Design, synthesis, and evaluation of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-ribitylaminolumazines as inhibitors of riboflavin synthase and lumazine synthase

Article abstract of DOI:10.1021/jo0201631

A series of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-D-ribityllumazine were synthesized as inhibitors of both Escherichia coli riboflavin synthase and Bacillus subtilis lumazine synthase. The compounds were designed to bind to both the ribitylpurine binding site and the phosphate binding site of lumazine synthase. In the carboxyalkyl series, maximum activity against both enzymes was observed with the 3′-carboxypropyl compound 22. Lengthening or shortening the chain linking the carboxyl group to the lumazine by one carbon resulted in decreased activity. In the phosphonoxyalkyl series, the 3′-phosphonoxypropyl compound 33 was more potent than the 4′-phosphonoxybutyl derivative 39 against lumazine synthase, but it was less potent against riboflavin synthase. Molecular modeling suggested that the terminal carboxyl group of 6-(3′-carboxypropyl)-7-oxo-8-D-ribityllumazine (22) may bind to the side chains of Arg127 and Lys135 of the enzyme. A hypothetical molecular model was also constructed for the binding of 6-(2′-carboxyethyl)-7-oxolumazine (15) in the active site of E. coli riboflavin synthase, which demonstrated that the active site could readily accommodate two molecules of the inhibitor.

Full text of DOI:10.1021/jo0201631

Design , Syn th esis, a n d Eva lu a tion of 6-Ca r boxya lk yl a n d
6-P h osp h on oxya lk yl Der iva tives of 7-Oxo-8-r ibityla m in olu m a zin es
a s In h ibitor s of Ribofla vin Syn th a se a n d Lu m a zin e Syn th a se
Mark Cushman,*,† Donglai Yang,† Stefan Gerhardt,‡,§ Robert Huber,§ Markus Fischer,‡
Klaus Kis,‡ and Adelbert Bacher‡
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal
Sciences, Purdue University, West Lafayette, Indiana 47907, Lehrstuhl fu¨r Organische Chemie und
Biochemie, Technische Universita¨t Mu¨nchen, D-85747 Garching, Germany, and Max-Planck-Institut fu¨r
Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, Germany
cushman@pharmacy.purdue.edu
Received March 8, 2002
A series of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-D-ribityllumazine were
synthesized as inhibitors of both Escherichia coli riboflavin synthase and Bacillus subtilis lumazine
synthase. The compounds were designed to bind to both the ribitylpurine binding site and the
phosphate binding site of lumazine synthase. In the carboxyalkyl series, maximum activity against
both enzymes was observed with the 3′-carboxypropyl compound 22. Lengthening or shortening
the chain linking the carboxyl group to the lumazine by one carbon resulted in decreased activity.
In the phosphonoxyalkyl series, the 3′-phosphonoxypropyl compound 33 was more potent than the
4′-phosphonoxybutyl derivative 39 against lumazine synthase, but it was less potent against
riboflavin synthase. Molecular modeling suggested that the terminal carboxyl group of 6-(3′-
carboxypropyl)-7-oxo-8-D-ribityllumazine (22) may bind to the side chains of Arg127 and Lys135 of
the enzyme. A hypothetical molecular model was also constructed for the binding of 6-(2′-
carboxyethyl)-7-oxolumazine (15) in the active site of E. coli riboflavin synthase, which demonstrated
that the active site could readily accommodate two molecules of the inhibitor.
In tr od u ction
from 5 produces the enol 6, which tautomerizes to the
ketone 7. Intramolecular nucleophilic attack of the ribi-
tylamino group of 7 on the ketone affords the carbinola-
mine 8, which dehydrates to yield the product 3.6,7
A hypothetical model of the phosphate 5 bound in the
active site of lumazine synthase has been proposed on
the basis of the X-ray structure of a complex formed
between the substrate analogue 9 and reconstituted,
The last two steps in the biosynthesis of riboflavin are
catalyzed by lumazine synthase and riboflavin synthase
(Scheme 1).1-5 Lumazine synthase catalyzes the penul-
timate step, in which the four-carbon phosphate 2
condenses with the pyrimidinedione 1 to form 6,7-
dimethyl-8-D-ribityllumazine (3). In an unusual dismu-
tation reaction, riboflavin synthase catalyzes the transfer
of a four-carbon unit between two molecules of 3, result-
ing in the formation of riboflavin 4 and the lumazine
synthase substrate 1, which can then be recycled.
A mechanism for the lumazine synthase-catalyzed
reaction is outlined in Scheme 2. Schiff base formation
involving the primary amino group of 1 and the ketone
of 2 leads to the imine 5. Elimination of phosphoric acid
† Purdue University.
‡ Technische Universita¨t Mu¨nchen.
§ Max-Planck-Institut.
(1) Plaut, G. W. E.; Smith, C. M.; Alworth, W. L. Annu. Rev.
Biochem. 1974, 43, 899-922.
(2) Plaut, G. W. E. In Comprehensive Biochemistry; Florkin, M.,
Stotz, E. H., Eds.; Elsevier: Amsterdam, 1971; Vol. 21, pp 11-45.
(3) Beach, R. L.; Plaut, G. W. E. J . Am. Chem. Soc. 1970, 92, 2913-
2916.
(4) Bacher, A.; Eberhardt, S.; Richter, G. In Escherichia coli and
Salmonella: Cellular and Molecular Biology, 2nd ed.; Neidhardt, F.
C., Ed.; ASM Press: Washington, DC, 1996; pp 657-664.
(5) Bacher, A.; Fischer, M.; Kis, K.; Kugelbrey, K.; Mo¨rtl, S.;
Scheuring, J .; Weinkauf, S.; Eberhardt, S.; Schmidt-Ba¨se, K.; Huber,
R.; Ritsert, K.; Cushman, M.; Ladenstein, R. Biochem. Soc. Trans.
1996, 24, 89-94.
icosahedral, â60 Bacillus subtilis lumazine synthase
capsid.8 By overlapping the phosphate group of 5 with a
solvent-derived, inorganic phosphate molecule in the
X-ray structure of the complex, a model of bound 5 could
be constructed in which the conformation of the phosphate-
(6) Volk, R.; Bacher, A. J . Am. Chem. Soc. 1988, 110, 3651-3653.
(7) Kis, K.; Volk, R.; Bacher, A. Biochemistry 1995, 34, 2883-2892.
(8) Ritsert, K.; Huber, R.; Turk, D.; Ladenstein, R.; Schmidt-Ba¨se,
K.; Bacher, A. J . Mol. Biol. 1995, 253, 151-167.
10.1021/jo0201631 CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/17/2002
J . Org. Chem. 2002, 67, 5807-5816
5807

Cushman et al.
SCHEME 1
SCHEME 2
containing side chain is held roughly as shown in
structure 5. This model has been supported by the X-ray
structure of a complex of Saccharomyces cerevisiae lu-
mazine synthase with a metabolically stable analogue 10
the chain would have to swing toward the nucleophilic
amine, and the enol would have to isomerize to a ketone.
