A new series of 3-alkyl phosphate derivatives of 4,5,6,7-tetrahydro-1-D- ribityl-1H-pyrazolo[3,4-d]pyrimidinedione as inhibitors of lumazine synthase: Design, synthesis, and evaluation

Article abstract of DOI:10.1021/jo070982r

(Chemical Equation Presented) Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin. A homologous series of three pyrazolopyrimidine analogues of a hypothetical intermediate in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. The key steps of the synthesis were C-5 deprotonation of 4-chloro-2,6-dimethoxypyrimidine, acylation of the resulting anion, and conversion of the product to a pyrazolopyrimidine with hydrazine. Alkylation of the pyrazolopyrimidine with a substituted ribityl iodide and deprotection of the ribityl chain afforded the final set of three products. All three compounds were extremely potent inhibitors of the lumazine synthases of Mycobacterium tuberculosis, Magnaporthe grisea, Candida albicans, and Schizosaccharomyces pombe lumazine synthase, with inhibition constants in the low nanomolar to subnanomolar range. Molecular modeling of one of the homologues bound to Mycobacterium tuberculosis lumazine synthase suggests that both the hypothetical intermediate in the lumazine synthase-catalyzed reaction pathway and the metabolically stable analogues bind similarly.

Full text of DOI:10.1021/jo070982r

A New Series of 3-Alkyl Phosphate Derivatives of
4,5,6,7-Tetrahydro-1-D-ribityl-1H-pyrazolo[3,4-d]pyrimidinedione as
Inhibitors of Lumazine Synthase: Design, Synthesis, and Evaluation
Yanlei Zhang,† Guangyi Jin,† Boris Illarionov,‡ Adelbert Bacher,‡ Markus Fischer,§ 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, Lehrstuhl fu¨r Organische Chemie und Biochemie, Technische UniVersita¨t Mu¨nchen, Lichtenbergstr
4, D-85747 Garching, Germany, and Institut fu¨r Lebensmittelchemie, Abteilung Lebensmittelchemie,
UniVersita¨t Hamburg, D-20146 Hamburg, Germany
cushman@pharmacy.purdue.edu
ReceiVed May 18, 2007
Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin. A homologous series
of three pyrazolopyrimidine analogues of a hypothetical intermediate in the lumazine synthase-catalyzed
reaction were synthesized and evaluated as lumazine synthase inhibitors. The key steps of the synthesis
were C-5 deprotonation of 4-chloro-2,6-dimethoxypyrimidine, acylation of the resulting anion, and
conversion of the product to a pyrazolopyrimidine with hydrazine. Alkylation of the pyrazolopyrimidine
with a substituted ribityl iodide and deprotection of the ribityl chain afforded the final set of three products.
All three compounds were extremely potent inhibitors of the lumazine synthases of Mycobacterium
tuberculosis, Magnaporthe grisea, Candida albicans, and Schizosaccharomyces pombe lumazine synthase,
with inhibition constants in the low nanomolar to subnanomolar range. Molecular modeling of one of
the homologues bound to Mycobacterium tuberculosis lumazine synthase suggests that both the hypothetical
intermediate in the lumazine synthase-catalyzed reaction pathway and the metabolically stable analogues
bind similarly.
Introduction
been shown to play an essential role in different Salmonella
disease models.5,6 Since animals lack the riboflavin biosynthetic
pathway, inhibitors of the pathway should therefore be selec-
tively toxic to the pathogen and not the host. The resistance of
bacteria to antibiotics is increasing at a sobering rate, and it is
therefore imperative that medicinal chemists develop new types
of antibiotics. Riboflavin synthase and lumazine synthase, the
last two enzymes in the riboflavin biosynthetic pathway, are
promising targets for the development of new antibiotics.
Riboflavin (vitamin B2) plays an essential role in metabolism.
Animals, including humans, obtain it from their diet, while a
variety of pathogenic Gram-negative Enterobacteria and Can-
dida- and Saccharomyces-type yeasts are dependent on endog-
enous biosynthesis because they do not have an efficient uptake
system.1-4 The riboflavin biosynthesis gene ribB has recently
† Purdue University.
‡ Technische Universita¨t Mu¨nchen.
§ Universita¨t Hamburg.
(4) Neuberger, G.; Bacher, A. Biochem. Biophys. Res. Commun. 1985,
127, 175-181.
(5) Rollenhagen, C.; Bumann, D. Infect. Immunol. 2006, 74, 1649-1660.
(6) Becker, D.; Selbach, M.; Rollenhagen, C.; Ballmaier, M.; Meyer, T.
F.; Mann, M.; Bumann, D. Nature 2006, 440, 303-307.
(1) Wang, A. I Chuan Hsueh Pao 1992, 19, 362-368.
(2) Oltmanns, O.; Lingens, F. Z. Naturforsch. 1967, 22b, 751-754.
(3) Logvinenko, E. M.; Shavlovsky, G. M. Mikrobiologiya 1967, 41,
978-979.
10.1021/jo070982r CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/18/2007
7176
J. Org. Chem. 2007, 72, 7176-7184

3-Alkyl DeriVatiVes as Inhibitors of Lumazine Synthase
SCHEME 1
SCHEME 2
Lumazine synthase catalyzes the condensation of 5-amino-
6-D-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) with 3,4-di-
hydroxybutanone 4-phosphate (2) to afford 6,7-dimethyl-8-D-
ribityllumazine(3).7,8 Riboflavinsynthasecatalyzesamechanistically
unusual dismutation of two molecules of 3 to form one molecule
of riboflavin (4) and one molecule of the lumazine synthase
substrate 1 (Scheme 1).9-13
The details of the reaction catalyzed by lumazine synthase
have not been completely elucidated. The currently proposed
mechanism outlined in Scheme 2 involves the 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, tautomerization of the enol 6 to the ketone
7, ring closure, and dehydration of the covalent hydrate 8 to
provide the product 3.14 Although the mechanism listed in
Scheme 2 is certainly very reasonable, the details of the pathway,
such as the timing of phosphate elimination relative to the
conformational reorganization of the side chain to allow
cyclization, remain unknown.14
concentration of substrate 1 and variable concentration of
substrate 2 in phosphate buffer, the inhibitor dissociation
constant Ki is 46 µM and the Kis is 250 µM.15 The installation
of a C-4 or C-5 phosphate side chain resulted in 11 and 12,
both of which proved to be less potent than purinetrione 9 on
Bacillus subtilis lumazine synthase in phosphate buffer. When
tested in the presence of variable concentrations of substrate 2
in phosphate buffer, 11 displayed a Kis of 852 µM and 12
displayed a Ki of 852 µM and a Kis of 817 µM.16 In contrast,
when they were tested on Mycobacterium tuberculosis lumazine
synthase in Tris buffer, they proved to be very potent inhibitors.
The phosphate 11 displayed a Ki of 4.1 nM, and 12 displayed
a Ki of 4.7 nM.16 By comparing with 9 (Ki ) 9.1 µM) in Tris
buffer,16 the potent contribution of the alkylphosphate side chain
toward inhibition of Mycobacterium tuberculosis lumazine
synthase can be discerned.
The purinetrione 9 is a moderate inhibitor of Bacillus subtilis
lumazine synthase. When tested in the presence of a fixed
(7) Neuberger, G.; Bacher, A. Biochem. Biophys. Res. Commun. 1986,
139, 1111-1116.
The study of purinetrione 9 and its alkylphosphonate deriva-
tives suggests that another series of compounds, the pyrazolopy-
rimidinediones 13, 14, and 15, could be effective inhibitors of
Mycobacterium tuberculosis lumazine synthase. The prior study
of alkylphosphonate derivatives of purinetrione 9 showed that
the C-4 derivative 11 is more potent than the others, which
(8) Kis, K.; Volk, R.; Bacher, A. Biochemistry 1995, 34, 2883-2892.
