Synthesis of Dihydrouracils Spiro-Fused to Pyrrolidines: Druglike Molecules Based on the 2-Arylethyl Amine Scaffold

The synthesis of a small library of dihydrouracils spiro-fused to pyrrolidines is described. These compounds are synthesized from β-aryl pyrrolidines, providing products with the 2-arylethyl amine moiety, a structural feature often encountered in compounds active in the central nervous system. The β-aryl pyrrolidines are synthesized through a three-step methodology that includes a Knoevenagel condensation reaction, a 1,3-dipolar cycloaddition reaction, and a nitrile reduction.

It is known that the difference in activity and receptor selectivity of drugs might be explained by the conformation of the contained privileged structure. Generation of semi-rigid drugs facilitates the study of their interactions with the receptors, may lead to more selective interactions with fewer side effects, and permits the rational design of more potent and selective drugs in the future [18,19]. Herein we present the parallel synthesis of a library comprised of compounds combining all the above features (Scheme 1). First, these compounds possess a β-aryl pyrrolidine with a conformationally constrained 2-arylethyl amine. Second, they are semirigid structures because of the spiro fusion to a dihydrouracil. It is known that the combination of privileged structures can lead to new chemical entities that may have pharmacological relevance [20,21] and increase the structural diversity. Scheme 1. Combination of two privileged structures to generate a product with increased rigidity.

Results and Discussion
We envisioned that a suitable strategy to synthesize these compounds may proceed as shown retrosynthetically in Scheme 2. The spiro dihydrouracils 5 could be synthesized by an annulation reaction of α-aminomethyl esters 6 and an isocyanate. The aminomethyl group of compounds 6 could be derived from a masked amino function, such as a cyano group, by reduction. Compounds 7 possess two electron-withdrawing groups at the carbon in the 3-position of the pyrrolidine, rendering it a perfect pattern for preparation by a 1,3-cycloaddition reaction of an azomethine ylide and an electron-deficient alkene. This would leave the 3-aryl-2-cyanoacrylates 8 as starting materials, which could be obtained by the Knoevenagel condensation reaction of methyl 2-cyanoacetate (9) and an aromatic aldehyde.

Scheme 2.
Retrosynthetic analysis for the synthesis of spiro dihydrouracils 5.

Knoevenagel condensation reaction
The synthesis of compound class 5 commenced with the condensation reaction of methyl 2cyanoacetate (9) and aromatic aldehydes 10. Twelve aldehydes 10{1-12} (electron-rich aromatic, electron-rich heteroaromatic, and electron-poor heteroaromatic aldehydes; Figure 2) were selected for the formation of the scaffolds.  The reaction conditions for the Knoevenagel condensation reaction are critically dependent on the electron-withdrawing groups bound to the activated methylene [22] and need to be optimized in every case. Initially, these reactions were performed using EtOH as solvent, but transesterification (up to 3%) was observed and the resulting mixture of methyl and ethyl esters was impossible to separate. These reactions also took place in THF (see entry 8, Table 1), but required a longer reaction time. Finally, treatment of 9 with a catalytic amount of piperidine in MeOH (except for entry 8 because of the low solubility of 10{8} in MeOH) at room temperature produced the desired acrylates 8{1-12} in excellent yields (Table 1). All these compounds are only sparingly soluble in MeOH, allowing the pure crystalline products to be easily collected by filtration. These products can also be recrystallized from MeOH yielding crystals of >99.5% purity. The reaction was completely stereoselective in all cases [23], only the E alkenes were observed as could be inferred from the 13 C-NMR coupling constants between the olefinic proton and the carbon atoms of the ester and the nitrile [24,25]. These values are 3 J = 6.6-6.9 Hz for the carbonyl group and 3 J = 13.6-13.9 Hz for the cyano group. The Knoevenagel adducts are stable at room temperature and unreactive towards the regular atmosphere, therefore remain unchanged for months.

