PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Org Chem. Author manuscript; available in PMC 2010 August 7.
Published in final edited form as:
PMCID: PMC2760403
NIHMSID: NIHMS132543

Discovery and Development of a Small Molecule Library with Lumazine Synthase Inhibitory Activity

Abstract

(E)-5-Nitro-6-(2-hydroxystyryl)pyrimidine-2,4(1H,3H)-dione (9) was identified as a novel inhibitor of Schizosaccharomyces pombe lumazine synthase by high-throughput screening of a 100,000 compound library. The Ki of 9 vs. Mycobacterium tuberculosis lumazine synthase was 95 μM. Compound 9 is a structural analog of the lumazine synthase substrate, 5-amino-6-(D-ribitylamino)-2,4-(1H,3H)pyrimidinedione (1). This indicates that the ribitylamino side chain of the substrate is not essential for binding to the enzyme. Optimization of the enzyme inhibitory activity through systematic structure modification of the lead compound 9 led to (E)-5-nitro-6-(4-nitrostyryl)pyrimidine-2,4(1H,3H)-dione (26), which has a Ki of 3.7 μM vs. M. tuberculosis lumazine synthase.

Introduction

Riboflavin (4, vitamin B2) plays a crucial role in many biological processes, including photosynthesis and mitochondrial electron transport. While animals obtain riboflavin from dietary sources, numerous microorganisms, including Gram-negative pathogenic bacteria and yeasts, lack an efficient riboflavin uptake system and are therefore absolutely dependent on endogenous riboflavin biosynthesis.1-4 Riboflavin biosynthesis therefore offers attractive targets for the design and synthesis of new antibiotics, which are urgently needed because pathogens are becoming drug-resistant at an alarming rate.

Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin (4) (Scheme 1). Lumazine synthase catalyzes the condensation of 3,4-dihydroxy-2-butanone 4-phosphate (2) with 5-amino-6-ribitylamino-2,4(1H,3H)pyrimidinedione (1), yielding 6,7-dimethyl-8-D-ribityllumazine (3).5,6 The final step in the biosynthesis involves a mechanistically unusual dismutation of two molecules of 3, resulting in the formation of one molecule of riboflavin (4) and one molecule of the pyrimidinedione derivative 1, which can then be recycled by lumazine synthase.7-15

Although the details of the reaction catalyzed by lumazine synthase have not been completely elucidated, a reasonable pathway can be outlined at the present time as depicted in Scheme 2. Condensation of the primary amino group of the substituted pyrimidinedione 1 with the ketone 2 to give Schiff base 5, elimination of phosphate to yield the enol 6, tautomerization of the enol 6 and isomerization of the imine to produce the ketone 7, ring closure, and dehydration of the covalent hydrate 8 provide the product 3.16 It can be assumed that the inorganic phosphate formed after elimination from 5 would remain enzyme bound, at least for some time, but that it would eventually have to be removed to make room for another molecule of the substrate 2. The present uncertainties revolve around the timing of phosphate elimination and the conformational reorganization of the side chain leading to intermediate 7.

A high-throughput screening (HTS) technique was developed based on competitive binding of lumazine synthase inhibitors and riboflavin to the active site of Schizosaccharomyces pombe lumazine synthase.17,18 The capacity of the S. pombe lumazine synthase to bind riboflavin is unique. Free riboflavin is fluorescent with high quantum yield while enzyme-bound riboflavin is not.19 Thus, displacement of riboflavin from the binding pocket results in a significant fluorescence increase of the system. The change in fluorescence caused by competitive binding between riboflavin and other ligands for S. pombe lumazine synthase was used to identify lumazine synthase inhibitors.17 All of the known inhibitors tested were positively identified, which confirmed the authenticity of this assay. HTS of a commercial 100,000 compound library yielded some interesting results, including the identification of a lead compound 9.

The thermodynamic HTS assay described above effectively bypasses the problems associated with the instabilities of the lumazine synthase substrates 1 and 2, and it also simplifies the assay by removing the time element. However, practically speaking, it had two limitations: 1) assay mixtures contained traces of free riboflavin that produced a fluorescent background; and 2) lumazine synthase inhibitors that bind outside the active site would probably be unable to release free riboflavin. In spite of these problems, the thermodynamic assay remained the method of choice for screening thousands of compounds, and the activities of the hit compounds were confirmed in secondary assays involving classical enzyme kinetics.

Our specific interest in compound 9 is the result of its structural similarity to the substrate 1. The identification of 9 as a lumazine synthase inhibitor represents an extension of ongoing work on the synthesis of metabolically stable intermediates in the lumazine synthase-catalyzed reaction.20-23 The HTS hit compound 9 demonstrates that the ribitylamino chain present in the substrate, which was thought to be necessary for binding to the enzyme, can be replaced by a simple hydroxystyryl moiety with retention of lumazine synthase inhibitory activity. The difference in physical properties between compounds 1 and 9 has significant implication for antibiotic drug development. Substrate analogs with ribityl side chains are not suitable as drug candidates because their hydrophilic nature can be expected to prevent them from penetrating bacterial cell walls. Gram negative bacterial cell walls contains an outer membrane composed of phospholipids and lipopolysaccharides that face toward external environment. Replacing the hydrophilic ribityl side chain of compound 1 with the styryl moiety renders compound 9 more lipophilic, which should facilitate the entry of the compound into bacteria. Compound 9 displayed Ki values of 210 μM vs. S. pombe lumazine synthase and 95 μM vs. Mycobacterium tuberculosis lumazine synthase. Thus, there is ample room for designing more potent lumazine synthase inhibitors based on the structure of the lead compound 9. Moreover, the lead compound 9 is a substrate analog of the lumazine synthase–catalyzed reaction, but it is also a product analog of the riboflavin synthase-catalyzed reaction. Structural analogs of 9 might therefore be expected to inhibit riboflavin synthase as well as lumazine synthase. Enzyme assays were therefore performed using the riboflavin synthases of M. tuberculosis and E. coli. In order to help define the spectrum of activity, lumazine synthase inhibition assays were also performed on M. tuberculosis lumazine synthase as well as S. pombe lumazine synthase.

An external file that holds a picture, illustration, etc.
Object name is nihms132543u1.jpg

Results and Discussion

The lumazine synthase inhibitory activity of the HTS hit compound 9 might be related to the presence of the phenolic hydroxy on the aromatic ring, which could possibly mimic one of the ribityl hydroxyl groups of the substrate 1. In addition, it is known that the substrate analog 10, which also contains a nitro group, has high affinity for the active site of lumazine synthase and has been crystallized in complex with Bacillus subtilis lumazine synthase.24,25 Molecular modeling was performed in order to investigate the binding mode of the hit compound 9 to the enzyme. The lead compound 9 was docked into the active site of the structure of Mycobacterium tuberculosis lumazine synthase26,27 using GOLD software (BST, version 3.0, 2005), and energy minimization was then performed with Sybyl 7.1. The resulting structure is displayed in Figure 1. According to this hypothetical model, the binding of the hit compound 9 in the active site of M. tuberculosis lumazine synthase is similar to that of substrate and product analogs.26-28 The structure involves hydrogen bonding of the phenolic hydroxyl group of 9 with Ala59, Ile60, and Glu61. In addition, stacking interactions involving Trp27 stabilize the binding. The molecular modeling results indicate that the binding of hit compound 9 in the active site of M. tuberculosis lumazine synthase is certainly plausible, and it most likely resembles the known crystal structure of the complex formed between the related substrate analog 10 and the enzyme.

Figure 1
Hypothetical model of the complex of the lead compound 9 with M. tuberculosis lumazine synthase. The distances shown are in Å. The diagram is programmed for wall-eyed (relaxed) viewing.

We have attempted to employ versatile methods that would allow the efficient synthesis of a wide variety of target molecules. Accordingly, an array of aromatic aldehydes were condensed with 6-methyl-5-nitrouracil to afford a focused library of alkenes with pure trans geometry.29-31 No attempt was made to synthesize cis alkenes because the preliminary ligand docking studies indicated unfavorable interactions in the active site. As outlined in Scheme 3, nitration of 6-methyluracil (11) in the presence of H2SO4 and fuming nitric acid furnished 6-methyl-5-nitrouracil (12).32 Condensation of compound 12 with aromatic aldehydes in the presence of piperidine provided the piperidine salts of 5-nitro-6-styryluracil derivatives 13. The piperidine salts were neutralized by the addition of excess hydrochloric acid to give pure 5-nitro-6-styryluracil derivatives. The presence of the nitro group at the 5-position activates the methyl group for the reaction with piperidine acting as the basic catalyst.