(9) Bacher, A.; Eberhardt, S.; Richter, G. In Escherichia coli and
Salmonella: Cellular and Molecular Biology, 2nd ed.; Neidhardt, F. C.,
Ed.; ASM Press: Washington, D.C., 1996; pp 657-664.
(10) 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.
(11) Illarionov, B.; Eisenreich, W.; Bacher, A. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 7224-7229.
(12) Illarionov, B.; Kemter, K.; Eberhardt, S.; Richter, G.; Cushman,
M.; Bacher, A. J. Biol. Chem. 2001, 276, 11524-11530.
(13) Plaut, G. W. E.; Harvey, R. A. Methods Enzymol. 1971, 18B, 515-
538.
(14) Volk, R.; Bacher, A. J. Am. Chem. Soc. 1988, 110, 3651-3653.
(15) Cushman, M.; Yang, D.; Kis, K.; Bacher, A. J. Org. Chem. 2001,
66, 8320-8327.
(16) Cushman, M.; Sambaiah, T.; Jin, G.; Illarionov, B.; Fischer, M.;
Bacher, A. J. Org. Chem. 2004, 69, 601-612.
J. Org. Chem, Vol. 72, No. 19, 2007 7177

Zhang et al.
SCHEME 3 a
indicates that the C-4 derivative 14 should be the best inhibitor
in this new series of compounds.16 Crystallography has previ-
ously shown that the C-6 carbonyl of the purinetriones 9-12
hydrogen bonds with the side chain amino group of Lys138 of
lumazine synthase.17 The hypothesis motivating the present
study was that the N-2 nitrogen of the pyrazolo[3,4-b]pyrimidine
system in the target compounds 13-15 would also hydrogen
bond to Lys138 and thus stabilize their complexes with the
enzyme.
a Reagents and conditions: (a) AgOPO(OBn)2, toluene, reflux (24 h);
(b) 4-chloro-2,6-dimethoxypyrimidine, n-BuLi, THF, -78 °C (20 min), -40
°C (1 h); (c) hydrazine, methanol, reflux (18 h); (d) K2CO3, DMF, 72 h;
(e) concd HCl, MeOH, reflux, 5 h.
midinedione series of inhibitors. The syntheses of the ribityl
pyrazolopyrimidinediones 13-15 are outlined in Scheme 3. The
alkyl phosphate side chains were synthesized and installed on
the commercially available 4-chloro-2,6-dimethoxypyrimidine.
Nucleophilic displacement reactions on the three commercially
available starting materials 16, 17, and 18 with silver diben-
zylphosphate resulted in the phosphates 19, 20, and 21 having
chain lengths of three, four, and five carbons. Commercially
available 4-chloro-2,6-dimethoxypyrimidine was deprotonated
with n-butyllithium at low temperature to afford an anion that
reacted with the three ethyl esters 19, 20, and 21 to provide
intermediates 22, 23, and 24. These reactions of 19, 20, and 21
were performed at different temperatures. The temperature must
be lower for lower homologue 19, otherwise, the desired reaction
did not occur. Reaction of intermediates 22, 23, and 24 with
hydrazine provided the dimethoxypyrazolopyrimidines bearing
chains of different lengths. A fully protected acyclic ribose
Results and Discussion
(17) Morgunova, K.; Meining, W.; Illarionov, B.; Haase, I.; Jin, G.;
Bacher, A.; Cushman, M.; Fischer, M.; Ladenstein, R. Biochemistry 2005,
44, 2746-2758.
On the basis of the previous study of 9-12, 3-, 4- and
5-carbon linker chains were chosen for the new pyrazolopyri-
7178 J. Org. Chem., Vol. 72, No. 19, 2007

3-Alkyl DeriVatiVes as Inhibitors of Lumazine Synthase
TABLE 1. Inhibition Constants versus Lumazine Synthases from M. tuberculosis, M. grisea, C. albicans, and S. pombe
S. pombeh
phosphate
buffer
Tris
buffer
compd
parameter
M. tuberculosise
M. griseaf
C. albicansg
13
Ksa (µM)
55 ( 8
14.4 ( 1.1
3.2 ( 0.3
0.61 ( 0.08
0.90 ( 0.03
0.021 ( 0.004
2.4 ( 0.2
Kcatb (min-1
Kic (µM)
)
0.16 ( 0.01
0.015 ( 0.004
2.69 ( 0.07
7.2 ( 0.2
0.71 ( 0.01
0.0020 ( 0.0007
0.0014 ( 0.0003
0.00089 ( 0.00045 0.00045
Kisd (µM)
mechanism
competitive
competitive
competitive
competitive
competitive
14
15
11
12
Ks (µM)
73 ( 8
22.6 ( 2.7
3.2 ( 0.3
0.73 ( 0.06
0.95 ( 0.02
0.026 ( 0.003
2.4 ( 0.2
K
cat (m-1
)
0.29 ( 0.02
0.030 ( 0.003
3.6 ( 0.2
7.2 ( 0.2
0.70 ( 0.01
0.0005 ( 0.0002
Ki (µM)
0.00080 ( 0.00058 0.00058
0.0009 ( 0.0003
K
is (µM)
mechanism
competitive
competitive
competitive
competitive
competitive
Ks (µM)
30 ( 3
0.18 ( 0.01
0.040 ( 0.003
14.4 ( 1.1
2.68 ( 0.07
0.018 ( 0.006
0.101 ( 0.039
mixed
3.2 ( 0.3
1.0 ( 0.1
0.90 ( 0.04
0.12 ( 0.03
0.32 ( 0.09
partial
2.4 ( 0.2
0.70 ( 0.01
0.020 ( 0.002
K
cat (min-1
)
7.2 ( 0.2
Ki (µM)
0.0037 ( 0.0005
K
is (µM)
mechanism
competitive
competitive
competitive
Ks (µM)
63 ( 6
5.2 ( 0.4
3.6 ( 0.3
2.5 ( 0.1
1.31 ( 0.05
0.0040 ( 0.0006
K
cat (min-1
)
1.4 ( 0.1
7.6 ( 0.2
2.1 ( 0.1
Ki (µM)
0.0041 ( 0.0023
0.0013 ( 0.0002
0.008 ( 0.001
K
is (µM)
mechanism
competitive
competitive
competitive
competitive
Ks (µM)
63 ( 5
1.40 ( 0.04
0.0047 ( 0.0019
4.0 ( 0.3
4.7 ( 0.5
2.3 ( 0.1
0.12 ( 0.02
2.5 ( 0.2
1.3 ( 0.1
0.044 ( 0.011
0.36 ( 0.13
mixed
K
cat (min-1
)
6.9 ( 0.2
Ki (µM)
is (µM)
mechanism
0.0077 ( 0.0013
K
competitive
competitive
competitive
b
a Ks is the substrate dissociation constant for the equilibrium E + S a ES. Kcat is the rate constant for the process ES f E + P. c Ki is the inhibitor
d
dissociation constant for the process E + I a EI. Kis is the inhibitor dissociation constant for the process ES + I a ESI. e Recombinant lumazine synthase
from M. tuberculosis, assay performed in Tris hydrochloride buffer. f Recombinant lumazine synthase from M. grisea, assay performed in Tris hydrochloride
buffer. g Recombinant lumazine synthase from C. albicans, assay performed in Tris hydrochloride buffer. h Recombinant lumazine synthase from S. pombe.
derivative 28 was prepared as described in the literature.18 The
ribityl iodide 28 reacted with 25, 26, and 27 to provide the fully
protected products 29, 30, and 31. The protecting groups were
removed with HCl in methanol at reflux to produce the final
products 13, 14, and 15.
rimidinediones 14 and 15 are approximately 8 times less
powerful inhibitors than the corresponding purinetriones 11 and
12 (Table 1). On the other hand, in tests versus pentameric
lumazine synthase of the pathogenic yeast C. albicans, the
difference in inhibitory potency of these two classes is only
about a factor of 2. Very high inhibitory potencies were
determined for pyrazolopyrimidinedione 14 versus the pentam-
eric lumazine synthase of fission yeast S. pombe (0.5 nM).