The details of this structural reorganization remain to
be determined.10 Although an E-imine is shown in
structure 5, the imine geometry in the actual intermedi-
ate is not known, and both Z- and E-imines can be
modeled to fit the active site of lumazine synthase.
However, a Z-imine is required in intermediate 7 for
cyclization to occur.
A variety of Gram-negative bacteria and yeasts have
been shown to lack an efficient riboflavin uptake system
and are consequently dependent on endogenous syn-
thesis.11-15 The enzymes involved in the biosynthesis of
riboflavin are therefore rational targets for antibiotic
drug development.
The reaction catalyzed by riboflavin synthase is thought
to be initiated by a nucleophilic attack of the lumazine
anion 11, formed at the acceptor site of the enzyme, on
C-6 of the lumazine derivative 12, formed at the donor
of the hypothetical Schiff base intermediate 5.9,10 In this
structure, the end of the phosphate-containing side chain
is distant from the nucleophilic ribitylamino group, which
implies that the enol group of 6 would also be generated
away from the nucleophilic amine. To cyclize, the end of
(11) Bandrin, S. V.; Beburov, M. Y.; Rabinovich, P. M.; Stepanov,
A. I. Genetika 1979, 15, 2063-2065.
(12) Sircar, J . C.; Capiris, T.; Kesten, S. J . J . Med. Chem. 1981, 24,
735.
(13) Oltmanns, O.; Lingens, F. Z. Naturforsch. 1967, 22, 751-754.
(14) Logvinenko, E. M.; Shavlovsky, G. M. Mikrobiologiya 1967, 41,
978-979.
(15) Roberts, J . C.; Gao, H.; Gopalsami, A.; Kongsjahju, A.; Patch,
R. J . Tetrahedron Lett. 1997, 38, 355-358.
(9) Cushman, M.; Mihalic, J . T.; Kis, K.; Bacher, A. J . Org. Chem.
1999, 64, 3838-3845.
(10) Meining, W.; Mo¨rtl, S.; Fischer, M.; Cushman, M.; Bacher, A.;
Ladenstein, R. J . Mol. Biol. 2000, 299, 181-197.
5808 J . Org. Chem., Vol. 67, No. 16, 2002

Derivatives of 7-Oxo-8-ribitylaminolumazines
SCHEME 3a
a
Reagents and conditions: (a) (1) ethyl 1,3-dithiane-2-carboxy-
late, n-BuLi, THF, -78 °C (30 min), (2) ethyl 5-bromovalerate,
-78 to 23 °C; (b) NBS, AgNO3, 2,6-lutidine, aq CH3CN, 0-23 °C
(30 min).
difficult for microorganisms to develop resistance to drugs
that target both enzymes.
Resu lts a n d Discu ssion
site by addition of water to substrate 3.3,16-20 The
riboflavin synthase inhibitors 1321-23 and 1418 were
designed and synthesized as analogues of the anion 11
formed at the acceptor site. The 7-oxolumazine derivative
15 was also synthesized as part of a program to prepare
affinity chromatography columns for riboflavin synthase
purification.21-23 Both 13 and 15 were effective inhibitors
of baker’s yeast riboflavin synthase.24 Although 15 (Ki
10 µM) was less potent than 13 (Ki 0.5 µM), the fact that
15 inhibited riboflavin synthase indicates a degree of
tolerance for larger substituents at C-6 on the lumazine
ring system. It is possible that lumazine derivatives
related to 15 might also function as lumazine synthase
inhibitors because the carboxylate anion could conceiv-
ably occupy the phosphonate binding site of 10 and the
presumed phosphate binding site of 5. To test this
hypothesis, the 7-oxolumazine 15 and some of its chain-
extended carboxyalkyl homologues have been synthe-
sized. Several analogues incorporating phosphates in-
stead of carboxylates at the end of the chain have also
been prepared and tested as inhibitors of both B. subtilis
lumazine synthase and Escherichia coli riboflavin syn-
thase. A long-range goal of this research has been to
obtain potential antibiotics that inhibit both lumazine
synthase and riboflavin synthase, since it would be more
An R-ketoester 19 required as an intermediate for the
synthesis of one of the desired 7-oxolumazines 23 was
prepared by modification of a procedure reported by
Whitman et al. (Scheme 3).25 Deprotonation of ethyl 1,3-
dithiane-2-carboxylate (17) with n-butyllithium in THF
afforded the expected lithium anion, which was alkylated
with ethyl 5-bromovalerate (16) to provide the product
18. Hydrolysis of the dithiane in aqueous acetonitrile in
the presence of silver nitrate, NBS, and 2,6-lutidine
afforded the desired compound 19.
Syntheses of three homologous 6-carboxyalkyl-7-oxo-
8-D-ribityllumazines 15, 22, and 23 as well as the lactone
25 are outlined in Scheme 4. Catalytic hydrogenation of
5-nitro-6-D-ribitylaminouracil (9)26 over palladium on
charcoal afforded the unstable intermediate 1. Since 1
is very easily oxidized when it is present as the free base,
resulting in complex mixtures of products, acetic acid and
2-ketoglutaric acid (20) were added directly to the
hydrogenation reaction mixture, and the subsequent
transformation was carried out at elevated temperature
to yield the product 15. Similarly, addition of ethanolic
HCl and 2-oxoadipic acid (21) to the hydrogenation
reaction mixture containing 1, followed by heating at
reflux, resulted in the expected oxolumazine derivative
22. In a comparable procedure, intermediate 1 was
reacted with diethyl 2-oxopimelate (19) to produce com-
pound 23. Reaction of 1 with R-ethoxalylbutyrolactone
(24)27 in hot acetonitrile in the presence of acetic acid
led to the oxolumazine 25, having a lactone substituent
in the 6-position.
(16) Paterson, T.; Wood, H. C. S. J . Chem. Soc., Chem. Commun.
1969, 290-291.
(17) Gould, K. L.; Nurse, P. Nature 1989, 342, 39-45.
(18) Cushman, M.; Patel, H. H.; Scheuring, J .; Bacher, A. J . Org.
Chem. 1992, 57, 5630-5643.
(19) Brand, S.; J ones, M. F.; Rayner, C. M. Tetrahedron Lett. 1995,
36, 8493-8496.
The synthesis of the 7-oxo-8-D-ribityllumazine analogue
33, having a phosphate connected to the lumazine ring
system by a three-carbon linker, is outlined in Scheme
5. Deprotonation of γ-butyrolactone (26) with sodium
(20) Scheuring, J .; Kugelbrey, K.; Weinkauf, S.; Cushman, M.;
Bacher, A.; Fischer, M. J . Org. Chem. 2001, 66, 3811-3819.
(21) Kulick, R. J .; Wood, H. S. C.; Wrigglesworth, R. J . Chem. Soc.,
Chem. Commun. 1975, 464-465.
(22) Ginger, C. D.; Wrigglesworth, R.; Inglis, W. D.; Kulick, R. J .;
Suckling, C. J .; Wood, H. S. C. J . Chem. Soc., Perkin Trans. 1 1984,
953-958.
