Design, synthesis, and biological evaluation of fluoronitrophenyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase

Article abstract of DOI:10.1016/S0960-894X(00)00271-7

The examination results of a novel series of potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. These agents incorporate an electrophilic fluoronitropheny

Full text of DOI:10.1016/S0960-894X(00)00271-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1471±1475
Design, Synthesis, and Biological Evaluation of Fluoronitrophenyl
Substituted Folate Analogues as Potential Inhibitors of GAR
Transformylase and AICAR Transformylase
Dale L. Boger,a,* Thomas H. Marsilje,a Rene A. Castro,a Michael P. Hedrick,a
Qing Jin,a Stephen J. Baker,b Jae Hoon Shimb and Stephen J. Benkovicb
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 16 March 2000; accepted 3 May 2000
AbstractÐThe examination results of a novel series of potential inhibitors of glycinamide ribonucleotide transformylase (GAR
Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. These agents incorporate an electrophilic
¯uoronitrophenyl group that can potentially react with an active site nucleophile or the substrate GAR/AICAR amine via
nucleophilic aromatic substitution. # 2000 Elsevier Science Ltd. All rights reserved.
Glycinamide ribonucleotide transformylase (GAR Tfase)
and aminoimidazole carboxamide ribonucleotide trans-
formylase (AICAR Tfase) are folate-dependent enzymes
central to the de novo purine biosynthetic pathway.
GAR Tfase and AICAR Tfase catalyze the transfer of the
N10-formyl group from (6R)-N10-formyltetrahydrofolate
to their respective substrates: GAR and AICAR.1 The
discovery that (6R)-5,10-dideazatetrahydrofolate (Lome-
trexol, DDATHF) 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 In previous stu-
dies, we examined numerous folate-based inhibitors that
incorporated electrophilic functional groups, which could
potentially interact either with active site nucleophiles or
the GAR/AICAR substrate amine.4 Herein, we report the
synthesis and evaluation of a novel class of 5,8-dideaza-
folate (DDAF) and monocyclic 5-deazatetrahydrofolate
(DACTHF) GAR Tfase and AICAR Tfase inhibitors
that incorporate an electrophilic ¯uoronitrophenyl group.
rapid aromatic nucleophilic displacement reactions even at
room temperature.5 In our own studies, we have observed
that ¯uoronitroaromatics stable to LiOH capture imida-
zole at room temperature in the presence of a tertiary
amine base.6 Fluoronitroaromatics have gained wide use
in protein sequencing and protein cross-linking due to
their reactivity toward amino acids with nucleophilic
side chains.7 Despite this potential, there have been few
reports of the use of ¯uoronitroaromatic-based enzyme
inhibitors.8
Our initial e€orts entailed the examination of two DDAF-
based inhibitors (Fig. 1). In contrast to expectations, nei-
ther exhibited time-dependent or irreversible GAR or
AICAR Tfase inhibition. Rather, they served as simple
competitive inhibitors, displaying Ki's comparable to
those observed with typical DDAF or TDAF-based
inhibitors.4 Encouragingly, this indicated that the large
¯uoronitrophenyl groups were not hindering or desta-
bilizing inhibitor binding at the two enzyme active sites,
just that they were not participating in an aromatic
nucleophilic substitution reaction within the active sites.