Crystallographic studies have previously shown that inorganic
phosphate, as well as organic phosphate or phosphonate groups
that are part of the structures of substrate 2 analogues, can
occupy the binding site for 2 in several lumazine synthases and
should act as competitive inhibitors of lumazine synthases.17,19,20
For this reason, the influence of inorganic phosphate (100 mM)
on the inhibitory potencies of compounds 11-15 has been
determined for both the homopentameric lumazine synthase of
S. pombe (Table 1) and the icosahedral (60-mer) lumazine
synthase of B. subtilis (Table 2). Indeed, inorganic phosphate
reduces the inhibitory potencies of all compounds tested versus
the S. pombe enzyme. Particularly, the difference in the
inhibitory potency of pyrazolopyrimidinedione compounds
reaches a factor of 50 with 14, whereas the maximum difference
for purinetrione compounds reaches a factor of 3 for 12. A
similar tendency is observed for purinetrione compounds tested
versus B. subtilis lumazine synthase (variable concentration of
1). For example, compound 11 reveals a Ki value of 12 µM in
Tris hydrochloride and 168 µM in phosphate buffer. In contrast
The pyrazolopyrimidinedione phosphates 13, 14, and 15 were
tested as inhibitors of recombinant M. tuberculosis lumazine
synthase, recombinant M. grisea lumazine synthase, recombinant
C. albicans lumazine synthase, recombinant S. pombe lumazine
synthase, recombinant B. subtilis lumazine synthase â60 capsids,
recombinant E. coli riboflavin synthase, and recombinant M.
tuberculosis riboflavin synthase. Representative Lineweaver-
Burk plots for inhibition of B. subtilis lumazine synthase, C.
albicans lumazine synthase, M. grisea lumazine synthase, and
S. pombe lumazine synthase by inhibitor 14 are presented in
Figure S33 (Supporting Information). The inhibition constants
and inhibition mechanisms for 13, 14, and 15 are listed in Tables
1 and 2. The data for the purinetrione phosphates 11 and 12
are also listed in Tables 1 and 2 for comparison.16
Comparative analysis of the kinetic data shows that imida-
zolone ring replacement in purinetriones with a pyrazole ring
leads to substantial changes in binding affinity. The most striking
effects were observed in tests versus riboflavin synthases of E.
coli and M. tuberculosis: pyrazolopyrimidinediones 13, 14, and
15 are essentially incapable of binding to either enzyme, whereas
purinetriones 11 and 12 show inhibition activity in the 2-330
µM range (Table 2).
With respect to the tests versus pentameric lumazine synthase
of the pathogenic mycobacterium M. tuberculosis, pyrazolopy-
(19) Ladenstein, R.; Ritsert, K.; Richter, G.; Bacher, A. Eur. J. Biochem.
1994, 223, 1007-1017.
(20) Meining, W.; Mo¨rtl, S.; Fischer, M.; Cushman, M.; Bacher, A.;
Ladenstein, R. J. Mol. Biol. 2000, 299, 181-197.
(18) Aslani-Shotorbani, G.; Buchanan, J. G.; Edgar, A. R.; Shahidi, P.
K. Carbohydr. Res. 1985, 136, 37-52.
J. Org. Chem, Vol. 72, No. 19, 2007 7179

Zhang et al.
TABLE 2. Inhibition Constants versus B. subtilis Lumazine Synthase and Riboflavin Synthases from E. coli and M. tuberculosis
B. subtilise lumazine synthase
phosphate buffer
Tris buffer
riboflavin synthases
E. colih M. tuberculosisi
variable
variable
variable
compd
parameter
1 concnf
2 concng
1 concnf
13
Ksa (µM)
6.2 ( 0.6
1.30 ( 0.05
9.9 ( 2.3
72 ( 30
3.8 ( 0.6
0.76 ( 0.03
8.2 ( 0.9
7.6 ( 0.6
3.6 ( 0.1
12 ( 4
49 ( 23
mixed
2.6 ( 0.2
3.4 ( 0.1
7.7 ( 1.1
Kcatb (min-1
Kic (µM)
)
0.84 ( 0.05
Kisd (µM)
mechanism
>1000
uncompetitive
>1000
uncompetitive
mixed
competitive
14
15
11
12
Ks (µM)
5.5 ( 0.5
1.4 ( 0.1
58 ( 17
113 ( 30
mixed
76 ( 6
5.5 ( 0.7
3.1 ( 0.1
148 ( 62
391 ( 132
mixed
2.6 ( 0.8
3.5 ( 0.2
4.6 ( 0.04
K
cat (min-1
)
1.39 ( 0.04
98 ( 19
335 ( 94
mixed
0.76 ( 0.02
Ki (µM)
K
is (µM)
> 1000
uncompetitive
> 1000
uncompetitive
mechanism
Ks (µM)
5.5 ( 0.5
1.42 ( 0.04
79 ( 17
118 ( 18
mixed
77 ( 9
8.0 ( 0.5
3.2 ( 0.1
57 ( 3
2.5 ( 0.3
3.2 ( 0.1
6.6 ( 0.7
0.63 ( 0.0
K
cat (min-1
)
1.40 ( 0.06
181 ( 68
297 ( 98
mixed
Ki (µM)
K
is (µM)
>1000
uncompetitive
>1000
uncompetitive
mechanism
competitive
Ks (µM)
3.2 ( 0.4
3.1 ( 0.1
168 ( 26
26 ( 2
5.4 ( 0.1
6.5 ( 0.7
2.1 ( 0.1
12 ( 1
2.1 ( 0.2
16.7 ( 0.4
332 ( 83
6.9 ( 0.7
1.10 ( 0.03
10.3 ( 0.9
K
cat (min-1
)
Ki (µM)
K
is (µM)
852 ( 103
uncompetitive
mechanism
competitive
competitive
competitive
competitive
Ks (µM)
3.8 ( 0.4
3.1 ( 0.1
271 ( 85
653 ( 163
mixed
40 ( 4
7.1 ( 0.7
2.4 ( 0.1
11 ( 2
55 ( 20
partial
2.0 ( 0.2
16.7 ( 0.4
2.4 ( 0.2
4.9 ( 0.7
1.01 ( 0.03
1.6 ( 0.2
K
cat (min-1
)
3.7 ( 0.1
852 ( 388
817 ( 212
mixed
Ki (µM)
is (µM)
mechanism
K
competitive
competitive
b
a Ks is the substrate dissociation constant for the equilibrium E + S a ES. Kcat is the rate constant for the process ES f E + P. c Ki is the inhibitor
d
dissociation constant for the process E + I a EI. Kis is the inhibitor dissociation constant for the process ES + I a ESI. e Recombinant â60 capsids from
B. subtilis. f The concentration of the dihydroxybutanone phosphate substrate 2 was held constant during the assay, while the concentration of the pyrimidinedione
substrate 1 was varied. g The concentration of the pyrimidinedione substrate 1 was held constant during the assay, while the concentration of the
dihydroxybutanone phosphate 2 was varied. h Recombinant riboflavin synthase from E. coli. i Recombinant riboflavin synthase from M. tuberculosis.
to this observation, the pyrazolopyrimidinedione compounds do
not show the regularities observed for purinetrione inhibitors.
