Solid phase synthesis of 5,6-disubstituted pyrimidinone and pyrimidindione derivatives

Solid phase synthesis of 5,6-disubstituted pyrimidinone and pyrimidindione derivatives by the use of the Syncore ® Reactor is described starting from easily available building blocks


Introduction
In the last few years pyrimidinone and pyrimidindione derivatives substituted either at the C-5 and C-6 position have emerged in the field of chemotherapy. 1 Among the important 6-substituted uracil derivative HEPT and its analogues, 2 Emivirine (EMV) 3 has been chosen as a candidate for clinical trials, and DABOs, 4 showed a potent and selective activity against HIV-1 (Figure 1).
In this context, the finding that C-6 substituted pyrimidinone and pyrimidindione derivatives showed selective antitumor, 5 antiviral, 6 antitubercular, 7 antifungal activity, 8 suggests the importance of testing this family of compounds as broad-spectrum drugs.In the last few years, we have been involved in the synthesis and biological evaluation of new pyrimidindione and pyrimidinone derivatives as potential antitumor and antiviral agents. 9We have described 10 one of the most reliable synthesis of 2-methoxy and 2-thiomethyl 5,6disubstituted pyrimidinone derivatives by condensation of O-methylisourea, or Smethylisothiourea with β-ketoesters in H 2 O/EtOH mixture in the presence of Ca(OH) 2 .Their versatility to be transformed into uracils, 9i,10 and thiouracils 9i has also been shown.By modification of the substitution in virtually all the six positions of the pyrimidinone nucleus we were able to synthesize compounds provided with interesting activity against HIV-1, 9g ASFV, 11 Sendai virus 9g and Rubella virus.Combinatorial chemistry, a technique that allows the synthesis of large numbers of molecules by varying combinations and permutations of modular components, has changed the nature of chemical discovery. 13Solid-phase synthesis (SPS) 14 is playing a decisive role in the ongoing development of combinatorial chemistry, mainly because it offers striking advantages in terms of synthetic flexibility, such as: (i) the ease of chemistry; (ii) the possibility of using high concentrations of reagents to drive reactions to completion; (iii) the elimination of purification steps en route, since impurities and excess reagents can be removed by simple washing of the solid support; (iv) the straightforward nature of parallel SPS; (v) the possibility of automation.
Based on these observations, our efforts were directed towards the elaboration of combinatorial methods, to be developed on solid support, which take advantage of the synthetic strategy elaborated in our laboratories, and the use of a semiautomatic synthesizer for generating pyrimidinone derivatives substituted either at C-5 and C-6.

Results and Discussion
We have already described 9f a versatile solid phase approach for the synthesis of a series of pyrimidinone derivatives.In the key step, a polymer-bound thiouronium salt 1 is condensed with different β-ketoesters by adding an excess of Ca(OH) 2 in water-ethanol solution (Scheme 1).As result, the corresponding pyrimidinone system 2 is anchored to the solid support (Merrifield resin) through a tetramethylene spacer which is important in order to create a "chemical distance" between the uracil nucleus and the polymer backbone, to confer more "solution like" properties and better solvent compatibility to the resin.The resulting thio-ether bond is stable to alkaline conditions, nucleophilic attack and is fairly stable to acids, so that the uracil moiety can be easily derivatized before being cleaved from the solid support.It is well known that n-alkyl thioethers are difficult to be cleaved and hence have not been extensively used as protecting groups; as a result, the most important issue to be addressed was the identification of a suitable cleavage methodology.In the literature many cleavage methodologies have been reported 7b,15 which proved to be unsuccessfull for the cleavage of polymer-bound pyrimidinones 2. Oxone ® , potassium hydrogen persulfate, is a reagent used for the oxidation of sulfides to sulfones.Treating 2 with Oxone ® , in a solution of dioxane/H 2 O at reflux temperature, a very reactive sulphone intermediate is obtained which is then subjected to nucleophilic attack by water leading to the cleavage of the uracil derivative 3. 9e Accordingly, when the oxidation with Oxone ® is performed in a methanolic solution, 2-methoxy derivatives 4 are obtained.