(23) Wrigglesworth, R.; Inglis, W. D.; Livingstone, D. B.; Suckling,
C. J .; Wood, H. S. C. J . Chem. Soc., Perkin Trans. 1 1984, 959-963.
(24) Al-Hassan, S. S.; Kulick, R. J .; Livingston, D. B.; Suckling, C.
J .; Wood, H. C. S.; Wrigglesworth, R.; Ferone, R. J . Chem. Soc., Perkin
Trans. 1 1980, 2645-2656.
(25) Burks, E. A.; J ohnson, W. H., J r.; Whitman, C. P. J . Am. Chem.
Soc. 1998, 120, 7665-7675.
(26) Cresswell, R. M.; Wood, H. S. C. J . Chem. Soc. 1960, 4768-
4775.
(27) Moloney, G. P.; Martin, G. R.; Mathews, N.; Milne, A.; Hobbs,
H.; Dodsworth, S.; Sang, P. Y.; Knight, C.; Williams, M.; Maxwell, M.;
Glen, R. C. J . Med. Chem. 1999, 42, 2504-2526.
J . Org. Chem, Vol. 67, No. 16, 2002 5809

Cushman et al.
SCHEME 4a
SCHEME 5a
a
Reagents and conditions: (a) NaOEt, EtOH, diethyl oxalate,
0 °C (1 h); (b) HBr, AcOH, 120 °C (4 h); (c) MeOH, 23 °C (12 h);
(d) (MeO)3CH, H2SO4, 23 °C (12 h); (e) AgOPO(OBn)2, C6H5CH3,
reflux (12 h); (f) H2, Pd(OH)2/C, ethanol (5 h); (g) HCl, EtOH, reflux
(12 h).
a
Reagents and conditions: (a) H2, Pd/C, H2O (12 h); (b) AcOH,
90-100 °C (4 h); (c) HCl, EtOH, reflux (12 h); (d) AcOH, CH3CN,
80 °C (2 h).
outlined in Scheme 6. Deprotonation of ethyl 1,3-dithiane-
2-carboxylate (17) with n-butyllithium afforded the anion,
which was alkylated with 1,4-dibromobutane (34), result-
ing in intermediate 35. Displacement of the bromide with
dibenzylphosphate led to the protected phosphate deriva-
tive 36. Hydrolysis of the dithiane 36 resulted when it
was subjected to NBS, silver nitrate, and 2,6-lutidine in
aqueous acetone. Debenzylation of 37 was effected using
palladium hydroxide on carbon, and the resulting product
38 yielded the substituted lumazine 39 after reaction
with the lumazine synthase substrate 1.
ethoxide and reaction of the resulting anion with diethyl
oxalate (27) afforded R-ethoxalylbutyrolactone (24).28
Reaction of 24 with HBr in acetic acid yielded 5-bromo-
2-oxovaleric acid (28), which was converted to the cor-
responding ester, methyl 5-bromo-2-oxovalerate (29).
Treatment of intermediate 29 with trimethyl orthofor-
mate provided the ketal 30. Displacement of the bromide
30 with dibenzyl phosphate gave the expected substitu-
tion product 31. Deprotection of the phosphate by hy-
drogenolysis over palladium hydroxide on carbon afforded
the expected phosphate 32, which was reacted with the
lumazine synthase substrate 1 to provide the desired
lumazine analogue 33.
The 6-substituted 7-oxo-8-ribityllumazines 15, 22, 23,
25, 33, and 39 were evaluated as inhibitors of recombi-
nant B. subtilis lumazine synthase â60 capsids and
recombinant E. coli riboflavin synthase, and the results
are listed in Table 1. Several additional inhibitors which
were previously designed and synthesized to target both
the phosphate binding site and the ribitylpyrimidine
binding site of lumazine synthase are also listed in Table
1 for comparison purposes. These include the benzamides
40 and 41,29 the homologous phosphonates 10, 42, and
Since we were somewhat unsure about the optimal
distance between the lumazine ring system and the
phosphate for enzyme inhibitory activity, a synthesis of
the higher homologue 39 of 33 was carried out, as
(28) Snyder, H. R.; Brooks, L. A.; Shapiro, S. H. In Organic
Syntheses; Blatt, A. H., Ed.; J ohn Wiley and Sons: New York, 1943;
Collect. Vol. II, pp 531-538.
5810 J . Org. Chem., Vol. 67, No. 16, 2002

Derivatives of 7-Oxo-8-ribitylaminolumazines
SCHEME 6a
TABLE 1. In h ibition Con sta n ts vs Lu m a zin e Syn th a se
a n d Ribofla vin Syn th a se
compd
lumazine synthasea Ki (µM) riboflavin synthaseb Ki (µM)
10
180 ( 88 (Ki, mixed inhib)
>1000
350 ( 22 (Kis)c
15
94 ( 28 (mixed inhib)
180 ( 35 (Kis)c
650 ( 140 (comp inhibd)
87 ( 17 (Ks)
210 ( 22 (kcat)
30.1 ( 2.1 (kcat
29 ( 5.9 (Ks)
)
22
23
84 (uncomp inhib)
49 ( 6.7 (Ks)
20 ( 6.4 (comp inhib)
84 ( 8.5 (Kis)c
200 ( 69 (mixed inhib)
57 ( 9.5 (Ks)
56 ( 6.9 (comp inhib)
18 ( 2.4 (Kis)
192 ( 5.7 (kcat)
93.3 ( 6.4 (kcat
)
270 ( 75 (Kis)c
25
33
39
220 ( 93 (partial inhibition)
45 ( 8.9 (Ks)
170 ( 26 (comp inhib)
160 ( 45 (Ks)
290 ( 5 (kcat
78.7 ( 5.9 (kcat
)
)
230 ( 60 (Kis)c
120 (uncomp inhib)
>1600
100 ( 21 (Ks)
150 ( 20 (kcat
)
120 ( 24 (Kis)c
160 ( 32 (comp inhib)
27 ( 4.8 (Ks)
150 ( 46 (comp inhib)
130 ( 14 (kcat
)
73.1 ( 3.7 (kcat
)
40
41
42
200
360
440 ( 200 (Ki, mixed inhib)
>1000
>1000
640 ( 300 (Kis)c
43
130 ( 33 (Ki, mixed inhib)
140 ( 15 (Kis)c
44
45
290 ( 120 (Ki, comp inhib)
690 ( 290 (Ki, mixed inhib)
1500 ( 640 (Kis)c
>1000
>1000
46
47
290 ( 130 (Ki, mixed inhib)
580 ( 170 (Kis)
46 ( 5 (Ki, partial inhib)
0.61 ( 0.5 (Ki, comp inhib)
9.6 ( 0.9 (Ks)
39.9 ( 0.7 (kcat
250 ( 42 (Kis)c
)
a
b
Recombinant â60 capsids from B. subtilis. Recombinant
c
riboflavin synthase from E. coli. Kis is the equilibrium constant
for the reaction EI + S h EIS. Competitive inhibition.
d
a
Reagents and conditions: (a) (1) n-BuLi, ethyl 1,3-dithiane-
2-carboxylate, -78 to 23 °C, (2) 1,4-dibromobutane, 23 °C (3 h);
(b) AgOPO(OBn)2, C6H5CH3, reflux (5 h); (c) NBS, AgNO3, 2,6-
lutidine, aq acetone, 0-23 °C (30 min); (d) H2, Pd(OH)2, EtOH (5
h); (e) EtOH, reflux (12 h).