The protocol did indeed seem to be versatile until the condensation was attempted with 2-nitrobenzaldehyde, 4-nitrobenzaldehyde, and 2,3-dihydroxybenzaldehyde. In those cases, the reaction did not produce the desired products, which is consistent with earlier literature reports.33 The protocol was therefore modified (Scheme 4) to provide the required 2-nitro derivative 14 and the other desired condensation products. Instead of using piperidine as the solvent, which initially resulted in the piperidine salt of the condensation product, the reaction was performed in 1-butanol, a high-boiling alcohol, in the presence of one equivalent of piperidine.

Two classes of compounds were synthesized as shown in structures 9, 15-18 (Class 1) and 19-24 (Class 2). The enzyme inhibitory activities of these compounds were determined using lumazine synthases from M. tuberculosis and S. pombe, and riboflavin synthases from Escherichia coli and M. tuberculosis. The results are listed in Table 1.

TABLE 1
Inhibition Constants vs. S. pombe Lumazine Synthase, M. tuberculosis Lumazine Synthase, M. tuberculosis Riboflavin Synthase, E. coli Riboflavin Synthase.a

The hit compound 9 exhibits a Ki of 210 μM vs. S. pombe lumazine synthase and a Ki of 95 μM vs. M. tuberculosis lumazine synthase. To determine the effect of the position of the hydroxy group in the phenyl ring on the enzyme inhibitory activity, Class I compounds were synthesized. Compound 15, with a 3-hydroxy group, and compound 16, with a 4-hydroxy group, showed marked increases in inhibitory activity, with the 3-hydroxy compound 15 exhibiting a Ki of 7.1 μM and 4-hydroxy compounds 16 exhibiting a Ki 12 μM vs. M. tuberculosis lumazine synthase. Both compounds 15 and 16 displayed remarkable selectivity for inhibition of M. tuberculosis lumazine synthase, being completely ineffective against S. pombe lumazine synthase and E. coli riboflavin synthase. Thus, changing the position of the phenol improved both the inhibitory activity vs. M. tuberculosis lumazine synthase as well as the selectivity.

The presence of two hydroxyl groups on compounds 17 and 18 changed the inhibitory activity profile drastically. Both of these compounds were found to be potent against M. tuberculosis lumazine synthase, M. tuberculosis riboflavin synthase, S. pombe lumazine synthase, and E. coli riboflavin synthase, with compound 17 being more potent than compound 18. Compound 17 exhibited a broader spectrum enzyme-inhibitory profile than the hit compound 9, with a Ki of 12 μM vs. M. tuberculosis lumazine synthase, a Ki of 19 μM vs. M. tuberculosis riboflavin synthase, a Ki of 4.9 μM vs. S. pombe lumazine synthase, and Ki of 10 μM vs. E. coli riboflavin synthase.

The hypothetical structural model of hit compound 9 in M. tuberculosis lumazine synthase (Figure 1) indicates that the hydroxy group may hydrogen bond with the Ala59, Ile60 and Glu61 amino acid residues. Class II compounds were synthesized in order to determine the importance of hydrogen bond donor and acceptor properties. Class II compounds with OCH3 groups displayed slightly better potencies than their counterparts with OH groups. Compound 21 exhibited a Ki of 9.6 μM vs. M. tuberculosis lumazine synthase, whereas compound 17 showed a Ki of 12 μM vs. M. tuberculosis lumazine synthase. Similarly, compound 19 exhibited Ki of 28 μM vs. M. tuberculosis lumazine synthase, whereas hit compound 9 displayed a Ki of 95 μM vs. M. tuberculosis lumazine synthase.

In Class III compounds, the hydroxyl groups have been replaced with different functional groups that can also participate in hydrogen bonding. Compounds with a NO2 group showed a marked increase in inhibitory activity as compared to the lead compound 9. Compound 14 exhibited a Ki of 16 μM vs. M. tuberculosis lumazine synthase, whereas the hit compound 9 had a Ki of 95 μM vs. M. tuberculosis lumazine synthase. The 4-NO2 derivative 26 displayed the best inhibitory activity, with a Ki of 3.7 μM vs. M. tuberculosis lumazine synthase. Among the compounds with different halogens, the 2-fluoro derivative 28 had the best activity with Ki of 7.8 μM vs. M. tuberculosis lumazine synthase. Thus, improved inhibitors of M. tuberculosis lumazine synthase were obtained by changing the OH group in the hit compound 9 to a NO2 group or to F.

Classes I-III yielded two compounds 26 and 15 with optimized inhibitory activity. Based on these results, class IV compounds were designed with two different hydrogen bonding groups in the benzene ring. This may result in the phenyl substituent more closely mimicking the ribityl chain of the substrate, with more opportunities for hydrogen bonding to the protein. These inhibitors were prepared following the standard protocol, with 1-butanol as the solvent and one equivalent of piperidine as the base.

The enzyme inhibitory activities of the Class IV compounds were disappointing. In fact, they had less inhibitory activity than their counterparts with single functional groups. Compound 35 showed the best inhibitory profile, with a Ki of 12 μM against M. tuberculosis lumazine synthase. These discouraging results prompted a reevaluation of the hypothesis that the functional groups on the aromatic ring hydrogen bond with the amino acid residues in the active site of M. tuberculosis lumazine synthase. This led to the synthesis of compounds 39 and 43 in the series of Class V compounds, which contain unsubstituted aromatic rings. Compound 39 was expected to be a very weak inhibitor, but to the contrary, it was found to have good inhibitory activity, with a Ki of 15 μM vs. M. tuberculosis lumazine synthase, which is much better than the hit compound 9 (Ki 95 μM vs. M. tuberculosis lumazine synthase). Compound 43, with a naphthyl substituent, exhibited even better inhibitory activity, with a Ki of 11 μM vs. M. tuberculosis lumazine synthase.

In Class VI compounds, the alkene linker is replaced by more flexible ethylene and aminomethylene connectors. The increase in conformational freedom should confer greater similarity with the substrate 1.

Compounds 45 and 46 were synthesized (Scheme 5) by treating 6-chloro-5-nitrouracil (52) with benzylamines 53 and 54 in presence of triethylamine. The free bases were liberated from their triethylamine salts by dissolving them in aq KOH and neutralizing with dilute HCl solution.

Compounds 47 and 48 were prepared by selective reduction of the trans alkene linkers present in 9 and 16. The presence of the reducible NO2 group and conjugated double bond in compounds 9 and 16 made these alkenes difficult to selectively reduce. The usual protocol involving Pd/C reduced both the NO2 group and the double bond. After unsuccessful attempts to selectively reduce either the double bond or the nitro group with Fe or Zn in acetic acid, Zn and hydrazinium monoformate, and Na2S2O4, it was discovered that hydrogenation over Lindlar catalyst resulted in selective reduction of the alkene linker without reduction of the NO2 group.

Compound 49 was accidentally formed during the condensation reaction of compound 12 with 2-formylbenzoic acid. Evidently, the alcohol intermediate formed from 12 and 2-formylbenzoic acid lactonizes to 49 instead of dehydrating to the alkene. The structure of the lactone 49 suggests that 5-nitro-6-styryluracil derivatives might be susceptible to nucleophilic attack on the exocyclic double bond, but no instance of this was observed in the present series of compounds, which in general were quite stable.

The NO2 group and the trans double bond in 9 and 39 were simultaneously reduced by hydrogenation using Pd/C to obtain compounds 50 and 51. This completes the synthesis of Class VI compounds.

All compounds in Class VI are either completely inactive or have very weak inhibitory activity. Molecular modeling was performed in order to investigate the possible bonding mode of compound 46 to the enzyme. The compound 46 was docked into the M. tuberculosis lumazine synthase structure using Gold software (BST, version 3.0, 2005). Energy minimization was then performed using Sybyl 7.1. The resulting structure is displayed in Figure 2. According to the theoretical model, the binding of the 2-hydroxyphenyl moiety of 46 in the active site of M. tuberculosis lumazine synthase is similar to the high-throughput screening compound 9. However, the model indicates that the uracil ring of 46 does not stack with Trp 27, which may explain the inactivity of 45-48.

