Asymmetric Total Syntheses of Both Enantiomers of Plymuthipyranone B and Its Unnatural Analogues: Evaluation of anti-MRSA Activity and Its Chiral Discrimination

Chiral total syntheses of both enantiomers of the anti-MRSA active plymuthipyranone B and all of the both enantiomers of three unnatural and synthetic analogues were performed. These two pairs of four chiral compounds are composed of the same 3-acyl-5,6-dihydro-2H-pyran-2-one structure. The starting synthetic step utilized a privileged asymmetric Mukaiyama aldol addition using Ti(OiPr)4/(S)-BINOL or Ti(OiPr)4/(R)-BINOL catalysis to afford the corresponding (R)- and (S)-δ-hydroxy-β-ketoesters, respectively, with highly enantiomeric excess (>98%). Conventional lactone formation and successive EDCI-mediated C-acylation produced the desired products, (R)- and (S)-plymuthipyranones B and three (R)- and (S)- synthetic analogues, with an overall yield of 42–56% with a highly enantiomeric excess (95–99%). A bioassay of the anti-MRSA activity against ATCC 43300 and 33591 revealed that (i) the MICs of the synthetic analogues against ATCC 43300 and ATCC 33591 were between 2 and 16 and 4 and 16 μg/mL, respectively, and those of vancomycin (reference) were 1 μg/mL. (ii) The natural (S)-plymuthipyranone B exhibited significantly higher activity than the unnatural (R)-antipode against both AACCs. (iii) The natural (R)-plymuthipyranone B and (R)-undecyl synthetic analogue at the C6 position exhibited the highest activity. The present work is the first investigation of the SAR between chiral R and S forms of this chemical class.


Introduction
The chiral discrimination of bioactivity between enantiomers has occupied a central position in modern research and the development of pharmaceuticals and agrochemicals. 3-Acyl-5,6-dihydro-2H-pyran-2-ones are unique heterocyclic molecules with a tricarbonyl moiety at the C(3)-position and an asymmetric center at the C(6)-position [1]. Several natural products contain these compounds, as depicted in Figure 1.

Synthesis of Three Sets of Natural Plymuthipyranone B and Two Sets of Synthetic Analogues
Our synthesis commenced with the privileged asymmetric Mukaiyama aldol addition using Ti(OiPr) 4 /(S)-BINOL catalysis originally developed by Soriente and Scettri's group [25][26][27][28], which consistently produced the (R)-aldol adducts. In addition, (S)-aldol adducts can be obtained in a similar and stereocomplementary manner by switching the chiral catalysis from (S)-BINOL to (R)-BINOL.

Synthesis of Three Sets of Natural Plymuthipyranone B and Two Sets of Synthetic Analogues
Our synthesis commenced with the privileged asymmetric Mukaiyama aldol addition using Ti(OiPr)4/(S)-BINOL catalysis originally developed by Soriente and Scettri's group [25][26][27][28], which consistently produced the (R)-aldol adducts. In addition, (S)-aldol adducts can be obtained in a similar and stereocomplementary manner by switching the chiral catalysis from (S)-BINOL to (R)-BINOL.
Eventually, (R)-and (S)-stereocomplementary syntheses were performed by only switching (S)-and (R)-BINOL catalysts, respectively, both of which are commercially available with nearly the same prices. Scheme 1. Asymmetric total synthesis of (R)-and (S)-plymuthipyranones B.
We next turned our attention to the syntheses of the two enantiomer sets of unnatural (synthetic) analogues of (R)-4c and (S)-4c, (R)-4d and (S)-4d, respectively (Scheme 2). A racemic compound of 4d exhibited the highest anti-MRSA activity among a series of these compounds [16]. Novel analogues (R)-, (S)-4d containing a terminal double bond in the substituent at the C(6)-position were selected as candidates, because (i) podoblastin B with a similar terminal double bond exhibited higher antifungal activity than podoblastins A and C with simple alkyl groups and (ii) starting 10-undecenal was commercially available.
Eventually, (R)-and (S)-stereocomplementary syntheses were performed by only switching (S)-and (R)-BINOL catalysts, respectively, both of which are commercially available with nearly the same prices.

