Development of Diversified Methods for Chemical Modification of the 5,6-Double Bond of Uracil Derivatives Depending on Active Methylene Compounds

The reaction of 5-halogenouracil and uridine derivatives 1 and 7 with active methylene compounds under basic conditions produced diverse and selective C-C bond formation products by virtue of the nature of the carbanions. Three different types of reactions such as the regioselective C-C bond formation at the 5- and 6-positions of uracil and uridine derivatives (products 2, 5, 8, 17, 20 and 21), and the formation of fused heterocycle derivatives 2,4-diazabicyclo[4.1.0]heptane (15) and 2,4-diazabicyclo-[4.1.0]nonane (16) via dual C-C bond formations at both the 5- and 6-positions were due to the different active methylene compounds used as reagents.

While 2 gave satisfactory spectral and microanalytical results consistent with the chemical structure, it was transformed into the known compound 1,3-dimethyluracil-5-acetic acid (3) [23], in a refluxing 47% HBr aqueous solution for 1 h in 95% yield to further confirm the structure. The results in Table 2 show that the rate of the reaction can be significantly affected by the type of halogens (Br, Cl and F). It is noteworthy that the reaction smoothly proceeded by the use of even 5-fluoro-1,3-dimethyluracil (1c) as the substrate although a prolonged reaction time was necessary (Entry 3). Table 2. Formation of 5-bis(ethoxycarbonyl)methyl-1,3-dimethyluracil (2). Although the highly stable C-F bond at the 5-position of the uracil ring severely retards the substitution between the F atom and malonate carbanion, there is a significant acceleration effect by the strongly electron-withdrawing nature of the F atom to reduce the electron density at the conjugated 6-position of the uracil ring, which suggests that the carbon at the 6-position undergoes a decrease in electron density compared to Br and Cl. A balance of these opposite properties seems to influence the reactivity of the 5-fluoro-1,3-dimethyluracil (1c) in a subtle way.
In relation to these results, we detected the presence of an intermediate 4 during the reaction of 1a and diethyl malonate carbanion by TLC analysis (invisible under a UV-lamp, but the spot was directly stained by iodine absorption). The intermediate 4 was isolated in 41% yield along with 33% of unchanged 1a by interruption of the reaction after a short time (2 h). The structure of the intermediate 4 was assigned by spectral and microanalytical results to 5,6-di-[bis(ethoxycarbonyl)methyl]-5,6dihydro-1,3-dimethyluracil with a trans-configuration based on the 1 H-NMR spectral data. The isolated intermediate 4 could be transformed by only stirring with sodium ethoxide in anhydrous EtOH at rt for 8 h to give the corresponding 5-bis(ethoxycarbonyl)methyl-1,3-dimethyluracil (2) in 67% yield (Scheme 2). Scheme 2. Formation of 5,6-di-[bis(ethoxycarbonyl)methyl]-5,6-dihydro-1,3-dimethyluracil (4) and its reactivity.
Based on these results, the plausible reaction mechanism for the formation of 2 involving a Michael 1,4-addition is indicated in Scheme 3. The intermediate 4 could be obtained via the generation of the C-6 malonate adduct (A) by the nucleophilic attack of a diethyl malonate carbanion on the 6-position of the uracil ring and nucleophilic substitution between the bromine atom at the 5-position (sp 3 -carbon) of A and the remaining malonate carbanion (SN 2 reaction). Subsequent C-C bond cleavage at the 6-position of 4 under basic conditions (E 2 reaction) is a key and rate-determing step for the formation of 2, and this is why the 5,6-di-[bis(ethoxycarbonyl)methyl]-5,6-dihydro-1,3-dimethyluracil (4) could be isolated (Scheme 3) [24]. Scheme 3. Plausible reaction mechanism.
Since these reactions using active methylene compounds as nucleophile sources were essential to perform under strong basic conditions due to the generation of carbanions, the use of the unprotected 5-bromouridine (6a) or 5-bromo-2'-deoxyuridine (6b) at the 3-position as a substrate were not suitable due to the formation of the inactive uracil-anion (Scheme 5).
