Multicomponent reactions (MCRs) have emerged as powerful and efficient tools for constructing various novel organic molecules of potential interest. These reactions are particularly useful in drug discovery owing to their flexibility and effectiveness compared with conventional multistep synthesis [1-3]. MCRs offer fast and elegant routes to target molecules via one-pot synthetic reaction schemes. These may be either simultaneous reactions or sequential-addition procedures that have high atom economy, bond-forming efficiency, and selectivity. Moreover, MCRs obviate the need for isolation and purification of intermediates, thereby minimizing waste, saving time, and reducing the cost of purification. Pyrano[2, 3-c] pyrazoles are an important class of heterocyclic compounds that have been widely explored as they form the essential core of emerging drugs with diverse bioactivity, including a potential inhibitor of human Chk1 kinase [4], as well as antitumor [5], analgesic [6], antibacterial [7], and anti-inflammatory [6-8] agents. Various MCRs, including three-component reactions (3CRs), four-component reactions (4CRs), and even five- component reactions (5CRs), have been reported for the synthesis of pyrano[2, 3-c]pyrazole derivatives. 3CRs of carbonyl compounds, malononitrile, and the corresponding pyrazolin-5-ones using various basic catalysts, such as trimethylamine [9], triethanolamine [10], piperidine [11], N-methylmorpholine [12], D/L-proline [13], MgO [14], silica sodium carbonate [15], and electrogenerated bases [16], have been reported. Furthermore, 4CRs have been developed for the reaction of aldehydes, malononitrile, hydrazine hydrate, and ethylacetoacetate using trimethylamine [17], Amberlyst A21 [18], cetyltrimethylammonium chloride [19], molecular sieves [20], magnetic Fe3O4 nanoparticles [21], SnO2 quantum dots [22], L-proline [23, 24], glycine [25], meglumine [26], per-6-amino-β- cyclodextrin [27], and lipase [28] as catalysts. More recently, overall 5CRs have been established involving Suzuki coupling of 4-bromobenzaldehyde and arylboronic acids followed by 4CRs involving readily available ethyl acetoacetate, malononitrile, and hydrazine hydrate [29]. However, in spite of their merits, most of these methods suffer from one or more drawbacks, such as harsh reaction conditions, expensive or unobtainable reagents, stoichiometric amounts of catalyst, long reaction time, unsatisfactory product yields, high waste generation, or laborious work-up. Considering the importance of pyrano[2, 3-c]pyrazole derivatives, more convenient and practical methodologies for their synthesis are still needed.
In light of the above, we considered that biocatalysts might be effective for this class of 4CRs owing to their promiscuous activity. Exploiting the ability of a biocatalyst to achieve the synthesis of pyrano[2, 3-c]pyrazole derivatives via 4CRs has long been challenging. To date, only one previous study [28], using lipase from Aspergillus niger, has reported the successful use of a biocatalyst for this purpose.
In the past decade, considerable effort has been expended on the development of biocatalysts, including proteins, enzymes, and whole cells, for use in organic synthesis, owing to their high catalytic activity and selectivity, as well as operational simplicity [30, 31]. Among them, bovine serum albumin (BSA), a ubiquitous, inexpensive, non-enzymatic transport protein derived from cows, with no natural catalytic function in biological systems, has proven an efficient biocatalyst because of its remarkable versatility for many kinds of catalytic activities and biotransformations [32]. It is capable of binding organic molecules by reversible non-covalent complexation in its hydrophobic pockets, thus providing a microenvironment for a number of organic transformations including reduction [33], Knoevenagel condensation [34, 35], aldol condensation [34], nitroaldol addition [36], Gewald reactions [37], Morita-Baylis- Hillman reactions [38], Biginelli reactions [39], thio-Michael addition [40], oxidative coupling of thiols [41], thiosulfination [42], and oxidation of tertiary amines to N-oxides [43].
Inspired by these results, we tested the use of BSA as a new, efficient and recyclable catalyst for the synthesis of pyrano[2, 3-c]pyrazole derivatives via 4CRs (Scheme 1). To the best of our knowledge, there has been no report of the use of BSA as a biocatalyst for 4CRs of this kind to date. Thus, exploiting the promiscuous activity of the abundant and readily available BSA to catalyze MCRs should extend the synthetic utility of biocatalysis.