43,9 as well as the homologous sulfonates 44, 45, and 46,9
and the purinetrione 47.9,30
The most potent inhibitor of lumazine synthase in the
present series was the 6-(3′-carboxypropyl)lumazine de-
rivative 22 (Ki 84 µM), followed closely by the corre-
sponding 6-(2′-carboxyethyl)lumazine analogue 15 (Ki 94
µM). Both of these compounds were more potent than
the 6-(4′-carboxybutyl)lumazine homologue 23 (Ki 200
µM) and the lactone 25 (Ki 220 µM). Evidently, the
optimal distance between the carboxyl group and the
lumazine ring system is afforded by a three-carbon linker.
Turning to the phosphates 33 (Ki 120 µM) and 39 (Ki
160 µM), we can see that connection of the phosphate to
the lumazine ring system by a three-carbon linker results
in a more potent inhibitor of lumazine synthase than a
four-carbon connection. However, both of these phos-
phates were less potent than the carboxylates 15 and 22.
It should be noted that the phosphate 33 was also more
(29) Cushman, M.; Mavandadi, F.; Kugelbrey, K.; Bacher, A. J . Org.
Chem. 1997, 62, 8944-8947.
(30) Cushman, M.; Yang, D.; Kis, K.; Bacher, A. J . Org. Chem. 2001,
66, 8320-8327.
potent than the phosphonates 42 (Ki 440 µM), 43 (Ki 180
µM), and 10 (Ki 130 µM), as well as the sulfonates 44 (Ki
130 µM), 45 (Ki 690 µM), and 46 (Ki 290 µM), in which
J . Org. Chem, Vol. 67, No. 16, 2002 5811

Cushman et al.
F IGURE 1. Binding of 5-nitro-6-ribitylaminouracil to B. subtilis lumazine synthase. The figure was constructed using published
X-ray coordinates and is programmed for walleyed viewing.
F IGURE 2. Hypothetical model of the binding of compound 22 to B. subtilis lumazine synthase. The figure is programmed for
walleyed viewing.
the phosphonates or sulfonates are connected through a
linker chain to a pyrimidine ring system instead of a
lumazine ring system.
6 Å sphere surrounding the ligand to remain flexible with
the remainder of the protein structure frozen. The
resulting Figure 2 was constructed by displaying the
amino acid residues involved in hydrogen bonding of the
protein with ligand 22, using a maximum hydrogen
bonding distance of 2.8 Å. The calculated distances
between the hydrogen bond donors and acceptors in the
complex of 22 with the protein are provided in Figure 3
(top), along with the corresponding distances derived
from the X-ray structure of the complex of the protein
with the nitropyrimidine 9 (bottom). Figure 3 also shows
the distances for hydrogen bondings of inorganic phos-
phate with lumazine synthase, which were derived from
the X-ray structure of the complex of the protein with 9.
The calculated structure of 22 with lumazine synthase
indicates that it is likely that the terminal carboxylate
of the side chain occupies a site that is near to, but not
A 2.4 Å resolution X-ray structure is available of the
substrate analogue 9 complexed with B. subtilis lumazine
synthase (Figure 1).8 The related 1.85 Å resolution X-ray
structure of the phosphonate inhibitor 109 (Ki 180 µM vs
that of B. subtilis lumazine synthase) bound to Saccha-
romyces cerevisiae lumazine synthase has also been
published.10 These structures allow the construction of
a hypothetical model of the binding of 22 to lumazine
synthase (Figure 2), which was produced by overlapping
the structure of 22 with the structure of 9 in the active
site of B. subtilis lumazine synthase. The structure of 9
was then removed, and the energy of the complex was
minimized using the MMFF94 force field while allowing
the ligand and the protein structure contained within a
5812 J . Org. Chem., Vol. 67, No. 16, 2002

Derivatives of 7-Oxo-8-ribitylaminolumazines
therefore seems to be optimal for inhibition of both
riboflavin synthase and lumazine synthase. Interestingly,
the replacement of the carboxylate of 22 with a phosphate
resulted in compound 33, which was inactive against
riboflavin synthase (Ki > 1600 µM). However, lengthen-
ing the linker chain by one carbon atom resulted in the
riboflavin synthase inhibitor 39 (Ki 150 µM).
An X-ray structure of E. coli riboflavin synthase, which
contains three identical subunits, has been published
along with a hypothetical model of bound 6,7-dimethyl-
8-D-ribityllumazine (3).31 According to the model, as well
as NMR studies of the structure of the N-terminal
domain of riboflavin synthase,32 the active site of the
enzyme is derived from the intermolecular juxtaposition
of residues belonging to both of the barrels in the
C-terminal and N-terminal domains of two monomer
units, with one substrate molecule bound to each barrel.
A model was constructed for the binding of inhibitor 15
to E. coli riboflavin synthase by manually positioning its
structure, as suggested by the published model.31 The
initial orientations of the ligand molecules in the active
site were also consistent with the X-ray structure of
monomeric Schizosaccharomyces pombe riboflavin syn-
thase complexed with two molecules of 15.33 Energy
minimization then led to the structure of the enzyme-
inhibitor complex displayed at the top of Figure 4.
According to this hypothetical structure of the inhibitor-
enzyme complex, one molecule of 15 (magenta) binds to
the C-barrel (orange residues), while another molecule
of the inhibitor (white) binds to the N-barrel (blue-green
residues). The empty active site at the bottom of Figure
4 was constructed from the published X-ray coordinates.31
There are 18 water molecules included in or near the
cavity that constitutes the active site, and many of them
are involved in hydrogen bonding to the protein and the
ligand molecules in the occupied active site.
F IGURE 3. Top: hydrogen bonds and distances in the
calculated model of inhibitor 22 bound in the active site of B.
subtilis lumazine synthase. Bottom: the corresponding dia-
gram based on the X-ray structure of the complex of 9 with
the enzyme.
An antibiotic that would be active against both ribo-
flavin synthase and lumazine synthase would be less
likely to lead to resistant strains, since the pathogen
would have to select two different resistant mutant
enzymes simultaneously to become resistant. The present
series of lumazine derivatives features several com-
pounds that are active against both enzymes.
identical with, the site occupied by inorganic phosphate
in the X-ray structure of the complex of 9 with lumazine
synthase. Although Arg127 both is involved in binding
to inorganic phosphate and is calculated to bind to the
carboxylate of 22, the second residue calculated to be
involved in hydrogen bonding is different in each case.
With phosphate, the backbone nitrogen of Thr86 is
involved in bonding to phosphate, whereas, in the
calculated structure of 22, it is Lys135. Comparison of
the location of Lys135 in the X-ray structure (Figure 1)
with that of the hypothetical structure in Figure 2
suggests that the side chain of Lys135 may undergo a
conformational change in which the terminal nitrogen
moves toward Arg127, resulting in a binding pocket that
is ideally suited for binding to the carboxylate. In the
X-ray structure (Figure 1), the distance from the side
chain amino nitrogen of Lys135 to the central carbon
atom of the guanidine moiety of Arg127 is 9.7 Å, while,
in the model of the binding of 22, it is 6.1 Å.