The maximal difference in Ki values reaches a factor of 3 for
compound 14 (58 and 148 µM in the presence or absence of
100 mM phosphate in the assay buffer, respectively). A plausible
explanation for this effect would be that the contribution of the
alkyl phosphate moiety to the inhibitory potencies versus 60-
mer B. subtilis lumazine synthase is much greater in the case
of pyrazolopyrimidinediones than purinetrione compounds.
Indeed, in experiments with variable concentrations of dihy-
droxybutanone phosphate 2, the Ki values for pyrazolopyrim-
idinediones vary from 8.2 to 181 µM (13 and 15, respectively,
Table 2), whereas the inhibitory potencies of the purinetriones
11 and 12 were above 800 µM.
The effect of the linker chain length connecting the phosphate
to the heterocyclic system was not great, varying from enzyme
to enzyme used in kinetic assays. With M. tuberculosis lumazine
synthase, the shortest linker chain is the best, but with M. grisea,
C. albicans, and S. pombe (in Tris buffer), the medium chain
length is the best. With B. subtilis lumazine synthase, the shortest
chain length is the best, and the medium chain length (C-4) is
better than the longer one (C-5) in phosphate buffer, but worse
in Tris buffer.
present case, a hypothetical model was constructed of the
binding of the lumazine synthase inhibitor 14 to M. tuberculosis
lumazine synthase (Figure 1). This model was produced by
overlapping the structure of 14 with that of 11 in one of the
five equivalent active sites of M. tuberculosis lumazine synthase.
The structure of 11 was then removed and the energy of the
complex minimized using the MMFF94s force field. The
resulting Figure 1 was constructed by displaying the amino acid
residues calculated to be involved in hydrogen bonding of the
protein with ligand 14, using a maximum distance of 3.5 Å
between the donor and acceptor atoms to be considered a
hydrogen bond. The calculated structure shows the pyrazol-
opyrimidinedione ring of the ligand 14 stacked with the indole
ring of Trp27. The phosphate of the ligand is extensively
hydrogen bonded with the one water molecule, the side chain
nitrogens of Arg128, as well as the backbone nitrogens of Gln86
and Thr87 and the side-chain hydroxyl of Thr87. The ribityl
hydroxyl groups are hydrogen bonded to the backbone nitrogen
and oxygen of Asn114, the side-chain oxygens of Glu61, and
the backbone nitrogen of Ile60. The pyrazolopyrimidinedione
ring of the ligand is hydrogen bonded to the backbone nitrogen
of Ala59, the backbone nitrogen of Ile83, backbone oxygen of
Val81, and the side-chain nitrogen of Lys138. These contacts
are summarized in Figure 2. It should be noted that the contacts
are either similar or identical to those seen in the crystal structure
of 11 bound to M. tuberculosis lumazine synthase (Figure 3).
In general, the medium linker chain length connecting the
phosphate to the heterocyclic system afforded the most potent
inhibitors. This may reflect the fact that the length of the chain
A crystal structure is available of the complex formed between
the substrate analogue 11 and M. tuberculosis lumazine synthase.
The crystal structure of the lower homologue 10 bound to M.
tuberculosis lumazine synthase has also been determined.17
These structures allow the rational docking and energy mini-
mization of additional lumazine synthase inhibitors. In the
7180 J. Org. Chem., Vol. 72, No. 19, 2007

3-Alkyl DeriVatiVes as Inhibitors of Lumazine Synthase
FIGURE 1. Hypothetical model for the binding of compound 14 to M. tuberculosis lumazine synthase. The figure is programmed for walleyed
viewing.
M. tuberculosis and C. albicans, have been synthesized. These
compounds can be considered as bisubstrate analogue inhibitors
since they contain the elements of the structures of both
substrates 1 and 2 and likely occupy the binding sites of both
substrates. They are also metabolically stable hypothetical
intermediate analogues that bind similarly to the proposed
intermediate 5. It can be anticipated that these new inhibitors
will contribute to the series of metabolically stable analogues
of the hypothetical intermediates in the lumazine synthase
reaction pathway that have continued to unravel the mechanism
of the reactions catalyzed by lumazine synthase and riboflavin
synthase.10,16,17,20-29
Experimental Section
3-[4,6-Dioxo-4,5,6,7-tetrahydro-1-D-ribityl-1H-pyrazolo[3,4-
d]pyrimidin-3-yl]propyl 1-Phosphate (13). Compound 29 (85 mg,
0.119 mmol) was dissolved in concentrated HCl (1.24 mL) and
FIGURE 2. Hydrogen bonds and distance in the calculated model of the
inhibitor 14 bound in the active site of M. tuberculosis lumazine synthase.
methanol (1.86 mL). The mixture was heated at reflux for 5 h.
The solvent was removed in vacuo to afford the crude product,
which was dissolved in water and freeze-dried to yield the product
13 (53 mg, 100%) as an amorphous white solid: 1H NMR (300
MHz, D2O) δ 4.06-3.38 (m, 9H), 2.61-2.56 (t, J ) 7.5 Hz, 2H),
1.82-1.76 (p, J ) 7 Hz, 2H); 13C NMR (75 MHz, D2O) δ 160.7,
(21) Goetz, J. M.; Poliks, B.; Studelska, D. R.; Fischer, M.; Kugelbrey,
K.; Bacher, A.; Cushman, M.; Schaefer, J. J. Am. Chem. Soc. 1999, 121,
7500-7508.
(22) Scheuring, J.; Kugelbrey, K.; Weinkauf, S.; Cushman, M.; Bacher,
A.; Fischer, M. J. Org. Chem. 2001, 66, 3811-3819.
(23) Mehta, A. K.; Studelska, D. R.; Fischer, M.; Giessauf, A.; Kemter,
K.; Bacher, A.; Cushman, M.; Schaefer, J. J. Org. Chem. 2002, 67, 2087-
2092.
(24) Gerhardt, S.; Haase, I.; Steinbacher, S.; Kaiser, J. T.; Cushman,
M.; Bacher, A.; Huber, R.; Fischer, M. J. Mol. Biol. 2002, 318, 1317-
1329.
(25) Cushman, M.; Yang, D.; Mihalic, J. T.; Chen, J.; Gerhardt, S.; Huber,
R.; Fischer, M.; Kis, K.; Bacher, A. J. Org. Chem. 2002, 67, 6871-6877.
(26) Zhang, X.; Meining, W.; Cushman, M.; Haase, I.; Fischer, M.;
FIGURE 3. The interactions between the M. tuberculosis lumazine
synthase and the ligand 11 in the crystal structure of the complex.17
Bacher, A.; Ladenstein, R. J. Mol. Biol. 2003, 328, 167-182.
(27) Cushman, M.; Jin, G.; Illarionov, B.; Fischer, M.; Ladenstein, R.;
Bacher, A. J. Org. Chem. 2005, 70, 8162-8170.
(28) Ramsperger, A.; Augustin, M.; Schott, A. K.; Gerhardt, S.; Krojer,
T.; Eisenreich, W.; Illarionov, B.; Cushman, M.; Bacher, A.; Huber, R.;
Fischer, M. J. Biol. Chem. 2006, 281, 1224-1232.
connecting the phosphate to the pyrimidinedione in the hypo-
thetical intermediate 5 is also four atoms in length.
In summary, several very potent inhibitors of the lumazine
synthases of a number of important human pathogens, including
(29) Morgunova, E.; Illarionov, B.; Sambaiah, T.; Haase, I.; Bacher, A.;
Cushman, M.; Fischer, M.; Ladenstein, R. FEBS J. 2006, 273, 4790-4804.
J. Org. Chem, Vol. 72, No. 19, 2007 7181

Zhang et al.