Albumin bovine V from bovine serum (BSA), hemoglobin from bovine erythrocytes (Lot# SG201402), egg white albumin, lysozyme from chicken egg (activity ≥ 20000 U/mg, Lot# KY201501), trypsin from porcine pancreas (activity ≥ 250 U/g, Lot# KY201305), and lipase from porcine pancreas (activity ≥ 30000 U/g, Lot# KY201312) were purchased from Sigma. Alpha amylase from bacillus submerged fermentation (activity ≥ 3000 U/g, pH = 5.5-7.5) was purchased from Damao, and silk fibroin was extracted from silk by a previously reported method [44]. All chemicals were obtained from commercial sources and used as received.
Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet 6700 spectrometer in KBr pellets in the range 4000-400 cm−1. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were obtained on a Bruker AC-300 using DMSO-d6 as the solvent and TMS as the internal standard. The melting points were measured on an Electrothermal X6 microscopic digital melting point apparatus.
A round-bottomed flask was charged with ethyl acetoacetate (1 mmol), 80% hydrazine hydrate (V/V, 1 mmol), malononitrile (1 mmol), the chosen carbonyl compound (1 mmol), BSA (50 mg), and 5 mL 90% aqueous ethanol solution (V/V). The reaction was incubated at 45 ℃ and stirred at 500 r/min for a specific time until completion. The progress was tracked by thin-layer chromatography. Upon completion, ethyl acetate preheated to 60 ℃ was added to quench the reaction. The BSA was recovered by filtration, washed adequately with ethanol and dried in preparation for the next run. The organic fraction was successively washed with brine and saturated sodium bisulfite solution and then concentrated on a rotary evaporator to give the crude product. The product was further purified by recrystallization with ethanol. The target products were identified by FT-IR, 1H NMR, 13C NMR, and melting point, all of which were in agreement with the literature values.
Initially, various biocatalysts, including enzymes and proteins, were screened using the 4CR of ethyl acetoacetate, hydrazine hydrate, malononitrile, and p-chlorobenzaldehyde as a model reaction. The results are summarized in Table 1. Among all the screened biocatalysts, BSA showed the most powerful catalytic activity, achieving a rapid reaction time of 0.75 h and affording the desired product in 95% yield (Table 1, entry 9). The use of other enzymes or proteins also resulted in appreciably greater yields (Table 1, entries 2-8). The lowest product yield was obtained in the absence of any catalyst (Table 1, entry 1), showing that catalysis plays a key role in this transformation. Encouraged by this result, we proceeded to explore in detail the influence of the catalyst dose, reaction medium, and reaction temperature on the model reaction using catalytic amounts of BSA.
The dosage of the biocatalyst was found to have an appreciable effect on the product yields. The 4CR model was studied with catalyst doses ranging from 0.0 to 60.0 mg of the substrate (Table 2). The 4CR model was found to proceed efficiently even with a low dose of BSA (15 mg in substrate), affording an 80% product yield (Table 2, entry 2). As can be seen from Table 2, with the catalyst dose increased from 15 to 50 mg, the apparent yield increased from 80% to 95% and the reaction time decreased from 1.5 to 0.75 h (Table 2, entries 2-5). Further increasing the catalyst dose to 55 or even 60 mg had no major effect on the product yield (Table 2, entries 6 and 7). Recall that, in the absence of BSA, the model reaction proceeded with a prolonged reaction time and a lower yield (Table 2, entry 1). In contrast, 50 mg of BSA biocatalyst was sufficient to drive the reaction to completion in short time.
The reaction medium generally plays an important role in biocatalysis. To identify the most suitable solvent, ten protic and aprotic solvents with varying polarities or other properties were screened. Several protic solvents, including EtOH, aqueous EtOH, and MeOH, rendered high product yields (Table 2, entries 2-15), while in aprotic solvents, including CH2Cl2, CH3CN, DMF, and DMSO, BSA exhibited low catalytic activity (Table 2, entries 18-21). Poor yields were also obtained in H2O, i-PrOH, and the absence of solvent (Table 2, entries 16-17 and 22). Based on the screening results, 90% EtOH (V/V) was chosen as the most suitable solvent for further study.