Figure 2
Hypothetical model showing hydrogen bonding between the phenolic hydroxyl group of compound 46 with Ala59 and Glu61 of M. tuberculosis lumazine synthase. The distances shown are in Å. The diagram is programmed for wall-eyed (relaxed) viewing.

In conclusion, high-throughput screening of a 100,000 compound commercial library led to the identification of the hit compound 9, which displayed a Ki of 95 μM vs. M. tuberculosis lumazine synthase. The design and synthesis of a focused array of structural analogs provided the optimized congener 26, which had a Ki of 3.7 μM vs. M. tuberculosis lumazine synthase. Both of these compounds are structural analogs of the lumazine synthase substrate 1 and the known lumazine synthase ligand 10. The results of this study show that the ribitylamino side chain of the ligand 10 can be replaced by substituted styryl moieties with retention of affinity for the enzyme. The circumvention of the ribitylamino side chain may contribute positively to antibiotic drug development because its polarity is expected to limit uptake by bacterial cells. As expected from the fact that the pyrimidinedione 1 is a substrate of the lumazine synthase-catalyzed reaction and an product of the riboflavin synthase-catalyzed reaction, many of the styryl derivatives in the present series, including 9, 17, 18, 29, 32, 33, 35, 40, 43, 44, 48, and 50, inhibited both enzymes. This is a potential advantage because drug resistance is less likely to emerge from target mutation with antibiotics that act on two targets, since resistance mutations would have to emerge in both targets at the same time for the organism to become drug resistant. Although it was previously demonstrated that the intermediate analog 55, in which the ribityl group is replaced by a chlorine atom, has affinity for lumazine synthase, compound 55 also has a phosphate moiety that contributes positively to binding.27 It therefore contrasts to the present series of inhibitors, which do not contain a phosphate moiety.

An external file that holds a picture, illustration, etc.
Object name is nihms132543u2.jpg

Experimental Section

General Procedure Method A

5-Nitro-6-methyluracil (12) (1.0 g, 5.85 mmol), benzaldehyde derivatives (29.2 mmol) and piperidine (10 mL) were heated at first on a boiling water-bath until the mixture had thickened and then for 30 min in an oil-bath at 150 °C. The mixture was diluted with methanol (15 mL), and the crystalline solid was filtered, washed with a methanol (5 mL) and ether (10 mL), and dried to give the piperidine salt. The salt was dissolved in dilute KOH solution. On the addition of excess of hydrochloric acid to the warm solution, a yellow powder precipitated. This was filtered and washed first with water (2 × 10 mL) and methanol (2 × 10 mL), and then with ether (2 × 10 mL) to give the desired condensation product.

(E)-6-(3-Hydroxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (15)

A yellow amorphous solid 15 (1.20 g. 75%): mp 238-240 °C (dec). 1H NMR (300 MHz, CDCl3) δ 7.53 (d, J = 16.4 Hz, 1 H), 7.16 (t, J = 7.8 Hz, 1 H), 6.97 (d, J = 7.6 Hz, 1 H), 6.92 (s, 1 H), 6.86 (d, J = 16.4 Hz, 1 H), 6.77 (d, J = 8.1 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 158.0, 156.9, 149.4, 147.8, 142.3, 135.8, 130.5, 126.7, 119.7, 118.3, 114.4, 113.8; EIMS m/z 275 (M+). Anal. Calcd for C12H9N3O5: C, 52.37; H, 3.30; N, 15.27. Found: C, 52.58; H, 3.14; N, 15.54.

(E)-6-(4-Hydroxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (16)

An orange amorphous solid 16 (1.25 g, 78%): mp 230-232 °C (dec). 1H NMR (300 MHz, CDCl3) δ 7.64 (d, J = 16.3 Hz, 1 H), 7.52 (d, J = 8.64 Hz, 2 H), 6.87 (J = 8.64 Hz, 2 H), 6.85 (d, J = 16.3 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 160.6, 156.9, 149.5, 148.0, 142.7, 130.8, 126.1, 125.7, 116.3, 109.9; negative ion EIMS m/z 549 [(2M − H+), 100], 275 (M, 11), 274 [(M − H+), 98], 144 (4). Anal. Calcd for C12H9N3O5: C, 52.37; H, 3.30; N, 15.27. Found: C, 52.45; H, 3.24; N, 15.58.

(E)-6-(3-Hydroxy-4-methoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (20)

A red amorphous solid 20 (625 mg, 67%): mp 300-304 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.34 (s, 1 H), 7.68 (d, J = 16.2 Hz, 1 H), 7.06 (s, 1 H), 7.04 (d, J = 7.8 Hz, 1 H), 6.98 (d, J = 7.8 Hz, 1 H), 6.69 (d, J = 16.2 Hz, 1 H), 3.81 (s, 3 H); 13C NMR (75 MHz, DMSO-d6) δ 156.8, 150.7, 149.4, 147.8, 147.0, 142.5, 127.1, 126.0, 121.8, 113.9, 112.2, 110.7, 55.7; negative ion EIMS m/z 609 [(2M − H+), 46], 304 [(M − H+), 100]. Anal. Calcd for C13H11N3O6: C, 51.15; H, 3.63; N, 13.77. Found: C, 50.80; H, 3.80, N, 13.43.

(E)-6-(2,3-Dimethoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (21)

An orange, amorphous solid 21 (655 mg, 70%): mp 280-283 °C. 1H NMR (300 MHz, DMSO-d6) δ 7.25 (d, J = 16.5 Hz, 1 H), 6.57 (m, 1 H), 6.48 (m, 1 H), 6.36 (d, J = 16.2 Hz, 1 H), 3.18 (s, 3 H), 3.14 (s, 3 H); 13C NMR (75 MHz, DMSO-d6) δ 157.1, 153.3, 149.8, 148.5, 148.1, 137.1, 128.4, 127.0, 124.9, 119.6, 115.5, 115.3, 61.2, 56.0; negative ion EIMS m/z 637 [(2M − H+), 100], 318 [(M − H+), 46]. Anal. Calcd for C14H13N3O6.·0.5H2O: C, 51.22; H, 4.30; N, 12.80. Found: C, 51.03; H, 3.91, N, 12.53.

(E)-6-(3,4-Dimethoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (22)

A red amorphous solid 22 (625 mg, 67%): mp 328-230 °C. 1H NMR (300 MHz, DMSO-d6) δ 7.67 (d, J = 16.2 Hz, 1 H), 7.23 (d, J = 1.6 Hz, 1 H), 7.16 (dd, J = 1.6 Hz, 8.4 Hz, 1 H), 7.00 (d, J = 8.4 Hz, 1 H), 6.85 (d, J = 16.2 Hz, 1 H), 3.80 (s, 3 H), 3.77 (s, 3 H); 13C NMR (75 MHz, DMSO-d6) δ 156.7, 151.5, 149.4, 149.1, 147.9, 142.3, 127.1, 126.0, 123.5, 111.7, 111.3, 110.0, 55.7; Negative ion EIMS m/z 637 [(2M − H+), 14], 318 [(M − H+), 100]. Anal. Calcd for C14H13N3O6: C, 52.67; H, 4.10; N, 13.16. Found: C, 52.69; H, 3.87, N, 13.01.

(E)-6-(2,3,4-Trimethoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (23)

A red amorphous solid 23 (610 mg, 60%): mp 308-310 °C. 1H NMR (300 MHz, DMSO-d6) δ 7.81 (d, J = 16.8 Hz, 1 H), 7.39 (d, J = 9.5 Hz, 1 H), 6.95 (d, J = 16.8 Hz, 1 H), 6.88 (d, J = 9.5 Hz, 1 H), 3.88 (s, 3 H), 3.86 (s, 3 H), 3.79 (s, 3 H); 13C NMR (75 MHz, DMSO-d6) δ 157.6, 155.6, 152.8, 149.4, 149.1, 141.9, 137.1, 126.1, 124.0, 120.7, 112.7, 108.3, 61.4, 60.6, 56.1; negative ion EIMS m/z 697 [(2M − H+), 100], 348 [(M − H+), 57]. Anal. Calcd for C15H15N3O7: C, 51.58; H, 4.33; N, 12.03. Found: C, 51.49; H, 4.35, N, 11.75.

