solid: 1H NMR (CDCl3, 400MHz) d 8.75 (d,
J=2.2 Hz, 1H), 8.58 (d, J=2.2 Hz, 1H), 8.15 (d, J=8.4 Hz,
2H), 7.91 (t, J=2.2 Hz, 1H), 7.66 (d, J=8.4 Hz, 2H), 3.95
(s, 3H), 3.82 (d, J=7.8 Hz, 2H), 1.87 (t, J=7.8 Hz, 1H);
MALDIHRMS (DHB) m/z 260.0735 (M+H+, C14H13
NO2S requires 260.0745).
Methyl 4-[4-[(2,4-diamino-6(1H)-pyrimidinon-5-yl)thio]-
methyl]-1H-imidazol-1-yl]methyl]benzoate (41). Argon
was bubbled through a solution of 40 (0.107g, 0.408mmol)
and
5-bromo-2,6-diamino-4(3H)-pyrimidinone
(38,
0.084g, 0.408mmol, 1.0 equiv) in DMF (2 mL) for 0.5 h.
The reaction mixture was warmed at 80 ꢀC for 2 h, cooled
to 25 ꢀC and concentrated under reduced pressure. Chro-
matography (SiO2, 2Â15 cm, 8% MeOH±CHCl3) a€or-
ded 41 (0.13 g, 81%) as a white solid: 1H NMR
(DMSO-d6, 500 MHz) d 9.93 (s, 1H), 7.95 (d, J=8.2 Hz,
2H), 7.67 (s, 1H), 7.28 (d, J=8.2 Hz, 2H), 6.97 (s, 1H),
6.26 (br s, 4H), 5.23 (s, 2H), 3.84 (s, 3H), 3.53 (s, 2H);
FABHRMS (NBA/NaI) m/z 387.1236 (M+H+,
C17H18N6O3S requires 387.1239).
Methyl 4-[5-[(2,4-diamino-6(1H)-pyrimidinon-5-yl)thio]-
methyl] - 3 - pyridinyl]benzoate (46). Obtained from 45
(0.032 g, 0.123 mmol) using the procedure described for
41. Chromatography (SiO2, 1Â15 cm, 20% MeOH±
1
CHCl3) a€orded 46 (0.044 g, 94%) as a white solid: H
NMR (DMSO-d6, 500 MHz) d 10.05 (s, 1H), 8.74 (s,
1H), 8.40 (s, 1H), 8.06 (d, J=8.2 Hz, 2H), 7.95 (s, 1H),
7.83 (d, J=8.2 Hz, 2H), 6.36 (br s, 2H), 6.04 (br s, 2H),
3.88 (s, 3H), 3.78 (s, 2H); FABHRMS (NBA/NaI) m/z
384.1137 (M+H+, C18H17N5O3S requires 384.1130).
4-[4-[(2,4-Diamino-6(1H)-pyrimidinon-5-yl)thio]methyl]-
1H-imidazol-1-yl]methyl]benzoic acid (42). Obtained from
41 (0.082 g, 0.212 mmol) using the procedure described for
1
11 to provide 42 (0.058 g, 74%): H NMR (DMSO-d6,
4-[5-[(2,4-Diamino-6(1H)-pyrimidinon-5-yl)thio]methyl]-3
-pyridinyl]benzoic acid (47). Obtained from 46 (0.062 g,
0.163 mmol) using the procedure described for 21. The
solution was concentrated under reduced pressure and
the residue was treated with toluene (3Â5 mL) to remove
traces of H2O to provide 46 (0.050 g, 91%) as a white
400 MHz) d 10.30 (br s, 1H), 9.05 (s, 1H), 7.97 (d, J=
8.4 Hz, 2H), 7.47 (s, 1H), 7.40 (d, J=8.4 Hz, 2H), 6.56 (br s,
4H), 5.44 (s, 2H), 3.68 (s, 2H); MALDIHRMS (DHB) m/z
395.0917 (M+Na+, C16H16N6O3S requires 395.0902).
1
di-tert-Butyl N-4-[4-[(2,4-diamino-6(1H)-pyrimidinon-5-
yl)thio]methyl]- 1H - imidazol -1 -yl]methyl] -L - glutamate
(43). Obtained from 42 (0.014 g, 0.038 mmol) using the
procedure described for 12. Chromatography (SiO2,
1Â15 cm, 10±40% MeOH±CH2Cl2) a€orded 43 (0.014 g,
solid: H NMR (DMSO-d6, 400 MHz) d 8.70 (s, 1H),
8.37 (s, 1H), 8.01 (d, J=8.1 Hz, 2H), 7.80 (s, 1H), 7.69 (d,
J=8.4 Hz, 2H), 6.55 (br s, 2H), 6.12 (br s, 2H), 3.78 (s,
2H); MALDIHRMS (DHB) m/z 370.0978 (M+H+,
C17H15N5O3S requires 370.0974).
