Total Synthesis of 6-Deoxydihydrokalafungin, a Key Biosynthetic Precursor of Actinorhodin, and Its Epimer

In this article, we report the total synthesis of 6-deoxydihydrokalafungin (DDHK), a key biosynthetic intermediate of a dimeric benzoisochromanequinone antibiotic, actinorhodin (ACT), and its epimer, epi-DDHK. Tricyclic hemiacetal with 3-siloxyethyl group was subjected to Et3SiH reduction to establish the 1,3-cis stereochemistry in the benzoisochromane, and a subsequent oxidation/deprotection sequence then afforded epi-DDHK. A bicyclic acetal was subjected to AlH3 reduction to deliver the desired 1,3-trans isomer in an approximately 3:1 ratio, which was subjected to a similar sequence to that used for the 1,3-cis isomer that successfully afforded DDHK. A semisynthetic approach from (S)-DNPA, an isolable biosynthetic precursor of ACT, was also examined to afford DDHK and its epimer, which are identical to the synthetic products.


Introduction
Actinorhodin (ACT, 1) is an aromatic polyketide belonging to the dimeric benzoisochromanequinone (BIQ) family [1] and is produced by Streptomyces coelicolor A3 (2), which is among the most genetically studied actinomycetes [2]. The biosynthesis of ACT (1) includes hydroxylation steps at the C-6 and C-8 positions of the benzoisochromane skeleton [3] mediated via the action of a two-component flavin-dependent monooxygenase (FMO), that is, the ActVA-ORF5/ActVB system comprising the oxygenase ActVA-5 and the flavin: NADH oxidoreductase ActVB. 6-Deoxydihydrokalafungin (DDHK, 3) was assumed to be the substrate of this FMO, undergoing sequential conversion to the trihydroxynaphthalene and tetrahydroxynaphthalene derivatives T3HN (4) and T4HN (5), respectively. DDHK (3) is presumed to be produced from (S)-DNPA (6) via reduction by ActVI-2, but has not yet been isolated from any biosynthetic strains of S. coelicolor or their mutants because of conversion to the shunt product actinoperylone [4]. To resolve the ambiguity regarding the intermediacy of DDHK (3) in ACT biosynthesis, we established a semisynthetic method for obtaining DDHK (3) and its epimer epi-DDHK (7) by the reduction of (S)-DNPA (6), an isolable biosynthetic precursor of ACT, and successfully clarified the function of the ActVA-ORF5/ActVB system in vitro using semisynthetic DDHK as the substrate [5].
We independently investigated the total synthesis of DDHK (3) and its epimer epi-DDHK (7) for the stereochemical correlation of the semisynthetic products. As shown in Scheme 1, DDHK (3) is composed of a benzoisochromane skeleton with two hydroxy groups on the C-9 and C-10 positions [6] and incorporates a disubstituted dihydropyran ring with 1,3-trans stereochemistry. In previous reports concerning the synthesis of the groups on the C-9 and C-10 positions [6] and incorporates a disubstituted dihydropyran ring with 1,3-trans stereochemistry. In previous reports concerning the synthesis of the biosynthetically related compounds kalafungin (8) and nanaomycin A (9), which contains a 1,3-trans-disubstituted benzoisochromane skeleton as well as a central quinone moiety, epimerization of the 1,3-cis isomer to the corresponding trans isomer under acidic conditions via conjugation with the C-5 carbonyl group was frequently employed [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. For example, Li and Ellison [8] reported the total synthesis of racemic kalafungin [(±)-8] and nanaomycin A [(±)-9], including acid-catalyzed epimerization of 1,3-cis 10 to the corresponding trans isomer 11 (Scheme 2a). However, this strategy is not suitable for the synthesis of DDHK (3) because of the lack of a carbonyl group or other oxygen functionality at the C-5 position. Therefore, a method for direct access to 1,3-trans-disubstituted benzoisochromanes is required. Zhang and O′Doherty reported a stereoselective oxa-Pictet-Spengler cyclization of quinol 13 and acetaldehyde for the formation of benzoisochromane 14 with 1,3-trans stereochemistry during their synthesis of nanaomycin A (9) [26] (Scheme 2b). Furthermore, the group of Suzuki and Ohmori successfully established the 1,3-trans stereochemistry in their total synthesis of ACT (1) by diastereoselective allylation at the C-3 position of hemiacetal 15 using allylsilane under acidic conditions, in which the stereochemistry was induced by the C-1 stereocenter [27,28] (Scheme 2c). In this article, we report the total synthesis of DDHK (3) and its epimer 7 using various reduction conditions for the stereoselective construction of the benzoisochromane moiety. First, we examined the total synthesis of epi-DDHK (7) with 1,3-cis stereochemistry via conventional reduction of cyclic hemiacetal 17, and we then explored reduction conditions for the construction of 1,3-trans-substituted benzoisochromane 18 to achieve the total synthesis of DDHK (3). Reduction of (S)-DNPA (6) to DDHK (3) and its epimer 7 was also examined to enable comparison of the spectral data of the synthetic and semisynthetic target compounds (Scheme 2d).