1,3-Dipolar cycloaddition reaction
The next step in the synthesis was the formation of the pyrrolidine-core structures by a 1,3-dipolar cycloaddition reaction using an azomethine ylide. This reaction is an important method for the formation of pyrrolidines [26] and has been used in the synthesis of natural products [27,28]. Among the vast number of procedures for making azomethine ylides [29][30][31][32][33][34][35][36][37][38][39][40][41], the decarboxylative condensation of α-amino acids with aldehydes, typically heated in toluene or DMF, was chosen [42]. Thus, the reaction of paraformaldehyde and sarcosine (N-methylglycine) in refluxing toluene in the presence of the 2-cyanoacrylates 8 cleanly provided the desired pyrrolidines 7, containing the 2arylethyl amine motif ( Table 2). The reaction was clean to such an extent that in some cases an extraction (H 2 O/Et 2 O) was all the purification needed (or just a short column chromatography). The reaction was totally stereospecific in most cases (entries 1-6 and 8), highly stereospecific for 8{9} (entry 9), and partially stereospecific for the electron-poor heteroaryls 8{10-12} (entries 10-12) [43]. The mixtures of diastereoisomers that arose could not be separated by column chromatography. The reaction did not take place with compound 8{7} (entry 7); after 4 h only some minor unidentified compounds were formed and most of the substrate was recovered, but there was no trace of compound 7{7}. The reaction of substrate 8{5} did form the product 7{5}, but with a lower yield compared to all the others. The reaction of substrate 8{8} formed the expected cycloadduct, but the indolic nitrogen was (dimethylamino)methylated during the reaction. After analysis of the results obtained so far, it was decided to continue the research only with the diastereomerically pure compounds 7{1-6} for the construction of the library scaffolds.

Reduction
The chemoselective reduction of the nitrile was best achieved by a heterogeneous catalytic hydrogenation using Raney nickel under a hydrogen atmosphere at room temperature (Table 3) [44]. We found that the addition of NH 3 or Et 3 N was crucial for the reaction to go to completion [45]. Eventually, Et 3 N was used since with NH 3 amide 12 (Figure 3) was formed alongside the product. Thus, compounds 7{1-6} were reacted under these conditions to complete the synthesis of the library scaffolds. After elimination of Raney nickel by filtration through diatomaceous earth and evaporation of MeOH and Et 3 N, the reaction cleanly gave the α-aminomethyl esters 6{1-6}.

