Efficient Synthesis of Boron-Containing α-Acyloxyamide Analogs via Microwave Irradiation

In this report, a Passerini three-component reaction utilizing boron-containing carboxylic acids or aldehydes is discussed. The reaction was carried out in water and facilitated by the use of microwave irradiation. This methodology allowed for the efficient formation of a broad range of boron-containing α-acyloxyamides under mild conditions within a short time. Two series of boron-containing α-acyloxyamides were synthesized and subsequently screened for cytotoxicity using the MTT cell viability assay. Two potential lead compounds were found to have potent activity against the HepG2 cancer cell line, demonstrating the potential of this methodology for use in the development of novel pharmaceuticals.


Introduction
Boron-based compounds possess a unique and potentially valuable feature, whereby the empty p-orbital on the boron atom is able to interact with a nucleophile from a biological target [1][2][3][4][5][6][7], forming a stable tetrahedral complex. This distinctive property provides great promise in the field of pharmaceuticals, as it allows boron-based compounds to react with a target of interest from a different perspective to their conventional carbon-based analogs. The FDA approval of bortezomib [Velcade TM , Figure 1a] in 2003 for the treatment of multiple myeloma and mantle cell lymphoma [8][9][10] represents one of the greatest examples of successful utilization of organoboron entities for treating human diseases. Encouraged by the success of bortezomib, several boron-containing molecules have since been developed to treat a wide range of diseases, a number of which are currently undergoing clinical trials (Figure 1b-e) [11][12][13][14][15][16][17]. Multicomponent reactions (MCRs) are convergent synthetic strategies in which three or more reagents are combined in one pot to produce the desired products [18,19]. It is an invaluable platform in the development of pharmaceutical agents as, owing to their versatility, MCRs are perfectly suited for producing boron-containing compounds for potential biological applications.
One of the most widely utilized MCRs is the isocyanide-based Passerini reaction, where an isocyanide, an aldehyde, and a carboxylic acid are condensed, generating α-acyloxyamides [20,21]. Although widely used by the organic and medicinal chemistry communities, the Passerini reaction has several shortcomings. One such drawback is that poor yields are often observed when a weakly acidic carboxylic acid [22] or no Lewis acid promoters [23] are involved in the reaction. In the last decades, tremendous progress has been made in the optimization of the Passerini reaction [24][25][26][27][28][29][30]. However, these studies mainly focused on improving the conditions for constructing molecules containing the elements carbon, hydrogen, oxygen, nitrogen, halogens, and sulfur. Reports on utilizing an MCR as a platform to generate boron-containing analogs are very limited, and those that have been published often demonstrate the requirement for a prolonged reaction time or excessive purification procedures [31]. Herein, we report the use of the Passerini reaction for the efficient synthesis of boron-containing α-acyloxyamides. This eco-friendly procedure could be carried out on a broad range of substrates at a moderate temperature under microwave irradiation conditions, with a reduced reaction time possible.

Chemistry
Initial experiments were carried out in order to determine the optimal conditions for the synthesis of boron-containing α-acyloxyamide analogs (Table 1). First, 1.0 equivalent of 4-carboxyphenylboronic acid pinacol ester (1a), 1.0 equivalent of benzaldehyde (2), and 1.0 equivalent of cyclohexyl isocyanide (3) were dissolved in the selected solvent and allowed to react at 45 °C for 90 min under microwave irradiation (150 W). It was found that while dichloromethane (entry 1) and methanol (entry 2) did not give desired product A1, using THF gave a moderate yield of 39% (entry 3). The yield was greatly improved to 77% when water was used as the solvent (entry 4). Increasing the reactant concentrations from 0.25 M to 1.0 M (entry 5) resulted in an increased isolated yield (84%), and extension of the reaction time from 90 min to 120 min (entry 6) gave an even higher yield (88%). In an attempt to improve the yield further, the temperature was increased to 55 °C for 120 min; however, rather than giving an increased amount of the desired product, a lower isolated yield was obtained (60%), suggesting product degradation due to overheating (entry 7). Thus, this series of optimization experiments highlighted a range of temperatures, reagent concentrations, and solvents that were effective for producing desired product A1 in good isolated yields. The desired product was purified using a simple precipitation procedure with an appropriate solvent system, and no additional column chromatography or reverse-phase high performance liquid chromatography was required for affording satisfactory purity. This finding could be of particular importance for the construction of boron-containing libraries, as one of the main challenges in boron chemistry is the purification of the final product, with excessive purification protocols often required for achieving satisfactory purity. Three carboxylphenylboronate esters 1a-c, seven aldehydes 2a-g, and cyclohexyl isocyanide (3) were then used to evaluate the scope of the reaction under the optimized conditions that were deduced from the results shown in Table 1. As demonstrated in Table 2, in all cases, the desired products A1-20 were isolated in moderate to good yields, ranging from 56%-88%. It was found that the position of the boronate ester did not influence the reaction as 4-and 3-carboxyphenylboronic acid pinacol esters 1a and 1b (entries 1 and 2) gave the desired products A1 and A2 in good yields, respectively. Apart from the result shown in entry 3, attachment of an electron-withdrawing group to the acid building block appeared to impede the reaction, as analogs A6, A9, A12, A15, and A18 were obtained in lower isolated yields. However, this trend was not observed when an electron-withdrawing group was attached to the aldehyde building block, as demonstrated by the good yields of analogs A4-9 obtained. Further, the attachment of an electron-donating group to the aldehyde building block was also tolerated, with analogs A10-15 achieved in good yields. Finally, as heteroaryl moieties are frequently found in many pharmaceutical agents [32][33][34][35], 3-pyridinecarboxaldehyde (2f), and furfural (2g) were used to construct the corresponding Passerini boronate esters. Five Passerini boronate esters containing heteroaryl motifs A16-20 were successfully synthesized in moderate to good yields using the optimized microwave-assisted conditions.  Encouraged by the results presented in Table 2, experiments were initiated in order to explore the possibility of using boron-containing aldehyde building blocks to synthesize Passerini analogs (Table 3). Although they are structurally similar, the optimal conditions for preparing A1 did not appear to be adequate for producing B1. For instance, the conditions of entries 4-6 in Table 1 were able to generate A1 in 77%, 84%, and 88% yields, while only affording B1 in 0%, 58%, and 65% (Table 3, entries 1-3). It was found that prolonged microwave irradiation was required to promote the reaction, with an improved yield of 75% achieved after 150 min of reaction time (entry 4). However, after an additional 60 min irradiation, the yield declined to 60% (entry 5), suggesting degradation of the product might have occurred under such extensive microwave treatment. After elucidating the optimal reaction conditions ( Table 3, entry 4), six acid building blocks, two boron-containing aldehydes, and cyclohexyl isocyanide were used to synthesize B1-12. In all cases, the desired products were isolated in good yields, ranging from 50%-89% (Table 4). It was found that the reactions were greatly influenced by the nature of the acid building blocks. For instance, reactions with benzoic acid (1d) gave higher yields (entries 1 and 2, 69%-75%) than for those achieved for the compound with an electron-withdrawing group (entries 3 and 4, 57%-59%). Furthermore, acids with an electron-donating group generally gave even higher yields than those without (entries 5-10, 72%-89%). Incorporation of a heteroaryl motif to the Passerini product was successfully accomplished in moderate yields of 50%-55% by using pyrazinecarboxylic acid (1i) (entries 11 and 12).