Antibacterial Evaluation against MRSA
The stereostructure-activity relationships between all the enantiomers of plymuthipyranone B (4b) and synthetic analogues 4c, 4d, 4e were assayed using two American Type Culture Collection (ATCC) cell lines (43300 and 33591) on the basis of their minimal inhibitory concentration (MIC) values against MRSA (Table 1). Broberg's group reported that the anti-MRSA activity order was 4d > 4c > plymuthipyranone B (4b) > 4e as "the racemic forms" [16].
The salient features are as follows: (i) The MICs of the synthetic analogues against ATCC 43300 and ATCC 33591 were between 2 and 16 and 4 and 16 μg/mL, respectively, and those of vancomycin (reference) were 1 μg/mL. (ii) As expected, natural plymuthipyranone B (S)-4b exhibited significantly higher activity than the unnatural antipode (R)-4b against both ATCCs. (iii) In clear contrast, reverse antipodal (S)-4c exhibited higher activity than (R)-4c against both ATCCs. (iv) Regarding the most active analogue 4d (the racemic form), however, the activity was (S)-4d = (R)-4d against ATCC 3300 and (S)-4d > (R)-4d against ATCC 33591. (v) These results are in reasonable accordance with the reported data of racemates 4b, 4c and 4d. (vi) (R)-4e and (S)-4e possessing a terminal double bond were quite less reactive in contrast to the SAR of podoblastins [15]. (vii) The MICs of the most active (R)-4c and (S)-4d were approximately half that of vancomycin.

Antibacterial Evaluation against MRSA
The stereostructure-activity relationships between all the enantiomers of plymuthipyranone B (4b) and synthetic analogues 4c, 4d, 4e were assayed using two American Type Culture Collection (ATCC) cell lines (43300 and 33591) on the basis of their minimal inhibitory concentration (MIC) values against MRSA (Table 1). Broberg's group reported that the anti-MRSA activity order was 4d > 4c > plymuthipyranone B (4b) > 4e as "the racemic forms" [16].
The salient features are as follows: (i) The MICs of the synthetic analogues against ATCC 43300 and ATCC 33591 were between 2 and 16 and 4 and 16 µg/mL, respectively, and those of vancomycin (reference) were 1 µg/mL. (ii) As expected, natural plymuthipyranone B (S)-4b exhibited significantly higher activity than the unnatural antipode (R)-4b against both ATCCs. (iii) In clear contrast, reverse antipodal (S)-4c exhibited higher activity than (R)-4c against both ATCCs. (iv) Regarding the most active analogue 4d (the racemic form), however, the activity was (S)-4d = (R)-4d against ATCC 3300 and (S)-4d > (R)-4d against ATCC 33591. (v) These results are in reasonable accordance with the reported data of racemates 4b, 4c and 4d. (vi) (R)-4e and (S)-4e possessing a terminal double bond were quite less reactive in contrast to the SAR of podoblastins [15]. (vii) The MICs of the most active (R)-4c and (S)-4d were approximately half that of vancomycin. the following SAR studies remain: (i) variation of 3-acyl substituent and (ii) isosterism for other related heterocycles such as 2H-pyrones, piperidine, etc.

Synthesis
All reactions were carried out in oven-dried glassware under an argon atmosphere. Flash column chromatography was performed with silica gel 60 (230-400 mesh ASTM, Merck, Darmstadt, Germany). TLC analysis was performed on Merck 0.25-millimeter Silicagel 60 F254 plates. Melting points were determined on a hot stage microscope apparatus (ATM-01, AS ONE, Osaka, Japan) and were uncorrected. NMR spectra were recorded on a JEOLRESONANCE EXC-400 or ECX-500 spectrometer (JEOL, Akishima, Japan) operating at 400 or 500 MHz for 1 H-NMR and 100 and 125 MHz for 13 C NMR. Chemical shifts (δ ppm) in CDCl3 were reported downfield from TMS (= 0) for 1 H-NMR. For 13 C-NMR, chemical shifts were reported in the scale relative to CDCl3 (77.00 ppm) as an internal reference. Mass spectra were measured on a JMS-T100LC spectrometer (JEOL, Akishima, Japan). HPLC data were obtained on a SHIMADZU (Kyoto, Japan) HPLC system (consisting of the following: LC-20AT, CMB20A, CTO-20AC and detector SPD-20A measured at 254 nm) using Chiracel AD-H or Ad-3 column (Daicel, Himeji, Japan, 25 cm) at 25 °C. Optical rotations were measured on a JASCO DIP-370 (Na lamp, 589 nm). All NMR spectra figures could be found in Supplementary Materials.