(E)-6-(3,4,5-Trimethoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (24)

A red amorphous solid 24 (610 mg, 60%): mp 293-296°C (dec). 1H NMR (300 MHz, DMSO-d6) δ 7.56 (d, J = 16.2 Hz, 1 H), 6.94 (d, J = 16.2 Hz, 1 H), 6.92 (s, 2 H), 3.76 (s, 3 H), 3.67 (s, 6 H); negative ion EIMS m/z 697 [(2M − H+), 43], 348 [(M − H+), 100]. Anal. Calcd for C15H15N3O7·1.25H2O: C, 48.46; H, 4.74; N, 11.30. Found: C, 48.27; H, 4.71, N, 11.09.

(E)-6-(2-Fluorostyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (28)

An orange, amorphous solid 28 (510 mg. 63%): mp 280-283 °C (dec). 1H NMR (300 MHz, MeOH-d4) δ 7.82 (d, J = 16.5 Hz, 1 H), 7.78 (m, 1 H), 7.50 (m, 1 H), 7.33 (d, J = 8.9 Hz, 1 H), 7.27 (d, J = 7.1 Hz, 1 H), 7.07 (d, J = 16.5 Hz, 1 H); 13C NMR (75 MHz, MeOH-d4) δ 162.5, 156.6, 149.3, 147.5, 133.8, 132.8, 129.1, 126.6, 125.2, 116.6, 116.5. Anal. Calcd for C12H8FN3O4: C, 51.99; H, 2.91; N, 15.16. Found: C, 51.79; H, 3.05, N, 15.25.

(E)-6-(4-Fluorostyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (29)

A red amorphous solid 29 (488 mg. 60%): mp 286-288 °C. 1H NMR (300 MHz, MeOH-d4) δ 7.73 (d, J = 16.0 Hz, 1 H), 7.45 (d, J = 8.3 Hz, 2 H), 6.94 (d, J = 8.4 Hz, 2 H), 6.66 (d, J = 16.0 Hz, 1 H); 13C NMR (75 MHz, MeOH-d4) δ 156.7, 152.7, 149.3, 147.9, 142.7, 130.3, 125.3, 122.8, 114.2, 107.6; negative ion EIMS m/z (rel intensity) 276 [(2M − H+), 100), 113 (2). Anal. Calcd for C12H8FN3O4·0.1H2O: C, 51.66; H, 2.96; N, 15.06. Found: C, 51.95; H, 3.22, N, 14.69.

5-Nitro-6-styryluracil (39)

A yellow amorphous solid 39 (5.1 g. 84%): mp 308-310 °C (lit.33 312-314 °C). 1H NMR (300 MHz, MeOH-d4) δ 7.78 (d, J = 16.2 Hz, 1 H), 7.63 (m, 2 H)(, 7.44 (m, 3 H), 6.98 (d, J = 16.2 Hz, 1 H); 13C NMR (75 MHz, MeOH-d4) δ 157.0, 149.6, 147.2, 134.6, 131.1, 129.5, 128.6, 126.8, 114.2; negative ion EIMS m/z 259 (M, 12), 258 [(M − H+), 98.5], 113 (2). Anal. Calcd for C12H9N3O4: C, 55.60; H, 3.50; N, 16.21. Found: C, 55.89; H, 3.41; N, 15.95.

(E)-5-Nitro-6-(2-(pyridine-2-yl)vinyl)pyrimidine-2,4(1H,3H)-dione (41)

An orange amorphous solid 41 (1.29 g. 85%): mp 290-292 °C (dec). 1H NMR (300 MHz, CDCl3) δ 8.63 (d, J = 4.8 Hz, 1 H), 7.92 (t, J = 8.6 Hz, 1 H), 7.73 (d, J = 15.9 Hz, 1 H), 7.60 (d, J = 7.8 Hz, 1 H), 7.44 (d, J = 15.9 Hz, 1 H), 7.43 (m, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 157.2, 150.6, 149.2, 148.3, 147.2, 141.3, 137.7, 128.0, 126.5, 126.4, 120.4; negative ion EIMS m/z 541 [(2MNa − 2H+), 100], 259 [(M − H+), 41]. Anal. Calcd for C11H8N4O4: C, 50.77; H, 3.10; N, 21.53. Found: C, 50.52; H, 3.16; N, 21.20.

(E)-5-Nitro-6-(3-(pyridine-3-yl)vinyl)pyrimidine-2,4(1H,3H)-dione (42)

A yellow amorphous solid 42 (1.22 g. 80%): mp 300-303 °C. 1H NMR (300 MHz, CDCl3) δ 8.76 (s, 1 H), 8.59 (d, J = 3.7 Hz, 1 H), 8.13 (d, J = 8.07 Hz, 1 H), 7.70 (d, J = 16.2 Hz, 1 H), 7.46 (dd, J = 4.8 Hz, 7.8 Hz, 1 H), 7.19 (d, J = 16.2 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.8, 151.03, 150.0, 149.8, 148.6, 137.8, 134.4, 130.3, 126.5, 124.2, 117.2; negative ion EIMS m/z 556.8 [(2M − 2H+), 100), 259 [(M − H+), 94], 156 (1). Anal. Calcd for C11H8N4O4: C, 50.77; H, 3.10; N, 21.53. Found: C, 50.47; H, 2.88; N, 21.71

General Procedure Method B

5-Nitro-6-methyluracil (12) (0.5 g, 2.92 mmol), benzaldehyde derivatives (5.84 mmol) and piperidine (0.3 mL, 3.21 mmol) in 1-butanol (10 mL) were heated at first to 100 °C until the mixture had thickened, and then at reflux for 6 h. The mixture was cooled to room temperature, filtered, and washed with a methanol (5 mL) and ether (10 mL). The salt was dissolved in dilute KOH solution, acidified with an excess of hydrochloric acid, and the solid was filtered and washed with water (2 × 10 mL) and methanol (2 × 10 mL), and then with ether (2 × 10 mL) and dried to give the desired condensation product.

(E)-6-(2-Nitrostyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (14)

A brown amorphous solid 14 (0.62 mg, 69%): mp 298-300 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.03 (d, J = 8.1 Hz, 1 H), 7.97 (d, J = 9.0 Hz, 1 H), 7.95 (d, J = 16.2 Hz, 1 H), 7.79 (t, J = 7.5 Hz, 1 H), 7.68 (d, J = 7.5 Hz, 1 H), 7.05 (d, J = 16.2 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.9, 149.3, 148.8, 147.0, 135.6, 134.0, 131.3, 129.2, 128.9, 127.0, 125.0, 119.3; negative ion EIMS m/z (rel intensity) 607 [(2M − H+), 100], 303 [(M − H+), 80], 240 (10). Anal. Calcd for C12H8N4O6: C, 47.38; H, 2.65; N, 18.42. Found: C, 47.45; H, 2.45; N, 18.10.

(E)-6-(2,3-Dihydroxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (17)

A brown amorphous solid 17 (0.49 g, 58%): mp 287-290 °C (dec). 1H NMR (300 MHz, DMSO-d6) δ 7.88 (d, J = 15.7 Hz, 1 H), 7.07 (d, J = 15.7 Hz, 1 H), 6.85 (m, 2 H), 6.67 (t, J = 7.8 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.1, 150.0, 149.5, 148.8, 147.4, 138.3, 125.4, 120.7, 119.3, 116.9, 114.2, 112.8; negative ion EIMS m/z 581 [(2M − H+), 62], 290 [(M − H+), 100]. Anal. Calcd for C12H9N3O6: C, 49.49; H, 3.12; N, 14.43. Found: C, 49.61; H, 3.22; N, 14.48.

(E)-6-(2,5-Dihydroxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (18)

A brown amorphous solid 18 (0.19 g, 23%): mp 279-282 °C (dec). 1H NMR (300 MHz, DMSO-d6) δ 9.78 (brs, 1 H), 7.80 (d, J = 15.8 Hz, 1 H), 7.04 (d, J = 15.8 Hz, 1 H), 6.81 (d, J = 1.97 Hz, 1 H), 6.70 (m, 2 H); 13C NMR (75 MHz, DMSO-d6) δ 156.3, 149.9, 149.5, 148.9, 147.4, 138.5, 125.5, 120.7, 119.3, 116.7, 114.2, 112.7; negative ion EIMS m/z (rel intensity) 581 [(2M − H+), 100], 290 [(M − H+), 54], 244 (1). Anal. Calcd for C12H9N3O6: C, 49.49; H, 3.12; N, 14.43. Found: C, 49.78; H, 3.14; N, 14.29.