Next we investigated the reaction of 1a with ethyl phenylacetate and benzyl cyanide in the presence of NaOEt as a base at rt. Surprisingly, and against all expectation, the 2,4-diazabicyclo[4.1.0]heptane derivatives 15a,b were obtained as the sole product (Scheme 8). The reaction was also found to proceed by the use of DBU instead of NaOEt. The structures of products 15a and 15b were supported by the spectral data and microanalytical results, and the characteristic AB pattern peaks of the bridgehead protons [15a: 3.20 (C-6-H), 3.72 (C-1-H), J 1,6 = 9.0 Hz; 15b:  3.03 (C-6-H), 3.58 (C-1-H), J 1,6 = 8.5 Hz] as a 2,4-diazabicyclo[4.1.0]heptane nucleus were also clearly observed in the 1 H-NMR spectrum [47]. Although the 2,4-diazabicyclo[4.1.0]heptane derivatives were likely the intermediates for the generation of the 5-substituted uracils [33,34], 15a was quite stable under basic conditions (NaOEt), even at the reflux temperature of EtOH.    As noted earlier in the Introduction, not so many chemical modification methods at the 6-position of uracil derivatives are reported in the literature, including the formation of 6-cyanouracil derivatives due to the cine-substitution reaction as shown in Scheme 1 [28,[30][31][32]. In other examples, Tanaka and Miyasaka et al. reported the electrophilic functionalization at the 6-position of 2',3'-isopropylidene-5'methoxymethyluridine via lithiation at the 6-position of the uracil ring [48], and the photochemicallyinduced nucleophilic substitution at the 6-position of 6-iodo-2'3'-isopropylidene-5'-methoxymethyluridine [49]. Needless to say, the normal nucleophilic substitution of the 6-halogenouracil derivatives under basic conditions has also been reported in the literature [50].
When 1a was allowed to react with ethyl acetoacetate in the presence of NaOEt in anhydrous EtOH at rt for 72 h, the 1,3-dimethyluracil-6-(α-acetyl)acetic acid ethyl ester (17) was obtained in 62% yield together with 32% of the unchanged 1a [Scheme 10; while the structure of 17 was indicated as the keto-form of the 6-substituent for the purpose of illustration, the actual structure in solution should be the enol-form with intramolecular hydrogen bond (17') based on 1 H-NMR analytical data]. The reaction also proceeded by the use of NaH as a base in anhydrous DMF. The structure of 17 was supported on the basis of the spectral data and microanalytical results, and confirmed by comparison of the spectral data with those of the product of the alternative synthesis based on the reaction using 6-chloro-1,3-dimethyluracil (19) and ethyl acetoacetate in the presence of NaH in anhydrous DMF at rt. Furthermore, 17 could be quantitatively converted into the well-known 1,3,6-trimethyluracil (18) in 98% yield after a 1 h reflux in a 47% HBr solution.

17'
The reaction sequence for the formation of 17 is outlined in Scheme 11. Although the Michael adduct (C) can be normally converted to the 6-substituted product 17 accompanied by the elimination of HBr (cine-substitution), it is rather reasonable to suggest that the reaction proceeded via the cyclic intermediate (E) generated by the intramolecular nucleophilic attack of the corresponding enolate anion of D on the 5-position under strong basic conditions [30][31][32]. We believe that is why the formation of the 6-substituted product 17 occurs when using ethyl acetoacetate. Scheme 11. Plausible reaction mechanism for the formation of 6-substituted product (17).
These diversified reactivities by virtue of the difference in the active methylene compounds may be controlled by the pK a values of the particular active methylene compounds. 5-Substituted products (i.e., compounds 2, 5 and 8) are obtained when using diethyl-and dimethyl malonate and benzylphenylketone possessing moderate acidities (pK a in DMSO: 16.4 [51], 15.9 [52] and 17.7 [53], respectively) due to the preference of the intermolecular nucleophilic attack at the 5-position of the adduct (A, Scheme 3) because of the comparatively longer life of the corresponding carbanions. On the other hand, cyclopropane and cyclopentane derivatives 15 and 16 are produced when using the less acidic active methylene compounds, such as ethyl phenylacetate, benzyl cyanide and dibenzylketone (pK a in DMSO: 22.7 [54], 21.9 [55] and 18.7 [53], respectively), due to the intramolecular nucleophilic attack at the 5-position of B (Scheme 9) in preference to the intermolecular nucleophilic attack based on excessive amounts of the active methylene compounds due to the unstable nature of the corresponding carbanions. In the case of ethyl acetoacetate (pK a in DMSO: 14.2 [56]) capable of forming an enolate (D, Scheme 11) under basic conditions, a special reactivity was observed since the intramolecular cyclization by the enolate-attack at the 5-position preferentially proceeded to give the 6-substituted products 17, 20 and 21. However, an exact rational explanation is difficult because many other active methylene compounds are not applicable to present reactions by reason of the frequent occurrence of side-reactions such as polymerization and decomposition under basic conditions.