Considering inhibition of riboflavin synthase, the data
presented in Table 1 show that the most potent riboflavin
synthase inhibitor in the present series is also the 6-(3′-
carboxypropyl)lumazine derivative 22 (Ki 20 µM). Either
shortening or lengthening the linker chain connecting the
carboxyl group to the lumazine ring resulted in less active
compounds (15, Ki 650 µM; 23, Ki 56 µM). A three-carbon
linker attaching the carboxyl group to the lumazine ring
Exp er im en ta l Section
Gen er a l. Melting points were determined in capillary tubes
and are uncorrected. The 1H NMR spectra were determined
at 300 MHz, and the 13C NMR spectra were recorded at 75.5
MHz. Microanalyses were performed at the Purdue Mi-
croanalysis Laboratory, and all values were within 0.4% of the
calculated compositions. Silica gel used for column chroma-
tography was 230-400 mesh.
6-(2-Car boxyleth yl)-7-oxo-8-D-r ibityllu m azin e (15). 5-Ni-
tro-6-(D-ribityl)aminouracil (9) (0.5 g, 1.6 mmol) was dissolved
in water (20 mL) and hydrogenated under H2 atmosphere for
12 h in the presence of Pd on activated carbon (50 mg, 10%).
Acetic acid (5 mL) and 2-ketoglutaric acid (20) (1.0 g, 6.8 mmol)
(31) Liao, D.-I.; Wawrzak, Z.; Calabrese, J . C.; Viitanen, P. V.;
J ordan, D. B. Structure 2001, 9, 399-408.
(32) Truffault, V.; Coles, M.; Diericks, T.; Abelmann, K.; Eberhardt,
S.; Lu¨ttgen, H.; Bacher, A.; Kessler, H. J . Mol. Biol. 2001, 309, 949-
960.
(33) Gerhardt, S.; Schott, A.-K.; Fischer, M.; Kairies, N.; Cushman,
M.; Illarionov, B.; Eisenreich, W.; Bacher, A.; Huber, R.; Steinbacher,
S. Unpublished results, 2002.
J . Org. Chem, Vol. 67, No. 16, 2002 5813

Cushman et al.
F IGURE 4. Top: hypothetical model of two molecules of compound 15 bound in the active site of E. coli riboflavin synthase.
Magenta: compound 15 bound to the N-barrel residues. Green: C-barrel residues. White: compound 15 bound to the C-barrel
residues. Orange: N-barrel residues. Red and green: water molecules. Yellow: hydrogen bonds. Both ligand molecules are viewed
approximately from within the planes of the lumazine rings, which are stacked. Bottom: X-ray structure of the empty active site
of E. coli riboflavin synthase.
were added, and the mixture was heated at 90-100 °C for 4
h. After cooling, the catalyst was removed by filtration and
the filtrate was concentrated under reduced pressure. The
residue was precipitated from EtOH to afford 15 (0.31 g, 50%)
as an amorphous solid: 1H NMR (300 MHz, D2O) δ 4.38 (m, 1
H), 4.25 (m, 1 H), 4.02 (m, 1 H), 3.72 (m, 1 H), 3.65 (m, 2 H),
3.58 (m, 1 H), 2.91 (t, J ) 6 Hz, 2 H), 2.70 (t, J ) 6 Hz, 2 H);
13C NMR δ 178.45, 163.23, 157.49, 151.85, 151.71, 145.02,
109.80, 73.67, 73.06, 70.16, 63.23, 46.29, 30.95, 28.41. Anal.
Calcd for C14H18N4O9‚1.0(H2O): C, 41.59; H, 4.99; N, 13.86.
Found: C, 41.64; H, 4.94; N, 13.75.
The ice bath was removed, and the mixture was stirred for 30
min at room temperature. The suspension was diluted with
diethyl ether (100 mL), washed with saturated NH4Cl, satu-
rated aqueous NaSO3, and saturated aqueous NaCl, dried, and
concentrated. The crude product was purified by silica gel
column chromatography and eluted with hexanes-ethyl ace-
tate (5:1-2:1) to provide 1925 as a colorless oil (0.35 g, 41%):
1H NMR (300 Hz, CDCl3) δ 4.43 (q, J ) 7.1 Hz, 2 H), 4.15 (q,
J ) 7.2 Hz, 2 H), 2.88 (m, 2 H), 2.34 (m, 2 H), 1.68 (quintet, J
) 3.4 Hz, 4 H), 1.38 (t, J ) 7.1 Hz, 3 H), 1.26 (t, J ) 7.2 Hz,
3 H).
E t h yl 2-(4-E t h oxyca r b on ylb u t yl)-1,3-d it h ia n e-2-ca r -
boxyla te (18). n-Butyllithium (6.5 mL, 1.6 M in hexane) was
added to a solution of ethyl 1,3-dithiane-2-carboxylate (17)
(1.92 g, 0.01 mol) in THF (20 mL) at -78 °C under N2
atmosphere. The reaction mixture was stirred for 30 min, and
then, ethyl 5-bromovalerate (16) (2.09 g, 0.01 mol) was added.
The mixture was warmed to room temperature and stirred for
a further 30 min. It was diluted with diethyl ether and washed
with 1 N HCl and H2O, dried, and concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography, eluted with hexanes-ethyl acetate (5:1-2:
1) to provide 1825 as a colorless oil (2.4 g, 75%): 1H NMR (300
Hz, CDCl3) δ 4.15 (q, J ) 7.1 Hz, 2 H), 4.05 (q, J ) 7.2 Hz, 2
H), 3.2 (m, 2 H), 2.55 (m, 2 H), 2.1-1.5 (m, 10 H), 1.26 (t, J )
7.1 Hz, 3 H), 1.18 (t, J ) 7.2 Hz, 3 H).
6-(3-Ca r boxylp r op yl)-7-oxo-8-(D-r ibityl)lu m a zin e (22).
5-Nitro-6-(D-ribityl)aminouracil (9) (0.5 g, 1.6 mmol) was
dissolved in water (15 mL) with a few drops of triethylamine
and hydrogenated under H2 atmosphere for 12 h in the
presence of Pd on activated carbon (100 mg, 10%). A solution
of 2-oxoadipic acid (21) (0.5 g, 2.1 mmol) in ethanol (10 mL)
and 2 N HCl (2 mL) was added, and the mixture was heated
at reflux for 12 h. After cooling, the catalyst was removed by
filtration and the filtrate was concentrated under reduced
pressure. The residue was dissolved in a solution of LiOH (0.1
g) in water (10 mL) and stirred at room temperature for 5 h.
