Discovery and development of a small molecule library with lumazine synthase inhibitory activity

Article abstract of DOI:10.1021/jo900238q

(Chemical Equation Presented) (E)-5-Nitro-6-(2-hydroxystyryl)pyrimidine-2, 4(1H,3H)-dione (9) was identified as a novel inhibitor of Schizosaccharomyces pombe lumazine synthase by high-throughput screening of a 100000 compound library. The K_i o

Full text of DOI:10.1021/jo900238q

pubs.acs.org/joc
Discovery and Development of a Small Molecule Library with Lumazine
Synthase Inhibitory Activity
Arindam Talukdar,† Meghan Breen,† Adelbert Bacher,‡ Boris Illarionov,§ Markus Fischer,§
Gunda Georg,^ Qi-Zhuang Ye,^ and Mark Cushman*,†
†Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and
Pharmaceutical Sciences, and The Purdue Cancer Center, Purdue University, West Lafayette,
‡
§
€
Indiana 47907, Ikosatec GmbH, Konigsbergerstr. 74, 85748 Garching, Germany, Institute of Food
Chemistry, University of Hamburg, D-20146 Hamburg, Germany, and ^Department of Medicinal Chemistry,
University of Kansas, Lawrence, Kansas 66047
cushman@pharmacy.purdue.edu
Received February 5, 2009
(E)-5-Nitro-6-(2-hydroxystyryl)pyrimidine-2,4(1H,3H)-dione (9) was identified as a novel inhibitor
of Schizosaccharomyces pombe lumazine synthase by high-throughput screening of a 100000
compound library. The Ki of 9 vs Mycobacterium tuberculosis lumazine synthase was
95 μM. Compound 9 is a structural analogue of the lumazine synthase substrate 5-amino-6-
(D-ribitylamino)-2,4-(1H,3H)pyrimidinedione (1). This indicates that the ribitylamino side chain
of the substrate is not essential for binding to the enzyme. Optimization of the enzyme inhibitory
activity through systematic structure modification of the lead compound 9 led to (E)-5-nitro-6-
(4-nitrostyryl)pyrimidine-2,4(1H,3H)-dione (26), which has a Ki of 3.7 μM vs M. tuberculosis
lumazine synthase.
Introduction
targets for the design and synthesis of new antibiotics, which
are urgently needed because pathogens are becoming drug-
resistant at an alarming rate.
Riboflavin (4, vitamin B2) plays a crucial role in many
biological processes, including photosynthesis and mito-
chondrial electron transport. While animals obtain ribofla-
vin from dietary sources, numerous microorganisms,
including Gram-negative pathogenic bacteria and yeasts,
lack an efficient riboflavin uptake system and are therefore
absolutely dependent on endogenous riboflavin biosynth-
esis.1-4 Riboflavin biosynthesis therefore offers attractive
Lumazine synthase and riboflavin synthase catalyze
the last two steps in the biosynthesis of riboflavin (4)
(Scheme 1). Lumazine synthase catalyzes the condensation
of 3,4-dihydroxy-2-butanone 4-phosphate (2) with 5-amino-
6-ribitylamino-2,4(1H,3H)pyrimidinedione (1), yielding 6,
7-dimethyl-8-D-ribityllumazine (3).5,6 The final step in
the biosynthesis involves a mechanistically unusual dismuta-
tion of two molecules of 3, resulting in the formation of
one molecule of riboflavin (4) and one molecule of the
(1) Wang, A. Yichuan Xuebao 1992, 19, 362–368.
(2) Oltmanns, O.; Lingens, F. Z. Naturforsch. 1967, 22 b, 751–754.
(3) Logvinenko, E. M.; Shavlovsky, G. M. Mikrobiologiya 1967, 41, 978–
979.
(4) Neuberger, G.; Bacher, A. Biochem. Biophys. Res. Commun. 1985,
127, 175–181.
(5) Neuberger, G.; Bacher, A. Biochem. Biophys. Res. Commun. 1986,
139, 1111–1116.
(6) Kis, K.; Volk, R.; Bacher, A. Biochemistry 1995, 34, 2883–2892.
DOI: 10.1021/jo900238q
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Published on Web 06/24/2009
J. Org. Chem. 2009, 74, 5123–5134 5123
2009 American Chemical Society

Talukdar et al.
JOCFeatured Article
SCHEME 1
SCHEME 2
pyrimidinedione derivative 1, which can then be recycled by
lumazine synthase.7-15
A high-throughput screening (HTS) technique was devel-
oped on the basis of competitive binding of lumazine
synthase inhibitors and riboflavin to the active site
of Schizosaccharomyces pombe lumazine synthase.17,18
The capacity of the S. pombe lumazine synthase to bind
riboflavin is unique. Free riboflavin is fluorescent with high
quantum yield, while enzyme-bound riboflavin is not.19
Thus, displacement of riboflavin from the binding pocket
results in a significant fluorescence increase of the system.
The change in fluorescence caused by competitive binding
between riboflavin and other ligands for S. pombe lumazine
synthase was used to identify lumazine synthase inhibitors.17
All of the known inhibitors tested were positively identified,
which confirmed the authenticity of this assay. HTS
of a commercial 100000 compound library yielded some
interesting results, including the identification of a lead
compound 9.
Although the details of the reaction catalyzed by lumazine
synthase have not been completely elucidated, a reasonable
pathway can be outlined at the present time as depicted in
Scheme 2. Condensation of the primary amino group of the
substituted pyrimidinedione 1 with the ketone 2 to give Schiff
base 5, elimination of phosphate to yield the enol 6, tautomer-
ization of the enol 6 and isomerization of the imine to produce
the ketone 7, ring closure, and dehydration of the covalent
hydrate 8 provide the product 3.16 It can be assumed that the
inorganic phosphate formed after elimination from 5 would
remain enzyme bound, at least for some time, but that it would
eventually have to be removed to make room for another
molecule of the substrate 2. The present uncertainties revolve
around the timing of phosphate elimination and the conforma-
tional reorganization of the side chain leading to intermediate 7.
(7) Fischer, M.; Haase, I.; Feicht, R.; Schramek, N.; Kohler, P.; Schie-
berle, P.; Bacher, A. Biol. Chem. 2005, 386, 417–428.
(8) Gerhardt, S.; Schott, A.-K.; Kairies, N.; Cushman, M.; Illarionov, B.;
Eisenreich, W.; Bacher, A.; Huber, R.; Steinbacher, S.; Fischer, M. Structure
2002, 10, 1371–1381.
(9) Illarionov, B.; Eisenreich, W.; Bacher, A. Proc. Natl. Acad. Sci. U.S.
A. 2001, 98, 7224–7229.
(10) Illarionov, B.; Eisenreich, W.; Schramek, N.; Bacher, A.; Fischer, M.
J. Biol. Chem. 2005, 280, 28541–28546.
The thermodynamic HTS assay described above effec-
tively bypasses the problems associated with the instabilities
of the lumazine synthase substrates 1 and 2, and it also
simplifies the assay by removing the time element. However,
practically speaking, it had two limitations: (1) assay mix-
tures contained traces of free riboflavin that produced a
(11) Illarionov, B.; Kemter, K.; Eberhardt, S.; Richter, G.; Cushman,
M.; Bacher, A. J. Biol. Chem. 2001, 276, 11524–11530.
(12) Maley, G. F.; Plaut, G. W. E. J. Biol. Chem. 1959, 234, 641–647.
(13) Plaut, G. W. E. J. Biol. Chem. 1963, 238, 2225–2243.
(14) Plaut, G. W. E.; Harvey, R. A. Methods Enzymol. 1971, 18B, 515–
538.
(15) Wacker, H.; Harvey, R. A.; Winestock, C. H.; Plaut, G. W. E. J.
Biol. Chem. 1964, 239, 3493–3497.
(16) Volk, R.; Bacher, A. J. Am. Chem. Soc. 1988, 110, 3651–3653.
(17) Chen, J.; Illarionov, B.; Bacher, A.; Fischer, M.; Haase, I.; Georg,
G.; Ye, Q.-z.; Ma, Z.; Cushman, M. Anal. Biochem. 2005, 338, 124–130.
(18) Kaiser, J.; Illarionov, B.; Rohdich, F.; Eisenreich, W.; Saller, S.; Van
den Brulle, J.; Cushman, M.; Bacher, A.; Fischer, M. Anal. Biochem. 2007,
365, 52–61.
(19) Fischer, M.; Haase, I.; Feicht, R.; Richter, G.; Gerhardt, S.;
Changeux, J.-P.; Huber, R.; Bacher, A. Eur. J. Biochem. 2002, 269, 519–526.
5124 J. Org. Chem. Vol. 74, No. 15, 2009

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˚
FIGURE 1. Hypothetical model of the complex of the lead compound 9 with M. tuberculosis lumazine synthase. The distances shown are in A.
The diagram is programmed for wall-eyed (relaxed) viewing.
fluorescent background, and (2) lumazine synthase inhibi-
tors that bind outside the active site would probably be
unable to release free riboflavin. In spite of these problems,
the thermodynamic assay remained the method of choice for
screening thousands of compounds, and the activities of the
hit compounds were confirmed in secondary assays invol-
ving classical enzyme kinetics.
synthase. Enzyme assays were therefore performed using
the riboflavin synthases of M. tuberculosis and E. coli. In
order to help define the spectrum of activity, lumazine
synthase inhibition assays were also performed on M. tuber-
culosis lumazine synthase as well as S. pombe lumazine
synthase.
Our specific interest in compound 9 is the result of its
structural similarity to the substrate 1. The identification of 9
as a lumazine synthase inhibitor represents an extension of
ongoing work on the synthesis of metabolically stable inter-
mediates in the lumazine synthase-catalyzed reaction.20-23
The HTS hit compound 9 demonstrates that the ribitylamino
chain present in the substrate, which was thought to be
necessary for binding to the enzyme, can be replaced by a
simple hydroxystyryl moiety with retention of lumazine
synthase inhibitory activity. The difference in physical prop-
erties between compounds 1 and 9 has significant implica-
tions for antibiotic drug development. Substrate analogues
with ribityl side chains are not suitable as drug candidates
because their hydrophilic nature can be expected to prevent
them from penetrating bacterial cell walls. Gram-negative
bacterial cell walls contain an outer membrane composed of
phospholipids and lipopolysaccharides that face toward the
external environment. Replacing the hydrophilic ribityl side
chain of compound 1 with the styryl moiety renders com-
pound 9 more lipophilic, which should facilitate the entry
of the compound into bacteria. Compound 9 displayed
Ki values of 210 μM vs S. pombe lumazine synthase and
95 μM vs Mycobacterium tuberculosis lumazine synthase.