152.6, 151.5, 146.2, 97.7, 72.8, 72.2, 70.4, 66.3 (2JPC ) 5.7 Hz),
62.7, 50.7, 28.6, 23.6; ESIMS m/z 429/430/431 (C13H17D4N4O10-
PH+/C13H16D5N4O10PH+/C13H15D6N4O10PH+). Anal. Calcd for
C13H21N4O10P‚H2O: C, 35.30; H, 5.24; N, 12.67. Found: C, 35.40;
H, 5.22; N, 12.51.
) 7 Hz, 2H), 4.16-4.10 (dt, 3JHH ) 6 Hz, 3JPH ) 6 Hz, 2H), 2.43-
2.39 (t, J ) 7.5 Hz, 2H), 1.79-1.49 (m, 6H), 1.42-1.37 (t, J ) 7
3
Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 173.9, 136.3 (d, JPC
)
2
2
6.8 Hz), 129.0, 128.3, 69.6 (d, JPC ) 5.6 Hz), 68.0 (d, JPC ) 6
3
Hz), 60.7, 34.5, 30.3 (d, JPC ) 7 Hz), 25.4, 24.8, 14.7; ESIMS
m/z 457 (MNa+). Anal. Calcd for C22H29O6P: C, 62.85; H, 6.95.
Found: C, 62.57; H, 7.21.
4-[4,6-Dioxo-4,5,6,7-tetrahydro1-D-ribityl-1H-pyrazolo[3,4-d]-
pyrimidin-3-yl]butyl 1-Phosphate (14). Compound 30 (35.0 mg,
0.0480 mmol), methanol (0.75 mL), and concd HCl (0.5 mL) were
added to a flask. The reaction mixture was stirred under reflux for
4.5 h. The solvent was removed under reduced pressure. Water (4
mL) was added, and the solution was filtered and frozen. The water
was removed by freeze drying to generate the product (7.8 mg,
37.0%) as an amorphous white solid: 1H NMR (300 MHz, D2O)
4-(6-Chloro-2,4-dimethoxypyrimidin-5-yl)-4-oxobutanyl 1-Di-
benzylphosphate (22). Dry THF (45 mL) was stirred and cooled
to -30 °C under argon. n-BuLi (2.66 mL, 4.25 mmol) was added
into the cooled THF, and the mixture was warmed to -10 °C for
30 min. The solution was cooled to -30 °C, and a solution of
4-chloro-2,6-dimethoxypyrimidine (0.74 g, 4.25 mmol) in THF (5
mL) was added. The reaction mixture was stirred at -30 °C for
another 1.5 h. The mixture was cooled to -78 °C, compound 19
(1.67 g, 4.25 mmol) was added, and stirring was continued for 1 h
at -78 °C. Hydrolysis was carried out at -70 °C using a mixture
of 35% HCl (2 mL) and THF (10 mL). The reaction mixture was
gently warmed to room temperature and made slightly basic with
saturated sodium bicarbonate solution. The organic layer was
separated. The aqueous layer was extracted with CH2Cl2 (3 × 10
mL). The extract was combined with the organic layer and dried
with Na2SO4 overnight. The solvent was removed to provide a crude
product, which was purified by silica gel flash chromatography,
eluting with hexane/ethyl acetate 1.5:1/1:1 to generate the product
(0.53 g, 31%) as a clear oil: 1H NMR (300 MHz, CDCl3) δ 7.51-
δ 4.13-3.55 (m, 9H), 2.62-2.60 (m, 2H), 1.61-1.52 (m, 4H); 13
C
NMR (75 MHz, D2O) δ 161.1, 152.8, 152.5, 146.3, 97.8, 72.9,
72.3, 70.5, 66.6 (2JPC ) 5.6 Hz), 62.8, 50.7, 29.4, 29.3, 26.7, 24.3;
ESIMS m/z 763 (MNa+); ESIMS m/z 439 (MH+). Anal. Calcd for
C14H23N4O10P‚4.5H2O: C, 35.77; H, 5.68; N, 11.92. Found: C,
35.89; H, 5.33; N, 11.36.
5-[4,6-Dioxo-4,5,6,7-tetrahydro-1-D-ribityl-1H-pyrazolo[3,4-
d]pyrimidin-3-yl]pentyl 1-Phosphate (15). Compound 31 (59.2
mg, 0.080 mmol), methanol (1.25 mL), and concd HCl (0.83 mL)
were added to a flask. The reaction mixture was stirred under reflux
for 5 h. The solvent was removed under reduced pressure. Water
(4 mL) was added, and the solution was filtered and frozen. The
water was removed by freeze drying to generate the product (9.30
mg, 25.7%) as an amorphous white solid: 1H NMR (300 MHz,
D2O) δ 4.13-3.52 (m, 9H), 2.61-2.59 (t, J ) 5 Hz, 2H), 1.52-
1.22 (m, 6H); 13C NMR (75 MHz, D2O) δ 160.9, 152.7, 146.2,
97.6, 72.9, 72.2, 70.5, 67.0, 66.9 (2JPC ) 5.6 Hz), 62.8, 50.6, 29.6,
29.5, 27.6, 26.9, 24.5; ESIMS m/z 475 (MNa+). Anal. Calcd for
C15H25N4O10P‚H2O: C, 38.30; H, 5.79; N, 11.91. Found: C, 38.72;
H, 5.58; N, 11.58.
Ethyl 3-[Bis(benzyloxy)phosphoryloxy]propanoate (19). Ethyl
4-bromobutyrate (1.17 g, 6.00 mmol) and the silver salt of dibenzyl
phosphate (2.31 g, 6.00 mmol) were heated at reflux in dry toluene
(50 mL) for 36 h under an atmosphere of argon. The solvent was
filtered and evaporated to afford an oil. The oil was purified by
silica gel flash chromatography, eluting with 3:2/2:1/2:1.5 hexane/
ethyl acetate to afford the product (1.71 g, 72.8%) as a clean oil:
1H NMR (300 MHz, CDCl3) δ 7.57-7.42 (m, 10H), 5.24-5.12
3
2
7.46 (m, 10H), 5.27-5.16 (dd, JPH ) 8 Hz, JHH ) 12 Hz, 4H),
4.30-4.22 (dt, 3JHH ) 6 Hz, 3JPH ) 6 Hz, 2H), 4.18 (s, 3H), 4.14
(s, 3H) 3.02-2.98 (t, J ) 7 Hz, 2H), 2.22-2.14 (p, J ) 6 Hz,
2H); 13C NMR (75 MHz, CDCl3) δ 119.4, 169.2, 164.3, 157.8,
3
2
136.2 (d, JPC ) 6.8 Hz), 129.0, 128.4, 115.0, 69.7 (d, JPC ) 5.5
2
3
Hz), 67.2 (d, JPC ) 6 Hz), 56.1, 55.6, 40.2, 24.6 (d, JPC ) 7.5
Hz); ESIMS m/z 543 (MNa+). Anal. Calcd for C24H26ClN2O7P:
C, 55.34; H, 5.03; N, 5.38. Found: C, 55.47; H, 5.10; N, 5.27.
5-(6-Chloro-2,4-dimethoxypyrimidin-5-yl)-5-oxopentanyl1-Di-
benzylphosphate (23). Dry 4-chloro-2,6-dimethoxypyrimidine
(0.744 g, 4.26 mmol) was dissolved in dry THF (14 mL) under
argon. The reaction mixture was cooled to -78 °C for 15 min, and
a 1.6 M solution of n-BuLi in hexane (2.69 mL, 4.30 mmol) was
added dropwise while maintaining the temperature below -70 °C.