Consequently, these studies were extended to the
¯uoronitroaromatic inhibitors shown in Table 1. The
acyclic pyrimidinone analogue of the tetrahydrofolate ring
system, 5,10 - dideaza - acyclic - 5,6,7,8 - tetrahydrofolic
(DACTHF) has been shown to retain potent cytotoxic
and enzyme inhibitory properties of the DDATHF ring
system exempli®ed by Lometrexol.9 The DACTHF core
Fluoronitroaromatic compounds are known to be unu-
sually susceptible to nucleophilic attack by soft nucleo-
philes. Thus, while water and hydroxide fail to displace
¯uoride, soft nucleophiles including phenols, imidazoles,
amines, thiols, and selected amides are able to undergo
*Corresponding author. Tel.: +858-784-7522; fax: +858-784-7550;
e-mail: boger@scripps.edu
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00271-7

1472
D. L. Boger et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1471±1475
With the exception of 12 and 17, which were equally
e€ective in the presence or absence of GAR, the remain-
ing inhibitors were more potent when pre-incubated with
only GAR Tfase than when they were pre-incubated
with both GAR Tfase and GAR. This indicates that the
inhibitors examined probably derive their activity via direct
adduct formation with GAR Tfase itself and the pre-incu-
bation conducted with GAR may protect the enzyme from
inactivation. A number of compounds (2, 7, 9, 11, 12, 14±
17, 22, 27, 35 and 40) inactivated the enzyme entirely after
6±8 h of pre-incubation at 250 mM. The remainder of the
inhibitors examined show only moderate activity or were
inactive. With the simplest inhibitors (1±6), only one (2)
showed strong activity. It appears that without the added
binding interaction of an appended benzyl side chain (as
seen in 7±40), the potency relies strongly on the sub-
stitution pattern of the ¯uoronitrophenyl portion of the
molecule. The most potent inhibitor within this group
was 2 which is also the only inhibitor that had two acti-
vating nitro groups enhancing the electrophilicity of the
¯uoronitroaromatic. This requirement for two activating
nitro groups was only seen in the simplest inhibitors (1±6).
Inhibitors 7±16 all share an appended methyl benzoate
group. The structural requirement for activity appears to
be a ¯uorine meta to the amino attachment point with a
nitro group para to the ¯uorine. Other factors including
alkyl chain length appear to be largely inconsequential.
Inhibitors 7±16 are the most consistently active sub-class
of inhibitors examined. Notably, after only 1 min of
incubation, compound 14 inhibited GAR Tfase 94% and
75% in the absence and presence of GAR respectively.
The rapid and potent inhibitory activity of 14 is espe-
cially interesting since 14 only contains one activating
nitro group. The di-t-butyl protected glutamate inhibitors
17±28 appear to have much more stringent structural
requirements for activity with only three inhibitors of this
class (17, 22 and 27) inactivating the GAR Tfase enzyme
after 6±8 h of pre-incubation. All three contain two nitro
activating groups and the less e€ective behavior of this
subclass was expected. In fact, it is surprising that any
members of this class are capable of enzyme inactivation.
Finally, the diacid inhibitors 29±40 were surprisingly
less active inhibitors than the equivalent di-t-butyl ester
inhibitors examined (17±28). In general, the activity of
the diacid inhibitors closely follows the pattern seen in
the corresponding di-t-butyl ester group of inhibitors.
Figure 1.
ring system was chosen in order to provide the inhibi-
tors greater ¯exibility facilitating the active site placement
of the ¯uoronitrophenyl group for nucleophilic attack.
The simplest inhibitors were obtained by reaction of the
Cbz-protected bromoalkyl amine10 with ethyl cyanoacte-
tate (1.5 equiv, K2CO3 (3 equiv), anhyd DMF, 70 ꢀC, 2
h) followed by condensation with guanidine (1.2 equiv,
NaOMe (2 equiv), anhyd CH3OH, 25ꢀC, 2 h) to furnish
the pyrimidinone core. Deprotection of the primary amine
(H2, Pd±C, CH3OH, 25 ꢀC, 6 h) followed by arylation of
the free amine with the appropriate ¯uoronitrobenzene (1.5
equiv, 2,6-lutidine (2 equiv), anhyd DMF, 25ꢀC, 12 h)
generated the inhibitor structures 1±6. The more complex
agents incorporating the benzoate and benzoyl glutamate
groups were obtained similarly. The same alkylamine
pyrimidinones generated above were alkylated (0.75 equiv,
anhyd. DMF, 60ꢀC, 8 h) with either methyl 4-(bromo-
methyl)benzoate or the benzylic bromide derived from the
coupling of a-bromo-p-toluic acid with di-t-butyl gluta-
mate via its acid chloride intermediate (oxalyl chloride,
(1.1 equiv), cat. anhyd DMF, anhyd. THF, 25 ꢀC, 1 h
followed by iPr2NEt (3 equiv), di-t-butyl glutamate (1.1
equiv), anhyd DMF, 25 ꢀC, 12 h). Arylation of the sec-
ondary amine produced with the appropriate ¯uoroni-
trobenzene (1.5 equiv, 2,6-lutidine (2 equiv), anhyd DMF,
25ꢀC, 12 h) generated the inhibitor structures 7±28.