Thus, there is ample room for designing more potent luma-
zine synthase inhibitors based on the structure of the lead
compound 9. Moreover, the lead compound 9 is a substrate
analogue of the lumazine synthase-catalyzed reaction, but it
is also a product analogue of the riboflavin synthase-cata-
lyzed reaction. Structural analogues of 9 might therefore be
expected to inhibit riboflavin synthase as well as lumazine
Results and Discussion
The lumazine synthase inhibitory activity of the HTS hit
compound 9 might be related to the presence of the phenolic
hydroxy on the aromatic ring, which could possibly mimic
one of the ribityl hydroxyl groups of the substrate 1. In
addition, it is known that the substrate analogue 10, which
also contains a nitro group, has high affinity for the active
site of lumazine synthase and has been crystallized in com-
plex with Bacillus subtilis lumazine synthase.24,25 Molecular
modeling was performed in order to investigate the binding
mode of the hit compound 9 to the enzyme. The lead
compound 9 was docked into the active site of the structure
of M. tuberculosis lumazine synthase26,27 using GOLD soft-
ware (BST, version 3.0, 2005), and energy minimization was
then performed with Sybyl 7.1. The resulting structure is
displayed in Figure 1. According to this hypothetical model,
the binding of the hit compound 9 in the active site of
M. tuberculosis lumazine synthase is similar to that
of substrate and product analogues.26-28 The structure
(24) Bacher, A.; Ludwig, H. C. Eur. J. Biochem. 1982, 127, 539–545.
€
(25) Ritsert, K.; Huber, R.; Turk, D.; Ladenstein, R.; Schmidt-Base, K.;
Bacher, A. J. Mol. Biol. 1995, 253, 151–167.
(26) Morgunova, K.; Meining, W.; Illarionov, B.; Haase, I.; Jin, G.;
Bacher, A.; Cushman, M.; Fischer, M.; Ladenstein, R. Biochemistry 2005,
44, 2746–2758.
(20) Zhang, Y. L.; Illarionov, B.; Morgunova, E.; Jin, G. Y.; Bacher, A.;
Fischer, M.; Ladenstein, R.; Cushman, M. J. Org. Chem. 2008, 73, 2715–
2724.
(21) Talukdar, A.; Illarionov, B.; Bacher, A.; Fischer, M.; Cushman, M.
J. Org. Chem. 2007, 72, 7167–7175.
(22) Zhang, Y. L.; Jin, G. Y.; Illarionov, B.; Bacher, A.; Fischer, M.;
Cushman, M. J. Org. Chem. 2007, 72, 7176–7184.
(23) Chen, J.; Sambaiah, T.; Illarionov, B.; Fischer, M.; Bacher, A.;
Cushman, M. J. Org. Chem. 2004, 69, 6996–7003.
(27) Morgunova, E.; Illarionov, B.; Sambaiah, T.; Haase, I.; Bacher, A.;
Cushman, M.; Fischer, M.; Ladenstein, R. FEBS J. 2006, 273, 4790–4804.
(28) Gerhardt, S.; Haase, I.; Steinbacher, S.; Kaiser, J. T.; Cushman, M.;
Bacher, A.; Huber, R.; Fischer, M. J. Mol. Biol. 2002, 318, 1317–1329.
J. Org. Chem. Vol. 74, No. 15, 2009 5125

Talukdar et al.
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SCHEME 3
solvent, which initially resulted in the piperidine salt of the
condensation product, the reaction was performed in
1-butanol, a high-boiling alcohol, in the presence of 1 equiv
of piperidine.
Two classes of compounds were synthesized as shown
in structures 9, 15-18 (class I), and 19-24 (class II).
The enzyme-inhibitory activities of these compounds
were determined using lumazine synthases from M. tubercu-
losis and S. pombe and riboflavin synthases from Escherichia
coli and M. tuberculosis. The results are listed in Table 1.
SCHEME 4
involves hydrogen bonding of the phenolic hydroxyl
group of 9 with Ala59, Ile60, and Glu61. In addition,
stacking interactions involving Trp27 stabilize the bind-
ing. The molecular modeling results indicate that the bind-
ing of hit compound 9 in the active site of M. tuberculosis
lumazine synthase is certainly plausible, and it most
likely resembles the known crystal structure of the complex
formed between the related substrate analogue 10 and the
enzyme.
We have attempted to employ versatile methods that
would allow the efficient synthesis of a wide variety of target
molecules. Accordingly, an array of aromatic aldehydes were
condensed with 6-methyl-5-nitrouracil to afford a focused
library of alkenes with pure trans geometry.29-31 No attempt
was made to synthesize cis alkenes because the preliminary
ligand docking studies indicated unfavorable interactions
in the active site. As outlined in Scheme 3, nitration of
6-methyluracil (11) in the presence of H2SO4 and fuming
nitric acid furnished 6-methyl-5-nitrouracil (12).32 Conden-
sation of compound 12 with aromatic aldehydes in the
presence of piperidine provided the piperidine salts of 5-
nitro-6-styryluracil derivatives 13. The piperidine salts were
neutralized by the addition of excess hydrochloric acid to
give pure 5-nitro-6-styryluracil derivatives. The presence of
the nitro group at the 5-position activates the methyl group
for the reaction with piperidine acting as the basic catalyst.
The protocol did indeed seem to be versatile until the
condensation was attempted with 2-nitrobenzaldehyde,
4-nitrobenzaldehyde, and 2,3-dihydroxybenzaldehyde. In
those cases, the reaction did not produce the desired pro-
ducts, which is consistent with previous literature reports.33
The protocol was therefore modified (Scheme 4) to provide
the required 2-nitro derivative 14 and the other desired
condensation products. Instead of using piperidine as the
(29) Grahner, B.; Winiwarter, S.; Lanzner, W.; Muller, C. E. J. Med.
Chem. 1994, 37, 1526–1534.
(30) Nishigaki, S.; Kanamori, Y.; Senga, K. Chem. Pharm. Bull. 1980, 28,
1636–1641.
(31) Senga, K.; Ichiba, M.; Nishigaki, S. J. Org. Chem. 1979, 44, 3830–
3834.
(32) Cresswell, R. M.; Wood, H. S. C. J. Chem. Soc. 1960, 4768–4775.
(33) Ross, W. C. J. J. Chem. Soc. 1948, 1128–1135.
The hit compound 9 exhibits a Ki of 210 μM vs S. pombe
lumazine synthase and a Ki of 95 μM vs M. tuberculosis
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TABLE 1. Inhibition Constants vs S. pombe Lumazine Synthase, M. tuberculosis Lumazine Synthase, M. tuberculosis Riboflavin Synthase, E. coli Riboflavin
Synthasea
compd
enzyme
Ks,b μM
kcat,c min-1
Ki,d μM
Kis,e μM
mode of inhibition
9
S. pombe LS
2.0 ( 0.2
13 ( 1
1.38 ( 0.03
0.26 ( 0.01
4.1 ( 0.2
0.31 ( 0.01
0.23 ( 0.01
0.43 ( 0.01
5.1 ( 0.1
1.57 ( 0.02
0.36 ( 0.01
0.27 ( 0.02
5.1 ( 0.1
210 ( 30
95 ( 6
competitive
competitive
uncompetitive
partial
competitive
partial
mixed
partial
mixed
partial
uncompetitive
uncompetitive
uncompetitive
uncompetitive
mixed
M. tuberculosis LS
M. tuberculosis RS
M. tuberculosis LS
M. tuberculosis LS
M. tuberculosis LS
E. coli RS
63 ( 6
917 ( 212
49 ( 12
14
15
16
17
38 ( 3
16 ( 3
7.1 ( 0.8
12 ( 5
50 ( 7
17 ( 2
17 ( 5
26 ( 4
16 ( 3
81 ( 29
37 ( 16
122 ( 10
53 ( 5
2.6 ( 0.3
1.2 ( 0.1
20 ( 2
10 ( 3
S. pombe LS
4.9 ( 0.9
12 ( 4
19 ( 9
M. tuberculosis LS
M. tuberculosis RS
E. coli RS
7.2 ( 1.0
3.4 ( 0.3
2.5 ( 0.2
22 ( 3
18
S. pombe LS
1.47 ( 0.04
0.17 ( 0.01
0.22 ( 0.02
1.71 ( 0.01
0.322 ( 0.004
1.41 ( 0.02
0.28 ( 0.01
2.21 ( 0.02
0.33 ( 0.01
0.33 ( 0.01
0.34 ( 0.01
1.40 ( 0.03
0.27 ( 0.01
1.14 ( 0.02
0.33 ( 0.01
1.30 ( 0.02
0.45 ( 0.01
1.39 ( 0.03
0.353 ( 0.004
0.27 ( 0.01
0.39 ( 0.02
0.40 ( 0.01
1.23 ( 0.01
0.34 ( 0.01
0.37 ( 0.01
0.64 ( 0.03
0.47 ( 0.01
0.59 ( 0.02
0.45 ( 0.01
1.09 ( 0.02
0.38 ( 0.01
4.3 ( 0.1
M. tuberculosis LS
M. tuberculosis RS
S. pombe LS
M. tuberculosis LS
S. pombe LS
M. tuberculosis LS
S. pombe LS
M. tuberculosis LS
M. tuberculosis LS
M. tuberculosis LS
S. pombe LS
M. tuberculosis LS
S. pombe LS
M. tuberculosis LS
S. pombe LS
M. tuberculosis LS
S. pombe LS
M. tuberculosis LS
M. tuberculosis LS
M. tuberculosis LS
M. tuberculosis RS
S. pombe LS
M. tuberculosis LS
M. tuberculosis LS
M. tuberculosis LS
M. tuberculosis RS
M. tuberculosis LS
M. tuberculosis RS
S. pombe LS
M. tuberculosis LS
E. coli RS
S. pombe LS
M. tuberculosis LS
S. pombe LS
M. tuberculosis LS
S. pombe LS
M. tuberculosis LS
S. pombe LS
M. tuberculosis LS
S. pombe LS
M. tuberculosis LS
E. coli RS
S. pombe LS
420 ( 168
398 ( 156
733 ( 227
44 ( 10
38 ( 2
7.7 ( 1.3
1.03 ( 0.04
6.4 ( 0.5
1.4 ( 0.1
36 ( 3
19
20
21
205 ( 91
28 ( 7
partial
uncompetitive
mixed
competitive
mixed
mixed
52 ( 19
243 ( 57
9.6 ( 4.5
32 ( 16
74 ( 12
1.7 ( 0.1
31 ( 3
22 ( 6
32 ( 3
22