Total Synthesis of epi-DDHK (7)
Construction of the key benzoisochromane skeleton of DDHK (3) and its epimer 7 was accomplished via Staunton-Weinreb annulation [29,30] using toluate 18 and α,β-unsaturated lactone 19 derived from L-aspartic acid [31], as reported by Donner [22,24] (Scheme 3). The MOM group was selected as the protecting group for phenol 20 to allow for more facile final-stage deprotection under mild acidic conditions compared with methyl group protection. Methylation of lactone 21 with excess CH3Li afforded cyclic hemiacetal 22 as a mixture of diastereomers. Treatment of 22 with Et3SiH in the presence of TFA in CH2Cl2 [14,15] followed by deprotection of the TBS group with HCl delivered the corresponding 1,3-cis-substituted benzoisochromane 23 in 40% yield from lactone 21. The stereochemistry of 23 was confirmed by the similarity of its NMR data (see supplementary material) to the corresponding methoxy derivative 24 [24]. In this reaction, cyclic enone 25 bearing a hydroxyethyl side chain was obtained as a byproduct, the formation of which was ascribed to dehydration of unreacted hemiacetal 22. After MOM protection of the phenolic OH in 23, stepwise oxidation of alcohol 26 using TEMPO followed by Pinnick oxidation [24] afforded carboxylic acid 27. Finally, removal of the MOM groups under acidic conditions furnished the desired epi-DDHK (7, 14%) alongside mono-MOM-protected epi-DDHK (28, 37%).

Total Synthesis of epi-DDHK (7)
Construction of the key benzoisochromane skeleton of DDHK (3) and its epimer 7 was accomplished via Staunton-Weinreb annulation [29,30] using toluate 18 and α,βunsaturated lactone 19 derived from L-aspartic acid [31], as reported by Donner [22,24] (Scheme 3). The MOM group was selected as the protecting group for phenol 20 to allow for more facile final-stage deprotection under mild acidic conditions compared with methyl group protection. Methylation of lactone 21 with excess CH 3 Li afforded cyclic hemiacetal 22 as a mixture of diastereomers. Treatment of 22 with Et 3 SiH in the presence of TFA in CH 2 Cl 2 [14,15] followed by deprotection of the TBS group with HCl delivered the corresponding 1,3-cis-substituted benzoisochromane 23 in 40% yield from lactone 21. The stereochemistry of 23 was confirmed by the similarity of its NMR data (see Supplementary Material) to the corresponding methoxy derivative 24 [24]. In this reaction, cyclic enone 25 bearing a hydroxyethyl side chain was obtained as a byproduct, the formation of which was ascribed to dehydration of unreacted hemiacetal 22. After MOM protection of the phenolic OH in 23, stepwise oxidation of alcohol 26 using TEMPO followed by Pinnick oxidation [24] afforded carboxylic acid 27. Finally, removal of the MOM groups under acidic conditions furnished the desired epi-DDHK (7, 14%) alongside mono-MOM-protected epi-DDHK (28, 37%).

Stereoselective Reduction Trials Using Modified Silane Reduction Conditions
Next, the diastereoselective reduction of hemiacetal 22 was examined using several silane reagents for the formation of trans-substituted benzoisochromanes. The attempted utilization of Ph3SiH instead of Et3SiH for the reduction of 22 afforded no reduced products, which was presumably attributable to steric hindrance (data not shown). Similarly, efforts to introduce the silane group into the phenolic OH moiety of 21 using diphenylchlorosilane to prepare silane 29 in the presence of bases such as imidazole and Hünig's base resulted in no reaction (Scheme 4). When NaH was applied as the base, desilylated alcohol 30 and disilane 31 [32], the formation of which was ascribed to reaction between the TBS group of 21 and the diphenylchlorosilyl anion, were obtained. Desilylated alcohol 30 was further modified by the attachment of a diisopropylsilyl group, and the resulting lactone 32 was methylated and then treated with TFA in order to promote intramolecular delivery of hydride from the silane moiety to the α face of the presumed oxonium ion derived from lactol. However, only bicyclic acetal 33 was obtained, rather than the desired reduced product 34. Scheme 4. Trials for alternative silane reduction.

