Cinchona Alkaloid Derivative-Catalyzed Enantioselective Synthesis via a Mannich-Type Reaction and Antifungal Activity of β-Amino Esters Bearing Benzoheterocycle Moieties

An efficient synthesis of highly functionalized chiral β-amino ester derivatives containing benzothiophene and benzothiazole moieties is developed by a Mannich-type reaction using a cinchona alkaloid-derived thiourea catalyst. The desired products were obtained in good yields and high enantioselectivities (~86% yield, >99% ee) using to the optimized reaction conditions. The synthesized compounds were characterized by 1H-NMR, 13C-NMR, IR, and HREI-MS analyses. The bioassays identified that compound 5dr has excellent antifungal activity, with a 60.53% inhibition rate against F. oxysporum, higher than that of the commercial agricultural fungicide hymexazol, whose inhibition rate was 56.12%.

Benzothiazoles have varied biological activities [38][39][40]. They are widely found in bioorganic and medicinal chemistry with applications in drug discovery and are still of great scientific interest nowadays [41]. Benzothiazole moieties are part of compounds showing numerous biological activities such as antibacterial, antimicrobial, anthelmintic, antitumor, anti-inflammatory properties [42,43].
Recently, we independently reported a chiral cinchona alkaloid-derived thiourea catalyst for enantioselective synthesis of chiral β-amino esters by Mannich-type reaction [44][45][46][47]. Herein, we report an extension of our previous study by using potentially bioactive benzothiophene and benzothiazole moieties [48][49][50] as the building blocks for the synthesis of chiral β-amino acid ester derivatives. The structures of these newly synthesized compounds were confirmed by 1 H-NMR, 13 C-NMR, IR spectra, and MS (HREI) analysis. The desired products 5a-5p were obtained in good yields and high enantioselectivities (~86% yield, >99% ee) according to the optimized reaction conditions. Bioassays identified these newly compounds possessing weak to good antifungal activity. The inhibition rate of 5dr (we used the postfixes "r" and "c" to distinguish between racemic and chiral compounds) against F. oxysporum was 60.53%, higher than the commercial agricultural fungicide hymexazol whose inhibition rate was 56.12%. Further experimental and mechanism of antifungal activity are underway.

Optimization of Reaction Conditions
The general synthetic strategy for the preparation of imines by reaction of aldehydes with amino benzothiophene derivatives is outlined in Scheme 1. All products 3a-d were characterized by spectroscopic methods.

Scheme 1. Synthesis of imines 3a-d.
Then, synthetically designed cinchona alkaloid thiourea Q (Figure 3) was used as the catalyst for asymmetric catalytic Mannich-type reaction of the imines and malonate ester. Catalyst Q bearing strong electron-withdrawing trifluoromethyl substituent on the benzene ring exhibited excellent catalytic activity because of its ability to promote the reaction through intermolecular hydrogen bond activation of the substrates. The effect of the reaction temperature, solvent, and catalyst loading was evaluated using catalyst Q (Table 1). Temperature had a pronounced effect on yield and enantioselectivity of the reactions. Use of other solvents, such as THF, acetone, and toluene, resulted in lower enatioselectivities compared to the use of dichloromethane (DCM) ( Table 1, entries 1-5). The best result was achieved at room temperature using 10 mol% of catalyst Q in DCM.  Under the optimized reaction conditions, the synthetic scope of the reaction was investigated using different imines and malonate esters, and the results are listed in Table 2.  The desired products 5a-p were obtained in good yields and high enantioselectivities (~86% yield, >99% ee) using to the optimized reaction conditions. In addition, the enantioselectivity of 5dc, 5hc, 5lc and 5pc was higher than that of the other compounds, probably because of malonate ester was a benzyl ester, and steric hindrance affected the Mannich-type reaction between imines and benzyl esters.

Antifungal Activity
The antifungal activity of compounds 5 was assayed by the reported method [51,52]. As it can be seen from the results presented in Table 3, compound 5dr possess medium antifungal activity against G. zeae, C. mandshurica and F. oxysporum, with inhibition rates of 40.67%, 41.44% and 60.53%, respectively.