After removal of the solvent, the residue was applied onto a
DEAE cellulose (anion-exchange) column and eluted with
water followed by 5% formic acid. The corresponding fractions
were combined and concentrated. The residue was precipitated
by AcOEt to afford 22 (300 mg, 47%) as an amorphous solid:
1H NMR (500 MHz, D2O) δ 4.30 (m, 1 H), 4.15 (m, 1 H), 3.96
(m, 1 H), 3.71 (m, 1 H), 3.60 (m, 2 H), 3.48 (m, 1 H), 2.60 (t, J
) 6 Hz, 2 H), 2.25 (t, J ) 6 Hz, 2 H), 1.80 (quintet, J ) 6 Hz,
2 H); ESIMS (negative ion mode) m/z 399 (M - H)-. Anal.
Dieth yl 2-Oxop im ela te (19). A solution of 18 (1.2 g, 3.7
mmol) in acetonitrile (3 mL) was added quickly, dropwise, to
a solution of N-bromosuccinimide (4.27 g, 0.024 mol), silver
nitrate (2.72 g, 0.016 mol), and 2,6-lutidine (0.86 g, 0.008 mol)
in acetonitrile-water (40:10) that was cooled in an ice bath.
5814 J . Org. Chem., Vol. 67, No. 16, 2002

Derivatives of 7-Oxo-8-ribitylaminolumazines
Calcd for C15H20N4O9‚0.6(AcOEt)‚0.4(HCO2H): C, 45.33; H,
5.47; N, 11.88. Found: C, 45.38; H, 5.40; N, 11.81.
) 7.0 Hz, 2 H), 2.16 (m, 2 H); 13C NMR (CDCl3) δ 192.70,
160.86, 52.89, 37.47, 32.25, 25.59.
6-(4-Ca r b oxylb u t yl)-7-oxo-8-(D-r ib it yl)lu m a zin e (23).
5-Nitro-6-(D-ribityl)aminouracil (9) (0.35 g, 1.1 mmol) was
dissolved in water (15 mL) with a few drops of triethylamine
and hydrogenated under H2 atmosphere for 12 h in the
presence of Pd on activated carbon (100 mg, 10%). A solution
of 19 (0.35 g, 1.5 mmol) in ethanol (10 mL) and 2 N HCl (2
mL) was added, and the mixture was heated at reflux for 12
h. After cooling, the catalyst was removed by filtration and
the filtrate was concentrated under reduced pressure. The
residue was dissolved in a solution of LiOH (0.1 g) in water
(10 mL) and stirred at room temperature for 5 h. After removal
of the solvent, the residue was applied onto an anion-exchange
resin column (Dowex 1 × 2-400) and eluted with water
followed by 5% formic acid. The corresponding fractions were
combined and concentrated. The residue was triturated with
a small amount of acetonitrile to afford 23 (350 mg, 77%) as
an amorphous solid: 1H NMR (300 Hz, D2O) δ 4.34 (m, 1 H),
4.23 (m, 1 H), 4.02 (m, 1 H), 3.75-3.65 (m, 3 H), 3.57 (m, 1
H), 2.62 (m, 2 H), 2.29 (m, 2 H), 1.52 (m, 4 H). Anal. Calcd for
Meth yl 5-Br om o-2,2-d im eth oxyp en ta n oa te (30). Methyl
5-bromo-2-oxopentanoate (29) (1.0 g, 4.8 mmol), trimethyl-
orthoformate (1.0 g, 9.6 mmol), and concentrated sulfuric acid
(2 drops) were stirred under nitrogen at room temperature for
12 h. Sodium bicarbonate (0.5 g) was added, and the mixture
was stirred for 0.5 h, filtered, and concentrated to give 3035
as a colorless oil (1.0 g, 81%): 1H NMR (300 MHz, CDCl3) δ
3.75 (s, 3 H), 3.30 (t, J ) 6.4 Hz, 2 H), 3.22 (s, 6 H), 1.98 (m,
2 H), 1.75 (m, 2 H); 13C NMR (100 MHz, CDCl3) δ 169.17,
102.00, 51.06, 49.68, 32.84, 32.06, 26.47.
Met h yl 5-Dib en zylp h osp h on oxy-2,2-d im et h oxyp en -
ta n oa te (31). A mixture of 30 (1.0 g, 3.9 mmol) and silver
dibenzylphosphate (1.5 g, 3.9 mmol) in toluene (20 mL) was
heated to reflux for 12 h. After cooling, the precipitate was
removed by filtration and the filtrate was concentrated. The
residue was purified by silica gel (50 g) column chromatogra-
phy, eluted with hexane-ethyl acetate (1:1-1:2), to give a
colorless oil 31 (0.6 g, 34%): 1H NMR (300 MHz, CDCl3) δ 7.37
(m, 10 H), 5.08-4.97 (m, 4 H), 3.99 (q, J ) 6.4 Hz, 2 H), 3.77
(s, 3 H), 3.22 (s, 6 H), 1.92 (m, 2 H), 1.60 (m, 2 H). Anal. Calcd
for C22H29O8P: C, 58.40; H, 6.46. Found: C, 58.23; H, 6.43.
C
16H22N4O9‚0.1(H2O)‚0.5(HCO2H): C, 45.12; H, 5.32; N, 12.76.
Found: C, 45.10; H, 5.25; N, 12.86.
r-Eth oxa lylbu tyr ola cton e (24).27 Sodium (3.7 g, 0.16 mol)
was cautiously added to absolute ethanol (80 mL) cooled by
an ice bath. Diethyl oxalate (22.0 g, 0.15 mol) was added to
the resultant solution of sodium ethoxide. Then, a solution of
butyrolactone (13.0 g, 0.15 mol) in ethanol (20 mL) was
dropwise added over 30 min, and the reaction mixture was
stirred at ice temperature for 1 h. Upon removal of the cooling
bath, the mixture was further stirred overnight at room
temperature. The solvent was removed under reduced pres-
sure, and the pasty residue was partitioned between ether and
water. The aqueous layer was acidified with 2 N HCl,
saturated with NaCl, and extracted with dichloromethane (4
× 100 mL). The extracts were combined, dried, and concen-
trated to yield the desired compound 2434 (27 g, 96%) as a
colorless oil: 1H NMR (300 Hz, CDCl3) δ 10.8 (br s, 1 H, OH),
4.43 (t, J ) 7.6 Hz, 2 H), 4.29 (q, J ) 7.1 Hz, 2 H), 3.21 (t, J
) 7.6 Hz, 2 H), 1.31 (t, 7.1 Hz, 3 H).
6-(γ-Bu tyr olacton -5-yl)-7-oxo-8-(D-r ibityl)lu m azin e (25).
5-Nitro-6-(D-ribityl)aminouracil (9) (0.5 g, 1.6 mmol) was
dissolved in water (20 mL) and hydrogenated under H2
atmosphere for 12 h in the presence of Pd on activated carbon
(100 mg, 10%). Acetic acid (1 mL) and R-ethoxalylbutyrolactone
(24)27 (1.0 g, 5.4 mmol) in CH3CN (10 mL) were added, and
the mixture was heated at 80 °C for 2 h. After cooling, the
catalyst was removed by filtration and the filtrate was
concentrated under reduced pressure. The residue was crystal-
lized from EtOH to afford 25 (0.4 g, 62%): 1H NMR (300 MHz,
D2O) δ 4.55 (m, 1 H), 4.40 (m, 1 H), 4.30 (m, 2 H), 4.15 (m, 1
H), 4.02 (m, 1 H), 3.75 (m, 1 H), 3.65 (m, 2 H), 3.56 (m, 1 H),
2.52 (m, 2 H); MS ESIMS (negative ion mode) m/z 397 (M -
H)-. Anal. Calcd for C15H18N4O9‚1.2(H2O): C, 42.90; H, 4.90;
N, 13.34. Found: C, 42.74; H, 4.99; N, 13.58.