After stirring at -78 °C for 20 min, compound 20 (1.73 g, 4.26
mmol) in dry THF (4.5 mL) was added quickly, and the reaction
mixture was allowed to slowly warm to -40 °C and stirred at -40
°C for 1 h. Brine (15 mL) was then added. The entire solution was
extracted with ethyl acetate (30 mL) and dried with Na2SO4
overnight. After concentrating, the oil was purified by silica gel
flash column chromatography, eluting with hexane/ethyl acetate
2:1/1:1 to afford the product (0.582 g, 25.6%) as a clean oil: 1H
NMR (300 MHz, CDCl3) δ 7.34-7.31 (m, 10H), 5.05-5.00 (dd,
3JPH ) 8 Hz, 2JHH ) 12 Hz, 4H), 4.01-3.96 (dt, 3JHH ) 6 Hz, 3JPH
) 6 Hz, 2H), 4.0 (s, 3H), 3.97 (s, 3H), 2.77-2.72 (t, J ) 6 Hz,
2H), 1.71-1.68 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 199.5,
168.7, 163.7, 157.2, 135.8 (d, 3JPC ) 6.8 Hz), 128.4, 127.8, 114.6,
3
2
(dd, JPH ) 8 Hz, JHH ) 12 Hz, 4H), 4.29-4.22 (q, J ) 7 Hz,
2H), 4.21-4.14 (dt, 3JHH ) 6 Hz, 3JPH ) 6 Hz, 2H), 2.51-2.46 (t,
J ) 3 Hz, 2H), 2.11-2.04 (p, J ) 7 Hz, 2H), 1.43-1.37 (t, J ) 7
Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 172.5, 135.7 (d, JPC
)
3
6.7 Hz), 128.4, 127.8, 69.1 (d, 2JPC ) 5.6 Hz), 66.6 (d, 2JPC ) 5.9
3
Hz), 60.3, 30.0, 25.3 (d, JPC ) 7.4 Hz), 14.0; ESIMS m/z 415
(MNa+). Anal. Calcd for C20H25O6P: C, 61.22; H, 6.42. Found:
C, 61.22; H, 6.41.25
Ethyl 4-[Bis(benzyloxy)phosphoryloxy]butanoate (20). Ethyl
5-bromovalerate (2.59 g, 12.4 mmol) and the silver salt of dibenzyl
phosphate (4.78 g, 12.4 mmol) were heated at reflux in dry toluene
(98 mL) for 24 h under an atmosphere of argon. The solvent was
filtered and evaporated to afford an oil. The oil was purified by
silica gel flash chromatography eluting with 1:2/1:1 ethyl acetate/
hexane to yield the product as a clean oil (2.97 g, 60%): 1H NMR
2
2
69.1 (d, JPC ) 5.5 Hz), 67.3 (d, JPC ) 6 Hz), 55.5, 55.0, 43.0,
29.2 (d, 3JPC ) 7.5 Hz), 19.3; ESIMS m/z 535 (MH+). Anal. Calcd
for C25H28ClN2O7P: C, 56.13; H, 5.28; N, 5.24. Found: C, 55.75;
H, 5.06; N, 5.16.
(300 MHz, CDCl3) δ 7.33-7.30 (m, 10H), 5.07-4.98 (dd, 3JPH
8 Hz, JHH ) 12 Hz, 4H), 4.13-4.06 (q, J ) 7 Hz, 2H), 4.00-
3.93 (dt, 3JHH ) 6 Hz, 3JPH ) 6 Hz, 2H), 2.27-2.23 (t, J ) 7 Hz,
2H), 1.63-1.60 (m, 4H), 1.25-2.0 (t, J ) 7 Hz, 3H).25
)
2
6-(6-Chloro-2,4-dimethoxypyrimidin-5-yl)-6-oxohexanyl 1-Di-
benzylphosphate (24). Dry 4-chloro-2,6-dimethoxypyrimidine
(0.748 g, 4.29 mmol) was dissolved in dry THF (12 mL) under
argon. The reaction mixture was cooled to -78 °C for 10 min, and
a 1.6 M solution of n-BuLi in hexane (2.90 mL, 4.64 mmol) was
added dropwise while maintaining the temperature below -70 °C.
After stirring at -78 °C for 30 min, compound 21 (1.80 g, 4.29
mmol) in dry THF (2 mL) was added quickly, and the reaction
mixture was allowed to slowly warmed to -40 °C and stirred at
-40 °C for 1.5 h. The reaction mixture was slowly warmed to
room temperature and stirred for 0.5 h. Brine (15 mL) was then
added. The entire solution was extracted with ethyl acetate (35 mL)
Ethyl 5-[Bis(benzyloxy)phosphoryloxy]pentanoate (21). 6-Bro-
mohexanoic acid ethyl ester (4.01 g, 18.0 mmol) and the silver
salt of dibenzyl phosphate (6.93 g, 18.0 mmol) were heated at reflux
in dry toluene (100 mL) for 24 h under an atmosphere of argon.
The solvent was removed to provide a residue. The residue was
purified by silica gel flash chromatography, eluting with 1:2/1:1
ethyl acetate/hexane to provide the product (5.78 g, 79.2%) as a
clean oil: 1H NMR (300 MHz, CDCl3) δ 7.53-7.49 (m, 10H),
3
2
5.18-5.17 (dd, JPH ) 8 Hz, JHH ) 12 Hz, 4H), 4.30-4.23 (q, J
7182 J. Org. Chem., Vol. 72, No. 19, 2007

3-Alkyl DeriVatiVes as Inhibitors of Lumazine Synthase
and dried with Na2SO4 overnight. The organic solution was filtered,
and the solvent was removed under reduced pressure to generate
an oil. The oil was purified with silica gel flash chromatography,
eluting with hexane/ethyl acetate 3:1/2:1 to afford the product (0.605
g, 26%) as a clean oil: 1H NMR (300 MHz, CDCl3) δ 7.55-7.54
mixture was stirred at room temperature for 3 days. The solvent
was removed under reduced pressure. The residue was purified by
silica gel TLC with hexane/ethyl acetate 1:3 as the mobile phase
to afford product 29 (95 mg, 56%) as a clean oil: 1H NMR (300
3
MHz, CDCl3) δ 7.40-7.34 (m, 10H), 5.04-5.01 (dd, JPH ) 8
3
2
2
(m, 10H), 5.29-5.22 (dd, JPH ) 8 Hz, JHH ) 12 Hz, 4H), 4.22
Hz, JHH ) 12 Hz, 4H), 4.57-4.54 (dd, J ) 4, 9 Hz, 1H), 4.24-
3
3
(s, 3H), 4.19-4.15 (dt, JHH ) 6 Hz, JPH ) 6 Hz, 2H), 4.17 (s,
3H), 2.97-2.92 (t, J ) 7 Hz, 2H), 1.91-1.55 (m, 6H); 13C NMR
(75 MHz, CDCl3) δ 201.6, 137.2 (d, 3JPC ) 6.7 Hz), 129.8, 129.1,
4.21 (m, 2H), 4.13-4.00 (m, 9H), 3.99-3.85 (m, 3H), 2.94-2.89
(t, J ) 7 Hz, 2H), 2.08-2.03 (p, J ) 7 Hz, 2H), 1.37 (s, 3H), 1.24
(s, 3H), 1.16 (s, 3H), 0.54 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
165.9, 164.7, 157.9, 144.9, 136.3 (d, 3JPC ) 6.8 Hz), 128.9, 128.3,
2
2
70.1 (d, JPC ) 5.7 Hz), 68.5 (d, JPC ) 6.3 Hz), 56.4, 55.9, 44.5,
30.5 (d, 3JPC ) 7.5 Hz), 25.4, 23.5; ESIMS m/z 571 (MNa+). Anal.
Calcd for C26H30ClN2O7P: C, 56.89; H, 5.51; N, 5.10. Found: C,
57.27; H, 5.50; N, 4.77.