Deprotectioꢀn of the glutamate side chain (1:1 v/v TFA±
CH2Cl2, 25 C, 1.5 h) provided inhibitors 29±40.11
The most consistently active series examined against
GAR Tfase (2, 12, 22 and 35) all contain the 2,4-dinitro-
5-¯uoro substitution pattern in the ¯uoronitrophenyl
group. All four agents showed inactivation of GAR
Tfase within 6 h. The most active member of this series
(12) also completely inhibited GAR Tfase in the presence
of GAR following 6 h of incubation. Importantly, the
activity of 12, which lacks the glutamate side chain,
indicates the potential of simpli®ed antifolate inhibitors
which do not incorporate this side chain.
The activity of inhibitors 1±40 against GAR and
AICAR Tfase as well as their cytotoxic activity (CCRF-
CEM) is shown in Table 2. Two separate GAR Tfase
inhibition experiments were performed to determine if the
time-dependent inhibition was due to inhibitor adduct
formation with either GAR or the enzyme itself. As shown
in Table 2, a number of the inhibitors examined exhibited
time-dependant inhibition of the GAR Tfase at a con-
centration of 250 mM. Agent 2 was the only compound
examined which showed signi®cantly higher potency in
the presence of GAR than in its absence. This indicates
that compound 2 probably gains its inhibitory activity
via direct adduct formation with the substrate GAR.
Five of the compounds (2, 12, 14, 17 and 22) that showed
the most signi®cant inhibition at 250 mM were further
tested against GAR Tfase at a 10Â lower concentration
(25 mM). As expected, these agents still exhibited inhi-
bition albeit at a lower level.

D. L. Boger et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1471±1475
Table 1. Fluoronitrophenyl structural analogues synthesized and evaluated
1473
Compound
n
R1
R2
R3
R4
R5
1
2
3
4
5
6
7
8
1
2
2
2
2
2
1
1
1
1
1
2
2
2
2
2
1
1
1
1
1
2
2
2
2
2
1
2
1
1
1
1
1
1
2
2
2
2
2
2
NO2
NO2
F
NO2
NO2
NO2
NO2
F
NO2
NO2
NO2
NO2
F
NO2
NO2
NO2
NO2
F
NO2
NO2
NO2
NO2
F
NO2
NO2
NO2
NO2
NO2
NO2
F
NO2
NO2
NO2
NO2
NO2
F
NO2
NO2
NO2
NO2
H
H
F
H
F
H
H
F
F
NO2
NO2
F
H
H
NO2
NO2
H
H
F
NO2
NO2
H
H
F
NO2
NO2
H
H
F
NO2
NO2
H
H
F
NO2
NO2
NO2
NO2
H
H
F
NO2
NO2
NO2
H
H
F
NO2
F
F
H
F
F
F
F
H
F
F
F
F
H
F
F
F
F
H
F
F
F
F
H
F
F
F
H
H
F
H
F
F
F
H
F
H
F
F
F
H
H
H
H
H
H
H
CH2-para-Ph-COOMe
CH2-para-Ph-COOMe
CH2-para-Ph-COOMe
CH2-para-Ph-COOMe
CH2-para-Ph-COOMe
CH2-para-Ph-COOMe
CH2-para-Ph-COOMe
CH2-para-Ph-COOMe
CH2-para-Ph-COOMe
CH2-para-Ph-COOMe
CH2-para-Ph-Glu(O-t-Bu)2
CH2-para-Ph-Glu(O-t-Bu)2
CH2-para-Ph-Glu(O-t-Bu)2
CH2-para-Ph-Glu(O-t-Bu)2
CH2-para-Ph-Glu(O-t-Bu)2
CH2-para-Ph-Glu(O-t-Bu)2
CH2-para-Ph-Glu(O-t-Bu)2
CH2-para-Ph-Glu(O-t-Bu)2
CH2-para-Ph-Glu(O-t-Bu)2
CH2-para-Ph-Glu(O-t-Bu)2
CH2-para-Ph-Glu(O-t-Bu)2
CH2-para-Ph-Glu(O-t-Bu)2
CH2-para-Ph-Glu-OH
CH2-para-Ph-Glu-OH
CH2-para-Ph-Glu-OH
CH2-para-Ph-Glu-OH
CH2-para-Ph-Glu-OH
CH2-para-Ph-Glu-OH
CH2-para-Ph-Glu-OH
CH2-para-Ph-Glu-OH
CH2-para-Ph-Glu-OH
CH2-para-Ph-Glu-OH
CH2-para-Ph-Glu-OH
CH2-para-Ph-Glu-OH
9
F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
H
H
H
F
F
H
H
H
F
F
H
H
H
F
F
H
H
F
F
H
F
F
H
H
F
H
F
F
H
H
F
The activity of these agents against AICAR Tfase after
12 h pre-incubation was also examined (Table 2). In gen-
eral, the agents were less potent AICAR Tfase inhibitors.