23
24
21 ( 2
34 ( 4
48 ( 23
97 ( 24
47 ( 4
mixed
1.2 ( 0.2
37 ( 3
uncompetitive
mixed
mixed
mixed
partial
mixed
mixed
partial
mixed
mixed
mixed
uncompetitive
mixed
mixed
competitive
uncompetitive
partial
uncompetitive
mixed
mixed
uncompetitive
mixed
competitive
uncompetitive
mixed
mixed
mixed
mixed
mixed
competitive
partial
mixed
mixed
partial
partial
uncompetitive
mixed
partial
uncompetitive
mixed
mixed
17 ( 4
42 ( 10
11 ( 2
37 ( 5
25
26
27
1.1 ( 0.1
6.0 ( 0.6
0.85 ( 0.08
28 ( 3
496 ( 219
173 ( 50
15 ( 5
13 ( 4
3.7 ( 0.9
41 ( 18
35 ( 10
7.8 ( 4.6
86 ( 51
361 ( 179
37 ( 17
187 ( 43
78 ( 38
13 ( 3
0.63 ( 0.08
8.1 ( 0.5
33 ( 4
36 ( 6
14 ( 1
0.62 ( 0.02
7.2 ( 0.6
8.7 ( 0.7
26 ( 3
10 ( 0.5
21 ( 2
9.7 ( 0.4
1.1 ( 0.1
6.4 ( 0.6
3.0 ( 0.1
0.94 ( 0.06
8.4 ( 0.6
0.72 ( 0.04
7.9 ( 0.4
0.85 ( 0.07
6.7 ( 0.3
0.93 ( 0.06
6.4 ( 0.6
4.3 ( 0.6
30 ( 4
2.9 ( 0.2
0.83 ( 0.07
7.6 ( 0.5
19 ( 2
2.2 ( 0.1
0.59 ( 0.03
11 ( 1
28
29
25 ( 8
776 ( 294
604 ( 54
234 ( 59
188 ( 38
30
26 ( 5
45 ( 13
31 ( 3
31
32
800 ( 107
72 ( 19
936 ( 138
682 ( 409
225 ( 59
224 ( 22
497 ( 159
33
34
35
42 ( 11
85 ( 39
23 ( 5
1.13 ( 0.01
0.38 ( 0.01
1.32 ( 0.01
0.37 ( 0.01
1.64 ( 0.02
0.40 ( 0.01
0.99 ( 0.01
0.35 ( 0.01
2.2 ( 0.1
94 ( 34
12 ( 1
36
37
38
39
40
276 ( 16
461 ( 150
921 ( 640
468 ( 113
390 ( 90
89 ( 11
87 ( 40
130 ( 59
197 ( 112
151 ( 81
24 ( 6
143 ( 54
15 ( 7.3
114 ( 44
13 ( 2
30 ( 8
26 ( 11
4.3 ( 0.2
3.6 ( 0.1
15 ( 4
806 ( 401
248 ( 67
42 ( 12
1.04 ( 0.01
0.38 ( 0.01
0.42 ( 0.01
3.32 ( 0.05
2.58 ( 0.02
0.37 ( 0.01
3.40 ( 0.04
1.96 ( 0.04
0.40 ( 0.01
0.56 ( 0.01
0.62 ( 0.02
0.34 ( 0.01
1.12 ( 0.02
4.2 ( 0.1
M. tuberculosis LS
M. tuberculosis LS
E. coli RS
42
43
24 ( 10
270 ( 32
476 ( 89
72 ( 31
S. pombe LS
94 ( 30
11 ( 3
M. tuberculosis LS
E. coli RS
S. pombe LS
M. tuberculosis LS
M. tuberculosis LS
M. tuberculosis LS
M. tuberculosis RS
S. pombe LS
44
2.3 ( 0.1
1.2 ( 0.1
7.8 ( 0.6
15 ( 1
426 ( 64
376 ( 127
488 ( 233
823 ( 128
904 ( 193
449 ( 45
154 ( 13
788 ( 148
141 ( 87
70 ( 31
45
46
48
49
50
uncompetitive
uncompetitive
uncompetitive
uncompetitive
uncompetitive
18 ( 1
8.5 ( 0.4
1.4 ( 0.1
2.7 ( 0.3
E. coli RS
aExperiments were conducted with recombinant lumazine synthases of S. pombe and M. tuberculosis and recombinant riboflavin synthases of
M. tuberculosis and E. coli. The assays with lumazine synthase were performed with dihydroxybutanone phosphate substrate concentration held
constant, while the concentration of the pyrimidinedione substrate was varied. bKs is the substrate dissociation constant for the equilibrium E þ S h ES.
cKcat is the rate constant for the process ES f E þ P. dKi is the inhibitor dissociation constant for the process E þ I h EI. eKis is the inhibitor dissociation
constant process ES þ I h ESI. Partial and mixed modes of inhibition are similar, except the partial mode contains an additional product-formation step
ESI f EI þ P. Reaction mixtures contained 50 mM Tris HCl, pH 7.0, 100 mM NaCl, and 5 mM DTT. The following compounds were inactive vs M.
3
tuberculosis LS: 41, 47-51; vs S. pombe LS: 14-16, 22, 23, 28, 29, 31, 41, 42, 50; vs M. tuberculosis RS: 14-16, 19-28, 30, 31, 34-47, 49-51 and vs E. coli
RS: 14-16, 19-31, 34, 36-39, 41, 42, 49. The following compounds were not tested vs S. pombe LS: 32, 33, 45, 46, 48 and vs E. coli RS: 33, 45, 46, 48.
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Talukdar et al.
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SCHEME 5
the hydroxy group may hydrogen bond with the
Ala59, Ile60, and Glu61 amino acid residues. Class II
compounds were synthesized in order to determine the
importance of hydrogen bond donor and acceptor proper-
ties. Class II compounds with OCH3 groups displayed
slightly better potencies than their counterparts with OH
groups. Compound 21 exhibited a Ki of 9.6 μM vs
M. tuberculosis lumazine synthase, whereas compound 17
showed a Ki of 12 μM vs M. tuberculosis lumazine synthase.
Similarly, compound 19 exhibited a Ki of 28 μM vs
M. tuberculosis lumazine synthase, whereas hit compound
9 displayed a Ki of 95 μM vs M. tuberculosis lumazine
synthase.
SCHEME 6
In class III compounds, the hydroxyl groups have
been replaced with different functional groups that can
also participate in hydrogen bonding. Compounds with
a NO2 group showed a marked increase in inhibitory activity
as compared to the lead compound 9. Compound 14
exhibited a Ki of 16 μM vs M. tuberculosis lumazine synthase,
whereas the hit compound 9 had a Ki of 95 μM vs
M. tuberculosis lumazine synthase. The 4-NO2 derivative 26
displayed the best inhibitory activity, with
a Ki of
3.7 μM vs M. tuberculosis lumazine synthase. Among
the compounds with different halogens, the 2-fluoro deri-
vative 28 had the best activity with a Ki of 7.8 μM vs
M. tuberculosis lumazine synthase. Thus, improved inhibi-
tors of M. tuberculosis lumazine synthase were obtained by
changing the OH group in the hit compound 9 to a NO2 group
or to F.
SCHEME 7
lumazine synthase. To determine the effect of the position of
the hydroxy group in the phenyl ring on the enzyme inhibi-
tory activity, class I compounds were synthesized. Com-
pound 15, with a 3-hydroxy group, and compound 16, with
a 4-hydroxy group, showed marked increases in inhibitory
activity, with the 3-hydroxy compound 15 exhibiting a Ki of
7.1 μM and 4-hydroxy compound 16 exhibiting a Ki 12 μM
vs M. tuberculosis lumazine synthase. Both compounds 15
and 16 displayed remarkable selectivity for inhibition of M.
tuberculosis lumazine synthase, being completely ineffective
against S. pombe lumazine synthase and E. coli riboflavin
synthase. Thus, changing the position of the phenol im-
proved both the inhibitory activity vs M. tuberculosis luma-
zine synthase as well as the selectivity.
The presence of two hydroxyl groups on compounds 17
and 18 changed the inhibitory activity profile drastically.
Both of these compounds were found to be potent against
M. tuberculosis lumazine synthase, M. tuberculosis riboflavin
synthase, S. pombe lumazine synthase, and E. coli riboflavin
synthase, with compound 17 being more potent than com-
pound 18. Compound 17 exhibited a broader spectrum
enzyme-inhibitory profile than the hit compound 9, with a
Ki of 12 μM vs M. tuberculosis lumazine synthase, a Ki of
19 μM vs M. tuberculosis riboflavin synthase, a Ki of 4.9 μM
vs S. pombe lumazine synthase, and a Ki of 10 μM vs E. coli
riboflavin synthase.
Classes I-III yielded two compounds 26 and 15 with
optimized inhibitory activity. Based on these results, class
IV compounds were designed with two different hydrogen
bonding groups in the benzene ring. This may result in the
The hypothetical structural model of hit compound 9 in
M. tuberculosis lumazine synthase (Figure 1) indicates that
5128 J. Org. Chem. Vol. 74, No. 15, 2009

Talukdar et al.
JOCFeatured Article
naphthyl substituent, exhibited even better inhibitory activ-
phenyl substituent more closely mimicking the ribityl chain
of the substrate, with more opportunities for hydrogen
bonding to the protein. These inhibitors were prepared
following the standard protocol, with 1-butanol as the
solvent and 1 equiv of piperidine as the base.
ity, with a Ki of 11 μM vs M. tuberculosis lumazine synthase.
In Class VI compounds, the alkene linker is replaced by
more flexible ethylene and aminomethylene connectors. The
increase in conformational freedom should confer greater
similarity with the substrate 1.
The enzyme inhibitory activities of the class IV com-
pounds were disappointing. In fact, they had less inhibitory
activity than their counterparts with single functional
groups. Compound 35 showed the best inhibitory profile,
with a Ki of 12 μM against M. tuberculosis lumazine
synthase. These discouraging results prompted a reevalua-
tion of the hypothesis that the functional groups on the
aromatic ring hydrogen bond with the amino acid residues in
the active site of M. tuberculosis lumazine synthase. This led
to the synthesis of compounds 39 and 43 in the series of class
V compounds, which contain unsubstituted aromatic rings.