2
109.2, 102.2, 98.1, 78.0, 69.6 (d, JPC ) 5.5 Hz), 69.3, 67.8 (d,
2JPC ) 6 Hz), 58.7, 55.4, 54.5, 49.1, 29.8 (d, 3JPC ) 7.5 Hz), 28.9,
26.3, 25.0, 24.3, 23.9; ESIMS m/z 713 (MH+). Anal. Calcd for
C35H45N4O10P: C, 58.98; H, 6.36; N, 7.86. Found: C, 59.09; H,
6.45; N, 7.70
3-(4,6-Dimethoxy-1H-pyrazolo[3,4-d]pyrimidin-3-yl)propyl
1-Dibenzylphosphate (25). Compound 22 (0.386 g, 0.742 mmol)
and hydrazine (0.028 mL, 0.891 mmol) were added to methanol
(65 mL). The mixture was kept at reflux under argon for 6 h. The
solvent was removed in vacuo to give a residue. The residue was
purified by column chromatography with hexane/ethyl acetate 1:1/
1:1.5 as eluent to give pure product 25 (0.207 g, 56%) as a clear
oil: 1H NMR (300 MHz, CDCl3) δ 7.14-7.08 (m, 10H), 4.92-
4-[4,6-Dimethoxy-1-(2,2,6,6-tetramethyl-tetrahydro[1,3]diox-
olo[4,5-e][1,3]dioxepin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl]-
butyl 1-Dibenzylphosphate (30). Compound 26 (54.8 mg, 0.107
mmol), compound 28 (37.0 mg, 0.107 mmol), and potassium
carbonate (44.3 mg, 0.321 mmol) were added into a flask. DMF
(anhydrous, 2 mL) was added to the flask, and the reaction mixture
was stirred under nitrogen at room temperature for 38 h. CHCl3
(10 mL) was added, and the reaction mixture was filtered, washed
with brine (8 mL), and then dried over Na2SO4 for 4 h. The solvent
was removed under reduced pressure to yield the crude product
(99 mg). It was purified by TLC with hexane/ethyl acetate 1:3 as
the mobile phase to afford the product (35.0 mg, 45.1%) as an oil:
1H NMR (300 MHz, CDCl3) δ 7.14 (m, 10H), 4.84-4.82 (dd, 3JPH
4.81 (dd, 3JPH ) 8 Hz, 2JHH ) 12 Hz, 4H), 3.98-3.91 (dt, 3JHH
)
3
6 Hz, JPH ) 6 Hz, 2H), 3.89 (s, 3H), 3.87 (s, 3H), 2.85-2.80 (t,
J ) 7 Hz, 2H), 2.01-1.92 (p, J ) 6 Hz, 2H); 13C NMR (75 MHz,
3
CDCl3) δ 166.1, 165.0, 159.1, 146.2, 136.3 (d, JPC ) 6.8 Hz),
2
2
128.9, 128.3, 97.9, 69.7 (d, JPC ) 5.6 Hz), 67.8 (d, JPC ) 6.2
3
Hz), 55.6, 54.7, 29.4 (d, JPC ) 7.2 Hz), 25.0; ESIMS m/z 499
(MH+). Anal. Calcd for C24H27N4O6P: C, 57.83; H, 5.46; N, 11.24.
Found: C, 57.82; H, 5.31; N, 11.25.
2
) 8 Hz, JHH ) 12 Hz, 4H), 4.42-4.38 (dd, J ) 4, 9 Hz, 1H),
4.07-3.71 (m, 14H), 2.69-2.64 (t, J ) 7 Hz, 2H), 1.57-1.51 (m,
4H), 1.44 (s, 3H), 1.19 (s, 3H), 0.98 (s, 3H), 0.37 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ 165.9, 164.7, 157.9, 145.7, 136.3 (d,
3JPC ) 6.8 Hz), 129.0, 128.9, 128.3, 109.2, 102.2, 98.1, 78.1, 69.6
4-(4,6-Dimethoxy-1H-pyrazolo[3,4-d]pyrimidin-3-yl)butyl 1-Di-
benzylphosphate (26). Compound 23 (0.582 g, 1.09 mmol),
methanol (25 mL), and hydrazine (41.8 mg, 1.31 mmol) were added
to a flask. The mixture was stirred under reflux for 18 h. The
reaction mixture was cooled to room temperature, and the solvent
was removed under reduced pressure. The oil was purified by silica
gel flash chromatography, eluting with hexane/ethyl acetate 1:4 to
afford pure product (324 mg, 58.1%) as an oil: 1H NMR (300 MHz,
CDCl3) δ 7.35-7.29 (m, 10H), 5.07-5.03 (dd, 3JPH ) 8 Hz, 2JHH
) 12 Hz, 4H), 4.11 (s, 3H), 4.09 (s, 3H), 4.07-4.03 (dt, 3JHH ) 6
2
2
(d, JPC ) 5.6 Hz), 69.3, 68.0 (d, JPC ) 6 Hz), 58.7, 55.3, 54.5,
3
49.2, 30.1, 30.0 (d, JPC ) 7 Hz), 28.2, 26.3, 25.3, 24.3, 24.0;
ESIMS m/z 727 (MH+).
5-[4,6-Dimethoxy-1-(2,2,6,6-tetramethyl-tetrahydro[1,3]dioxolo-
[4,5-e][1,3]dioxepin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-
3-yl]pentyl 1-Dibenzylphosphate (31). Compound 27 (100 mg,
0.190 mmol), 28 (65.4 mg, 0.190 mmol), and potassium carbonate
(78.7 mg, 0.570 mmol) were added to a flask. Anhydrous DMF
(2.5 mL) was added to the flask, and the reaction mixture was stirred
under nitrogen for 3 days. CH2Cl2 (10 mL) was added, and the
reaction mixture was filtered and washed with brine (5 mL). The
aqueous phase was extracted with CH2Cl2 (3 × 5 mL). The organic
phases were combined and dried over Na2SO4 for 4 h. The solvent
was removed under reduced pressure to generate an oil. The oil
was purified by silica gel flash chromatography, eluting with
hexane/ethyl acetate 1:1/1.5:1 to afford the product (63.0 mg,
44.8%) as a clean oil: 1H NMR (300 MHz, CDCl3) δ 7.30-7.24
(m, 10H), 5.01-4.97 (dd, 3JPH ) 8 Hz, 2JHH ) 12 Hz, 4H), 4.56-
4.53 (dd, J ) 4, 9 Hz, 1H), 4.23-3.85 (m, 14H), 2.82-2.77 (t, J
3
Hz, JPH ) 6 Hz, 2H), 2.96-2.91 (t, J ) 7 Hz, 2H), 1.83-1.70
(m, 4H); 13C NMR (75 MHz, CDCl3) δ 166.2, 165.1, 159.1, 147.2,
3
2
136.3 (d, JPC ) 6.8 Hz), 128.9, 128.3, 97.9, 69.6 (d, JPC ) 5.6
2
3
Hz), 68.1 (d, JPC ) 6.1 Hz), 55.6, 54.7, 30.1 (d, JPC ) 7 Hz),
28.3, 25.0; ESIMS m/z 513 (MH+). Anal. Calcd for C25H29N4O6P:
C, 58.59; H, 5.70; N, 10.93. Found: C, 58.52; H, 5.56; N, 10.78.