1
62%) as a white solid: H NMR (CDCl3, 400 MHz) d
7.77 (d, J=8.1 Hz, 2H), 7.53 (d, J=7.56, 1H), 7.46 (s,
1H), 7.06 (d, J=8.1 Hz, 2H), 6.76 (br s, 2H), 6.58 (s, 1H),
5.60 (br s, 2H), 5.01 (s, 2H), 4.66±4.61 (m, 1H), 3.66 (d,
J=12.3Hz, 1H), 3.60 (d, J=13.2 Hz, 1H), 2.46±2.29 (m,
2H), 2.24±2.17 (m, 1H), 2.10±2.01 (m, 1H), 1.46 (s, 9H),
1.39 (s, 9H); FABHRMS (NBA/CsI) m/z 746.1719
(M+Cs+, C29H39N7O6S requires 746.1737).
di-tert-Butyl N-{4-[5-[(2,4-diamino-6(1H)-pyrimidinon-5-
yl)thio]methyl] - 3 - pyridinyl]benzoyl} - L - glutamate (48).
Obtained from 47 (0.052 g, 0.141 mmol) using the proce-
dure described for 23. Chromatography (SiO2, 1Â15 cm,
20% MeOH±CHCl3) a€orded 48 (0.031 g, 36%) as a
1
white solid: H NMR (CDCl3, 400 MHz) d 9.95 (br s,
1H), 8.60 (s, 1H), 8.40 (s, 1H), 7.83 (d, J=8.1 Hz, 2H),
7.64 (s, 1H), 7.51 (d, J=8.4 Hz, 2H), 7.47 (d, J=7.6Hz,
2H), 6.12 (br s, 2H), 5.38 (br s, 2H), 4.70±4.64 (m, 1H),
3.75 (s, 2H), 2.49±2.34 (m, 1H), 2.12±2.03 (m, 1H), 1.50 (s,
9H), 1.42 (s, 9H); FABHRMS (NBA/CsI) m/z 743.1605
(M+Cs+, C30H38N6O6S requires 743.1605).
N-4-[4-[(2,4-Diamino-6(1H)-pyrimidinon-5-yl)thio]me-
thyl]-1H-imidazol-1-yl]methyl]-L -glutamic acid (3c).
Obtained from 43 (5.1 mg, 0.008 mmol) using the proce-
dure described for 3a to provide 3c±CF3CO2H (2.8 mg,
69%) as a white solid: 1H NMR (DMSO-d6, 400 MHz) d
10.40 (br s, 1H), 9.19 (s, 1H), 8.63 (d, J=7.8 Hz, 2H),
7.92 (d, J=8.1 Hz, 2H), 7.51 (s, 1H), 7.42 (d, J=8.1 Hz,
2H), 6.63 (br s, 4H), 5.43 (s, 2H), 4.41±4.38 (m, 1H), 3.69
(s, 3H), 2.34 (t, J=7.3 Hz, 2H), 2.14±2.07 (m, 1H), 1.99±
1.90 (m, 1H); FABHRMS (NBA/CsI) m/z 634.0468
(M+Cs+, C21H23N7O6S requires 634.0485).
N-{4-[5-[(2,4-Diamino-6(1H)-pyrimidinon-5-yl)thio]me-
thyl]-3-pyridinyl]benzoyl}-L-glutamic acid (4c). Obtained
from 48 (3.0 mg, 0.006 mmol) using the procedure described
for 4a to provide 4c±CF3CO2H (2.5 mg, 84%) as a white
solid: 1H NMR (DMSO-d6, 400 MHz) d 8.92 (s, 1H), 8.75
(d, J=7.8 Hz, 1H), 8.55 (s, 1H), 8.19 (s, 1H), 8.03 (d, J=
8.4 Hz, 2H), 7.84 (d, J=8.4Hz, 2H), 6.66 (br s, 2H), 6.42
(br s, 2H), 4.44±4.41 (m, 1H), 2.38 (t, J=7.6Hz, 2H), 2.16±
2.08 (m, 1H), 2.02±1.92 (m, 1H); FABHRMS (NBA/NaI)
m/z 499.1388 (M+H+, C22H22N6O6S requires 499.1400).
Methyl 4-[5-(acetylthio)methyl-3-pyridinyl]benzoate (44).
Obtained from 19 (0.200 g, 0.764 mmol) using the pro-
cedure described for 39. Chromatography (SiO2,
3Â15 cm, 5% MeOH±CHCl3 a€orded 44 (0.20 g, 87%)
1
as a white solid: H NMR (CDCl3, 500 MHz) d 8.75 (s,
GAR and AICAR Tfase inhibition. This was conducted
as previously detailed23 with the exception that the
AICAR Tfase inhibition was conducted in the absence
of 5 mM b-mercaptoethanol and screened with 10 mM
enzyme, 25 mM inhibitor and 22.5 mM of cofactor.
1H), 8.57 (s, 1H), 8.14 (d, J=8.1 Hz, 2H), 7.85 (s, 1H),
7.64 (d, J=8.1 Hz, 2H), 4.17 (s, 2H), 3.95 (s, 3H), 2.38 (s,
3H); FABHRMS (NBA/NaI) m/z 302.0854 (M+H+,
C16H15NO3S requires 302.0851).

D. L. Boger et al. / Bioorg. Med. Chem. 8 (2000) 1075±1086
1085
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 ®nancial support of the
National Institutes of Health (CA 63536) and the
Skaggs Institute for Chemical Biology.
23. For related analogues and studies see: Caperelli, C. A. J.
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