Synthesis
All reactions were carried out in oven-dried glassware under an argon atmosphere. Flash column chromatography was performed with silica gel 60 (230-400 mesh ASTM, Merck, Darmstadt, Germany). TLC analysis was performed on Merck 0.25-millimeter Silicagel 60 F254 plates. Melting points were determined on a hot stage microscope apparatus (ATM-01, AS ONE, Osaka, Japan) and were uncorrected. NMR spectra were recorded on a JEOLRESONANCE EXC-400 or ECX-500 spectrometer (JEOL, Akishima, Japan) operating at 400 or 500 MHz for 1 H-NMR and 100 and 125 MHz for 13 C NMR. Chemical shifts (δ ppm) in CDCl3 were reported downfield from TMS (= 0) for 1 H-NMR. For 13 C-NMR, chemical shifts were reported in the scale relative to CDCl3 (77.00 ppm) as an internal reference. Mass spectra were measured on a JMS-T100LC spectrometer (JEOL, Akishima, Japan). HPLC data were obtained on a SHIMADZU (Kyoto, Japan) HPLC system (consisting of the following: LC-20AT, CMB20A, CTO-20AC and detector SPD-20A measured at 254 nm) using Chiracel AD-H or Ad-3 column (Daicel, Himeji, Japan, 25 cm) at 25 °C. Optical rotations were measured on a JASCO DIP-370 (Na lamp, 589 nm). All NMR spectra figures could be found in Supplementary Materials.

Synthesis
All reactions were carried out in oven-dried glassware under an argon atmosphere. Flash column chromatography was performed with silica gel 60 (230-400 mesh ASTM, Merck, Darmstadt, Germany). TLC analysis was performed on Merck 0.25-millimeter Silicagel 60 F254 plates. Melting points were determined on a hot stage microscope apparatus (ATM-01, AS ONE, Osaka, Japan) and were uncorrected. NMR spectra were recorded on a JEOLRESONANCE EXC-400 or ECX-500 spectrometer (JEOL, Akishima, Japan) operating at 400 or 500 MHz for 1 H-NMR and 100 and 125 MHz for 13 C NMR. Chemical shifts (δ ppm) in CDCl3 were reported downfield from TMS (= 0) for 1 H-NMR. For 13 C-NMR, chemical shifts were reported in the scale relative to CDCl3 (77.00 ppm) as an internal reference. Mass spectra were measured on a JMS-T100LC spectrometer (JEOL, Akishima, Japan). HPLC data were obtained on a SHIMADZU (Kyoto, Japan) HPLC system (consisting of the following: LC-20AT, CMB20A, CTO-20AC and detector SPD-20A measured at 254 nm) using Chiracel AD-H or Ad-3 column (Daicel, Himeji, Japan, 25 cm) at 25 °C. Optical rotations were measured on a JASCO DIP-370 (Na lamp, 589 nm). All NMR spectra figures could be found in Supplementary Materials.

Synthesis
All reactions were carried out in oven-dried glassware under an argon atmosphere. Flash column chromatography was performed with silica gel 60 (230-400 mesh ASTM, Merck, Darmstadt, Germany). TLC analysis was performed on Merck 0.25-millimeter Silicagel 60 F254 plates. Melting points were determined on a hot stage microscope apparatus (ATM-01, AS ONE, Osaka, Japan) and were uncorrected. NMR spectra were recorded on a JEOLRESONANCE EXC-400 or ECX-500 spectrometer (JEOL, Akishima, Japan) operating at 400 or 500 MHz for 1 H-NMR and 100 and 125 MHz for 13 C NMR. Chemical shifts (δ ppm) in CDCl3 were reported downfield from TMS (= 0) for 1 H-NMR. For 13 C-NMR, chemical shifts were reported in the scale relative to CDCl3 (77.00 ppm) as an internal reference. Mass spectra were measured on a JMS-T100LC spectrometer (JEOL, Akishima, Japan). HPLC data were obtained on a SHIMADZU (Kyoto, Japan) HPLC system (consisting of the following: LC-20AT, CMB20A, CTO-20AC and detector SPD-20A measured at 254 nm) using Chiracel AD-H or Ad-3 column (Daicel, Himeji, Japan, 25 cm) at 25 °C. Optical rotations were measured on a JASCO DIP-370 (Na lamp, 589 nm). All NMR spectra figures could be found in Supplementary Materials.