Only four of the agents examined (29, 34, 35 and 40)
strongly inhibited AICAR Tfase after the 12 h pre-incu-
bation. These all contain the free glutamic acid side chain
and are the same four agents of this subclass that sig-
ni®cantly inhibited GAR Tfase. In contrast, the simpler
agents (2, 7, 9, 11, 12 and 14±16) that were inhibitors of
GAR Tfase exhibited only slight or no inhibition of
AICAR Tfase. Given the general reactivity of ¯uoroni-
trophenyl compounds, it is interesting to note the selectivity
seen between the related GAR Tfase and AICAR Tfase.
CCRF-CEM cells (Table 2). The cytotoxic activity of
most of the agents was relatively nonpotent and uniform
against the CCRF-CEM cell line regardless of whether
media purines (hypoxanthine) or pyrimidines (thymidine)
were present or absent. This indicates a lack of activity
due to speci®c inhibition of the purine or pyrimidine bio-
synthetic pathways. The one exception to this absence of
selectivity was 32, which in addition to being one of the
most cytotoxic compounds in the series examined, also
showed a signi®cant increase in potency in the absence of
media hypoxanthine indicating its selective cytotoxicity
may be derived from inhibition of either GAR or
AICAR transformylase.
Agents 1±40 as well as Lometrexol were examined for
cytotoxic activity both in the presence (+) and absence
( ) of added hypoxanthine and thymidine against
In preliminary studies to elucidate the nature of the
activity of this class of inhibitors, 12 (500 mM) was
incubated with GAR Tfase (6.4 mM). Electrospray mass

1474
D. L. Boger et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1471±1475
Table 2. In vitro biological activity against GAR Tfase, AICAR Tfase and CCRF-CEM cells12
Pre-incubation (time)
Gar Tfase+GARa
GAR Tfasea
30 min
AICAR Tfasea
12 h
CCRF-CEM IC50 (mM)b
) T, (+) T,
( ) H
Agent
3 min
30 min
(h)
3 min
(h)
(+) T,
(+) Hc
(
(+) H
(
(
) T,
) H
1
2
NAd
100
NA
NA
NA
NA
NA
66
79
32
81
35
64
NA
82
25
NA
75
68
25
81
42
40
66
42
76
NA
97
39
98
72
68
96
91
NA
75
NA
2 (6)
56 (6)
88 (6)
NA
Ki=35 mM
76
76
80
87
80
86
47
68
16
78
17
46
83
61
6
NA
64
58
1.2
37
1.0
43
1.1
55
1.00
73
71
77
82
70
69
24
60
9
43
3
9
77
57
5
76
41
33
11
68
47
57
52
53
29
74
61
24
56
55
22
58
nt
96
84
78
94
67
nt
NA
80
78
62
71
39 (6)
58 (6)
59 (6)
63 (6)
53 (6)
64 (7)
2 (6)
30 (6)
0 (6)
12 (6)
2 (6)
0 (6)
65 (6)
41 (6)
0 (6)
39 (6)
0 (6)
0 (6)
2 (25 mM)
3
4
5
6
7
8
9
10
NA
NA
NA
NA
NA
54
85
29
62
31
22
83
78
12
NA
44
54
12
98
47
48
57
40
65
NA
94
42
93
80
59
93
nt
NA
92
n.t.d
NA
98
73
97
94
76
97
97
68
76
97
97
>100
97
>100
NA
NA
NA
NA
75
73
NA
94
NA
77
n.t.