While the synthesized 5-and 6-substituted deoxyuridine derivatives 8g-8i, 9g, 9h, 13, 14, 21b and 22b were evaluated for their antiviral activities against the herpes simplex virus (HSV), human cytomegalovirus (HCMV) and influenza A virus and cytostatic activity, these compounds unfortunately indicated no or minimal activities.

General
All reagents were obtained from commercial sources and used without further purification. Analytical thin-layer chromatography (TLC) was carried out on pre-coated Silica gel 60 F-254 plates (32-63 µm particle size) and visualized with UV light (254 nm). The 10% Pd/C was obtained from Merck KGaA or Aldrich. Flash column chromatography was performed with Silica gel 60 (40-63 µm particle size, Merck KGaA) or Silica gel 60N (100-210 µm, Kanto Chemical). The 1 H and 13 C-NMR spectra were recorded by a JEOL AL 400 spectrometer (Tokyo, Japan), JEOL EX 400 spectrometer (400 MHz for 1 H-NMR and 100 MHz for 13 C-NMR) or JEOL TNG-GX270 spectrometer (270 MHz for 1 H-NMR) with tetramethylsilane or residual protonated solvent used as a reference. Elemental analyses were carried out at the Microanalytical Laboratory of our university (YANACO CHN CORDER MT-5 instrument, Tokyo, Japan). The EI and FAB Mass spectra were obtained using a JEOL JMS-SX102A instrument (Tokyo, Japan). The UV spectra were obtained in ethanol using a Shimadzu UV-260 spectrophotometer (Kyoto, Japan). Table 2 30.0 mmol) in absolute EtOH (100 mL)] was stirred for 16 h at room temperature and then treated as described above to give 2 (2.00 g, 67%); (c) A solution of 5-fluoro-1,3-dimethyluracil (1c, 474 mg, 3.00 mmol) and diethyl malonate (1.59 g, 9.90 mmol) in ethanolic NaOEt [prepared from Na (207 mg, 9.00 mmol) in absolute EtOH (30 mL)] was stirred for 24 h at room temperature and then treated as described above to give 2 (582 mg, 65%). (3) [23]. A mixture of 5-bis(ethoxycarbonyl)methyl-1,3dimethyluracil (2) (349 mg, 1.17 mmol) in a 47% HBr solution was refluxed for 1 h. The solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel with CHCl 3 -MeOH (5:1) as the eluant to give 3 (220 mg, 95%), which was identical to the authentic sample. (4). A solution of 5-bromo-1,3dimethyluracil (1a) (657 mg, 3.00 mmol) and diethyl malonate (1.59 g, 9.90 mmol) in ethanolic NaOEt [prepared from Na (207 mg, 9.00 mmol) in absolute EtOH (30 mL)] was stirred at room temperature for 2 h. The mixture was neutralized with Amberlite CG-50 (H + ) and filtered. The ion exchange resin was washed with ethanol, and the combined filtrates were concentrated under reduced pressure. The residue was treated with H 2 O (30 mL). The aqueous solution was extracted with CHCl 3 and the extract was dried over MgSO 4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel with benzene-EtOAc (6:1) as the eluant to give the starting material 1a (217 mg, 33%) and the 5,6-dihydrouracil 4 (564 mg, 41%), which was recrystallized from EtOH. m.p. Reaction of 4 with sodium ethoxide: A solution of 4 (101 mg, 0.220 mmol) in ethanolic NaOEt [prepared from Na (14.9 mg, 0.650 mmol) in absolute EtOH (5 mL)] was stirred at room temperature for 8 h. The solvent was removed under reduced pressure and the residue was treated with H 2 O (20 mL). The solution was neutralized with c.HCl and the aqueous solution was extracted with CHCl 3 . The extract was dried over MgSO 4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel with benzene-EtOAc (6:1) as the eluant to give 1,3-dimethyluracil-5malonic acid