HN
Considering the difficulties encountered in the cleavage step, we turned our attention to the use of the Wang resin characterized by the presence of a benzylic spacer, extensively used as a protecting group for thio groups.The polymer-bound thiouronium salt 5, obtained following a procedure developed in our laboratories, 9e,f was condensed with different β-ketoesters (50 equiv) in a water-ethanol mixture affording the corresponding pyrimidinones 6. Oxone ® cleavage procedure applied to 6 gave compounds 3 and 4 in comparable yields.As a consequence of be using the acid labile Wang linker, the corresponding 2-thio uracils 7 (Scheme 2) could be accessed from 6, in similar yield and purity that 3 and 4, by a benzylic-type cleavage using 5% TFA in CH 2 Cl 2 .
The cleavage with greater amounts of TFA (10% and 25%) gave mixtures of compounds 7a and 8 (Scheme 3) characterized by the presence of growing percentages of 8. Considering that Merrifield and Wang resins are hydrophobic in nature, whereas the cyclization reaction and the Oxone ® cleavage were performed in polar solvent (water, ethanol), Tentagel ® resin was finally investigated for pyrimidinones synthesis as this polymer is quite hydrophilic and readily solvated by polar solvents.Unfortunately, Oxone ® cleavage methodology applied to Tentagel ® resin supported pyrimidinones afforded the desired compounds in poor yields together with byproducts derived from the degradation of the solid support itself.As described in Scheme 1, treating the polymer supported thiouronium salt 5 with different βketoesters afforded highly diverse pyrimidinones 6 substituted at positions 5 and 6, due to the incorporation of different β-ketoester residues.The use of three different cleavage methodologies gave differently modified pyrimidinones at position 2.This provides a good centre of diversity for producing libraries in the search of lead identification and lead optimization.
The use of three different cleavage methodologies to pyrimidinones 6 gave differently modified pyrimidinones at 2. This provides a good centre of diversity for producing libraries in the search of lead identification and lead optimization.
The availability of a large number of diverse β-ketoesters, which can be either commercially available or easily prepared in a few steps from commercial starting materials, makes them good building-blocks.Moreover, the easiness of synthetic methodologies of this solid phase protocol provides the possibility of automation of reactions.
As a result, we have focused our attention on a parallel version of this synthesis by using a Buchi Syncore ® Reactor.This semiautomatic synthesizer is an instrument able to provide up to 24 reactions in parallel, comprehending the vacuum evaporation steps, filtration and washing of the resins, without cross contamination problems and avoiding some boring passages, with final huge time recovery.The vortex/shaking system provided for the Syncore ® ensures complete mixing of the samples without the use of a stirrer which could damage delicate solid-phase beads.
Our first approach was the search of the ideal conditions to optimize the cyclization reaction on the resin in the Syncore ® .This is, in fact, a tricky point being the step from the flask to the Syncore ® not so straightforward.
In a classical experiment, cyclization was performed in the presence of Ca(OH) 2 in a 1:1 mixture ethanol: water (25 ml for 500 mg of Wang resin) at 25°C for six days using 50 equivalents of β-ketoester.Under these conditions 6-methyluracil was obtained in 95% yield after oxidation and nucleophilic cleavage by water.
Therefore the cyclization step suffers from some drawbacks such as: (i) long time of reaction (six days), (ii) large excess of β-ketoesters (50 equivalent are very expensive!),(iii) large amount of solvent.Cyclization performed under the same experimental conditions by using Syncore ® (the vortex/shaking system provided for the Syncore ® is unable to stir a great mass of solvent) afforded 6-methyl uracil in low yield (62%) after Oxone ® cleavage in dioxane/H 2 O.
It was therefore noticed that the Syncore ® vortex/shaking system was unable to maintain the polymer and Ca(OH) 2 suspended in the solvent mixture and three different layers could be clearly observed in the reaction vessel: the resin on the bottom, a white layer of Ca(OH) 2 and a large mass of solvent on the top.The pH of the reaction was observed to be 5 all the time.
A few points were then taken into consideration to improve the yield: (i) reduction of the solvent amount and β-ketoester equivalents, (ii) study of the effect of a soluble base (NaOH) on the reaction course, with constant monitoring of the pH of the solution, to avoid hydrolysis of βketoesters, (iii) use of dioxane as co-solvent in order to improve the swelling of the resin, (iv) study of the effect of the time on the cyclization reaction.
