PEG 400/Cerium Ammonium Nitrate Combined with Microwave-Assisted Synthesis for Rapid Access to Beta-Amino Ketones. An Easy-to-Use Protocol for Discovering New Hit Compounds

Compound libraries are important requirement in target-based drug discovery. In the present work, a small focused compound library based on β-aminoketone scaffold has been prepared combining microwave-assisted organic synthesis (MAOS) with polymer-assisted solution phase synthesis (PASPS) and replacing reaction workup standard purification procedures with solid phase extraction (SPE). Specifically, the effects of solvent, such as dioxane, dimethylformamide (DMF), polyethylene glycol 400 (PEG 400), temperature, irradiation time, stoichiometric ratio of reagents, and catalysts (HCl, acetic acid, cerium ammonium nitrate (CAN)) were investigated to maximize both conversion and yield. The optimized protocol generally afforded the desired products in satisfying yields and purities. The designed library is a part of our current research on sigma 1 receptor modulators, a valuable tool for the identification of novel potential hit compounds.


Introduction
Identifying hit compounds is the first step in the complex drug-discovery process, and the degree of structural diversity is an important element, enhancing the rate of success in finding a potential lead candidate. In this context, β-amino carbonyl compounds represent a class of important pharmacophores and useful building blocks for the synthesis of diverse classes of biologically active molecules [1,2].
In light of these considerations and as part of our ongoing research, we herein focus on the development of an efficient protocol based on the three-component one-pot Mannich reaction for the preparation of a β-amino ketone small library endowed with general formula A (Figure 2), consisting of a tertiary amine bridged to an aromatic ring by a propylenic chain. The final aim is to discover new potential sigma receptor (SR) modulators [29][30][31][32]. We set up an efficient, clean, quick, and scalable protocol based on microwave-assisted organic synthesis (MAOS), using cerium ammonium nitrate (CAN) as a catalyst and polyethylene glycol 400 (PEG 400) as a solvent, combined with polymer-assisted solid phase synthesis (PASPS). Purification of final compounds occurred by solid phase extraction (SPE). Overall, our strategy led us to obtain the desired β-amino ketones efficiently and quickly.

Results and Discussion
Through this procedure, a small focused library of 36 β-amino ketones derived from the coupling of aryl-ketones 1-6 with amines a-f (Figure 3) was prepared. Relying on our long experience in the SR field, both building blocks were selected by taking into account the state-of the-art structure activity relationship (SAR) of SR ligands [33,34]. We exploited aromatic or heterocyclic methyl-ketones (1-6) and cyclic (a, d, f) or benzyl acyclic (b, c, e) secondary amines ( Figure 3).

Setup and Optimization of Synthetic Protocol
According to data in the literature concerning the different reactivities of secondary amines related to their structures and experimental conditions in the Mannich reaction [25,35], we set up a novel protocol using the cyclic and acyclic amines a and b as "building block" models. First, compounds 1a and 1b were synthesized with conventional heating, applying an existing protocol ( Figure 4, condition A), and were properly purified [36]. Molar extinction coefficients of acetophenones 1, 1a, and 1b were determined (1.265 × 10 4 , 6.327 × 10 3 , and 6.703 × 10 3 L·mol −1 ·cm −1 , respectively) and high performance liquid chromatography-ultraviolet-photodiode array detector (HPLC-UV-PAD) methods were devecloped to determine the percentage of conversion and purity of new compounds. Afterwards, based on our own experience, we set up a microwave-assisted synthetic protocol ( Figure 4) to obtain our β-amino ketones 1-6, a-f. Of note, MAOS has already been successfully employed in Mannich reactions [37,38]. Microwave oven parameters (i.e., temperature, irradiation power, and time) were explored and different solvents, such as dioxane, dimethylformamide (DMF), tetrahydrofuran (THF), methanol (MeOH), ethanol (EtOH) tested. Temperature and irradiation power varied from 35 • C to 200 • C and from 60 W to 200 W, respectively, as did irradiation time. Lastly, both type and amount of protic acidic additive were evaluated (HCl, HBF 4 , HClO 4 , acetic acid). Unfortunately, no satisfying results were obtained. Therefore, we considered the use of ceric ammonium nitrate (CAN) as a catalyst in PEG 400, as it had already been used in a three-component Mannich reaction to access β-amino ketones of general structure B ( Figure 2) under conventional heating [39]. Accordingly, we employed this catalyst/solvent combination in our microwave-assisted protocol to access the designed compounds of general structure A ( Figure 2). Compounds 1a and 1b were obtained under microwave irradiation (60 W, 90 • C for 10 min) using 5% mol of CAN in PEG 400. The HPLC analysis (see Appendix A) showed that the reaction was clean and quick, affording the desired products with 80% conversion. Interestingly, using hydrochloride amines as reagents led to the best results. A schematic comparison between the old and new protocols (path A and path B, respectively) is shown in Figure 4. With these promising results, we moved forward to determine the effect of stoichiometric ratio of reagents on both conversion percentage and crude purity. Results are reported in Tables 1 and 2. Reactions conducted with excess amine (entries 2-4 and 10-13) led to high conversion percentages, even if the products had lower purities. An opposite trend was observed using an excess of ketone (entries 5-8 and 14-17). Accordingly, conditions of entries 6 and 15 (i.e., 2 equivalents (eq.) of ketone and 1 eq. of amine) were considered the best compromise and were extended to the other substrate for preparation of the whole library.  Reagents and reaction conditions: CAN (0.05 eq.), paraformaldehyde (1.0 eq.), PEG 400, (MW: 90 • C, 60 W, 10 min).