Instruments and Chemicals
Melting points were determined using a XT-4 binocular microscope (Beijing Tech Instrument Co., Beijing, China) and were not corrected. IR spectra were recorded using a Bruker VECTOR 22 spectrometer in KBr disks. 1 H-and 13 C-NMR spectra were recorded using a JEOL-ECX 500 MHz NMR spectrometer at room temperature in CDCl 3 or DMSO-d 6 solvent using tetrametylsilane as the internal standard. Elemental analyses were performed using an Elementar Vario-III CHN analyzer. MS spectra were recorded using a VG Autospec-3000 spectrometer. Analytical TLC was performed using silica gel GF254 plates. Column chromatographic purifications were carried out using column chromatographic silica gel. All the reagents were purchased from commercial sources and used as received, unless otherwise noted. Reactions were performed under a positive dry argon pressure in oven-dried or flame-dried glassware equipped with a magnetic stir bar. Standard inert atmosphere techniques were used in handling all air and moisture sensitive reagents. All the reagents were of analytical reagent grade or in chemically pure form. All the solvents were dried, deoxygenated, and distilled prior to use.

General Methods for Preparation of 3a-d
6-Substituted-2-aminobenzothiazole (10 mmol) was dissolved in toluene (10 mL), and the resulting solution was magnetically stirred, followed by dropwise addition of benzothiophene-2-methanal (10 mmol) dissolved in toluene (10 mL) at room temperature. The resulting reaction mixture was refluxed after adding acetic acid (1.0 mL), and complete consumption of the starting materials was observed after 24 h. After the completion of the reaction, the solvent was removed by distillation under reduced pressure. The resulting residue was recrystallized using ethyl alcohol to afford products 3a-3d.

General Method for the Preparation of 5a-p
To a magnetically stirred solution of imine (0.50 mmol) in DCM (3 mL) in the presence of 10% catalyst Q, the malonate ester (0.7 mmol) was added dropwise at room temperature. The complete consumption of starting materials was observed after 72-96 h. After removing the solvent by reduced pressure distillation, the reaction mixture was subjected to column chromatography on silica gel (EA/PE = 1:7) to afford compounds 5a-p.

Antifungal Activity Section
The antifungal activity of all synthesized compounds was tested against six pathogenic fungi, G. azeae, F. oxysporum, C. mandshurica, P. sasakii, P. infestans and S. sclerotiorum through the poison plate technique. All the compounds were dissolved in dimethyl sulfoxide (DMSO, 10 mL) before mixing with potato dextrose agar (PDA, 90 mL). The compounds were tested at a concentration of 50 μg/mL. All fungal species were incubated in PDA at 27 ± 1 °C for 5 day to obtain new mycelium for antifungal assay. Mycelia dishes approximately 4 mm in diameter were cut from the culture medium. One of the specimens was picked up with a sterilized inoculation needle and then inoculated in the center of the PDA plate aseptically. The inoculated plates were incubated at 27 ± 1°C for 5 day. DMSO in sterile distilled water served as the control, whereas hymexazole acted as the positive control. Three replicates were conducted for each treatment. The radial growth of the fungal colonies was measured, and the data were statistically analyzed. The inhibitory effects of the test compounds in vitro against these fungi were calculated as follows: where C represents the diameter of fungal growth on untreated PDA, T represents the diameter of fungi on treated PDA, and I is the inhibitory rate.

Conclusions
Sixteen pairs of chiral β-amino acid ester derivatives containing benzothiophene and benzothiazole units were designed and synthesized. The desired products 5a-p were obtained in good yields and high enantioselectivities (~86% yield, >99% ee) according to the optimized reaction conditions. The enantioselectivity of 5dc, 5hc, 5lc and 5pc was higher than the other compounds, probably because the malonate ester was a benzyl ester, and steric hindrance affected the Mannich-type reaction between the imines and benzyl esters. Bioassays identified these new compounds as possessing weak to good antifungal activity. The inhibition rate of 5dr against F. oxysporum was 60.53%, higher than the commercial agricultural fungicide hymexazole whose inhibition rate was 56.12% at a concentration of 50 μg/mL. Further experimental studies of the mechanism of antifungal activity are underway.