(E)-6-(2-Methoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (19)

A yellow amorphous solid 19 (0.56 g, 66%): mp 289-300 °C. 1H NMR (300 MHz, DMSO-d6) δ 7.90 (d, J = 16.4 Hz, 1 H), 7.57 (d, J = 7.6 Hz, 1 H), 7.42 (t, J = 7.6 Hz, 1 H), 7.09 (d, J = 7.6 Hz, 1 H), 7.08 (d, J = 16.4 Hz, 1 H), 7.00 (d, J = 7.6 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 158.3, 156.8, 149.5, 148.1, 137.6, 132.4, 129.5, 126.4, 122.8, 121.0, 114.6, 112.0, 55.8; negative ion EIMS m/z 577 [(2M − H+), 100], 288 [(M − H+), 58]. Anal. Calcd for C13H11N3O5: C, 53.98; H, 3.83; N, 14.53. Found: C, 53.92; H, 3.92; N, 14.17.

(E)-5-Nitro-6-(3-nitrostyryl)pyrimidine-2,4(1H,3H)-dione (25)

A yellow amorphous solid 25 (0.56 g, 63%): mp 278-280 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.44 (s, 1 H), 8.23 (dd, J = 1.44 Hz, 8.06 Hz, 1 H), 8.06 (d, J = 7.7 Hz, 1 H), 7.76 (d, J = 16.3 Hz, 1 H), 7.70 (d, J = 8.06 Hz, 1 H), 7.25 (d, J = 16.3 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.6, 149.5, 148.3, 147.8, 138.9, 136.0, 134.3, 130.6, 126.8, 124.7, 122.5, 117.7; negative ion EIMS m/z 607 [(2M − H+), 100], 303 [(M − H+), 37]. Anal. Calcd for C12H8N4O6: C, 47.38; H, 2.65; N, 18.42. Found: C, 47.02; H, 2.73; N, 18.13.

(E)-5-Nitro-6-(4-nitrostyryl)pyrimidine-2,4(1H,3H)-dione (26)

A brown amorphous solid 26 (0.62 mg, 69%): mp 304-308 °C (dec). 1H NMR (300 MHz, DMSO-d6) δ 8.23 (d, J = 8.8 Hz, 2 H), 7.89 (d, J = 8.8 Hz, 2 H), 7.68 (d, J = 15.8 Hz, 1 H), 7.38 (d, J = 15.8 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.7, 149.3, 148.1, 147.6, 140.6, 138.9, 129.3, 127.0, 124.3, 118.7. Anal. Calcd for C12H8N4O6: C, 47.38; H, 2.65; N, 18.42. Found: C, 47.52; H, 2.70, N, 18.19.

(E)-4-(2-(5-Nitro-2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-yl)vinyl)benzoic Acid (27)

A pale-yellow amorphous solid 27 (0.28 mg, 64%): mp 342-345 °C. 1H NMR (300 MHz, DMSO-d6) δ 11.83 (brs, 1 H), 7.98 (brs, 1 H), 7.95 (brs, 1 H), 7.72 (d, J = 16.3 Hz, 1 H), 7.71 (m, 2 H), 7.07 (d, J = 16.3 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 167.2, 156.8, 149.9, 147.7, 140.8, 138.2, 130.4, 128.2, 127.0, 116.8; negative ion EIMS m/z 605 [(2M − H+), 100], 302 [(M − H+), 70]. Anal. Calcd for C13H9N3O6 0.5H2O: C, 50.01; H, 3.23; N, 13.46. Found: C, 50.29; H, 2.89; N, 13.22.

(E)-5-Nitro-6-(4-bromostyryl)pyrimidine-2,4(1H,3H)-dione (30)

A yellow amorphous solid 30 (0.62 g, 63%): mp 296-298 °C (dec). 1H NMR (300 MHz, DMSO-d6) δ 7.68 (d, J = 16.3 Hz, 1 H), 7.62 (dd, J = 8.4 Hz, 18.0 Hz, 4 H), 7.03 (d, J = 16.3 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.7, 149.4, 147.7, 140.4, 133.5, 132.2, 130.2, 126.6, 124.2, 115.1. Anal. Calcd for C12H8BrN3O4: C, 42.63; H, 2.38; N, 12.43; Br, 23.63. Found: C, 42.41; H, 2.37; N, 12.27; Br, 23.55.

(E)-6-(4-Chlorostyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (31)

A yellow amorphous solid 31 (0.58 g, 67%): mp 326-328 °C. 1H NMR (300 MHz, DMSO-d6) δ 7.68 (d, J = 16.2 Hz, 1 H), 7.60 (d, J = 8.4 Hz, 2 H), 7.51 (d, J = 8.4 Hz, 2 H), 7.03 (d, J = 16.3 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.7, 149.6, 148.1, 140.0, 135.2, 133.3, 130.0, 129.2, 115.4. Anal. Calcd for C12H8ClN3O4: C, 49.08; H, 2.75; Cl, 12.07; N, 14.35. Found: C, 48.89; H, 2.79; Cl, 11.89; N, 13.92.

(E)-6-(2-Hydroxy-5-nitrostyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (32)

A brickred amorphous solid 32 (625 mg, 67%): mp 298-300 °C. 1H NMR (300 MHz, DMSO-d6) δ 11.83 (brs, 2 H), 8.38 (d, J = 2.8 Hz, 1 H), 8.14 (dd, J = 2.8, 9.0 Hz, 1 H), 7.83 (d, J = 16.4 Hz, 1 H), 7.34 (d, J = 16.4 Hz, 1 H), 7.07 (d, J = 9.0 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 162.8, 156.8, 149.3, 147.8, 138.9, 136.2, 127.1, 126.8, 126.0, 121.7, 156.9, 156.8; negative ion EIMS m/z (rel intensity) 639 [2M − H+), 80], 319 [(M − H+), 100]. Anal. Calcd for C12H8N4O7·0.75H2O: C, 43.19; H, 2.87; N, 16.79. Found: C, 43.38; H, 2.81; N, 16.68.

(E)-6-(2-Hydroxy-3-nitrostyryl)-3-nitropyrimidine-2,4(1H,3H)-dione (33)

A yellowish-brown amorphous solid 33 (705 mg, 75%): mp 305-307 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.04 (dd, J = 2.8 Hz, 1 H), 7.97 (d, J = 7.8 Hz, 1 H), 7.90 (d, J = 16.4 Hz, 1 H), 7.24 (d, J = 16.4 Hz, 1 H), 7.09 (t, J = 8.0 Hz, 1 H); EIMS m/z (rel intensity) 321 (MH+, 32), 359 (MK+, 50), 659 (100); negative ion EIMS m/z (rel intensity) 319 [(M − H+), 100], 256 (11). Anal. Calcd for C12H8N4O7: C, 45.01; H, 2.52; N, 17.50. Found: C, 45.34; H, 2.55; N, 17.56.

(E)-6-(2-Methoxy-5-nitrostyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (34)

A yellow amorphous solid 34 (630 mg, 65%): mp 278-280 °C. 1H NMR (300 MHz, DMSO-d6) δ 11.88 (s, 1 H), 11.73 (s, 1 H), 8.46 (d, J = 2.6 Hz, 1 H), 8.29 (dd, J = 2.6, 9.2 Hz, 1 H), 7.84 (d, J = 16.4 Hz, 1 H), 7.33 (d, J = 9.2 Hz, 1 H), 7.27 (d, J = 16.4 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 162.9, 156.7, 149.2, 147.8, 141.0, 134.9, 127.3, 126.8, 124.6, 123.5, 117.9, 112.9, 57.0; negative ion EIMS m/z 667 [(2M − H+), 100], 333 [(M − H+), 23]. Anal. Calcd for C13H10N4O7·0.5H2O: C, 45.49; H, 3.23; N, 16.32. Found: C, 45.31; H, 3.04; N, 16.20.