In Vitro Biological Evaluation
The thirty three boron-containing α-acyloxyamides mentioned above were subsequently screened for anti-proliferative activity against the HepG2 (human hepatocellular carcinoma) cancer cell line using the MTT cell viability assay. It was found that A4 and A5 gave IC 50 values of 33.6 μM and 27.5 μM, respectively, where non-boron analog A21 was observed to be inactive. Further boron-containing α-acyloxyamides are currently being synthesized using the method developed in the present study, and their structure-activity relationships will be reported in due course.

General
All starting materials were obtained from commercial suppliers and used without further purification unless otherwise noted. Reactions were performed on a CEM Co., Discover microwave reactor using sealed vessels. 1 H-, 13 C-, 11 B-NMR spectra were recorded on a Bruker Avance 600 FT-NMR spectrometer at 600. 13, 150.90, and 192.54 MHz, respectively. All 11 B chemical shifts were referenced to external BF 3 ·OEt 2 (0.0 ppm). Data are represented as follows: chemical shifts (ppm), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, br = broad), coupling constant J (Hz). Melting points were determined by using a Fargo MP-2D melting point apparatus and were uncorrected. High resolution ESI mass spectra were obtained by Finnigan MAT 95S.

In Vitro Biological Evaluation
In Vitro Cytotoxicity Assay The anti-tumor activities of boron-containing analogs against lung (A549), breast (MDA-MB-231), and liver (HepG2) cancer cell lines were evaluated using a microculture tetrazolium test (MTT, Sigma-Aldrich, Saint Louis, MO, USA). Briefly, tumor cells or fibroblasts (5000 cells in 100 μL complete medium per well) were seeded into a 96-well plate (Nunc, Roskilde, Denmark). After incubation at 37 °C for 24 h, 100 μL of culture medium with or without boron-containing analogs was added to each well in triplicate for 48 h of consecutive incubation at 37 °C. In the treatment of each cell line, cells were incubated for another 24, 48 and 72 h. MTT solution 50 μL (Sigma) was added to each well. Following incubation at 37 °C for an additional 4 h, supernatants were removed and 100 μL DMSO was added to dissolve the MTT-formazan product. The plate was read using a microplate reader (Labsystems, Helsinki, Finland) at 550 nm. The cell inhibitions at 20, 10, 5, 2.5 μg/mL of boron-containing analogs were estimated and the IC50 for boron-containing analogs calculated for the control group was set to 100% [36].

Conclusions
In conclusion, a convenient and efficient microwave-assisted Passerini MCR under aqueous conditions was developed. Broad ranges of boron-containing α-acyloxyamides were synthesized in moderate to good yields using this method. In addition, a simple acid/base extraction protocol was developed, which enabled simple and effective purification of these boron-containing compounds. This is a major achievement that renders this synthetic strategy suitable for use in the library synthesis of boron molecules. All of the synthesized analogs were screened using the MTT assay, with two compounds found to be active against the HepG2 cell line. Further structure-activity relationship evaluations based on these two analogs is currently ongoing, and the results will be reported in due course.