5-(4,6-Dimethoxy-1H-pyrazolo[3,4-b]pyrimidin-3-yl)pentyl
1-Dibenzylphosphate (27). Compound 24 (0.482 g, 0.880 mmol),
methanol (20 mL), and hydrazine (56.32 mg, 1.76 mmol) were
added to a flask. The mixture was stirred under reflux for 4 h, and
then kept stirring at 45 °C for 12 h. The reaction mixture was cooled
to room temperature. The solvent was removed under reduced
pressure to afford an oil. The oil was purified by silica gel flash
column chromatography, eluting with hexane/ethyl acetate 1:1/1:2
to yield the product (284.6 mg, 61.5%) as a clean oil: 1H NMR
) 7 Hz, 2H), 1.69-1.34 (m, 12H), 1.14 (s, 3H), 0.52 (s, 3H); 13
C
NMR (75 MHz, CDCl3) δ 165.9, 164.6, 157.8, 146.1, 136.4 (d,
3JPC ) 6.8 Hz), 128.9, 128.8, 128.3, 109.2, 102.2, 98.1, 78.0, 69.6
(d, 2JPC ) 5.6 Hz), 69.3, 68.2 (d, 2JPC ) 6.1 Hz), 58.7, 55.3, 54.5,
(300 MHz, CDCl3) δ 7.37-7.30 (m, 10H), 5.08-5.03 (dd, 3JPH
)
2
3
3
8 Hz, JHH ) 12 Hz, 4H), 4.18-4.12 (dt, JHH ) 6 Hz, JPH ) 6
Hz, 2H), 4.14 (s, 3H), 4.10 (s, 3H), 2.95-2.90 (t, J ) 7 Hz, 2H),
1.81-1.44 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 167.3, 166.1,
160.1, 148.6, 137.2 (d, 3JPC ) 6.8 Hz), 129.7, 129.1, 98.5, 70.0 (d,
3
49.1, 30.3 (d, JPC ) 7.1 Hz), 29.0, 28.9, 28.6, 26.3, 25.5, 24.3,
23.9; ESIMS m/z 763 (MNa+). Anal. Calcd for C37H49N4O10P: C,
59.99; H, 6.67; N, 7.56. Found: C, 59.61; H, 6.69; N, 7.39.
Kinetic Assays. All assays have been performed in 96-well
microtiter plates using a computer-controlled SpectraMax 2 mi-
croplate reader (Molecular Devices GmbH, Ismaning, Germany).
Enzymes used in kinetic assays are specified in the Table 3.
Lumazine Synthase Assay. Assay mixtures with variable
concentrations of 1 contained 50 mM Tris hydrochloride, pH 7.0,
100 mM NaCl, 2% (v/v) DMSO, 5 mM dithiothreitol, 100 µM 2,
lumazine synthase, and 1 (3-150 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,
2JPC ) 5.6 Hz), 68.6 (d, 2JPC ) 6.1 Hz), 55.9, 55.0, 30.5 (d, 3JPC
)
7.1 Hz), 28.9, 28.7, 25.6; ESIMS m/z 527 (MH+). Anal. Calcd for
C26H31N4O6P: C, 59.31; H, 5.93; N, 10.64. Found: C, 59.29; H,
5.80; N, 10.39.
3-[4,6-Dimethoxy-1-(2,2,6,6-tetramethyltetrahydro[1,3]dioxolo-
[4,5-e][1,3]dioxepin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-
3-yl]propyl 1-Dibenzylphosphate (29). The protected ribityl iodide
28 (100 mg, 0.29 mmol) was added to the substituted pyrazolo-
[3,4-d]pyrimidine 25 (118 mg, 0.24 mg) in DMF (3 mL). K2CO3
(98 mg, 0.15 mmol) was added to the reaction mixture. The reaction
J. Org. Chem, Vol. 72, No. 19, 2007 7183

Zhang et al.
regression method using the program DynaFit.30 Different inhibition
models were considered for the calculation. Ki and Kis values (
standard deviations were obtained from the fit under consideration
of the most likely inhibition model as described earlier.27
Lumazine Synthase Molecular Modeling. Using Sybyl (Tripos,
Inc., version 7.1, 2005), the X-ray crystal structure of the complex
of 3-(1,3,7-trihydro-9-D-ribityl-2,6,8-purinetrione-7-yl)butane 1-phos-
phate (11) and the lumazine synthase of M. tuberculosis (1w29)
was downloaded and hydrogen atoms were added to the complex.
The complex was first minimized using the steepest descent method
to a termination gradient of 0.05 kcal/mol employing the MMFF94s
force field and MMFF94 charges, and then the complex was
minimized using the conjugate gradient method to a termination
gradient of 0.05 kcal/mol. The structure of the inhibitor 14 was
overlapped with the structure of 3-(1,3,7-trihydro-9-D-ribityl-2,6,8-
purinetrione-7-yl)butane 1-phosphate (11), which was then deleted.
The energy of the complex was first minimized using the steepest
descent method to a termination gradient of 0.05 kcal/mol while
employing the MMFF94s force field and MMFF94 charges, and
then the complex was minimized using conjugate gradient method
to a termination gradient of 0.05 kcal/mol. Figure 1 was constructed
by displaying the amino acid residues of the enzyme that are
involved in hydrogen bonding and stacking with the inhibitor 14.
The maximum distance between donor and acceptor atoms con-
tributing to the hydrogen bonds shown in Figure 1 was set to 3.5
Å.
TABLE 3. Enzymes Used in Kinetic Assays
specific
concn of enzyme
in reaction
activity
enzyme
organism
µM mg-1 h-1 mixture, µg mL-1
lumazine synthases: B. subtilis
S. pombe
8.7
4.2
2.0
0.9
1.5
1.0
30
1.1
4.0
C. albicans
4.4
M. grisea
14.8
1.1
20.6
4.1
M. tuberculosis
riboflavin synthases: E. coli
M. tuberculosis
and lumazine synthase in 51 mM Tris hydrochloride, pH 7.0, was
added to 4 µL of inhibitor in 100% (v/v) DMSO (inhibitor
concentration window, 0-300 µM) 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-ribityllumazine (3) was measured online for the
period of 40 min at 27 °C at 408 nm (ꢀlumazine ) 10 200 M-1 cm-1).
For kinetic assays with variable concentrations of 2, the
concentration of 1 was set to 15 µM whereas the concentration of
2 in reaction mixture was varied between 30 and 400 µM. For
kinetic assays in phosphate buffer, Tris hydrochloride and NaCl in
reaction mixtures were replaced by 100 mM K/Na-phosphate pH
7.0. All other assay parameters were the same as described above.
Riboflavin Synthase Assay. Assay mixtures contained 50 mM
Tris hydrochloride, pH 7.0, 100 mM NaCl, 1% (v/v) DMSO, 5
mM dithiothreitol, enzyme, and variable concentrations of 3 (3-
50 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, and riboflavin synthase in 51 mM Tris hydrochloride,
pH 7.0, was added to 5 µL of inhibitor in 40% (v/v) DMSO
(inhibitor concentration window, 0-400 µM) 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 (30-500 µM) in 51 mM Tris hydrochloride, pH 7.0.
The formation of riboflavin was measured online for the period of
40 min at 27 °C at 470 nm (ꢀriboflavin ) 9600 M-1 cm-1).
Acknowledgment. This research was made possible by NIH
Grant GM51469, as well as by support from the Fonds der
Chemischen Industrie and the Hans Fischer Gesellschaft. This
investigation was conducted in a facility constructed with
support from Research Facilities Improvement Program Grant
Number C06-14499 from the National Center for Research
Resources of the National Institutes of Health.
Supporting Information Available: 1H and 13C NMR spectra
of all new compounds, and Lineweaver-Burk plots for inhibition
of lumazine synthases by inhibitor 14. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO070982R
Evaluation of Experimental Data. The velocity-substrate data
were fitted for all inhibitor concentrations with a nonlinear
(30) Kuzmic, P. Anal. Biochem. 1996, 237, 260-273.
7184 J. Org. Chem., Vol. 72, No. 19, 2007