(E)-6-(3-Hydroxy-4-nitrostyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (35)

A pale yellow amorphous solid 35 (323 mg, 69%): mp 310-312 °C. 1H NMR (300 MHz, DMSO-d6) δ 11.73 (brs, 1 H), 11.19 (brs, 1 H), 7.94 (d, J = 8.6 Hz, 1 H), 7.66 (d, J = 16.3 Hz, 1 H), 7.30 (s, 1 H), 7.26 (d, J = 8.6 Hz, 1 H), 7.07 (d, J = 16.4 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.6, 152.3, 149.2, 147.2, 140.5, 139.1, 137.3, 127.0, 126.1, 118.6, 118.5, 118.1; negative ion EIMS m/z 639 [(2M − H+), 100], 319 [(M − H+), 78]. Anal. Calcd for C12H8N4O7: C, 45.01; H, 2.52; N, 17.50. Found: C, 44.98; H, 2.54; N, 17.45.

(E)-6-(4-Hydroxy-3-nitrostyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (36)

A yellow amorphous solid 36 (315 mg, 67%): mp 320-322 °C. 1H NMR (300 MHz, DMSO-d6) δ 11.83 (s, 1 H), 11.55 (brs, 1 H), 8.12 (d, J = 1.9 Hz, 1 H), 7.78 (dd, J = 1.9, 8.7 Hz, 1 H), 7.67 (d, J = 16.3 Hz, 1 H), 7.15 (d, J = 8.7 Hz, 1 H), 6.92 (d, J = 16.3 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.7, 153.8, 149.4, 147.5, 139.8, 137.4, 134.3, 126.4, 125.7, 125.4, 119.9; Pos EIMS m/z (rel intensity) 321 (M+, 100); negative ion EIMS m/z 639 [(2M − H+), 100], 319 [(M − H+), 30]. Anal. Calcd for C12H8N4O7: C, 45.01; H, 2.52; N, 17.50. Found: C, 44.70; H, 2.58; N, 17.44.

(E)-3-Hydroxy-4-(2-(5-nitro-2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-yl)vinyl)benzoic Acid (37)

A yellow red amorphous solid 37 (0.28 g, 60%): mp 347-349 °C (dec). 1H NMR (300 MHz, DMSO-d6) δ 11.91 (s, 1 H), 11.78 (s, 1 H), 10.82 (s, 1 H), 7.86 (d, J = 16.3 Hz, 1 H), 7.57 (d, J = 8.1 Hz, 1 H), 7.49 (s, 1 H), 7.41 (d, J = 8.1 Hz, 1 H), 7.27 (d, J = 16.3 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 167.2, 157.2, 157.1, 149.9, 148.0, 137.8, 133.5, 130.1, 126.8, 125.0, 120.4, 116.9, 116.1; negative ion EIMS m/z 637 [(2M − H+), 100], 318 [(M − H+), 27]. Anal. Calcd for C13H9N3O7·0.5H2O: C, 47.57; H, 3.07; N, 12.80. Found: C, 47.35; H, 2.85; N, 12.56.

(E)-6-(2-Fluoro-3-methoxystyryl)-5-nitropyrimidine-2,4(1H,3H)-dione (38)

A yellow amorphous solid 38 (115 mg, 65%): mp 318-320 °C. 1H NMR (300 MHz, DMSO-d6) δ 11.88 (s, 1 H), 11.85 (s, 1 H), 7.82 (d, J = 16.5 Hz, 1 H), 7.31 (t, J = 6.9 Hz, 1 H), 7.26 (m, 1 H), 7.21 (m, 1 H), 7.04 (d, J = 16.5 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.4, 151.2, 149.9, 147.8, 134.0, 127.0, 125.1, 122.9, 119.2, 116.4, 115.6, 56.5; negative ion EIMS m/z 613 [(2M − H+), 16], 306 [(M − H+), 100]. Anal. Calcd for C13H10FN3O5: C, 50.82; H, 3.28; N, 13.68; F, 6.18. Found: C, 50.46; H, 3.15; N, 13.44; F, 5.94.

(E)-5-Nitro-6-[2-(1H-pyrrol-2-yl)vinyl]pyrimidine-2,4(1H,3H)-dione (40)

A black amorphous solid 40 (0.49 g, 68%): mp 302-304 °C (dec). 1H NMR (300 MHz, DMSO-d6) δ 7.71 (d, J = 16.0 Hz, 1 H), 7.13 (m, 2 H), 6.58 (d, J = 16.0 Hz, 1 H), 6.53 (m, 1 H), 6.21 (m, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 156.8, 149.5, 147.9, 133.3, 132.4, 128.8, 117.52, 110.8, 105.2; negative ion EIMS m/z 495 [(2M − H+), 77], 247 [(M − H+), 100]. Anal. Calcd for C10H8N4O4: C, 48.39; H, 3.25; N, 22.57. Found: C, 48.17; H, 3.51; N, 22.85.

(E)-6-(2-(Naphthalene-2-yl)vinyl)-5-nitropyrimidine-2,4(1H,3H)-dione (43)

A yellow amorphous solid 43 (0.66 g, 73%): mp 320-322 °C. 1H NMR (300 MHz, DMSO-d6) δ 11.73 (brs, 2 H), 8.11 (s, 1 H), 7.99 (m, 1 H), 7.93 (m, 1 H), 7.91 (d, J = 16.2 Hz, 1 H), 7.79 (d, J = 8.5 Hz, 1 H), 7.55 (m, 2 H), 7.13 (d, J = 16.2 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 157.0, 150.2, 149.1, 141.2, 133.8, 132.9, 132.0, 129.8, 128.8, 128.6, 127.7, 127.5, 126.9, 126.3, 123.8, 115.4; negative ion EIMS m/z 639 [(2MNa − 2H+), 100], 617 [(2M − H+), 22], 308 [(M − H+), 30]. Anal. Calcd for C16H11N3O4·0.75H2O: C, 59.54; H, 3.90; N, 13.02. Found: C, 59.18; H, 3.46; N, 12.83.

(E)-6-(2-(3H-Indol-3-yl)vinyl)-5-nitropyrimidine-2,4(1H,3H)-dione (44)

A red amorphous solid 44 (0.62g, 71%): mp 340-342 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.10 (s, 1 H), 11.68 (s, 1 H), 11.53 (s, 1 H), 8.15 (d, J = 16.5 Hz, 1 H), 7.98 (d, J = 2.1 Hz, 1 H), 7.79 (d, J = 7.9 Hz, 1 H), 7.49 (d, J = 7.9 Hz, 1 H), 7.22 (m, 2 H), 6.85 (d, J = 16.5 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 157.0, 149.6, 148.7, 137.8, 137.5, 132.5, 125.2, 124.6, 123.1, 121.6, 119.5, 112.9, 112.8, 106.3; negative ion EIMS m/z 319 [(MNa − 2H+), 66], 297 [(M − H+), 8]. Anal. Calcd for C14H10N4O4: C, 56.38; H, 3.38; N, 18.78. Found: C, 56.10; H, 3.22; N, 18.76.

6-(Benzylamino)-5-nitropyrimidine-2,4(1H,3H)-dione (45)

6-Chloro-5-nitrouracil (52) (0.15 g, 0.78 mmol), benzylamine (0.09 mL, 0.86 mmol) and Et3N (0.3 mL, 2.35 mmol) in 1,4-dioxane (5 mL) were heated at reflux for 12 h. The mixture was cooled to room temperature, filtered, and washed with methanol (5 mL) and ether (10 mL). The salt was dissolved in dilute KOH solution, acidified with an excess of hydrochloric acid, and the solid was filtered and washed with water (2 × 10 mL), methanol (2 × 10 mL) and then with ether (2 × 10 mL) and dried to yield a white amorphous solid 45 (145 mg, 73%): mp 284-286 °C. 1H NMR (300 MHz, DMSO-d6) δ 7.30 (m, 5 H), 4.71 (s, 2 H); 13C NMR (75 MHz, DMSO-d6) δ 157.1, 154.2, 147.6, 136.3, 128.7, 127.3, 126.4, 109.2, 46.5; EIMS m/z (rel intensity) 285 (MNa+, 100), 263 (MH+, 7); negative ion EIMS m/z (rel intensity) 261 [(M − H+), 100], 227 (7). Anal. Calcd for C11H10N4O4: C, 50.38; H, 3.84; N, 21.37. Found: C, 50.04; H, 3.69; N, 21.05.