Stereoselective Reduction Trials with Bicyclic Acetal and Total Synthesis of DDHK (3)
For the construction of the benzoisochromane skeleton bearing 1,3-trans stereochemistry, we focused on the aforementioned bicyclic acetal 33 (Scheme 4). The similar bicyclic acetal 35 was reported by Donner as a byproduct of the Et3SiH reduction of the hemiacetal derived from lactone 36 to 1,3-cis benzoisochromane 37 during the total synthesis of 5-epi-Scheme 3. Construction of tricyclic lactone 21 and synthesis of epi-DDHK (7).

Stereoselective Reduction Trials Using Modified Silane Reduction Conditions
Next, the diastereoselective reduction of hemiacetal 22 was examined using several silane reagents for the formation of trans-substituted benzoisochromanes. The attempted utilization of Ph 3 SiH instead of Et 3 SiH for the reduction of 22 afforded no reduced products, which was presumably attributable to steric hindrance (data not shown). Similarly, efforts to introduce the silane group into the phenolic OH moiety of 21 using diphenylchlorosilane to prepare silane 29 in the presence of bases such as imidazole and Hünig's base resulted in no reaction (Scheme 4). When NaH was applied as the base, desilylated alcohol 30 and disilane 31 [32], the formation of which was ascribed to reaction between the TBS group of 21 and the diphenylchlorosilyl anion, were obtained. Desilylated alcohol 30 was further modified by the attachment of a diisopropylsilyl group, and the resulting lactone 32 was methylated and then treated with TFA in order to promote intramolecular delivery of hydride from the silane moiety to the α face of the presumed oxonium ion derived from lactol. However, only bicyclic acetal 33 was obtained, rather than the desired reduced product 34.

Stereoselective Reduction Trials Using Modified Silane Reduction Conditions
Next, the diastereoselective reduction of hemiacetal 22 was examined using several silane reagents for the formation of trans-substituted benzoisochromanes. The attempted utilization of Ph3SiH instead of Et3SiH for the reduction of 22 afforded no reduced products, which was presumably attributable to steric hindrance (data not shown). Similarly, efforts to introduce the silane group into the phenolic OH moiety of 21 using diphenylchlorosilane to prepare silane 29 in the presence of bases such as imidazole and Hünig's base resulted in no reaction (Scheme 4). When NaH was applied as the base, desilylated alcohol 30 and disilane 31 [32], the formation of which was ascribed to reaction between the TBS group of 21 and the diphenylchlorosilyl anion, were obtained. Desilylated alcohol 30 was further modified by the attachment of a diisopropylsilyl group, and the resulting lactone 32 was methylated and then treated with TFA in order to promote intramolecular delivery of hydride from the silane moiety to the α face of the presumed oxonium ion derived from lactol. However, only bicyclic acetal 33 was obtained, rather than the desired reduced product 34. Scheme 4. Trials for alternative silane reduction.

Stereoselective Reduction Trials with Bicyclic Acetal and Total Synthesis of DDHK (3)
For the construction of the benzoisochromane skeleton bearing 1,3-trans stereochemistry, we focused on the aforementioned bicyclic acetal 33 (Scheme 4). The similar bicyclic acetal 35 was reported by Donner as a byproduct of the Et3SiH reduction of the hemiacetal derived from lactone 36 to 1,3-cis benzoisochromane 37 during the total synthesis of 5-epi-Scheme 4. Trials for alternative silane reduction.