96
NA
n.t.
NA
94
>100
1.1
67
>100
72
11
>100
1.7
36
>100
72
11
>100
1.3
48
>100
72
27 (6)
67 (6)
30 (6)
47 (6)
22 (6)
0 (6)
2 (6)
75 (6)
6 (6)
84 (6)
8 (6)
14 (6)
7 (6)
79 (6)
47 (6)
49 (6)
58 (6)
37 (6)
40 (6)
76 (6)
88 (6)
51 (8)
85 (8)
66 (6)
42 (8)
63 (8)
68 (6)
NA
89 (7)
85 (8)
NA
66 (6)
43 (6)
92 (8)
79 (8)
79 (8)
81 (8)
45 (6)
1.9
61
91
66
13
11
12
12
2.5
3.5
4.3
3.9
12 (25 mM)
13
14
14 (25 mM)
15
45
>100
55
>100
45
>100
41
>100
78
70
44
19
75
52
82
65
57
38
79
71
26
78
53
32
71
77
93
90
80
87
91
85
87
78
96
66
89
30
53
15
55
66
12
43
55
4.3
45
59
13
16
17
0 (6)
87
n.t.
99
17 (25 mM)
18
19
20
21
22
22 (25 mM)
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
39 (6)
52 (3)
40 (6)
48 (6)
35 (6)
4 (6)
69 (6)
47 (6)
20 (8)
27 (8)
27 (6)
3 (8)
44 (8)
31 (6)
91 (7)
73 (7)
81 (8)
98 (8)
32 (6)
13 (6)
88 (8)
87 (8)
74 (8)
83 (8)
13 (6)
46
44
50
20
1.8
40
47
56
27
1.5
46
50
69
27
46
47
51
23
98
NA
NA
90
n.t.
NA
99
NA
NA
81
84
7
NA
NA
NA
NA
6
2.6
3.1
20
7.7
14
3.1
8.9
4.0
20
2.4
12
1.7
7.5
3.9
36
17
3.4
3.4
4.8
3.5
24
13
8.7
4.8
7.1
3.2
52
52
36
56
>100
>100
NA
89
78
NA
NA
NA
NA
79
>100
>100
>100
>100
54
>100
>100
>100
>100
50
>100
>100
15
>100
37
44
>100
>100
>100
>100
48
81
NA
90
nt
98
85
82
2.1
>100
49
40
>100
>100
>100
>100
36
11
43
51
NA
NA
NA
NA
11
>100
>100
>100
>100
36
>100
>100
>100
>100
46
38
39
91
66
96
59
97
40
Lometrexol
>100
>100
0.07
0.13
aGAR and AICAR Tfase inhibition following preincubation (%control activity remaining (normalized)). Agents were tested at a concentration of
250 mM.
bThe cytotoxic assays were conducted in media free of purines or pyrimidines in the presence of (+) or absence of ( ) thymidine or hypoxanthine.
cT=Thymidine, H=Hypoxanthine.
dNA=not active, nt=not tested.
spectrometry indicated the covalent attachment of 1
molar equivalent of 12 to GAR Tfase. Interestingly,
when 12 (250 mM) was incubated with GAR Tfase (6.4
mM) in the presence of b-GAR (1.25 mM), electrospray
mass spectrometry did not indicate covalent attachment
of 12 to the enzyme, although 12 is still an e€ective time-
dependent inhibitor of GAR Tfase in the presence of b-
GAR. This suggests that direct active site alkylation
occurs in the absence of b-GAR, but that b-GAR alky-
lation occurs within the active site when it is included in
the pre-incubation.
In conclusion, a novel class of folate based inhibitors has
been reported that incorporate the ¯uoronitrophenyl
group. Members of this new class of agents have been
shown to inhibit GAR Tfase in a time dependent fash-
ion. In the one case (12) examined to date, this occurs
via adduct formation with nucleophilic residues in the
GAR Tfase active site ( b-GAR) or by b-GAR alkyla-
tion (+ b-GAR). Further studies with the most active
series of compounds (2, 12, 22 and 35 as well as 14 and
32) are in progress and the results will be disclosed in
due course.