Synthesis
All reactions were carried out in oven-dried glassware under an argon atmosphere. Flash column chromatography was performed with silica gel 60 (230-400 mesh ASTM, Merck, Darmstadt, Germany). TLC analysis was performed on Merck 0.25-millimeter Silicagel 60 F254 plates. Melting points were determined on a hot stage microscope apparatus (ATM-01, AS ONE, Osaka, Japan) and were uncorrected. NMR spectra were recorded on a JEOLRESONANCE EXC-400 or ECX-500 spectrometer (JEOL, Akishima, Japan) operating at 400 or 500 MHz for 1 H-NMR and 100 and 125 MHz for 13 C NMR. Chemical shifts (δ ppm) in CDCl3 were reported downfield from TMS (= 0) for 1 H-NMR. For 13 C-NMR, chemical shifts were reported in the scale relative to CDCl3 (77.00 ppm) as an internal reference. Mass spectra were measured on a JMS-T100LC spectrometer (JEOL, Akishima, Japan). HPLC data were obtained on a SHIMADZU (Kyoto, Japan) HPLC system (consisting of the following: LC-20AT, CMB20A, CTO-20AC and detector SPD-20A measured at 254 nm) using Chiracel AD-H or Ad-3 column (Daicel, Himeji, Japan, 25 cm) at 25 °C. Optical rotations were measured on a JASCO DIP-370 (Na lamp, 589 nm). All NMR spectra figures could be found in Supplementary Materials.

Synthesis
All reactions were carried out in oven-dried glassware under an argon atmosphere. Flash column chromatography was performed with silica gel 60 (230-400 mesh ASTM, Merck, Darmstadt, Germany). TLC analysis was performed on Merck 0.25-millimeter Silicagel 60 F254 plates. Melting points were determined on a hot stage microscope apparatus (ATM-01, AS ONE, Osaka, Japan) and were uncorrected. NMR spectra were recorded on a JEOLRESONANCE EXC-400 or ECX-500 spectrometer (JEOL, Akishima, Japan) operating at 400 or 500 MHz for 1 H-NMR and 100 and 125 MHz for 13 C NMR. Chemical shifts (δ ppm) in CDCl3 were reported downfield from TMS (= 0) for 1 H-NMR. For 13 C-NMR, chemical shifts were reported in the scale relative to CDCl3 (77.00 ppm) as an internal reference. Mass spectra were measured on a JMS-T100LC spectrometer (JEOL, Akishima, Japan). HPLC data were obtained on a SHIMADZU (Kyoto, Japan) HPLC system (consisting of the following: LC-20AT, CMB20A, CTO-20AC and detector SPD-20A measured at 254 nm) using Chiracel AD-H or Ad-3 column (Daicel, Himeji, Japan, 25 cm) at 25 °C. Optical rotations were measured on a JASCO DIP-370 (Na lamp, 589 nm). All NMR spectra figures could be found in Supplementary Materials.

Synthesis
All reactions were carried out in oven-dried glassware under an argon atmosphere. Flash column chromatography was performed with silica gel 60 (230-400 mesh ASTM, Merck, Darmstadt, Germany). TLC analysis was performed on Merck 0.25-millimeter Silicagel 60 F254 plates. Melting points were determined on a hot stage microscope apparatus (ATM-01, AS ONE, Osaka, Japan) and were uncorrected. NMR spectra were recorded on a JEOLRESONANCE EXC-400 or ECX-500 spectrometer (JEOL, Akishima, Japan) operating at 400 or 500 MHz for 1 H-NMR and 100 and 125 MHz for 13 C NMR. Chemical shifts (δ ppm) in CDCl3 were reported downfield from TMS (= 0) for 1 H-NMR. For 13 C-NMR, chemical shifts were reported in the scale relative to CDCl3 (77.00 ppm) as an internal reference. Mass spectra were measured on a JMS-T100LC spectrometer (JEOL, Akishima, Japan). HPLC data were obtained on a SHIMADZU (Kyoto, Japan) HPLC system (consisting of the following: LC-20AT, CMB20A, CTO-20AC and detector SPD-20A measured at 254 nm) using Chiracel AD-H or Ad-3 column (Daicel, Himeji, Japan, 25 cm) at 25 °C. Optical rotations were measured on a JASCO DIP-370 (Na lamp, 589 nm). All NMR spectra figures could be found in Supplementary Materials.