Compound 39 was expected to be a very weak inhibitor, but
to the contrary, it was found to have good inhibitory activity,
with a Ki of 15 μM vs M. tuberculosis lumazine synthase,
which is much better than the hit compound 9 (Ki 95 μM vs
M. tuberculosis lumazine synthase). Compound 43, with a
Compounds 45 and 46 were synthesized (Scheme 5) by
treating 6-chloro-5-nitrouracil (52) with benzylamines 53
and 54 in the presence of triethylamine. The free bases were
liberated from their triethylamine salts by dissolving them in
aq KOH and neutralizing with dilute HCl solution.
Compounds 47 and 48 were prepared by selective reduc-
tion of the trans alkene linkers present in 9 and 16 (Scheme 6).
J. Org. Chem. Vol. 74, No. 15, 2009 5129

Talukdar et al.
JOCFeatured Article
The presence of the reducible NO2 group and conjugated
double bond in compounds 9 and 16 made these alkenes
difficult to selectively reduce. The usual protocol involving
Pd/C reduced both the NO2 group and the double bond.
After unsuccessful attempts to selectively reduce either the
double bond or the nitro group with Fe or Zn in acetic acid,
Zn and hydrazinium monoformate, and Na2S2O4, it was
discovered that hydrogenation over Lindlar catalyst resulted
in selective reduction of the alkene linker without reduction
of the NO2 group.
SCHEME 8
Compound 49 was accidentally formed during the con-
densation reaction of compound 12 with 2-formylbenzoic
acid (Scheme 7). Evidently, the alcohol intermediate formed
from 12 and 2-formylbenzoic acid lactonizes to 49 instead of
dehydrating to the alkene. The structure of the lactone 49
suggests that 5-nitro-6-styryluracil derivatives might be sus-
ceptible to nucleophilic attack on the exocyclic double bond,
but no instance of this was observed in the present series of
compounds, which in general were quite stable.
replaced by a chlorine atom, has affinity for lumazine
synthase, compound 55 also has a phosphate moiety that
contributes positively to binding.27 It therefore contrasts
with the present series of inhibitors, which do not contain a
phosphate moiety.
The NO2 group and the trans double bond in 9 and 39 were
simultaneously reduced by hydrogenation using Pd/C to
obtain compounds 50 and 51 (Scheme 8). This completes
the synthesis of class VI compounds.
All compounds in class VI are either completely inactive or
have very weak inhibitory activity. Molecular modeling was
performed in order to investigate the possible bonding mode
of compound 46 to the enzyme. The compound 46 was
docked into the M. tuberculosis lumazine synthase structure
using Gold software (BST, version 3.0, 2005). Energy mini-
mization was then performed using Sybyl 7.1. The resulting
structure is displayed in Figure 2. According to the theore-
tical model, the binding of the 2-hydroxyphenyl moiety of 46
in the active site of M. tuberculosis lumazine synthase is
similar to the high-throughput screening compound 9. How-
ever, the model indicates that the uracil ring of 46 does not
stack with Trp 27, which may explain the inactivity of 45-48.
In conclusion, high-throughput screening of a 100000
compound commercial library led to the identification of
the hit compound 9, which displayed a Ki of 95 μΜ vs
M. tuberculosis lumazine synthase. The design and synthesis
of a focused array of structural analogues provided the
optimized congener 26, which had a Ki of 3.7 μΜ vs
M. tuberculosis lumazine synthase. Both of these compounds
are structural analogs of the lumazine synthase substrate 1
and the known lumazine synthase ligand 10. The results of
this study show that the ribitylamino side chain of the ligand
10 can be replaced by substituted styryl moieties with reten-
tion of affinity for the enzyme. The circumvention of the
ribitylamino side chain may contribute positively to anti-
biotic drug development because its polarity is expected to
limit uptake by bacterial cells. As expected from the fact that
the pyrimidinedione 1 is a substrate of the lumazine
synthase-catalyzed reaction and a product of the riboflavin
synthase-catalyzed reaction, many of the styryl derivatives in
the present series, including 9, 17, 18, 29, 32, 33, 35, 40, 43, 44,
48, and 50, inhibited both enzymes. This is a potential
advantage because drug resistance is less likely to emerge
from target mutation with antibiotics that act on two targets,
since resistance mutations would have to emerge in both
targets at the same time for the organism to become drug
resistant. Although it was previously demonstrated that the
intermediate analogue 55, in which the ribityl group is
Experimental Section
General Procedure: Method A. 5-Nitro-6-methyluracil (12)
(1.0 g, 5.85 mmol), benzaldehyde derivatives (29.2 mmol), and
piperidine (10 mL) were heated at first on a boiling water bath
until the mixture had thickened and then for 30 min in an oil
bath at 150 ꢀC. The mixture was diluted with methanol (15 mL),
and the crystalline solid was filtered, washed with methanol
(5 mL) and ether (10 mL), and dried to give the piperidine salt.
The salt was dissolved in dilute KOH solution. On the addition
of excess of hydrochloric acid to the warm solution, a yellow
powder precipitated. This was filtered and washed first with
water (2ꢀ10 mL) and methanol (2ꢀ10 mL) and then with ether
(2 ꢀ10 mL) to give the desired condensation product.
(E)-6-(3-Hydroxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione
(15). A yellow amorphous solid 15 (1.20 g, 75%): mp 238-
240 ꢀC dec; 1H NMR (300 MHz, CDCl3) δ 7.53 (d, J= 16.4 Hz,
1 H), 7.16 (t, J= 7.8 Hz, 1 H), 6.97 (d, J= 7.6 Hz, 1 H), 6.92 (s,
1 H), 6.86 (d, J=16.4 Hz, 1 H), 6.77 (d, J=8.1 Hz, 1 H); 13C NMR
(75 MHz, DMSO-d6) δ 158.0, 156.9, 149.4, 147.8, 142.3, 135.8,
130.5, 126.7, 119.7, 118.3, 114.4, 113.8; EIMS m/z 275 (Mþ). Anal.
Calcd for C12H9N3O5: C, 52.37; H, 3.30; N, 15.27. Found: C,
52.58; H, 3.14; N, 15.54.
(E)-6-(4-Hydroxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione
(16). An orange amorphous solid 16 (1.25 g, 78%): mp 230-
232 ꢀC dec; 1H NMR (300 MHz, CDCl3) δ 7.64 (d, J= 16.3 Hz,
1 H), 7.52 (d, J=8.64 Hz, 2 H), 6.87 (J=8.64 Hz, 2 H), 6.85 (d,
J=16.3 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 160.6, 156.9,
149.5, 148.0, 142.7, 130.8, 126.1, 125.7, 116.3, 109.9; negative-ion
EIMS m/z549 [(2 M-Hþ)-, 100], 275 (M, 11), 274 [(M-Hþ)-, 98],
144 (4). Anal. Calcd for C12H9N3O5: C, 52.37; H, 3.30; N, 15.27.
Found: C, 52.45; H, 3.24; N, 15.58.
(E)-6-(3-Hydroxy-4-methoxystyryl)-5-nitropyrimidine-2,4
(1H,3H)-dione (20). A red amorphous solid 20 (625 mg, 67%): mp
300-304 ꢀC; 1H NMR (300 MHz, DMSO-d6) δ 9.34 (s, 1 H), 7.68
(d, J=16.2 Hz, 1 H), 7.06 (s, 1 H), 7.04 (d, J=7.8 Hz, 1 H), 6.98 (d,
J=7.8 Hz, 1 H), 6.69 (d, J=16.2 Hz, 1 H), 3.81 (s, 3 H); 13C NMR
(75 MHz, DMSO-d6) δ 156.8, 150.7, 149.4, 147.8, 147.0, 142.5,
127.1, 126.0, 121.8, 113.9, 112.2, 110.7, 55.7; negative-ion EIMS
5130 J. Org. Chem. Vol. 74, No. 15, 2009

Talukdar et al.
JOCFeatured Article
FIGURE 2. Hypothetical model showing hydrogen bonding between the phenolic hydroxyl group of compound 46 with Ala59 and Glu61 of
˚
M. tuberculosis lumazine synthase. The distances shown are in A. The diagram is programmed for wall-eyed (relaxed) viewing.
m/z 609 [(2 M-Hþ)-, 46], 304 [(M-Hþ)-, 100]. Anal. Calcd for
C13H11N3O6: C, 51.15; H, 3.63; N, 13.77. Found: C, 50.80; H, 3.80,
N, 13.43.
H); 13C NMR (75 MHz, MeOH-d4) δ 156.7, 152.7, 149.3, 147.9,
142.7, 130.3, 125.3, 122.8, 114.2, 107.6; negative-ion EIMS m/z (rel
intensity) 276 [(2 M - Hþ)-, 100), 113 (2). Anal. Calcd for
(E)-6-(2,3-Dimethoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-
dione (21). An orange, amorphous solid 21 (655 mg, 70%): mp
280-283 ꢀC; 1H NMR (300 MHz, DMSO-d6) δ 7.25 (d, J=16.5
Hz, 1 H), 6.57 (m, 1 H), 6.48 (m, 1 H), 6.36 (d, J=16.2 Hz, 1 H),
3.18 (s, 3 H), 3.14 (s, 3 H); 13C NMR (75 MHz, DMSO-d6) δ 157.1,
153.3, 149.8, 148.5, 148.1, 137.1, 128.4, 127.0, 124.9, 119.6, 115.5,
115.3, 61.2, 56.0; negative-ion EIMS m/z 637 [(2 M -Hþ)-, 100],
C12H8FN3O4 0.1H2O: C, 51.66; H, 2.96; N, 15.06. Found: C,
3
51.95; H, 3.22, N, 14.69.
5-Nitro-6-styryluracil (39). A yellowamorphoussolid39 (5.1 g,
1
84%): mp 308-310 ꢀC (lit.33 mp 312-314 ꢀC); H NMR (300
MHz, MeOH-d4) δ 7.78 (d, J=16.2 Hz, 1 H), 7.63 (m, 2 H), 7.44
(m, 3 H), 6.98 (d, J=16.2 Hz, 1 H); 13C NMR (75 MHz, MeOH-
d4) δ 157.0, 149.6, 147.2, 134.6, 131.1, 129.5, 128.6, 126.8, 114.2;
negative-ion EIMS m/z 259 (M, 12), 258 [(M-Hþ)-, 98.5], 113 (2).
Anal. Calcd for C12H9N3O4: C, 55.60; H, 3.50; N, 16.21. Found:
C, 55.89; H, 3.41; N, 15.95.
318 [(M - Hþ)-, 46]. Anal. Calcd for C14H13N3O6. 0.5H2O: C,
3
51.22; H, 4.30; N, 12.80. Found: C, 51.03; H, 3.91, N, 12.53.