Parallel synthesis of spiro dihydrouracils
The procedure followed for the formation of the spiro dihydrouracils was formation of a urea by addition of an isocyanate and subsequent cyclization by reaction with a base. The conversion of chemset 6 into chemset 5 was accomplished using reagent chemset 13. Eight isocyanates 13{1-8} (alkyl, electron-rich aryl, electron-poor aryl, and heteroaryl isocyanates; Figure 4) were selected for the generation of a 48-compound library.  The reactions for the formation of the α-ureidomethyl esters were run in either CH 2 Cl 2 or DMF depending on reagent solubility and reactivity. Thus, the reactions of 6{1-6} and 13{1,3-5} in CH 2 Cl 2 for 15 h at room temperature afforded the corresponding α-ureidomethyl esters. In order to reach full conversion to the α-ureidomethyl esters using isocyanates 13{2,6-8}, DMF at 80 °C for 15 h had to be used. After evaporation of the solvent, the crude mixture was dissolved in THF and 1 M KOBu t in THF (1 equiv) was added [46,47]. The reactions were stirred at room temperature for 15 h and the solvent was evaporated. Liquid-liquid extraction afforded two different types of compounds, depending on the isocyanate used: (1) the alkyl isocyanates 13{1-3} gave the expected 4-arylspiro[dihydrouracil-5,3′-pyrrolidines] 5{1-6,1-3} with yields ranging from 49 to 80% (61% average) and with purities ranging from 60 to 99% (83% average; Scheme 3 and Table 4) according to LC-MS analysis (also confirmed by 1 H-NMR spectroscopy) and (2) the aryl isocyanates 13{4-8} gave mostly the unexpected α-ureidomethyl acids 14{1-6,4-8} with yields ranging from 45 to 79% (64% average) and with purities ranging from 0 to >99% (82% average; Scheme 4 and The reactions carried out with reagent 13{1} resulted in high purities (89% average) for the formation of the spiro dihydrouracils, due to lack of competing reactions. The purities of the products from the reactions run in DMF (reagent 13{2}) were in the range 60 to 92% (70% average). These lower purities could be due to partial decomposition of the isocyanates at the temperature used for the reactions in DMF. The reactions carried out with reagent 13{3} gave a mixture of the expected ethyl esters 5{1-6,3}, the methyl esters 5{1-6,9} (from transesterification of the ethyl ester on the R group by methoxide, formed in the cyclization), the acids 5{1-6,10} (from hydrolysis of the esters), and the deorganylated compounds 5{1-6,11} ( Figure 5 and Table 4). The overall cyclization reaction worked well, since products 5{1-6,9}, 5{1-6,10}, and 5{1-6,11} were formed from 5{1-6,3}. Shorter reaction times should thus be used to avoid these side reactions.  The above-mentioned deorganylation side reaction could have taken place through an E1cB mechanism (Scheme 5). The substrate 5{1-6,3} (or 5{1-6,9}) is deprotonated to form the enolate 5{1-6,12}, which undergoes an elimination reaction to afford 5{1-6,11} after work-up.  The aryl-substituted dihydrouracils underwent hydrolysis (and not the alkyl-substituted dihydrouracils) because the electrophilicity of the ureide carbonyls is enhanced (with respect to the alkyl group) due to the conjugation of the imide-type nitrogen with the aryl group. Thus, residual H 2 O from KOBu t could have hydrolyzed the ureide to the ureido acid [48].    In order to find conditions for the exclusive formation of the spiro dihydrouracils using aryl isocyanates, the α-ureidomethyl ester 15{1,4} was synthesized, isolated, and reacted with several bases under different conditions for the formation of spiro dihydrouracil 5{1,4} (Table 6).
Firstly, the reaction was attempted with an easy-to-handle base because, if successful, it would make the work-up of the reactions easy-an important factor in parallel synthesis. All the amines used were found to have insufficient basicity for this transformation to take place (entries 1-5) [49][50][51]. The amidine DBU gave promising results, but the separation of the product 5{1,4} from DBU (and especially from the coreagent Bu 4 NBr, entry 8) was difficult and tedious, making these reaction conditions unsuitable for parallel synthesis [52,53]. Potassium tert-butoxide was the only base that caused >99% of the starting material to react [54], but it was the base that gave the largest amount of hydrolyzed product 14{1,4} (entries 9-12). Heating only (entry 15) resulted in decomposition of the starting material. To the best of our knowledge, there is no example in the literature of a dihydrouracil ring with such a tendency toward hydrolysis under basic conditions. This cyclization can also take place using acid catalysis [55][56][57][58], but this has not yet been attempted. The compounds 5{1-6,1-3}, 5{1,4}, 6{1-6}, 14{1-6,4-8}, and 15{1,4} were tested on different CNS targets, but the results cannot be published because of the patent policy of the companies involved in the project.

General
Reagents were obtained from commercial suppliers and were used without purification. Solvents were distilled from appropriate drying agents prior to use and were stored under nitrogen. Reactions were followed, and R F values were obtained, using thin-layer chromatography (TLC) on silica gelcoated plates (Merck 60 F254) with the indicated solvent mixture. Detection was performed with UV light and/or by charring at ca. 150 °C after dipping into a solution of KMnO 4 or ninhydrin. Column or flash chromatography was carried out using ACROS silica gel (0.035-0.070 mm, pore diameter ca. 6 nm). IR spectra were recorded on an ATI Mattson Genesis Series FTIR spectrometer. Highresolution mass spectra were recorded on a JEOL AccuTOF (ESI) or a MAT900 (EI, CI, and ESI). Low-resolution ESI mass spectra were recorded on a Thermo Finnigan LCQ Advantage Max Ion Trap mass spectrometer. Elemental analyses were carried out using a Carlo Erba Instruments CHNS-O EA 1108 element analyzer. Melting points were analyzed with a Büchi melting point B-545 and are not corrected. Gas chromatography (GC) was performed on a Hewlett Packard 5890, containing a HP1 column (25 m x 0.32 mm x 0.17 μm), FID detection, and equipped with a HP3393A integrator. NMR spectra were recorded at 298 K on a Bruker DMX 300 (300 MHz) or a Varian 400 (400 MHz) spectrometer in the solvent indicated. Chemical shifts are given in parts per million (ppm) with respect to tetramethylsilane (0.00 ppm) or CD 3 SOCHD 2 (2.50 ppm) as internal standard for 1 H-NMR; and CDCl 3 (77.16 ppm) or CD 3 SOCD 3 (39.52 ppm) as internal standard for 13 C-NMR [59]. Coupling constants are reported as J values in hertz (Hz). Multiplicity data are denoted by s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), b (broad), and app (apparent). Peak assignment in 13 C spectra are based on 2D gHSQC and gHMBC spectra, and DEPT 135 when needed. Chain numbering corresponds to IUPAC nomenclature, so unprimed atoms belong to the principal chain, primed atoms belong to the first named substituent, doubled-primed atoms to the second named substituent, etc. LC-MS measurements were run on a Shimadzu LC-10A VP series liquid chromatography system, equipped with an SPD-10A VP UV-vis detector and a LCMS-2010A mass spectrometer. The column used for the LC analysis was an Agilent Zorbax Extend C18 (3.5 μm, 4.6 × 150 mm), and it was eluted at 1 mL/min with a gradient made up of two solvent mixtures. Solvent A consisted of 0.1% trifluoroacetic acid in water and solvent B consisted of 0.1% trifluoroacetic acid in acetonitrile. The gradient was run as follows: t ) 0 min, 50% A; t ) 5 min, 5% A; t ) 10 min, 5% A; t ) 12.5 min, 50% A; t ) 20 min, 50% A. A wavelength of 215 nm was selected for the analysis of purity.