diethyl ester (2, 44.0 mg, 67%). (5). A mixture of 5-bromo-1,3-dimethyluracil (1a) (1.32 g, 6.00 mmol) and benzylphenylketone (3.69 g, 19.8 mmol) in ethanolic NaOEt [prepared from Na (414 mg, 18.0 mmol) in absolute EtOH (60 mL)] was stirred at room temperature for 1 h. The solvent was removed under reduced pressure and the residue was treated with H 2 O (40 mL). The solution was neutralized with conc. HCl and the aqueous solution was extracted with CHCl 3 . The extract was dried over MgSO 4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel with CHCl 3 as the eluant to give 5 (1.94 g, 96%), which was recrystallized from CHCl 3

7-Cyano-1,4-dimethyl-7-phenyl-2,4-diazabicyclo[4,1,0]heptane-3,5-dione
(15b). 5-Bromo-1,3dimethyluracil (1a, 657 mg, 3.00 mmol) was added to a stirred solution of benzylcyanide (1.16 g, 9.90 mmol) in ethanolic NaOEt [prepared from Na (207 mg, 9.00 mmol) in absolute EtOH (30 mL)] and the mixture was stirred at room temperature for 10 min. The mixture was neutralized with Amberlite CG-50 (H + ) and filtered. The ion exchanger was washed with EtOH. The combined filtrates were concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel with chloroform as the eluant to give 15b (720 mg, 94%), which was recrystallized from 2,4-Dimethyl-7,9-diphenyl-2,4-diazabicyclo [4,3,0]nonane-3,5,8-trione (16). 5-Bromo-1,3-dimethyluracil (1a) (657 mg, 3.00 mmol) was added to a stirred solution of dibenzylketone (2.08 g, 9.90 mmol) in ethanolic NaOEt [prepared from Na (207 g, 9.00 mmol) in absolute EtOH (30 mL)] and the mixture was stirred at room temperature for 30 min. The mixture was neutralized with Amberlite CG-50 (H + ) and filtered. The ion exchange resin was washed with EtOH. The combined filtrates were concentrated under reduced pressure, and the residue was purified by chromatography on silica gel with CHCl 3 as the eluant to give 16 (638 mg, 61%), which was recrystallized from chloroform-ether. m.p. 220-221 °C;   (17). (a) A solution of 5-bromo-1,3dimethyluracil (1a, 2.20 g, 10.0 mmol) and ethyl acetoacetate (4.32 g, 33.0 mmol) in ethanolic NaOEt [prepared from Na (690 mg, 30.0 mmol) in absolute EtOH (100 mL)] was stirred for 3 days at room temperature. The mixture was evaporated under reduced pressure, and the residue was treated with H 2 O. The resulting precipitate was filtered off, and the mother liquor was extracted with CHCl 3 . The extract was dried over Na 2 SO 4 , and the solvent was removed under reduced pressure. The residue was treated with Et 2 O and the resulting precipitate was filtered off. The combined precipitate was washed with Et 2 O to afford the recovered (1a) (700 mg, 32%), which was identical to the authentic sample. The water layer was neutralized with c.HCl, and the mixture was extracted with CHCl 3 . The extract was concentrated in vacuo, and the residue was purified by column chromatography on silica gel with CHCl 3 as the eluant to give 17 ( 6.60 mmol) in anhydrous DMF (5 mL) was added sodium hydride (60% in mineral oil) (240 mg, 6.00 mmol). The mixture was stirred at room temperature for 5 days, and the solvent was removed under reduced pressure. The residue was dissolved in H 2 O (20 mL) and then washed with CHCl 3 . The aqueous layer was neutralized with conc. HCl and extracted with CHCl 3 . The extract was dried over Na 2 SO 4 , and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with CHCl 3 as the eluant to give 17 (311 mg, 58%), which was identical to the sample prepared above.