(E)-6-(3,4-Dimethoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-
dione (22). A red amorphous solid 22 (625 mg, 67%): mp 328-
230 ꢀC; 1H NMR (300 MHz, DMSO-d6) δ 7.67 (d, J = 16.2 Hz,
1 H), 7.23 (d, J=1.6 Hz, 1 H), 7.16 (dd, J=1.6 Hz, 8.4 Hz, 1 H),
7.00 (d, J=8.4 Hz, 1 H), 6.85 (d, J=16.2 Hz, 1 H), 3.80 (s, 3 H),
3.77 (s, 3 H); 13C NMR (75 MHz, DMSO-d6) δ 156.7, 151.5, 149.4,
149.1, 147.9, 142.3, 127.1, 126.0, 123.5, 111.7, 111.3, 110.0, 55.7;
negative-ion EIMS m/z 637 [(2 M - Hþ)-, 14], 318 [(M - Hþ)-,
100]. Anal. Calcd for C14H13N3O6: C, 52.67; H, 4.10; N, 13.16.
Found: C, 52.69; H, 3.87, N, 13.01.
(E)-5-Nitro-6-(2-(pyridin-2-yl)vinyl)pyrimidine-2,4(1H,3H)-
dione (41). An orange amorphous solid 41 (1.29 g, 85%): mp 290-
292 ꢀC dec; 1H NMR (300 MHz, CDCl3) δ 8.63 (d, J=4.8 Hz,
1 H), 7.92 (t, J = 8.6 Hz, 1 H), 7.73 (d, J = 15.9 Hz, 1 H), 7.60 (d,
J=7.8 Hz, 1 H), 7.44 (d, J=15.9 Hz, 1 H), 7.43 (m, 1 H); 13C NMR
(75 MHz, DMSO-d6) δ 157.2, 150.6, 149.2, 148.3, 147.2, 141.3,
137.7, 128.0, 126.5, 126.4, 120.4; negative-ion EIMS m/z 541
[(2 MNa - 2Hþ)-, 100], 259 [(M - Hþ)-, 41]. Anal. Calcd for
C11H8N4O4: C, 50.77; H, 3.10; N, 21.53. Found: C, 50.52; H, 3.16;
N, 21.20.
(E)-5-Nitro-6-(3-(pyridin-3-yl)vinyl)pyrimidine-2,4(1H,3H)-
dione (42). A yellow amorphous solid 42 (1.22 g, 80%): mp 300-
303 ꢀC; 1H NMR (300 MHz, CDCl3) δ8.76 (s, 1 H), 8.59 (d, J=3.7
Hz, 1 H), 8.13 (d, J=8.07 Hz, 1 H), 7.70 (d, J=16.2 Hz, 1 H), 7.46
(dd, J=4.8 Hz, 7.8 Hz, 1 H), 7.19 (d, J=16.2 Hz, 1 H); 13C NMR
(75 MHz, DMSO-d6) δ 156.8, 151.03, 150.0, 149.8, 148.6, 137.8,
134.4, 130.3, 126.5, 124.2, 117.2; negative-ion EIMS m/z 556.8 [(2
M-2Hþ)-, 100), 259 [(M -Hþ)-, 94], 156 (1). Anal. Calcd for
C11H8N4O4: C, 50.77; H, 3.10; N, 21.53. Found: C, 50.47; H, 2.88;
N, 21.71
General Procedure: Method B. 5-Nitro-6-methyluracil (12)
(0.5 g, 2.92 mmol), benzaldehyde derivatives (5.84 mmol), and
piperidine (0.3 mL, 3.21 mmol) in 1-butanol (10 mL) were
heated at first to 100 ꢀC until the mixture had thickened and
then at reflux for 6 h. The mixture was cooled to room
temperature, filtered, and washed with methanol (5 mL) and
ether (10 mL). The salt was dissolved in dilute KOH solution
and acidified with an excess of hydrochloric acid, and the solid
was filtered, washed with water (2ꢀ10 mL) and methanol (2ꢀ
10 mL) and then with ether (2ꢀ10 mL), and dried to give the
desired condensation product.
(E)-6-(2-Nitrostyryl)-5-nitropyrimidine-2,4(1H,3H)-dione
(14). A brown amorphous solid 14 (0.62 mg, 69%): mp 298-
300 ꢀC; 1H NMR (300 MHz, DMSO-d6) δ 8.03 (d, J = 8.1 Hz,
1 H), 7.97 (d, J=9.0 Hz, 1 H), 7.95 (d, J=16.2 Hz, 1 H), 7.79 (t,
J=7.5 Hz, 1 H), 7.68 (d, J = 7.5 Hz, 1 H), 7.05 (d, J=16.2 Hz,
1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.9, 149.3, 148.8, 147.0,
135.6, 134.0, 131.3, 129.2, 128.9, 127.0, 125.0, 119.3; negative-ion
(E)-6-(2,3,4-Trimethoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-
dione (23). A red amorphous solid 23 (610 mg, 60%): mp 308-
310 ꢀC; 1H NMR (300 MHz, DMSO-d6) δ 7.81 (d, J=16.8 Hz,
1 H), 7.39 (d, J=9.5 Hz, 1 H), 6.95 (d, J=16.8 Hz, 1 H), 6.88 (d,
J=9.5 Hz, 1 H), 3.88 (s, 3 H), 3.86 (s, 3 H), 3.79 (s, 3 H); 13C NMR
(75 MHz, DMSO-d6) δ 157.6, 155.6, 152.8, 149.4, 149.1, 141.9,
137.1, 126.1, 124.0, 120.7, 112.7, 108.3, 61.4, 60.6, 56.1; negative-
ion EIMS m/z 697 [(2 M-Hþ)-, 100], 348 [(M - Hþ)-, 57]. Anal.
Calcd for C15H15N3O7: C, 51.58; H, 4.33; N, 12.03. Found: C,
51.49; H, 4.35, N, 11.75.
(E)-6-(3,4,5-Trimethoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-
dione (24). A red amorphous solid 24 (610 mg, 60%): mp 293-
296 ꢀC dec; 1H NMR (300 MHz, DMSO-d6) δ 7.56 (d, J = 16.2
Hz, 1 H), 6.94 (d, J=16.2 Hz, 1 H), 6.92 (s, 2 H), 3.76 (s, 3 H), 3.67
(s, 6 H); negative-ion EIMS m/z 697 [(2 M-Hþ)-, 43], 348 [(M -
Hþ)-, 100]. Anal. Calcd for C15H15N3O7 1.25H2O: C, 48.46; H,
3
4.74; N, 11.30. Found: C, 48.27; H, 4.71, N, 11.09.
(E)-6-(2-Fluorostyryl)-5-nitropyrimidine-2,4(1H,3H)-dione
(28). An orange, amorphous solid 28 (510 mg, 63%): mp 280-
283 ꢀC dec; 1H NMR (300 MHz, MeOH-d4) δ 7.82 (d, J=16.5 Hz,
1 H), 7.78 (m, 1 H), 7.50 (m, 1 H), 7.33 (d, J=8.9 Hz, 1 H), 7.27 (d,
J=7.1 Hz, 1 H), 7.07 (d, J=16.5 Hz, 1 H); 13C NMR (75 MHz,
MeOH-d4) δ 162.5, 156.6, 149.3, 147.5, 133.8, 132.8, 129.1, 126.6,
125.2, 116.6, 116.5. Anal. Calcd for C12H8FN3O4: C, 51.99; H,
2.91; N, 15.16. Found: C, 51.79; H, 3.05, N, 15.25.
(E)-6-(4-Fluorostyryl)-5-nitropyrimidine-2,4(1H,3H)-dione
(29). A red amorphous solid 29 (488 mg, 60%): mp 286-288 ꢀC;
1H NMR (300 MHz, MeOH-d4) δ 7.73 (d, J=16.0 Hz, 1 H), 7.45
(d, J=8.3 Hz, 2 H), 6.94 (d, J=8.4 Hz, 2 H), 6.66 (d, J=16.0 Hz, 1
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Talukdar et al.
JOCFeatured Article
EIMS m/z (rel intensity) 607 [(2 M-Hþ)-, 100], 303 [(M-Hþ)-,
80], 240 (10). Anal. Calcd for C12H8N4O6: C, 47.38; H, 2.65; N,
18.42. Found: C, 47.45; H, 2.45; N, 18.10.
1H NMR (300 MHz, DMSO-d6) δ 7.68 (d, J=16.2 Hz, 1 H), 7.60
(d, J=8.4 Hz, 2 H), 7.51 (d, J=8.4 Hz, 2 H), 7.03 (d, J=16.3 Hz,
1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.7, 149.6, 148.1, 140.0,
135.2, 133.3, 130.0, 129.2, 115.4. Anal. Calcd for C12H8ClN3O4: C,
49.08; H, 2.75; Cl, 12.07; N, 14.35. Found: C, 48.89; H, 2.79; Cl,
11.89; N, 13.92.
(E)-6-(2,3-Dihydroxystyryl)-5-nitropyrimidine-2,4(1H,3H)-
dione (17). A brown amorphous solid 17 (0.49 g, 58%): mp 287-
290 ꢀC dec; 1H NMR (300 MHz, DMSO-d6) δ 7.88 (d, J=15.7 Hz,
1 H), 7.07 (d, J=15.7 Hz, 1 H), 6.85 (m, 2 H), 6.67 (t, J=7.8 Hz,
1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.1, 150.0, 149.5, 148.8,
147.4, 138.3, 125.4, 120.7, 119.3, 116.9, 114.2, 112.8; negative-ion
EIMS m/z 581 [(2 M - Hþ)-, 62], 290 [(M - Hþ)-, 100]. Anal.
Calcd for C12H9N3O6: C, 49.49; H, 3.12; N, 14.43. Found: C,
49.61; H, 3.22; N, 14.48.
(E)-6-(2,5-Dihydroxystyryl)-5-nitropyrimidine-2,4(1H,3H)-
dione (18). A brown amorphous solid 18 (0.19 g, 23%): mp 279-
282 ꢀC dec; 1H NMR (300 MHz, DMSO-d6) δ 9.78 (brs, 1 H), 7.80
(d, J=15.8 Hz, 1 H), 7.04 (d, J=15.8 Hz, 1 H), 6.81 (d, J=1.97
Hz, 1 H), 6.70 (m, 2 H); 13C NMR (75 MHz, DMSO-d6) δ 156.3,
149.9, 149.5, 148.9, 147.4, 138.5, 125.5, 120.7, 119.3, 116.7, 114.2,
112.7; negative-ion EIMS m/z (rel intensity) 581 [(2 M - Hþ)-,
100], 290 [(M-Hþ)-, 54], 244 (1). Anal. Calcd for C12H9N3O6: C,
49.49; H, 3.12; N, 14.43. Found: C, 49.78; H, 3.14; N, 14.29.