6-(2-Hydroxybenzylamino)-5-nitropyrimidine-2,4(1H,3H)-dione (46)

6-Chloro-5-nitrouracil (52) (0.27 g, 1.4 mmol), 2-hydroxybenzylamine (0.19 g, 1.56 mmol) and Et3N (0.6 mL, 4.23 mmol) in 1,4-dioxane (6 mL) were heated at reflux for 12 h. The mixture was cooled to room temperature, filtered, and washed with methanol (5 mL) and ether (10 mL). The salt was dissolved in dilute KOH solution, acidified with an excess of hydrochloric acid, and the solid was filtered and washed with water (2 × 10 mL), methanol (2 × 10 mL) and then with ether (2 × 10 mL) and dried to give a pale-yellow amorphous solid 46 (0.25 g, 64%): mp 138-140 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.84 (t, J = 5.4 Hz, 1 H), 9.72 (brs, 1 H), 7.25 (dd, J = 1.3 Hz, 7.3 Hz, 1 H), 7.12 (m, 1 H), 6.75 (m, 2 H), 4.43 (d, J = 6.1 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6) δ 159.0, 158.7, 156.1, 130.7, 128.8, 125.4, 118.8, 116.7, 66.4; EIMS m/z (rel intensity) 579 (2MNa+, 20), 380 (100), 301 (MNa+, 10); negative ion EIMS m/z (rel intensity) 555 [(2M - H+), 100], 278 (M, 10), 277 [(M - H+), 85], 241. Anal. Calcd for C11H10N4O5: C, 47.49; H, 3.62; N, 20.14. Found: C, 47.61; H, 3.69; N, 19.83.

6-(2-Hydroxy-2-phenethyl)-5-nitropyrimidine-2,4(1H,3H)-dione (47)

Lindlar catalyst (5 mg) was added to a solution of compound 9 (100 mg, 0.36 mmol) in MeOH (5 mL). A hydrogen balloon was attached and the mixture was stirred at room temperature for 12 h. The reaction mixture was filtered through celite, which was then washed with MeOH (2 × 5 mL). The solution was concentrated and the residue was washed several times with CH2Cl2 (3 × 10 mL) and THF (3 × 10 mL). Finally, compound 47 was precipitated out by dissolving in MeOH and adding excess diethyl ether to furnish the pure product (78 mg, 78%) as pale yellow amorphous solid: mp 231-234 °C. 1H NMR (300 MHz, MeOH-d4) δ 7.06 (m, 2 H), 6.76 (m, 2 H), 2.88 (d, J = 6.8 Hz, 1 H), 2.85 (d, J = 5.6 Hz, 1 H), 2.70 (d, J = 5.6 Hz, 1 H), 2.67 (d, J = 6.8 Hz, 1 H); 13C NMR (75 MHz, MeOH-d4) δ 163.6, 156.4, 152.0, 135.8, 131.2, 128.9, 127.7, 120.7, 119.0, 116.0, 29.8, 29.1; negative ion ESI-MS m/z (rel intensity) 276 [(M − H+), 100], 258 (15); HRMS m/z calcd for C12H11N3O5 (M − H+) 276.0620, found 276.0619. Anal. Calcd for C12H11N3O5: C, 51.99; H, 4.00; N, 15.16. Found: C, 51.78; H, 4.28; N, 15.04.

6-(4-Hydroxy-2-phenethyl)-5-nitropyrimidine-2,4(1H,3H)-dione (48)

Lindlar catalyst (5 mg) was added to a solution of compound 16 (100 mg, 0.36 mmol) in MeOH (5 mL). A hydrogen balloon was attached and the mixture was stirred at room temperature for 12 h. The reaction mixture was filtered through celite, which was then washed with MeOH (2 × 5 mL). The solution was concentrated and the residue was washed several times with CH2Cl2 (3 × 10 mL) and THF (3 × 10 mL). Finally, compound 48 was precipitated out by dissolving in MeOH and adding excess diethyl ether to furnish the pure product (70 mg, 70%) as a yellowish amorphous solid: mp 235-237 °C. 1H NMR (300 MHz, MeOH-d4) δ 7.06 (d, J = 8.4 Hz, 2 H), 6.69 (d, J = 8.4 Hz, 2 H), 2.80 (d, J = 6.5 Hz, 1 H), 2.77 (d, J = 5.6 Hz, 1 H), 2.68 (d, J = 5.6 Hz, 1 H), 2.65 (d, J = 6.5 Hz, 1 H); 13C NMR (75 MHz, MeOH-d4) δ 163.7, 157.0, 152.0, 135.4, 132.4, 130.5, 119.0, 116.3, 33.0, 31.9; negative ion ESI-MS m/z (rel intensity) 276 [(M − H+), 100], 258 (33); negative íon HRMS m/z calcd for C12H11N3O5 (M − H+) 276.0620, found 276.0623. Anal. Calcd for C12H11N3O5: C, 51.99; H, 4.00; N, 15.16. Found: C, 51.91; H, 4.21; N, 14.95.

5-Nitro-6-[(3-oxo-1,3-dihydroisobenzofuran-1-yl)methyl]pyrimidine-2,4-(1H,3H)-dione (49)

a pale-yellow amorphous solid 49 (0.3 mg, 68%): mp 335-337 °C. 1H NMR (300 MHz, DMSO-d6) δ 7.74 (m, 2 H), 7.56 (m, 2 H), 5.92 (dd, J = 3.0, 9.6 Hz, 1 H), 3.50 (dd, J = 3.3, 13.8 Hz, 1 H), 2.86 (dd, J = 9.6, 13.8 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6) δ 169.1, 156.4, 151.7, 149.1, 148.2, 134.8, 130.0, 128.0, 125.2, 125.0 122.8, 78.2, 35.1; negative ion EIMS m/z (rel intensity) 302 [(M − H+), 100], 170 (61). Anal. Calcd for C13H9N3O6: C, 51.49; H, 2.99; N, 13.86. Found: C, 51.12; H, 3.08; N, 13.57.

5-Amino-(2-hydroxy-2-phenethyl)pyrimidine-2,4-(1H,3H)-dione (50)

Concentrated HCl (0.5 ml) and 10% Pd/C (30 mg) were added to a solution of 5-nitro-6-styryluracil (39) (300 mg, 1.16 mmol) in MeOH (10 mL). A hydrogen balloon was attached and the mixture was stirred at room temperature for 24 h. The reaction mixture was filtered through celite, which was then washed with 50% aq MeOH (5 mL) to provide compound 50 as a white solid (225 mg, 73%): mp 251-254 °C. 1H NMR (300 MHz, MeOH-d4/ D2O) δ 7.27 (m, 4 H), 7.23 (m, 1 H), 2.97 (m, 2 H), 2.85 (m, 2 H); 13C NMR (75 MHz, MeOH-d4) δ 160.1, 151.4, 150.1, 140.6, 129.8, 129.5, 127.9, 115.2, 34.4, 31.8; EIMS m/z (rel intensity) 231 (M+, 51), 140 (M+ - C7H7, 80), 91 (M+ - C5H6N3O2, 100); CIMS m/z (rel intensity) 232 (MH+, 100). Anal. Calcd for C12H14ClN3O2: C, 53.84; H, 5.27; N, 15.70. Found: C, 53.99; H, 5.01; N, 15.90.

5-Amino-6-(2-hydroxyphenethyl)pyrimidine-2,4(1H,3H)-dione Hydrochloride (51)

Pd/C (5 mg) was added to a solution of compound 9 (100 mg, 0.36 mmol) in MeOH (20 mL). The mixture was shaken in Parr apparatus under H2 atmosphere for 24 h. The reaction mixture was filtered through celite, which was then washed with MeOH (2 × 10 mL). The solution was concentrated and the residue was washed several times with CH2Cl2 (3 × 10 mL) and THF (3 × 10 mL). Finally, compound 51 was precipitated out by dissolving it in MeOH and adding excess diethyl ether to furnish pure compound 51 (65 mg, 72%) as yellow amorphous solid: mp 239-241 °C (dec). 1H NMR (300 MHz, MeOH-d4) δ 7.06 (m, 2 H), 6.75 (m, 2 H), 2.88 (d, J = 6.8 Hz, 1 H), 2.85 (d, J = 5.9 Hz, 1 H), 2.70 (d, J = 5.9 Hz, 1 H), 2.68 (d, J = 6.8 Hz, 1 H); EIMS m/z (rel intensity) 248 (MH+, 100), 194 (10), 230; HRMS m/z calcd for C12H14N3O3 (MH+) 248.1035, found 248.1034. Anal. Calcd for C12H14ClN3O3: C, 50.80; H, 4.97; N, 14.81. Found: C, 50.91; H, 4.81; N, 14.95.