Meth yl 5-P h osp h on oxy-2,2-d im eth oxyp en ta n oa te (32).
A mixture of compound 31 (0.6 g, 1.3 mmol) and Pd(OH)2 on
activated carbon (50 mg, 20%) in ethanol (20 mL) was
hydrogenated under H2 atmosphere for 5 h. The catalyst was
removed by filtration, and the filtrate was concentrated under
reduced pressure to give 32 as a colorless oil (0.35 g, 97%),
which was used without further purification. 1H NMR (300
MHz, CD3OD) 3.97 (q, J ) 6.4 Hz, 2 H), 3.75 (s, 3 H), 3.22 (s,
6 H), 2.00 (m, 2 H), 1.57 (m, 2 H); 13C NMR (100 MHz, CD3-
OD) δ 171.17, 103.49, 67.03 (d), 52.94, 50.08, 30.80, 25.57 (d).
7-O x o -6-(3-p h o s p h o n o x y p r o p y l)-8-(D -r i b i t y l)lu -
m a zin e (33). A mixture of 5-nitro-6-(D-ribityl)aminouracil (9)
(0.35 g, 1.1 mmol) and Pd on activated carbon (50 mg, 10%) in
water (15 mL) was hydrogenated under H2 atmosphere for 12
h. A solution of 32 (0.35 g, 1.3 mmol) in ethanol (15 mL) and
2 N HCl (2 mL) was added, and the mixture was heated at
reflux for 12 h. After cooling, the catalyst was removed by
filtration and the filtrate was concentrated under reduced
pressure. The residue was dissolved in ethanol, and the
insoluble material was filtered off. The ethanolic filtrate was
concentrated, and the residue was applied onto an anion-
exchange resin column (Dowex 1 × 2-400) and eluted with
water followed by 5% formic acid. The corresponding fractions
were combined and concentrated. The residue was precipitated
by i-PrOH to afford 33 (310 mg, 62%) as an amorphous solid:
1
ESIMS (negative ion mode) m/z 451 (M - H)-; H NMR (300
MHz, D2O) δ 4.37 (m, 1 H), 4.23 (m, 1 H), 4.04 (m, 1 H), 3.87
(m, 2 H), 3.8-3.6 (m, 3 H), 3.55 (m, 1 H), 2.74 (t, J ) 6 Hz,
2 H), 1.93 (m, 2 H). Anal. Calcd for C14H21N4O11P‚1(H2O)‚0.1-
(i-PrOH): C, 36.06; H, 5.04; N, 11.67. Found: C, 36.10; H, 4.95;
N, 11.57.
Eth yl 2-(4-Br om obu tyl)-1,3-dith ian e-2-car boxylate (35).
n-Butyllithium (6.5 mL, 1.6 M in hexane) was added to a
solution of ethyl 1,3-dithiane-2-carboxylate (17) (1.92 g, 0.01
mol) in THF (20 mL) at -78 °C under N2 atmosphere. The
solution was warmed to room temperature and added to a
solution of 1,4-dibromobutane (4.3 g, 0.02 mol) in THF (30 mL).
The reaction mixture was stirred for 3 h at room temperature.
It was diluted with diethyl ether, washed with 1 N HCl and
H2O, dried, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography,
eluted with hexanes-ethyl acetate (5:1) to provide 35 as a
colorless oil (2.5 g, 76%): 1H NMR (300 Hz, CDCl3) δ 4.25 (q,
J ) 7.1 Hz, 2 H), 3.41 (t, J ) 6.5 Hz, 2 H), 3.3 (m, 2 H), 2.65
(m, 2 H), 2.2 (m, 1 H), 1.92 (m, 2 H), 1.85 (m, 2 H), 1.66 (m, 2
H), 1.38 (t, J ) 7.1 Hz, 3 H), 0.92 (m, 1 H).
Meth yl 5-Br om o-2-oxop en ta n oa te (29). R-Ethoxalylbu-
tyrolactone (24) (9.0 g, 48 mol) was dissolved in a solution of
30% HBr in acetic acid (30 mL). The reaction mixture was
heated at 120 °C for 1 h. Then additional HBr-AcOH solution
(20 mL) was added, and the mixture was stirred at 120 °C for
3 h. The solution was concentrated, and residue 2827 was taken
into methanol (50 mL) and stirred overnight at room temper-
ature. The solvent was evaporated, and the residue was
dissolved in Et2O (100 mL), washed with water, and dried over
Na2SO4. After removal of the solvent, the residue was purified
by silica gel column chromatography (ethyl acetate-hexane,
2:1) to afford 2935 as a colorless oil (6.5 g, 48%): 1H NMR (300
Hz, CDCl3) δ 3.80 (s, 3 H), 3.44 (t, J ) 6.3 Hz, 2 H), 3.05 (t, J
(34) Knighton, D. R.; Cadena, D. L.; Zheng, J .; Eyck, L. F. T.; Taylor,
S. S.; Sowadski, J . M.; Gill, G. N. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 5001-5005.
Eth yl 2-(4-Diben zylp h osp h on oxybu tyl)-1,3-d ith ia n e-2-
ca r boxyla te (36). A mixture of 35 (1.5 g, 4.6 mmol) and silver
dibenzylphosphate (1.76 g, 4.6 mmol) in toluene (50 mL) was
(35) Yates, P.; Schwartz, D. A. Can. J . Chem. 1983, 61, 509-518.
J . Org. Chem, Vol. 67, No. 16, 2002 5815

Cushman et al.
heated at reflux for 5 h under N2. After cooling, the precipitate
was removed by filtration and the filtrate was concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography and eluted with hexanes-ethyl ace-
tate (2:1) to provide 36 as a colorless oil (1.5 g, 62%): 1H NMR
(300 Hz, CDCl3) δ 7.35 (m, 10 H), 5.05-5.00 (m, 4 H), 4.26 (q,
J ) 7.1 Hz, 2 H), 4.00 (q, J ) 6.4 Hz, 2 H), 3.3 (m, 2 H), 2.65
(m, 2 H), 2.2 (m, 1 H), 1.92 (m, 2 H), 1.85 (m, 2 H), 1.60 (m, 2
H), 1.38 (t, J ) 7.1 Hz, 3 H), 0.92 (m, 1 H).