All the effects of these variables were studied at the same time, by preparing nine resins and using Syncore ® according to Scheme 4.  Each trial was performed on 500 mg of resin, suspended in 6 ml of solvent and using 5 equivalents of ethyl acetoacetate.The loading of the resin 6a was, in all cases, established by the cleavage of 6-methyl uracil by treatment with Oxone ® using dioxane/H 2 O as solvents.In order to drive reaction to completion, the cleavage step, performed in a flask, was repeated twice under the same experimental conditions.After the cleavage the yield of 6-methyl uracil was determined and used as discriminant.
In three experiments the cyclization reaction was performed in an ethanol:water mixture in the presence of Ca(OH) 2 at 25°C.The reaction was carried on for 24 h (entry a) and 72 h (entry b) respectively; in the third experiment (entry c) the cyclization was performed for 72 h then the resin was filtered and the cyclization step was repeated under the same experimental condition for 48 h.
In another set of three experiments (entries d-f) the cyclization reaction was performed in a similar fashion but using dioxane as co-solvent in order to improve the swelling of the resin.We observed that Ca(OH) 2 is not completely soluble in this solvent mixture and that a pH of 9 was maintained all of the time.
In another two experiments the cyclization reaction was performed in the presence of NaOH in an ethanol:water:dioxane mixture at 25°C for 24h (entry g) and 72 h (entry h).NaOH is completely soluble in this solvent mixture; the pH was determined to be 10 the first day, decreasing to 8 during the other days.In a third experiment (entry i) the cyclization was performed for 72 h, then the resin was filtered and a further cyclization step was performed in the same experimental conditions for 48 h at pH 10.
As we can observe in Table 1, reduction of solvent amounts improved the loading of 6methyluracil on the resin, whereas a long time of reaction decreased the yield; in fact better yields were obtained after only one day under all experimental conditions.The use of dioxane as co-solvent in order to improve the swelling of the resin did not improve the loading significantly.
Considering that cyclization on a polymer-bound thiouronium salt 5 in presence of NaOH in H 2 O/EtOH/dioxane at 25°C for 24 h (entry g) resulted to be the best choice using a Syncore ® Reactor, we then focused our attention on the synthesis of a small collection of molecules using a parallel approach.Compound 5 was treated with different substituted β-ketoesters (Scheme 5) under the former conditions to afford compounds 6.The resulting polymer-bound 5,6disubstituted pyrimidinones were then cleaved from the resin employing the three multidirectional cleavage strategies (both in the flask and using Syncore ® ) leading to the final products 3, 4, and 7 in good to excellent yields.
In conclusion, diverse uracils and thiouracils were obtained with this methodology in comparable yields with that which has been described, with the advantage of using a smaller amount of β-ketoester (10 times less) and in a shorter time (1 day instead of 6).A second attempt to further functionalize the polymer supported pyrimidinone was made.To this aim compound 6a was first chlorinated at position 4 using a procedure already described in solution 16 and then cleaved using both TFA and Oxone ® strategies (Scheme 6).Treatment of the polymer-bound pyrimidine derivative 9 with 5% TFA proved to be unsuccessful whereas, using 25% TFA, 10 was isolated deriving from the cleavage of the ethereal bond of the linker, thus confirming the chlorination at position 4. On the other hand, treatment of 9 with Oxone ® afforded 6-methyluracil.In order to investigate the step of chlorine displacement, a polymer-supported pyrimidine 11 (already prepared in our laboratories) not susceptible to a nucleophilic attack was treated with Oxone ® (Scheme 7).After 48 h other 10 eq of β-ketoester were added.After 72 h the polymer was filtered, washed successively with hot H 2 O (3 x 10 mL), EtOH (3 x 10 mL), CH 2 Cl 2 (3 x 10 mL), and Et 2 O (3 x 10 mL).The reaction was repeated twice; after filtration and washing the polymer-was dried in vacuo at 20 °C for 4 h.
Polymer-bound pyrimidinones 6a-n: automatic procedure Polymer 5 was divided in 12 portions.Each sample of resin (500 mg, 0.5 mmol functional group) was swollen in dioxane (2 mL) for 1 h shaking at 200 rpm, then ethanol (2 mL) was added and reaction was shaked for an additional hour.A solution of NaOH (20 mg, 0.5 mmol) in water (2 mL) and the opportune β-ketoester (2.5 mmol) were added into 2 h time and the reaction mixtures were shaked at room temperature for 24 h.The resins were then filtered, washed successively with H 2 O (3 x 50 mL), MeOH (3 x 50 mL), CH 2 Cl 2 (3 x 50 mL) and hexane (50 mL) and dried in vacuo at 20 °C for 4 h using Syncore ® Filtration Unit and vacuum pump.