Lumazine Synthase Assay

Assay mixtures contained 50 mM Tris hydrochloride, pH 7.0, 100 mM NaCl, 2% (v/v) DMSO, 5 mM dithiothreitol, 100 μM 2, lumazine synthase (Table 2), variable concentrations of 1 (3 – 150 μM) and inhibitor (0 – 150 μM) in a volume of 0.2 mL. Assay mixtures were prepared as follows. A solution (175 μL) containing 103 mM NaCl, 5.1 mM dithiothreitol, 114 μM 2, lumazine synthase in 51 mM Tris hydrochloride, pH 7.0, was added to 4 μL of inhibitor in 100% (v/v) DMSO in a well of a 96-well microtiter plate. The reaction was started by adding 21 μL of a solution containing 103 mM NaCl, 5.1 mM dithiothreitol, and substrate 1 (30 – 1500 μM) in 51 mM Tris hydrochloride, pH 7.0. The formation of 6,7-dimethyl-8-D-ribityllumazine (3) was measured online for a period of 40 min at 27 °C with a computer-controlled plate reader at 408 nm (εLumazine = 10,200 M−1cm−1).

Table 2
Enzymes used in kinetic assays.

Riboflavin Synthase Assay

Assay mixtures contained 50 mM Tris hydrochloride, pH 7.0, 100 mM NaCl, 2% (v/v) DMSO, 5 mM dithiothreitol, enzyme (Table 2), variable concentrations of 3 (3 – 20 μM) and inhibitor (0 – 150 μM) in a volume of 0.2 mL. Assay mixtures were prepared as follows. A solution (175 μL) containing 103 mM NaCl, 5.1 mM dithiothreitol, and riboflavin synthase in 51 mM Tris hydrochloride, pH 7.0, was added to 4 μL of inhibitor in 100% (v/v) DMSO in a well of a 96-well microtiter plate. The reaction was started by adding 21 μL of a solution containing 103 mM NaCl, 5.1 mM dithiothreitol, and substrate 3 (30 – 200 μM) in 51 mM Tris hydrochloride, pH 7.0. The formation of riboflavin was measured online for a period of 40 min at 27 °C with a computer-controlled plate reader at 470 nm (εRiboflavin = 9600 M−1cm−1).

Details of the Assay Procedures

For both lumazine synthase and riboflavin synthase assays, four different inhibitor concentrations and seven different substrate concentrations were used. Therefore, a series of measurements for one compound included 28 sample reactions plus 4 reactions without substrate added (as control samples). The enzyme was added before the reaction mixture was pipetted into different wells on a 96-well microtiterplate. The enzyme concentration in all reaction mixtures was identical. After enzyme was added, the solution was divided into four parts, and a different amount of inhibitor was added to each of them. The accuracy of the measurements on the plate reader was checked regularly. An identical amount of substrate was added to every well on the microtiter plate containing identical enzyme solutions. The results of measurements were analyzed for all wells, and standard deviation values were between 3% and 8%. The standard deviation values in Table 1 are important criteria for determining the mode of inhibition, and do not reflect the accuracy of pipetting or photometric measurements.

Evaluation of Kinetic Data

The velocity-substrate data were fitted for all inhibitor concentrations with a non-linear regression method using the program DynaFit. (Kuzmich 1996) 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.17

Supplementary Material

1_si_001

Acknowledgments

This research was made possible by NIH grant GM51469, the Fonds der Chemischen Industrie, and the Hans Fischer Gesellschaft. The experimental work 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.

Footnotes

Supporting Information Available: 1H NMR and 13C NMR spectra of 9, 14-23 and 25-51. This material is available free of charge via the Internet at http://pubs.acs.org.

References

1. Wang A. I Chuan Hsueh Pao. 1992;19:362–368. [PubMed]
2. Oltmanns O, Lingens F. Z Naturforschung. 1967;22 b:751–754. [PubMed]
3. Logvinenko EM, Shavlovsky GM. Mikrobiologiya. 1967;41:978–979.
4. Neuberger G, Bacher A. Biochem Biophys Res Commun. 1985;127:175–181. [PubMed]
5. Neuberger G, Bacher A. Biochem Biophys Res Commun. 1986;139:1111–1116. [PubMed]
6. Kis K, Volk R, Bacher A. Biochemistry. 1995;34:2883–2892. [PubMed]
7. Fischer M, Haase I, Feicht R, Schramek N, Kohler P, Schieberle P, Bacher A. Biol Chem. 2005;386:417–428. [PubMed]
8. Gerhardt S, Schott AK, Kairies N, Cushman M, Illarionov B, Eisenreich W, Bacher A, Huber R, Steinbacher S, Fischer M. Structure. 2002;10:1371–1381. [PubMed]
9. Illarionov B, Eisenreich W, Bacher A. Proc Natl Acad Sci U S A. 2001;98:7224–7229. [PubMed]
10. Illarionov B, Eisenreich W, Schramek N, Bacher A, Fischer M. J Biol Chem. 2005;280:28541–28546. [PubMed]
11. Illarionov B, Kemter K, Eberhardt S, Richter G, Cushman M, Bacher A. J Biol Chem. 2001;276:11524–11530. [PubMed]
12. Maley GF, Plaut GWE. J Biol Chem. 1959;234:641–647. [PubMed]
13. Plaut GWE. J Biol Chem. 1963;238:2225–2243. [PubMed]
14. Plaut GWE, Harvey RA. Methods Enzymol. 1971;18B:515–538.
15. Wacker H, Harvey RA, Winestock CH, Plaut GWE. J Biol Chem. 1964;239:3493–3497. [PubMed]
16. Volk R, Bacher A. J Am Chem Soc. 1988;110:3651–3653.
17. Chen J, Illarionov B, Bacher A, Fischer M, Haase I, Georg G, Ye Qz, Ma Z, Cushman M. Anal Biochem. 2005;338:124–130. [PubMed]
18. Kaiser J, Illarionov B, Rohdich F, Eisenreich W, Saller S, Van den Brulle J, Cushman M, Bacher A, Fischer M. Anal Biochem. 2007;365:52–61. [PubMed]
19. Fischer M, Haase I, Feicht R, Richter G, Gerhardt S, Changeux JP, Huber R, Bacher A. Eur J Biochem. 2002;269:519–526. [PubMed]
20. Zhang YL, Illarionov B, Morgunova E, Jin GY, Bacher A, Fischer M, Ladenstein R, Cushman M. J Org Chem. 2008;73:2715–2724. [PubMed]
21. Talukdar A, Illarionov B, Bacher A, Fischer M, Cushman M. J Org Chem. 2007;72:7167–7175. [PubMed]
22. Zhang YL, Jin GY, Illarionov B, Bacher A, Fischer M, Cushman M. J Org Chem. 2007;72:7176–7184. [PubMed]
23. Chen J, Sambaiah T, Illarionov B, Fischer M, Bacher A, Cushman M. J Org Chem. 2004;69:6996–7003. [PubMed]
24. Bacher A, Ludwig HC. Eur J Biochem. 1982;127:539–545. [PubMed]
25. Ritsert K, Huber R, Turk D, Ladenstein R, Schmidt-Bäse K, Bacher A. J Mol Biol. 1995;253:151–167. [PubMed]
26. Morgunova K, Meining W, Illarionov B, Haase I, Jin G, Bacher A, Cushman M, Fischer M, Ladenstein R. Biochemistry. 2005;44:2746–2758. [PubMed]
27. Morgunova E, Illarionov B, Sambaiah T, Haase I, Bacher A, Cushman M, Fischer M, Ladenstein R. FEBS J. 2006;273:4790–4804. [PubMed]
28. Gerhardt S, Haase I, Steinbacher S, Kaiser JT, Cushman M, Bacher A, Huber R, Fischer M. J Mol Biol. 2002;318:1317–1329. [PubMed]
29. Grahner B, Winiwarter S, Lanzner W, Muller CE. J Med Chem. 1994;37:1526–1534. [PubMed]
30. Nishigaki S, Kanamori Y, Senga K. Chem Pharm Bull. 1980;28:1636–1641.
31. Senga K, Ichiba M, Nishigaki S. J Org Chem. 1979;44:3830–3834.
32. Cresswell RM, Wood HSC. J Chem Soc. 1960:4768–4775.
33. Ross WCJ. J Chem Soc. 1948:1128–1135. [PubMed]