Synthesis
All reactions were carried out in oven-dried glassware under an argon atmosphere. Flash column chromatography was performed with silica gel 60 (230-400 mesh ASTM, Merck, Darmstadt, Germany). TLC analysis was performed on Merck 0.25-millimeter Silicagel 60 F254 plates. Melting points were determined on a hot stage microscope apparatus (ATM-01, AS ONE, Osaka, Japan) and were uncorrected. NMR spectra were recorded on a JEOLRESONANCE EXC-400 or ECX-500 spectrometer (JEOL, Akishima, Japan) operating at 400 or 500 MHz for 1 H-NMR and 100 and 125 MHz for 13 C NMR. Chemical shifts (δ ppm) in CDCl3 were reported downfield from TMS (= 0) for 1 H-NMR. For 13 C-NMR, chemical shifts were reported in the scale relative to CDCl3 (77.00 ppm) as an internal reference. Mass spectra were measured on a JMS-T100LC spectrometer (JEOL, Akishima, Japan). HPLC data were obtained on a SHIMADZU (Kyoto, Japan) HPLC system (consisting of the following: LC-20AT, CMB20A, CTO-20AC and detector SPD-20A measured at 254 nm) using Chiracel AD-H or Ad-3 column (Daicel, Himeji, Japan, 25 cm) at 25 °C. Optical rotations were measured on a JASCO DIP-370 (Na lamp, 589 nm). All NMR spectra figures could be found in Supplementary Materials.

Synthesis
All reactions were carried out in oven-dried glassware under an argon atmosphere. Flash column chromatography was performed with silica gel 60 (230-400 mesh ASTM, Merck, Darmstadt, Germany). TLC analysis was performed on Merck 0.25-millimeter Silicagel 60 F 254 plates. Melting points were determined on a hot stage microscope apparatus (ATM-01, AS ONE, Osaka, Japan) and were uncorrected. NMR spectra were recorded on a JEOLRESONANCE EXC-400 or ECX-500 spectrometer (JEOL, Akishima, Japan) operating at 400 or 500 MHz for 1 H-NMR and 100 and 125 MHz for 13 C NMR. Chemical shifts (δ ppm) in CDCl 3 were reported downfield from TMS (= 0) for 1 H-NMR. For 13 C-NMR, chemical shifts were reported in the scale relative to CDCl 3 (77.00 ppm) as an internal reference. Mass spectra were measured on a JMS-T100LC spectrometer (JEOL, Akishima, Japan). HPLC data were obtained on a SHIMADZU (Kyoto, Japan) HPLC system (consisting of the following: LC-20AT, CMB20A, CTO-20AC and detector SPD-20A measured at 254 nm) using Chiracel AD-H or Ad-3 column (Daicel, Himeji, Japan, 25 cm) at 25 • C. Optical rotations were measured on a JASCO DIP-370 (Na lamp, 589 nm). All NMR spectra figures could be found in Supplementary Materials. [25].

Synthesis
All reactions were carried out in oven-dried glassware under an argon atmosphere. Flash column chromatography was performed with silica gel 60 (230-400 mesh ASTM, Merck, Darmstadt, Germany). TLC analysis was performed on Merck 0.25-millimeter Silicagel 60 F254 plates. Melting points were determined on a hot stage microscope apparatus (ATM-01, AS ONE, Osaka, Japan) and were uncorrected. NMR spectra were recorded on a JEOLRESONANCE EXC-400 or ECX-500 spectrometer (JEOL, Akishima, Japan) operating at 400 or 500 MHz for 1 H-NMR and 100 and 125 MHz for 13 C NMR. Chemical shifts (δ ppm) in CDCl3 were reported downfield from TMS (= 0) for 1 H-NMR. For 13 C-NMR, chemical shifts were reported in the scale relative to CDCl3 (77.00 ppm) as an internal reference. Mass spectra were measured on a JMS-T100LC spectrometer (JEOL, Akishima, Japan). HPLC data were obtained on a SHIMADZU (Kyoto, Japan) HPLC system (consisting of the following: LC-20AT, CMB20A, CTO-20AC and detector SPD-20A measured at 254 nm) using Chiracel AD-H or Ad-3 column (Daicel, Himeji, Japan, 25 cm) at 25 °C. Optical rotations were measured on a JASCO DIP-370 (Na lamp, 589 nm). All NMR spectra figures could be found in Supplementary Materials.

Conclusions
We performed asymmetric total syntheses of all of the both enantiomers of the anti-MRSA active plymuthipyranone B and three unnatural analogues. The present synthetic method utilized a privileged asymmetric Mukaiyama aldol addition using Ti(OiPr) 4 /(S)or (R)-BINOL catalysis as the key step, originally developed by Soriente and Scettri's group. The total syntheses were each implemented in three steps and an overall yield of 42-56% with a highly enantiomeric excess (95-99%). The bioassay of the anti-MRSA activity against ATCC 43300 and 33591 revealed that natural (R)-plymuthipyranone B and (R)-undecyl synthetic analogue at the C6 position exhibited the highest activity with low MIC values.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Informed consent was obtained from subjects involved in the study.
Data Availability Statement: Data is contained within article and supplementary material.