Eth yl 5-(Diben zylp h osp h on o)-2-oxoh exa n oa te (37). A
solution of 36 (2.1 g, 0.004 mol) in acetonitrile (10 mL) was
added quickly, dropwise, to a solution of N-bromosuccinimide
(4.27 g, 0.024 mol), silver nitrate (2.72 g, 0.016 mol), and 2,6-
lutidine (0.86 g, 0.008 mol) in acetonitrile-water (40:10) that
was cooled in an ice bath. The ice bath was removed, and the
mixture was stirred for 30 min at room temperature. The
suspension was diluted with diethyl ether (100 mL), washed
with saturated NH4Cl, saturated aqueous NaSO3, and satu-
rated aqueous NaCl, dried, and concentrated to give 37 (1.7
g, 100%) as an oil: 1H NMR (300 Hz, CDCl3) δ 7.57 (m, 10 H),
5.17-4.86 (m, 4 H), 4.24 (q, J ) 7.1 Hz, 2 H), 3.33 (m, 2 H),
1.8-1.4 (m, 6 H), 1.26 (t, J ) 7.1 Hz, 3 H).
of 0.05 kcal/mol while employing the MMFF94 force field.
During the minimization of the complex, inhibitor 22 and a 6
Å sphere surrounding it were allowed to remain flexible while
the remaining portion of the complex was held ridged using
the aggregate function. Figure 1 was constructed by displaying
the amino acid residues of the enzyme that are involved in
hydrogen bonding with inhibitor 22. The maximum distance
between donor and acceptor atoms contributing to the hydro-
gen bonds shown in Figure 2 was set to 2.8 Å.
Molecu la r Mod elin g on Ribofla vin Syn th a se. Using
Sybyl (Tripos, Inc., version 6.5, 1998), the X-ray crystal
structure of E. coli riboflavin synthase (1I8D) was downloaded
and two molecules of the ligand 15 were docked and oriented
as suggested by the published model of the binding of two
molecules of substrate 3 in the active site.31 The C- and
N-terminal groups were changed to neutral carboxylic acid and
amino groups, and hydrogens were added to the protein
structure and to the oxygens of the water molecules. MMFF94
charges were loaded, and a 6 Å spherical subset including and
surrounding the two ligand molecules was then energy mini-
mized using the Powell method to a termination gradient of
0.05 kcal/mol while employing the MMFF94 force field. The
remaining portion of the protein was held ridged using the
aggregate function during energy minimization. Figure 4 was
constructed by displaying the amino acid residues in the C-
and N-barrels surrounding the two ligand molecules. The
maximum distance between donor and acceptor atoms con-
tributing to the hydrogen bonds shown in Figure 4 was set to
2.8 Å.
Eth yl 2-Oxo-6-p h osp h on oxyh exa n oa te (38). A mixture
of compound 37 (0.9 g, 2.1 mmol) and Pd(OH)2 on activated
carbon (20 mg, 20%) in ethanol (20 mL) was hydrogenated
under H2 atmosphere for 5 h. The catalyst was removed by
filtration, and the filtrate was concentrated under reduced
pressure to give a colorless oil (0.55 g, 99%), which was used
without further purification.
Lu m a zin e Syn th a se Assa y.36 Reaction mixtures contained
100 mM potassium phosphate, pH 7.0, 5 mM EDTA, 5 mM
dithiothreitol, inhibitor (0-86 µM), 170 µM 5-amino-6-ribi-
tylamino-2,4(1H,3H)-pyrimidinedione (1), and lumazine syn-
thase (30 µg, specific activity 12.5 µmol mg-1 h-1) in a total
volume of 560 µL. The solution was incubated at 37 °C, and
the reaction was started by the addition of a small volume (20
µL) of L-3,4-dihydroxy-2-butanone 4-phosphate to a final
concentration of 50-310 µM. The velocity-substrate data were
fitted for all inhibitor concentrations with a nonlinear regres-
sion method.37 Different inhibition models were considered for
the calculation. Ki values ( standard deviations were obtained
from the fit under consideration of the most likely inhibition
model.
7-Oxo-6-(4-p h osp h on oxyb u t yl)-8-(D-r ib it yl)lu m a zin e
(39). A mixture of 5-nitro-6-D-ribitylaminouracil (9) (0.5 g, 1.6
mmol) and Pd on activated carbon (50 mg, 10%) in water (20
mL) was hydrogenated under H2 atmosphere for 12 h. A
solution of 38 (0.55 g, 2.1 mmol) in ethanol (15 mL) was added,
and the mixture was heated at reflux for 12 h. After cooling,
the catalyst was removed by filtration and the filtrate was
concentrated under reduced pressure. The residue was treated
with ethanol, and the precipitate was removed by filtration.
The ethanolic filtrate was concentrated, and the residue was
applied onto an anion-exchange resin column (Dowex 1 ×
2-400) and eluted with water followed by 5% formic acid. The
corresponding fractions were combined and concentrated. The
residue was precipitated by AcOEt to afford 39 (300 mg, 36%)
as an amorphous solid: ESIMS (negative ion mode) m/z 465
Ribofla vin Syn th a se Assa y.38 Reaction mixtures con-
tained buffer (100 mM potassium phosphate, 10 mM EDTA,
10 mM sodium sulfite), inhibitor (0-87 µM), and riboflavin
synthase (10 µg, specific activity 50 µmol mg-1 h-1). After
preincubation, the reactions were started by the addition of
various amounts of 6,7-dimethyl-8-ribityllumazine (3) (20-200
µM) to a total volume of 570 µL. The formation of riboflavin
(4) was measured online with a computer controlled photom-
eter at 470 nm (ꢀRiboflavin ) 9100 M-1 cm-1). The Ki evaluation
was performed in the same manner as described above.
1
(M - H)-; H NMR (500 MHz, D2O) δ 4.28 (m, 1 H), 4.16 (m,
1 H), 3.96 (m, 1 H), 3.78 (m, 2 H), 3.71 (m, 1 H), 3.64 (m, 2 H),
3.50 (m, 1 H), 2.61 (t, J ) 6 Hz, 2 H), 1.55 (m, 4 H); 13C NMR
(100 MHz, D2O) δ 162.83, 157.40, 154.21, 151.02, 143.97,
109.50, 73.64, 73.13, 70.07, 67.29 (d), 63.30, 46.54, 33.15, 30.15
(d), 23.23. Anal. Calcd for
(HCO2H): C, 38.89; H, 5.16; N, 10.86. Found: C, 38.77; H,
5.11; N, 10.96.
C15H23N4O11P‚0.3(AcOEt)‚0.5-
Molecu la r Mod elin g on Lu m a zin e Syn th a se. Using
Sybyl (Tripos, Inc., version 6.5, 1998), the X-ray crystal
structure of the complex of 5-nitro-6-ribitylamino-2,4-(1H,3H)-
pyrimidinedione (9) and the lumazine synthase of B. subtilis
(1RVV)8 was clipped to include information within a 15 Å
sphere of one of the 60 equivalent ligand molecules. The
residues that were clipped in this cut complex were capped
with either neutral amino or carboxyl groups. The structure
of inhibitor 22 was overlapped with the structure of 5-nitro-
6-ribitylamino-2,4-(1H,3H)pyrimidinedione (9), which was then
deleted. Hydrogen atoms were added to the complex. MMFF94
charges were loaded, and the energy of the complex was
minimized using the Powell method to a termination gradient
Ack n ow led gm en t. This research was made possible
by NIH Grant GM51469 as well as by support from the
Deutsche Forschungsgemeinschaft and Fonds der Che-
mischen Industrie.
J O0201631
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