5,6-Disubstituted pyrimidine-2,4(1H,3H)-diones 3a-d: classical procedure
Polymer 2 (1g, 1.2 mmol of functional group) or polymer 6 (1 g, 1 mmol of functional group) was suspended in dioxane (18 mL) and swollen for 10 min.A solution of Oxone ® (3 eq) in H 2 O (3 mL) was added and the reaction mixture was stirred overnight at reflux temperature.The polymer was then filtered, washed with MeOH (3 x 10 ml) and the organic solution evaporated under reduced pressure to give the desired products 3a-d.Reaction was repeated twice in the same conditions.3a.mp 317 °C (lit 17   The resins were filtered by using Syncore ® Filtration Unit and the solvent was evaporated by using a Syncore ® vacuum pump to yield compounds 3(a-l) in good purity.Reaction was repeated twice in the same conditions.3e.mp 330-332 °C (lit 20

Classical procedure
Polymer 2 (1g, 1.2 functional group) or polymer 6 (1 g, 1 mmol functional group) was suspended in methanol (18 mL) and swollen for 10 min.Oxone ® (3 eq) was added and the reaction mixture was stirred overnight at room temperature.The polymer was then filtered, washed with MeOH (3 x 10 mL) and the organic solution evaporated under reduced pressure to give the expected product 4a-d.Reaction was repeated twice in the same conditions.

Automatic procedure
Polymer 6 was divided in 4 portions.Each sample (500 mg, 0.5 mmol functional group) was swollen in methanol (6 mL) for 1 h, then Oxone ® (922 mg, 1.5 mmol) was added and the reaction was shaked overnight at 300 rpm at reflux temperature.The polymer was filtered by using Syncore ® Filtration Unit and the filtrate was evaporated by using Syncore ® vacuum pump to afford compounds 4a-d in good purity.The resin was filtered, washed with MeOH (3x10 mL), CHCl 3 (2x10 mL), CH 2 Cl 2 (2x10 mL) and Et 2 O (3x10 ml).The organic solution was concentrated in vacuo to give 7a-d in good purity.

Automatic procedure
Polymer 6 was divided in 4 portions.Each sample (500 mg, 0.5 mmol functional group) was swollen in dry CH 2 Cl 2 (5.7 mL) for 1h, then TFA (0.3 mL) was added and the reaction was shaked for 12 h at 200 rpm at room temperature.The resin was then filtered by using Syncore ® Filtration Unit and TFA was evaporated by using Syncore® vacuum pump to afford compounds 7a-d in good purity.

Scheme 4 .
Scheme 4. Optimization of the cyclization reaction by the use of Syncore ® Reactor.

Table 1 .
Results of the optimization of the cyclization reaction by the use of Syncore ® Reactor © ARKAT USA, Inc

in Honor of Prof. Vincenzo Tortorella ARKIVOC 2004 (v) 349-363 ISSN 1424-6376 Page 361
(1g, 1 mmol functional group) was suspended in dry CH 2 Cl 2 (19 mL) and swollen for 10 min.TFA (1 mL) was then added and the suspension was stirred at room temperature for 12 h.