D. L. Boger et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1471±1475
1475
References and Notes
9. (a) Taylor, E. C.; Harrington, P.M.; Shih, C. A. Hetero-
cycles 1989, 28, 1169. (b) Shih, C.; Gossett, L. S.; Worzalla, J.
F.; Rinzel, S. M.; Grindey, G. B.; Harrington, P. M.; Taylor,
E. C. J. Med. Chem. 1992, 35, 1109. (c) Bigham, E. C.;
Hodson, S. J.; Mallory, R.; Wilson, D.; Duch, D. S.; Smith, G.
K.; Ferone, R. J. Med. Chem. 1992, 35, 1399. (d) Taylor, E.
C.; Schrader, T. H.; Walensky, L. D. Tetrahedron 1992, 48, 19.
10. (a) Jacobson, A. R.; Tam, S. W.; Sayre, L. M. J. Med.
Chem. 1991, 34, 2816. (b) Khan, H. A.; Robins, D. J. J. Chem.
Soc. Perkin Trans. 1 1985, 101.
1. Warren, M. S.; Mattia, K. M.; Marolewski, A. E.;
Benkovic, S. J. Pure Appl. Chem. 1996, 68, 2029.
2. Jackson, R. C.; Harkrader, R. J. In Nucleosides and Cancer
Treatment; Tattersall, M. H. N., Fox, R. M., Eds.; Academic:
Sydney, 1981; pp 18±31.
3. (a) Beardsley, G. P.; Moroson, B. A.; Taylor, E. C.; Moran,
R. G. J. Biol. Chem. 1989, 264, 328. (b) Baldwin, S. W.; Tse,
A.; Gossett, L. S.; Taylor, E. C.; Rosowsky, A.; Shih, C.;
Moran, R. G. Biochemistry 1991, 30, 1997.
4. (a) Boger, D. L.; Haynes, N.-E.; Kitos, P. A.; Warren, M. S.;
Ramcharan, J.; Marolewski, A. E.; Benkovic, S. J. Bioorg. Med.
Chem. 1997, 5, 1817. (b) Boger, D. L.; Haynes, N.-E.; Warren, M.
S.; Gooljarsingh, L. T.; Ramcharan, J.; Kitos, P. A.; Marolewski,
A. E.; Benkovic, S. J. Bioorg. Med. Chem. 1997, 5, 1831. (c) Boger,
D. L.; Haynes, N.-E.; Warren, M. S.; Ramcharan, J.; Kitos, P. A.;
Benkovic, S. J. Bioorg. Med. Chem. 1997, 5, 1839. (d) Boger, D.
L.; Haynes, N.-E.; Warren, M. S.; Ramcharan, J.; Marolewski, A.
E.; Kitos, P. A.; Benkovic, S. J. Bioorg. Med. Chem. 1997, 5, 1847.
(e) Boger, D. L.; Kochanny, M. J.; Cai, H.; Wyatt, D.; Kitos, P.
A.; Warren, M. S.; Ramcharan, L. T.; Gooljarsingh, L. T.;
Benkovic, S. J. Bioorg. Med. Chem. 1998, 6, 643.
5. Terrier, F. In Nucleophilic Aromatic Displacement: The
In¯uence of the Nitro Group; VCH: New York, 1991.
6. Boger, D. L.; Zhou, J.; Borzilleri, R. M.; Nukui, S.; Castle,
S. L. J. Org. Chem. 1997, 62, 2059.
7. (a) Sanger, F. Biochem. J. 1945, 39, 507. (b) Zahn, H. Kol-
loid-Z. 1951, 121, 40. (c) Zahn, H.; Meienhofer, J. Makromol.
Chem. 1958, 26, 126.
8. (a) Molina, H. A.; Bistoletti, Y. Acta Bioquim. Clin. Lati-
noam. 1988, 22, 391. (b) Shanahan, M. F.; Jacquez, J. A.
Membr. Biochem. 1978, 1, 239. (c) Riva, F.; Giantoso, A.;
Voltattorni, C. B.; Turano, C. Methods Enzymol. 1979, 62,
510. (d) Riva, F.; Giartosio, A.; Turano, C. Methods Enzymol.