MW-Assisted Library Synthesis
The optimized protocol was then employed to synthesize the small focused library. The following table reports the yields and purities of each compound.
The developed protocol allowed production of the desired compounds (Table 3) with the exception of dibenzylaminic (1-6c) and 4 acetyl-pyridinic (4a-f) compounds ( Figure 3). Of note, cyclic amines (a, d, f) were well tolerated in this protocol; in particular, piperidines were the most versatile reagents, since they were able to react with the ketone counterpart, offering products in satisfying yields (Table 3). Relying on the molecules endowed with an acyclic amine, different reactivity was seen: N-benzylmethylamine > 3,4-dimethoxy-N-methylbenzylamine > dibenzylamine. The failure in the reaction involving dibenzylamine may be attributed to its low basicity, which may slow down the formation of the intermediate imine resulting from the reaction with formaldehyde. This result is not surprising. Indeed, it has been shown that the reactivity in the Mannich reaction is strictly related to the amine structure, and within a homologous series the reactivity may be different. An emblematic example is diethylamine, which is unable to be transformed into β-amino ketone, whereas the superior and inferior amine analogues react efficiently to give access to the desired products [35]. Regarding the ketone building block, the procedure was successfully applied to aromatic ketones, presenting benzene and thienyl nucleus, whereas no product was observed when ketone 4, bearing a pyridine moiety, was used. This behavior can be explained by considering the basic properties of the pyridine ring, which reduced the reactivity of methyl ketone 4. We performed additional model reactions on ketone 4 under traditional conditions at high temperature and for long times to force the reaction. Again, the desired products (compounds 4a and 4b) were not isolated, supporting our hypothesis that the low reactivity of 4 acetyl-pyridine compromised the reaction outcome.

Materials and Methods
Reactions performed under conventional heating were monitored by thin layer chromatography (TLC) with Fluka silica gel 60 F254 (Merck KGaA, Darmstadt, Germany) and purified by automatic flash chromatography with CombiFlash ® RF (AlfaTech, Teledyne Isco, Inc., Genoa, Italy).
All reactions conducted under microwave irradiation were performed in a microwave mono-mode oven specifically for organic synthesis (Discover ® LabMate instrument, CEM Corporation, Matthews, NC, USA). The obtained products were purified with Bond Elut SCX ® cartridge (Varian, Walnut Creek, CA, USA) and silica gel SPE cartridge (Varian, Walnut Creek, CA, USA).
UV spectra were recorded on a LAMBDA™ 25 UV/VIS spectrometer (Perkin Elmer Inc., Waltham, MA, USA). HPLC analyses were carried out on a Jasco HPLC system (Jasco Europe S.r.l., Cremella, Italy), consisting of a pump model PU 1580, a Reodyne 7125 injector (20 µL sample loop), and an MD-1510 diode array detector, combined with a Spectra AS3000 autosampler. Experimental data were acquired and interpreted with Borwin PDA and Borwin chromatograph software 1.5. Reversed-phase HPLC analyses were carried out at room temperature on an XTerra RP18 column (3.5 µm, 4.6 × 50 mm) (Waters, Milford, MA, USA) and a Hypersil ODS RP18 column (3 µm, 4.6 × 100 mm) (VWR, Milano, Italy). The mobile phase was phosphate buffer (pH 7.8) added with acetonitrile as organic modifier; the analysis was carried out using gradient elution (see Tables A1 and A2

General Procedure for the Synthesis of β-Aminoketones 1a-6f under Microwave Heating (Method B)
A mixture of ketone (2.0 eq.), amine hydrochloride (1 eq.), paraformaldehyde (1 eq.), and CAN (0.05 eq.) in PEG 400 (0.8 mL) was irradiated with a microwave power of 60 watts at 90 • C for 10 min. The reaction workup was performed as follows: the mixture was quenched in 2 M NaOH, then the solid was collected by centrifugation, dissolved in methanol or dichloromethane (depending on the solubility of the compound), and purified using SCX cartridge, eluting with a solution of 0.3 M NH 3 /MeOH in dichloromethane to remove the excess ketone. Finally, the product was isolated using silica gel SPE cartridge, eluting with dichloromethane to remove the nonreacted amine. Then the organic phase was evaporated to dryness.      3-(4-Benzylpiperidin-1-yl)-1-(thiophen-2-yl)propan-1-one (5a). Yield: 63%; 1

Conclusions
In summary, we have developed a rapid and easy-to-use microwave-assisted protocol based on a combination of PEG 400/CAN, PASPS, and SPE, obtaining the desired products faster than conventional procedures. The reaction optimized with respect to various parameters afforded most of the desired products with good yield and satisfying purity. Our approach could be adapted to a new library of compounds with different aromatic ketones. No less important, the obtained compounds could serve as key intermediates for further functionalization at the ketone group to allow scaffold modifications, suitable for disclosing novel potential hit compounds. We believe that simple reaction procedures and substrate compatibility along with environmentally friendly conditions make our protocol an important supplement to the existing methods.
Lastly, the small focused library we present aims at discovering new potential sigma 1 receptor modulators as part of our ongoing research in this field. From this consideration came our decision to add these products to the library of MuTaLig, an innovative ligand identification platform for the drug-discovery process.
Author Contributions: S.C. conceived the work, contributed to reviewing the manuscript, and was responsible for the correctness of the whole study. M.V.R. and G.R. wrote the manuscript. D.R., M.V.R., and M.D.G. were responsible for the experimental design and for data analysis of the whole study. G.R. and M.R. performed the experiments.

Conflicts of Interest:
The authors declare no conflict of interest.