(E)-6-(2-Methoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione
(19). A yellow amorphous solid 19 (0.56 g, 66%): mp 289-300 ꢀC;
1H NMR (300 MHz, DMSO-d6) δ 7.90 (d, J=16.4 Hz, 1 H), 7.57
(d, J=7.6 Hz, 1 H), 7.42 (t, J=7.6 Hz, 1 H), 7.09 (d, J=7.6 Hz,
1 H), 7.08 (d, J=16.4Hz, 1H), 7.00(d,J= 7.6 Hz, 1 H); 13CNMR
(75 MHz, DMSO-d6) δ 158.3, 156.8, 149.5, 148.1, 137.6, 132.4,
129.5, 126.4, 122.8, 121.0, 114.6, 112.0, 55.8; negative-ion EIMS
m/z 577 [(2 M-Hþ)-, 100], 288 [(M-Hþ)-, 58]. Anal. Calcd for
C13H11N3O5: C, 53.98; H, 3.83; N, 14.53. Found: C, 53.92; H, 3.92;
N, 14.17.
(E)-6-(2-Hydroxy-5-nitrostyryl)-5-nitropyrimidine-2,4(1H,3H)-
dione (32). A brick-red amorphous solid 32 (625 mg, 67%): mp
298-300 ꢀC; 1H NMR (300 MHz, DMSO-d6) δ 11.83 (brs, 2 H),
8.38 (d, J=2.8 Hz, 1 H), 8.14 (dd, J = 2.8, 9.0 Hz, 1 H), 7.83 (d,
J=16.4 Hz, 1 H), 7.34 (d, J=16.4 Hz, 1 H), 7.07 (d, J=9.0 Hz,
1 H); 13C NMR (75 MHz, DMSO-d6) δ 162.8, 156.8, 149.3, 147.8,
138.9, 136.2, 127.1, 126.8, 126.0, 121.7, 156.9, 156.8; negative-ion
EIMS m/z (rel intensity) 639 [2 M - Hþ)-, 80], 319 [(M - Hþ)-,
100]. Anal. Calcd for C12H8N4O7 0.75H2O: C, 43.19; H, 2.87; N,
3
16.79. Found: C, 43.38; H, 2.81; N, 16.68.
(E)-6-(2-Hydroxy-3-nitrostyryl)-3-nitropyrimidine-2,4(1H,3H)-
dione (33). A yellowish-brown amorphous solid 33 (705 mg, 75%):
mp 305-307 ꢀC; 1H NMR (300 MHz, DMSO-d6) δ 8.04 (dd, J=
2.8 Hz, 1 H), 7.97 (d, J=7.8 Hz, 1 H), 7.90 (d, J=16.4 Hz, 1 H),
7.24 (d, J=16.4 Hz, 1 H), 7.09 (t, J = 8.0 Hz, 1 H); EIMS m/z (rel
intensity) 321 (MHþ, 32), 359 (MKþ, 50), 659 (100); negative-ion
EIMS m/z (rel intensity) 319 [(M - Hþ)-, 100], 256 (11). Anal.
Calcd for C12H8N4O7: C, 45.01; H, 2.52; N, 17.50. Found: C,
45.34; H, 2.55; N, 17.56.
(E)-6-(2-Methoxy-5-nitrostyryl)-5-nitropyrimidine-2,4(1H,3H)-
dione (34). A yellow amorphous solid 34 (630 mg, 65%): mp
278-280 ꢀC; H NMR (300 MHz, DMSO-d6) δ 11.88 (s, 1 H),
1
11.73 (s, 1 H), 8.46 (d, J=2.6 Hz, 1 H), 8.29 (dd, J=2.6, 9.2 Hz,
1 H), 7.84 (d, J=16.4 Hz, 1 H), 7.33 (d, J=9.2 Hz, 1 H), 7.27 (d,
J = 16.4 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 162.9,
156.7, 149.2, 147.8, 141.0, 134.9, 127.3, 126.8, 124.6, 123.5,
117.9, 112.9, 57.0; negative-ion EIMS m/z 667 [(2 M - Hþ)-,
(E)-5-Nitro-6-(3-nitrostyryl)pyrimidine-2,4(1H,3H)-dione
(25). A yellow amorphous solid 25 (0.56 g, 63%): mp 278-280 ꢀC;
1H NMR (300 MHz, DMSO-d6) δ 8.44 (s, 1 H), 8.23 (dd, J=1.44
Hz, 8.06 Hz, 1 H), 8.06 (d, J = 7.7 Hz, 1 H), 7.76 (d, J = 16.3 Hz,
100], 333 [(M - Hþ)-, 23]. Anal. Calcd for C13H10N4O7 0.5-
3
H2O: C, 45.49; H, 3.23; N, 16.32. Found: C, 45.31; H, 3.04; N,
16.20.
1 H), 7.70 (d, J = 8.06 Hz, 1 H), 7.25 (d, J=16.3 Hz, 1 H); 13
C
NMR (75 MHz, DMSO-d6) δ 156.6, 149.5, 148.3, 147.8, 138.9,
136.0, 134.3, 130.6, 126.8, 124.7, 122.5, 117.7; negative-ion EIMS
m/z 607 [(2 M-Hþ)-, 100], 303 [(M-Hþ)-, 37]. Anal. Calcd for
C12H8N4O6: C, 47.38; H, 2.65; N, 18.42. Found: C, 47.02; H, 2.73;
N, 18.13.
(E)-6-(3-Hydroxy-4-nitrostyryl)-5-nitropyrimidine-2,4(1H,3H)-
dione (35). A pale yellow amorphous solid 35 (323 mg, 69%): mp
310-312 ꢀC; 1H NMR (300 MHz, DMSO-d6) δ 11.73 (brs, 1 H),
11.19 (brs, 1 H), 7.94 (d, J=8.6 Hz, 1 H), 7.66 (d, J=16.3 Hz, 1 H),
7.30 (s, 1 H), 7.26 (d, J=8.6 Hz, 1 H), 7.07 (d, J=16.4 Hz, 1 H);13
C
(E)-5-Nitro-6-(4-nitrostyryl)pyrimidine-2,4(1H,3H)-dione
(26). A brown amorphous solid 26 (0.62 mg, 69%): mp 304-
308 ꢀC dec; 1H NMR (300 MHz, DMSO-d6) δ 8.23 (d, J=8.8 Hz,
2 H), 7.89 (d, J=8.8 Hz, 2 H), 7.68 (d, J=15.8 Hz, 1 H), 7.38 (d,
J=15.8 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.7, 149.3,
148.1, 147.6, 140.6, 138.9, 129.3, 127.0, 124.3, 118.7. Anal. Calcd
for C12H8N4O6: C, 47.38; H, 2.65; N, 18.42. Found: C, 47.52; H,
2.70, N, 18.19.
NMR (75 MHz, DMSO-d6) δ 156.6, 152.3, 149.2, 147.2, 140.5,
139.1, 137.3, 127.0, 126.1, 118.6, 118.5, 118.1; negative-ion EIMS
m/z 639 [(2 M - Hþ)-, 100], 319 [(M - Hþ)-, 78]. Anal. Calcd for
C12H8N4O7: C, 45.01; H, 2.52; N, 17.50. Found: C, 44.98; H, 2.54;
N, 17.45.
(E)-6-(4-Hydroxy-3-nitrostyryl)-5-nitropyrimidine-2,4(1H,3H)-
dione (36). A yellow amorphous solid 36 (315 mg, 67%): mp 320-
322 ꢀC. 1H NMR (300 MHz, DMSO-d6) δ 11.83 (s, 1 H),
11.55 (brs, 1 H), 8.12 (d, J=1.9 Hz, 1 H), 7.78 (dd, J = 1.9, 8.7
Hz, 1 H), 7.67 (d, J=16.3 Hz, 1 H), 7.15 (d, J=8.7 Hz, 1 H), 6.92
(d, J=16.3 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.7,
153.8, 149.4, 147.5, 139.8, 137.4, 134.3, 126.4, 125.7, 125.4, 119.9;
Pos EIMS m/z (rel intensity) 321 (Mþ, 100); negative-ion EIMS
m/z 639 [(2 M - Hþ)-, 100], 319 [(M - Hþ)-, 30]. Anal. Calcd for
C12H8N4O7: C, 45.01; H, 2.52; N, 17.50. Found: C, 44.70; H, 2.58;
N, 17.44.
(E)-4-(2-(5-Nitro-2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-
yl)vinyl)benzoic Acid (27). A pale-yellow amorphous solid 27 (0.28
1
mg, 64%): mp 342-345 ꢀC; H NMR (300 MHz, DMSO-d6) δ
11.83 (brs, 1 H), 7.98 (brs, 1 H), 7.95 (brs, 1 H), 7.72 (d, J=16.3 Hz,
1 H), 7.71 (m, 2 H), 7.07 (d, J=16.3 Hz, 1 H); 13C NMR (75 MHz,
DMSO-d6) δ 167.2, 156.8, 149.9, 147.7, 140.8, 138.2, 130.4, 128.2,
127.0, 116.8; negative-ion EIMS m/z 605 [(2 M - Hþ)-, 100], 302
[(M - Hþ)-, 70]. Anal. Calcd for C13H9N3O6 0.5H2O: C, 50.01;
3
H, 3.23; N, 13.46. Found: C, 50.29; H, 2.89; N, 13.22.