1977, 46, 441. (e) Riva, F.; Giartosio, A.; Voltattorni, C. B.;
Orlacchio, A.; Turano, C. Biochem. Biophys. Res. Commun.
1975, 66, 863. (f) Riva, F.; Carotti, D.; Barra, D.; Giartosio,
A.; Turano, C. J. Biol. Chem. 1980, 255, 9230. (g) Carotti, D.;
Riva, F.; Santucci, R.; Ascoli, F.; Fasella, P. Eur. J. Biochem.
1982, 124, 589. (h) Ottonello, S.; Mozzarelli, A.; Rossi, G.
L.; Carotti, D.; Riva, F. Eur. J. Biochem. 1983, 133, 47. (i)
Carotti, D.; Andria, F.; Giartosio, A.; Turano, C.; Riva, F.
Eur. J. Biochem. 1985, 146, 619.
11. 1H NMR characterization of representative compounds is
as follows: 2: 1H NMR (DMSO-d6, 250 MHz) d 9.29 (bs, 1H),
8.87 (d, J=8.0 Hz, 1H), 7.16 (d, J=15.0 Hz, 1H), 6.12 (bs,
2H), 5.94 (bs, 2H), 3.18±3.10 (m, 2H), 2.26 (t, J=6.8 Hz, 2H),
1.65±1.54 (m, 2H); 12: 1H NMR (DMSO-d6, 400 MHz) d 8.57
(d, J=7.9 Hz, 1H), 7.92 (d, J=8.2 Hz, 2H), 7.41 (d, J=7.9
Hz, 2H), 7.31 (d, J=14.9 Hz, 1H), 6.08 (bs, 2H), 5.84 (bs, 2H),
4.73 (s, 2H), 3.83 (s, 3H), 3.21 (t, J=7.0 Hz, 2H), 2.09 (t,
1
J=7.9 Hz, 2H), 1.66±1.59 (m, 2H); 22: H NMR (DMSO-d6,
400 MHz) d 8.57 (d, J=7.7 Hz, 1H), 7.83 (d, J=8.0 Hz, 2H),
7.37 (d, J=8.0 Hz, 2H), 7.32 (d, J=15.0 Hz, 1H), 4.73 (s, 2H),
4.36±4.25 (m, 1H), 3.26±3.15 (m, 2H), 2.33 (t, J=6.6 Hz, 2H),
2.20±2.11 (m, 2H), 2.10±1.85 (m, 2H), 1.74±1.60 (m, 2H), 1.40
1
(s, 9H), 1.37 (s, 9H); 35: H NMR (DMSO-d6, 250 MHz) d
8.56 (d, J=8.4 Hz, 1H), 7.84 (d, J=9.1 Hz, 2H), 7.38 (d,
J=8.0 Hz, 2H), 7.31 (d, J=15.0 Hz, 1H), 4.73 (s, 2H), 4.46±
4.32 (m, 1H), 3.29±3.15 (m, 2H), 2.34 (t, J=8.0 Hz, 2H), 2.20±
2.05 (m, 2H), 2.03±1.88 (m, 2H), 1.74±1.60 (m, 2H).
12. The CCRF-CEM cytotoxicity assay, the AICAR Tfase
inhibition studies and the time-dependent GAR Tfase inhibi-
tion studies were performed as described in Boger, D. L.;
Haynes, N.-E.; Kitos, P. A.; Warren, M. S.; Ramcharan, J.;
Marolewski, A. E.; Benkovic, S. J. Bioorg. Med. Chem. 1997,
5, 1817 with the following changes: In the GAR Tfase assay,
solutions were made containing 50 nM purN GAR Tfase, 750
nM BSA, 1.25 mM GAR (if GAR was present during the pre-
incubation) and 250 mM inhibitor. These solutions were incu-
bated at room temperature. Aliquots of these stock solutions
were taken, diluted 25-fold in assay bu€er, and thermostated
to 26 ꢀC on a Cary 1 UV-Visible spectrophotometer. Assays
were initiated by the addition of 20 mM fDDF at the indicated
time points. In the AICAR Tfase assay, the assay solutions
were incubated at room temperature for 12 h before the reac-
tion was initiated by addition of AICAR.