Reaction temperature is an important parameter in biocatalysis. To further investigate the optimum conditions for the model MCR, we evaluated the effect of six different temperatures in the range 25-78 ℃ on the reaction yield and reaction time. Both these variables were found to be highly sensitive to temperature. As shown in Table 2, the product yield increased from 85% to 95% and the reaction time decreased from 2 to 0.75 h when the temperature was raised from 25 °C (room temperature) to 45 ℃ (Table 2, entries 8, 9 and 5). However, temperatures over 45 ℃ led to lower yields (Table 2, entries 10-12), possibly because of the partial deactivation of the biomolecular BSA at these temperatures, indicating the importance of an appropriate operating temperature to retain biocatalytic activity. Therefore, 45 ℃ was selected as the optimal temperature for the reaction.
To obtain further insight into the catalytic possibilities of BSA, we investigated the scope and generality of the 4CRs for which it displays activity. A variety of substituted aryl aldehydes carrying either electron-withdrawing or electron- donating substituents on the aromatic ring were reacted with malononitrile, hydrazine hydrate, and ethyl acetoacetate in 90% aqueous ethanol in the presence of catalytic amounts of BSA under the above-mentioned optimal conditions. As shown in Table 3, all of the reactions proceeded smoothly to produce the expected pyrano[2, 3-c]pyrazoles with excellent yields of 73%-96%. With both electron-donating and electron- withdrawing substituents, at either the para or the meta position on the aromatic ring of the aryl aldehyde, the reactions proceeded without marked effects on their progress and with high yields. Notably, the reactions of the aryl aldehydes bearing a substituent at the ortho position afforded slightly lower yields owing to the steric effect (Table 3, entries 2, 3, 5, and 8). Many sensitive functional groups were compatible with the BSA biocatalyst, such as methoxy, phenolic hydroxy, and N, N-dimethylamino, and gave high yields (Table 3, entries 11, 12, 14, and 15). Apart from the aryl aldehydes, various ketones were also used as components in the 4CR scheme, under similar conditions, to produce the desired pyrano[2, 3-c]pyrazole derivatives in excellent yields with slightly prolonged reaction time (Table 3, entries 16-21). All of the products were fully characterized by melting point and FT-IR, 1H NMR, and 13C NMR spectroscopy.
For practical application, the reusability of a catalyst is a crucial factor. To explore this aspect of BSA, catalytic recycling experiments were performed using the 4CR of 4-chlorobenzaldehyde, hydrazine hydrate, malononitrile and ethyl acetoacetate as a model reaction. After completing the reaction, the BSA could be conveniently and efficiently recovered from the reaction mixture by filtering, washing successively with acetone three times, and then drying. The recovered BSA was then reused directly in consecutive cycles of the reaction under the above mentioned optimized conditions. The results (Fig. 1) show that only a slight loss of the catalytic activity of BSA was observed after each cycle.
The protein BSA has no known natural catalytic function. Structurally, BSA contains 60 Lys, 41 Asp and 58 Glu residues [51] and, with an isoelectric point near pH = 4.5 [52], presents as mildly basic at neutral pH. The catalytic activity of BSA is determined by the basic character of the amino groups on the side chains of certain residues, especially lysine. Therefore, we propose the following mechanism for the synthesis of pyrano[2, 3-c]pyrazoles via a tandem process, as depicted in Scheme 2. First, an -NH2 group on BSA simultaneously promotes both the condensation of ethyl acetoacetate with hydrazine hydrate and the Knoevenagel condensation of the aryl aldehyde with malononitrile to produce the 3-methyl- 1H-pyrazol-5(4H)-one A and the intermediate olefin B. Subsequent Michael addition of A and B promoted by -NH2 on BSA produces the intermediate C. Finally, we propose that C undergoes intramolecular cyclization followed by tautomerization to give the dihydropyrano[2, 3-c]pyrazole derivatives.
We have developed a novel, efficient, and environmentally friendly protocol for the synthesis of a diverse range of pyrano[2, 3-c]pyrazole derivatives using BSA as a reusable biocatalyst. This method offers the advantages of environmental compatibility, mild reaction conditions, short reaction time, high yields and operational simplicity. Moreover, the method is distinguished by its multicomponent process and wide substrate scope, accepting a variety of carbonyl compounds as starting materials, which make it a useful and practical process for the synthesis of structurally diverse pyrano[2, 3-c]pyrazole derivatives.
2016,
Vol. 37 Issue (9): 1461-1468 
PDF
2016,
Vol. 37 Issue (9): 1461-1468 PDF