(E)-3-Hydroxy-4-(2-(5-nitro-2,6-dioxo-1,2,3,6-tetrahydropyr-
imidine-4-yl)vinyl)benzoic Acid (37). A yellow red amorphous
solid 37 (0.28 g, 60%): mp 347-349 ꢀC dec; 1H NMR (300 MHz,
DMSO-d6) δ 11.91 (s, 1 H), 11.78 (s, 1 H), 10.82 (s, 1 H), 7.86 (d,
J=16.3 Hz, 1 H), 7.57 (d, J=8.1 Hz, 1 H), 7.49 (s, 1 H), 7.41 (d,
J=8.1 Hz, 1 H), 7.27 (d, J=16.3 Hz, 1 H); 13C NMR (75 MHz,
DMSO-d6) δ 167.2, 157.2, 157.1, 149.9, 148.0, 137.8, 133.5,
130.1, 126.8, 125.0, 120.4, 116.9, 116.1; negative-ion EIMS m/z
637 [(2 M - Hþ)-, 100], 318 [(M - Hþ)-, 27]. Anal. Calcd for
(E)-5-Nitro-6-(4-bromostyryl)pyrimidine-2,4(1H,3H)-dione
(30). A yellow amorphous solid 30 (0.62 g, 63%): mp 296-298 ꢀC
dec; 1H NMR (300 MHz, DMSO-d6) δ 7.68 (d, J = 16.3 Hz, 1 H),
7.62 (dd, J=8.4 Hz, 18.0 Hz, 4 H), 7.03 (d, J=16.3 Hz, 1 H); 13
C
NMR (75 MHz, DMSO-d6) δ 156.7, 149.4, 147.7, 140.4, 133.5,
132.2, 130.2, 126.6, 124.2, 115.1. Anal. Calcd for C12H8BrN3O4: C,
42.63; H, 2.38; N, 12.43; Br, 23.63. Found: C, 42.41; H, 2.37; N,
12.27; Br, 23.55.
(E)-6-(4-Chlorostyryl)-5-nitropyrimidine-2,4(1H,3H)-dione
(31). A yellow amorphous solid 31 (0.58 g, 67%): mp 326-328 ꢀC;
C13H9N3O7 0.5H2O: C, 47.57; H, 3.07; N, 12.80. Found: C,
3
47.35; H, 2.85; N, 12.56.
5132 J. Org. Chem. Vol. 74, No. 15, 2009

Talukdar et al.
JOCFeatured Article
(E)-6-(2-Fluoro-3-methoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-
dione (38). A yellow amorphous solid 38 (115 mg, 65%): mp 318-
320 ꢀC; 1H NMR (300 MHz, DMSO-d6) δ 11.88 (s, 1 H), 11.85 (s,
1 H), 7.82 (d, J=16.5 Hz, 1 H), 7.31 (t, J=6.9 Hz, 1 H), 7.26 (m,
1 H), 7.21 (m, 1 H), 7.04 (d, J=16.5 Hz, 1 H); 13C NMR (75 MHz,
DMSO-d6) δ 156.4, 151.2, 149.9, 147.8, 134.0, 127.0, 125.1, 122.9,
119.2, 116.4, 115.6, 56.5; negative-ion EIMS m/z 613 [(2 M -
Hþ)-, 16], 306 [(M - Hþ)-, 100]. Anal. Calcd for C13H10FN3O5:
C, 50.82; H, 3.28; N, 13.68; F, 6.18. Found: C, 50.46; H, 3.15; N,
13.44; F, 5.94.
H), 7.25 (dd, J=1.3 Hz, 7.3 Hz, 1 H), 7.12 (m, 1 H), 6.75 (m, 2 H),
4.43 (d, J=6.1 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6) δ 159.0,
158.7, 156.1, 130.7, 128.8, 125.4, 118.8, 116.7, 66.4; EIMS m/z (rel
intensity) 579 (2 MNaþ, 20), 380 (100), 301 (MNaþ, 10); negative-
ion EIMS m/z (rel intensity) 555 [(2 M- Hþ)-, 100], 278 (M, 10),
277 [(M-Hþ)-, 85], 241. Anal. Calcd for C11H10N4O5: C, 47.49;
H, 3.62; N, 20.14. Found: C, 47.61; H, 3.69; N, 19.83.
6-(2-Hydroxy-2-phenethyl)-5-nitropyrimidine-2,4(1H,3H)-
dione (47). Lindlar catalyst (5 mg) was added to a solution of
compound 9 (100 mg, 0.36 mmol) in MeOH (5 mL). A hydrogen
balloon was attached, and the mixture was stirred at room
temperature for 12 h. The reaction mixture was filtered through
Celite, which was then washed with MeOH (2 ꢀ 5 mL). The
solution was concentrated, and the residue was washed several
times with CH2Cl2 (3 ꢀ 10 mL) and THF (3 ꢀ 10 mL). Finally,
compound 47 was precipitated out by dissolving in MeOH and
adding excess diethyl ether to furnish the pure product (78 mg,
78%) as a pale yellow amorphous solid: mp 231-234 ꢀC; 1H NMR
(300 MHz, MeOH-d4) δ 7.06 (m, 2 H), 6.76 (m, 2 H), 2.88 (d, J=
6.8 Hz, 1 H), 2.85 (d, J=5.6 Hz, 1 H), 2.70 (d, J=5.6 Hz, 1 H), 2.67
(d, J=6.8 Hz, 1 H); 13C NMR (75 MHz, MeOH-d4) δ 163.6, 156.4,
152.0, 135.8, 131.2, 128.9, 127.7, 120.7, 119.0, 116.0, 29.8, 29.1;
negative-ion ESI-MS m/z (rel intensity) 276 [(M-Hþ)-, 100], 258
(15); HRMS m/z calcd for C12H11N3O5 (M - Hþ)- 276.0620,
found 276.0619. Anal. Calcd for C12H11N3O5: C, 51.99; H, 4.00; N,
15.16. Found: C, 51.78; H, 4.28; N, 15.04.
6-(4-Hydroxy-2-phenethyl)-5-nitropyrimidine-2,4(1H,3H)-
dione (48). Lindlar catalyst (5 mg) was added to a solution of
compound 16 (100 mg, 0.36 mmol) in MeOH (5 mL). A hydrogen
balloon was attached, and the mixture was stirred at room
temperature for 12 h. The reaction mixture was filtered through
Celite, which was then washed with MeOH (2 ꢀ 5 mL). The
solution was concentrated, and the residue was washed several
times with CH2Cl2 (3 ꢀ 10 mL) and THF (3 ꢀ 10 mL). Finally,
compound 48 was precipitated out by dissolving in MeOH and
adding excess diethyl ether to furnish the pure product (70 mg,
70%) as a yellowish amorphous solid: mp 235-237 ꢀC; 1H NMR
(300 MHz, MeOH-d4) δ 7.06 (d, J=8.4 Hz, 2 H), 6.69 (d, J =
8.4 Hz, 2 H), 2.80 (d, J=6.5 Hz, 1 H), 2.77 (d, J= 5.6 Hz, 1 H), 2.68
(d, J=5.6 Hz, 1 H), 2.65 (d, J=6.5 Hz, 1 H); 13C NMR (75 MHz,
MeOH-d4) δ 163.7, 157.0, 152.0, 135.4, 132.4, 130.5, 119.0, 116.3,
33.0, 31.9; negative-ion ESI-MS m/z (rel intensity) 276 [(M-Hþ)-,
100], 258 (33); negative-ion HRMS m/z calcd for C12H11N3O5
(M-Hþ)- 276.0620, found 276.0623. Anal. Calcd for C12H11-
N3O5: C, 51.99; H, 4.00; N, 15.16. Found: C, 51.91; H, 4.21;
N, 14.95.
(E)-5-Nitro-6-[2-(1H-pyrrol-2-yl)vinyl]pyrimidine-2,4(1H,3H)-
dione (40). A black amorphous solid 40 (0.49 g, 68%): mp 302-304
ꢀC dec; 1H NMR (300 MHz, DMSO-d6) δ 7.71 (d, J=16.0 Hz, 1
H), 7.13 (m, 2 H), 6.58 (d, J=16.0 Hz, 1 H), 6.53 (m, 1 H), 6.21 (m,
1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.8, 149.5, 147.9, 133.3,
132.4, 128.8, 117.52, 110.8, 105.2; negative-ion EIMS m/z495 [(2 M
- Hþ)-, 77], 247 [(M-Hþ)-, 100]. Anal. Calcd for C10H8N4O4: C,
48.39; H, 3.25; N, 22.57. Found: C, 48.17; H, 3.51; N, 22.85.
(E)-6-(2-(Naphthalen-2-yl)vinyl)-5-nitropyrimidine-2,4(1H,3H)-
dione (43). A yellow amorphous solid 43 (0.66 g, 73%): mp 320-
322 ꢀC; 1H NMR (300 MHz, DMSO-d6) δ 11.73 (brs, 2 H), 8.11 (s,
1 H), 7.99 (m, 1 H), 7.93 (m, 1 H), 7.91 (d, J=16.2 Hz, 1 H), 7.79 (d,
J=8.5 Hz, 1 H), 7.55 (m, 2 H), 7.13 (d, J=16.2 Hz, 1 H);13C NMR
(75 MHz, DMSO-d6) δ 157.0, 150.2, 149.1, 141.2, 133.8, 132.9,
132.0, 129.8, 128.8, 128.6, 127.7, 127.5, 126.9, 126.3, 123.8, 115.4;
negative-ion EIMS m/z 639 [(2 MNa - 2Hþ)-, 100], 617 [(2 M-
Hþ)-, 22], 308 [(M-Hþ)-, 30]. Anal. Calcd for C16H11N3O4 0.75
3
H2O: C, 59.54; H, 3.90; N, 13.02. Found: C, 59.18; H, 3.46; N,
12.83.
(E)-6-(2-(3H-Indol-3-yl)vinyl)-5-nitropyrimidine-2,4(1H,3H)-
dione (44). A red amorphous solid 44 (0.62 g, 71%): mp 340-
342 ꢀC; 1H NMR (300 MHz, DMSO-d6) δ 12.10 (s, 1 H), 11.68
(s, 1 H), 11.53 (s, 1 H), 8.15 (d, J=16.5 Hz, 1 H), 7.98 (d, J=2.1
Hz, 1 H), 7.79 (d, J=7.9 Hz, 1 H), 7.49 (d, J = 7.9 Hz, 1 H), 7.22
(m, 2 H), 6.85 (d, J=16.5 Hz, 1 H); 13C NMR (75 MHz, DMSO-
d6) δ 157.0, 149.6, 148.7, 137.8, 137.5, 132.5, 125.2, 124.6, 123.1,
121.6, 119.5, 112.9, 112.8, 106.3; negative-ion EIMS m/z 319
[(MNa - 2Hþ)-, 66], 297 [(M - Hþ)-, 8]. Anal. Calcd for
C14H10N4O4: C, 56.38; H, 3.38; N, 18.78. Found: C, 56.10; H,
3.22; N, 18.76.