General procedure for Knoevenagel condensation reaction
Piperidine (5 drops) was added to a solution of methyl 2-cyanoacetate (9) and the aldehyde 10 (1.0 equiv) in MeOH. The resulting reaction mixture was stirred at room temperature for the time indicated in each case. The reaction mixture was filtered and the precipitate was recrystalized from MeOH. The filtrate was concentrated under reduced pressure and purified by recrystalization from MeOH.

General procedure for 1,3-dipolar cycloaddition reactions of 8
A round-bottomed flask fitted with a Dean-Stark apparatus, a reflux condenser, and a drying tube containing calcium chloride was charged with 2-cyanoacrylate 8 and toluene (0.20-0.25 M). When the mixture was under reflux, sarcosine (N-methylglycine; 1.2 equiv) and paraformaldehyde (3.6 equiv) were added. This addition was repeated every 40 min until the substrate had completely reacted. Water (20 mL) was then added and the layers were separated. The aqueous layer was extracted with Et 2 O (3 × 30 mL) and the combined organic layers were dried (MgSO 4 ), filtered, and concentrated in vacuo.

General procedure for reduction
An excess (7-8 heaped teaspoons) of freshly washed (with MeOH) Raney nickel was added to a solution of α-cyano ester 7 with Et 3 N (ca. 1 equiv) in MeOH (0.25-0.30 M). The mixture was stirred for 15 h at room temperature under a hydrogen atmosphere (1 atm). The catalyst was separated by filtration with suction through a glass filter with a 0.5 cm layer of diatomaceous earth. The catalyst was washed thoroughly with MeOH. The combined methanolic solutions were concentrated on a rotary evaporator.

Parallel synthesis
3.5.1. General procedure 1 for spiro dihydrouracil/α-ureidomethyl acid formation using parallel synthesis A solution of isocyanate 13 (0.12 mmol for 1, 0.10 mmol for 3-5) from a 0.3 M stock solution in CH 2 Cl 2 was added to a solution of α-aminomethyl ester (0.10 mmol) in CH 2 Cl 2 (1.5 mL). The resulting reaction mixture was stirred at room temperature for 15 h. After that time, the solvent was evaporated and THF (1.5 mL) and 1 M KOBu t in THF (0.10 mmol) were added. The reaction mixture was then stirred at room temperature for 15 h. A saturated solution of NH 4 Cl (1.0 mL) was added and the layers were separated (centrifugation was needed for the separation when aryl isocyanate was used). The aqueous layer was extracted with CH 2 Cl 2 (2 × 1.5 mL) and the combined organic layers were evaporated to dryness under vacuum.
We have also developed a method for the synthesis of a small library of 7-alkyldihydrouracils spirofused to pyrrolidines to the 3-position. The corresponding 7-aryl derivatives hydrolyzed under the conditions utilized for the cyclization and yielded α-ureidomethyl acids. The optimization of this cyclization should be further developed.