6-(Benzylamino)-5-nitropyrimidine-2,4(1H,3H)-dione (45). 6-
Chloro-5-nitrouracil (52) (0.15 g, 0.78 mmol), benzylamine (0.09
mL, 0.86 mmol), and Et3N (0.3 mL, 2.35 mmol) in 1,4-dioxane
(5 mL) were heated at reflux for 12 h. The mixture was cooled to
room temperature, filtered, and washed with methanol (5 mL)
and ether (10 mL). The salt was dissolved in dilute KOH
solution and acidified with an excess of hydrochloric acid, and
the solid was filtered and washed with water (2 ꢀ 10 mL),
methanol (2 ꢀ 10 mL), and then with ether (2 ꢀ 10 mL) and
dried to yield a white amorphous solid 45 (145 mg, 73%): mp
284-286 ꢀC; 1H NMR (300 MHz, DMSO-d6) δ 7.30 (m, 5 H),
4.71 (s, 2 H); 13C NMR (75 MHz, DMSO-d6) δ 157.1, 154.2,
147.6, 136.3, 128.7, 127.3, 126.4, 109.2, 46.5; EIMS m/z (rel
intensity) 285 (MNaþ, 100), 263 (MHþ, 7); negative-ion EIMS
m/z (rel intensity) 261 [(M - Hþ)-, 100], 227 (7). Anal. Calcd for
C11H10N4O4: C, 50.38; H, 3.84; N, 21.37. Found: C, 50.04; H,
3.69; N, 21.05.
5-Nitro-6-[(3-oxo-1,3-dihydroisobenzofuran-1-yl)methyl]pyri-
midine-2,4-(1H,3H)-dione (49). A pale-yellow amorphous solid
49 (0.3 mg, 68%): mp 335-337 ꢀC. 1H NMR (300 MHz,
DMSO-d6) δ 7.74 (m, 2 H), 7.56 (m, 2 H), 5.92 (dd, J = 3.0,
9.6 Hz, 1 H), 3.50 (dd, J=3.3, 13.8 Hz, 1 H), 2.86 (dd, J=9.6,
13.8 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 169.1, 156.4,
151.7, 149.1, 148.2, 134.8, 130.0, 128.0, 125.2, 125.0 122.8, 78.2,
35.1; negative-ion EIMS m/z (rel intensity) 302 [(M-Hþ)-, 100],
170 (61). Anal. Calcd for C13H9N3O6: C, 51.49; H, 2.99; N,
13.86. Found: C, 51.12; H, 3.08; N, 13.57.
5-Amino-(2-hydroxy-2-phenethyl)pyrimidine-2,4-(1H,3H)-
dione (50). Concentrated HCl (0.5 mL) and 10% Pd/C (30 mg)
were added to a solution of 5-nitro-6-styryluracil (39) (300 mg, 1.16
mmol) in MeOH (10 mL). A hydrogen balloon was attached, and
the mixture was stirred at room temperature for 24 h. The reaction
mixture was filtered through Celite, which was then washed with
50% aq MeOH (5 mL) to provide compound 50 as a white solid
6-(2-Hydroxybenzylamino)-5-nitropyrimidine-2,4(1H,3H)-dione
(46). 6-Chloro-5-nitrouracil (52) (0.27 g, 1.4 mmol), 2-hydroxy-
benzylamine (0.19 g, 1.56 mmol), and Et3N (0.6 mL, 4.23 mmol) in
1,4-dioxane (6 mL) were heated at reflux for 12 h. The mixture was
cooled to room temperature, filtered, and washed with methanol
(5 mL) and ether (10 mL). The salt was dissolved in dilute KOH
solution and acidified with an excess of hydrochloric acid, and the
solid was filtered and washed with water (2ꢀ 10 mL), methanol
(2ꢀ10 mL), and then with ether (2ꢀ10 mL) and dried to give a pale-
1
(225 mg, 73%): mp 251-254 ꢀC; H NMR (300 MHz, MeOH-
d4/D2O) δ 7.27 (m, 4 H), 7.23 (m, 1 H), 2.97 (m, 2 H), 2.85 (m, 2 H);
13C NMR (75 MHz, MeOH-d4/D2O) δ 160.1, 151.4, 150.1, 140.6,
129.8, 129.5, 127.9, 115.2, 34.4, 31.8; EIMS m/z (rel intensity) 231
(Mþ, 51), 140 (Mþ - C7H7, 80), 91 (Mþ - C5H6N3O2, 100); CIMS
1
yellow amorphous solid 46 (0.25 g, 64%): mp 138-140 ꢀC; H
NMR (300 MHz, DMSO-d6) δ 9.84 (t, J=5.4 Hz, 1 H), 9.72 (brs, 1
J. Org. Chem. Vol. 74, No. 15, 2009 5133

Talukdar et al.
JOCFeatured Article
TABLE 2. Enzymes Used in Kinetic Assays
Assay mixtures were prepared as follows. A solution (175 μL)
containing 103 mM NaCl, 5.1 mM dithiothreitol, and riboflavin
synthase in 51 mM Tris hydrochloride, pH 7.0, was added to
4 μL of inhibitor in 100% (v/v) DMSO in a well of a 96-well
microtiter plate. The reaction was started by adding 21 μL of a
solution containing 103 mM NaCl, 5.1 mM dithiothreitol, and
substrate 3 (30 - 200 μM) in 51 mM Tris hydrochloride, pH 7.0.
The formation of riboflavin was measured online for a period
of 40 min at 27 ꢀC with a computer-controlled plate reader at
470 nm (εriboflavin = 9600 M-1 cm-1).
concn of
enzyme in
reaction mixture
specific
activity
enzyme
organism
S. pombe
(μmol mg-1 h-1
)
(μg mL-1
)
lumazine
synthases:
6.8
1.0
M. tuberculosis
E. coli
0.8
21
30
0.8
riboflavin
synthases:
M. tuberculosis
4.0
4.0
Details of the Assay Procedures. For both lumazine synthase
and riboflavin synthase assays, four different inhibitor concen-
trations and seven different substrate concentrations were used.
Therefore, a series of measurements for one compound included
28 sample reactions plus four reactions without substrate added
(as control samples). The enzyme was added before the reaction
mixture was pipetted into different wells on a 96-well microtiter
plate. The enzyme concentration in all reaction mixtures was
identical. After enzyme was added, the solution was divided into
four parts, and a different amount of inhibitor was added to
each of them. The accuracy of the measurements on the plate
reader was checked regularly. An identical amount of substrate
was added to every well on the microtiter plate containing
identical enzyme solutions. The results of measurements were
analyzed for all wells, and standard deviation values were
between 3% and 8%. The standard deviation values in Table 1
are important criteria for determining the mode of inhibition
and do not reflect the accuracy of pipetting or photometric
measurements.
m/z(rel intensity) 232 (MHþ, 100). Anal. Calcd for C12H14ClN3O2:
C, 53.84; H, 5.27; N, 15.70. Found: C, 53.99; H, 5.01; N, 15.90.
5-Amino-6-(2-hydroxyphenethyl)pyrimidine-2,4(1H,3H)-
dione Hydrochloride (51). Pd/C (5 mg) was added to a solution of
compound 9 (100 mg, 0.36 mmol) in MeOH (20 mL). The mixture
was shaken in a Parr apparatus at 30 psi under H2 atmosphere for
24 h. The reaction mixture was filtered through Celite, which was
then washed with MeOH (2ꢀ10 mL). The solution was concentrated,
and the residue was washed several times with CH2Cl2 (3 ꢀ 10 mL)
and THF (3 ꢀ 10 mL). Finally, compound 51 was precipitated out
by dissolving it in MeOH and adding excess diethyl ether to furnish
pure compound 51 (65 mg, 72%) as a yellow amorphous solid: mp
239-241 ꢀC dec; 1H NMR (300 MHz, MeOH-d4) δ 7.06 (m, 2 H),
6.75 (m, 2 H), 2.88 (d, J = 6.8 Hz, 1 H), 2.85 (d, J = 5.9 Hz, 1 H),
2.70 (d, J = 5.9 Hz, 1 H), 2.68 (d, J = 6.8 Hz, 1 H); EIMS m/z (rel
intensity) 248 (MHþ, 100), 194 (10), 230; HRMS m/z calcd for
C12H14N3O3 (MHþ) 248.1035, found 248.1034. Anal. Calcd for
C12H14ClN3O3: C, 50.80; H, 4.97; N, 14.81. Found: C, 50.91; H,
4.81; N, 14.95.
Lumazine Synthase Assay. Assay mixtures contained 50 mM
Tris hydrochloride, pH 7.0, 100 mM NaCl, 2% (v/v) DMSO, 5
mM dithiothreitol, 100 μM 2, lumazine synthase (Table 2),
variable concentrations of 1 (3-150 μM), and inhibitor (0-
150 μM) in a volume of 0.2 mL. Assay mixtures were prepared as
follows. A solution (175 μL) containing 103 mM NaCl, 5.1 mM
dithiothreitol, 114 μM 2, and lumazine synthase in 51 mM Tris
hydrochloride, pH 7.0, was added to 4 μL of inhibitor in 100%
(v/v) DMSO in a well of a 96-well microtiter plate. The reaction
was started by adding 21 μL of a solution containing 103 mM
NaCl, 5.1 mM dithiothreitol, and substrate 1 (30-1500 μM) in
51 mM Tris hydrochloride, pH 7.0. The formation of 6,7-
dimethyl-8-D-ribityllumazine (3) was measured online for a
period of 40 min at 27 ꢀC with a computer-controlled plate
reader at 408 nm (εlumazine = 10200 M-1 cm-1).
Evaluation of Kinetic Data. The velocity-substrate data were
fitted for all inhibitor concentrations with a nonlinear regression
method using the program DynaFit.34 Different inhibition
models were considered for the calculation. Ki and Kis values
( standard deviations were obtained from the fit under con-
sideration of the most likely inhibition model as described
previously.17
Acknowledgment. This research was made possible by
NIH Grant No. GM51469, the Fonds der Chemischen
Industrie, and the Hans Fischer Gesellschaft. The experi-
mental work was conducted in a facility constructed with
support from Research Facilities Improvement Program
Grant No. C06-14499 from the National Center for Re-
search Resources of the National Institutes of Health.
Supporting Information Available: 1H NMR and 13C NMR
spectra of 9, 14-23, and 25-51. This material is available free of
charge via the Internet at http://pubs.acs.org.
Riboflavin Synthase Assay. Assay mixtures contained 50 mM
Tris hydrochloride, pH 7.0, 100 mM NaCl, 2% (v/v) DMSO, 5
mM dithiothreitol, enzyme (Table 2), variable concentrations of
3 (3-20 μM), and inhibitor (0-150 μM) in a volume of 0.2 mL.
(34) Kuzmic, P. Anal. Biochem. 1996, 237, 260–273.
5134 J. Org. Chem. Vol. 74, No. 15, 2009