Stereoselective Reduction Trials with Bicyclic Acetal and Total Synthesis of DDHK (3)
For the construction of the benzoisochromane skeleton bearing 1,3-trans stereochemistry, we focused on the aforementioned bicyclic acetal 33 (Scheme 4). The similar bicyclic acetal 35 was reported by Donner as a byproduct of the Et 3 SiH reduction of the hemiacetal derived from lactone 36 to 1,3-cis benzoisochromane 37 during the total synthesis of 5-epi-9-methoxykalafungin, where NaBH 4 reduction of 35 afforded 37 [24] (Scheme 5a). We examined an alternative method for the preparation of these bicyclic acetals. Methylation of lactone 21 followed by acid treatment delivered cyclic enone 25 in 77% yield over two steps (Scheme 5b). Subsequent treatment of 25 with excess NaH and MOMCl led to the formation of bicyclic acetal 33 (5%) and the corresponding bis-MOM ether 38 (47%). Therefore, reduction trials using major component 38 were performed to explore the construction of the desired 1,3-trans stereochemistry. s 2021, 26, x FOR PEER REVIEW 5 of 14 9-methoxykalafungin, where NaBH4 reduction of 35 afforded 37 [24] (Scheme 5a). We examined an alternative method for the preparation of these bicyclic acetals. Methylation of lactone 21 followed by acid treatment delivered cyclic enone 25 in 77% yield over two steps (Scheme 5b). Subsequent treatment of 25 with excess NaH and MOMCl led to the formation of bicyclic acetal 33 (5%) and the corresponding bis-MOM ether 38 (47%). Therefore, reduction trials using major component 38 were performed to explore the construction of the desired 1,3-trans stereochemistry. The reduction of 38 under acidic conditions, which would affect the acetal group, was examined. Application of NaBH3CN in the presence of aqueous HCl afforded a mixture of diastereomers (cis:trans = 81:19, as determined by 1 H-NMR for the crude product, see supplementary material), where the 1,3-cis isomer 26 and the desired trans isomer 39 were isolated in 69% and 13% yield, respectively (Table 1, run 1). The trans stereochemistry of 39 was confirmed by NOE experiments, where enhancement of the signals between the C-1 methyl group and C-3 H atom was observed. The trans selectivity was slightly improved (cis:trans = 74:26) when BH3·THF was used (run 2). Inspired by the trans selectivity reported by Yamamoto and co-workers for the reduction of aliphatic bicyclic acetals using aluminum hydrides [33], we next examined the use of DIBAL-H. The reaction in CH2Cl2 at −78 °C afforded an approximately 1:1 mixture of diastereomers (run 3), whereas the undesired cis isomer was preferentially obtained when Et2O was used as solvent (run 4). The reaction in THF exhibited improved selectivity with a cis:trans ratio of about 2:3 (run 5). As an alternative aluminum hydride reagent, AlH3 prepared from LiAlH4 and AlCl3 was next investigated. Reaction at −78 °C delivered the desired trans isomer in an improved cis:trans ratio of about 1:3, albeit with only low conversion (run 6). Increasing the temperature to −60 °C afforded a similar diastereoselectivity with improved chemical yield (run 7). Extending the reaction time did not further improve the yield (run 8). The reduction of 38 under acidic conditions, which would affect the acetal group, was examined. Application of NaBH 3 CN in the presence of aqueous HCl afforded a mixture of diastereomers (cis:trans = 81:19, as determined by 1 H-NMR for the crude product, see Supplementary Material), where the 1,3-cis isomer 26 and the desired trans isomer 39 were isolated in 69% and 13% yield, respectively (Table 1, run 1). The trans stereochemistry of 39 was confirmed by NOE experiments, where enhancement of the signals between the C-1 methyl group and C-3 H atom was observed. The trans selectivity was slightly improved (cis:trans = 74:26) when BH 3 ·THF was used (run 2). Inspired by the trans selectivity reported by Yamamoto and co-workers for the reduction of aliphatic bicyclic acetals using aluminum hydrides [33], we next examined the use of DIBAL-H. The reaction in CH 2 Cl 2 at −78 • C afforded an approximately 1:1 mixture of diastereomers (run 3), whereas the undesired cis isomer was preferentially obtained when Et 2 O was used as solvent (run 4). The reaction in THF exhibited improved selectivity with a cis:trans ratio of about 2:3 (run 5). As an alternative aluminum hydride reagent, AlH 3 prepared from LiAlH 4 and AlCl 3 was next investigated. Reaction at −78 • C delivered the desired trans isomer in an improved cis:trans ratio of about 1:3, albeit with only low conversion (run 6). Increasing the temperature to −60 • C afforded a similar diastereoselectivity with improved chemical yield (run 7). Extending the reaction time did not further improve the yield (run 8).
The obtained trans isomer 39 was subjected to a similar oxidation sequence as cis isomer 26 to afford carboxylic acid 40. Subsequent removal of the MOM groups using 10% HCl in THF for 3 h afforded only a trace amount of DDHK (3) alongside the monoprotected DDHK (41, 32%). However, adjustment of the acid concentration and reaction time improved the yield of DDHK (3) and 41 to 27% and 71%, respectively. Trial of deprotection of mono MOM ether 41 using aqueous HCl resulted in a formation of complex mixture. However, application of BCl 3 to 41 at −78 • C afforded DDHK (3) in 69% yield (Scheme 6). the undesired cis isomer was preferentially obtained when Et2O was used as solvent (run 4). The reaction in THF exhibited improved selectivity with a cis:trans ratio of about 2:3 (run 5). As an alternative aluminum hydride reagent, AlH3 prepared from LiAlH4 and AlCl3 was next investigated. Reaction at −78 °C delivered the desired trans isomer in an improved cis:trans ratio of about 1:3, albeit with only low conversion (run 6). Increasing the temperature to −60 °C afforded a similar diastereoselectivity with improved chemical yield (run 7). Extending the reaction time did not further improve the yield (run 8). The obtained trans isomer 39 was subjected to a similar oxidation sequence as cis isomer 26 to afford carboxylic acid 40. Subsequent removal of the MOM groups using 10% HCl in THF for 3 h afforded only a trace amount of DDHK (3) alongside the monoprotected DDHK (41, 32%). However, adjustment of the acid concentration and reaction time improved the yield of DDHK (3) and 41 to 27% and 71%, respectively. Trial of deprotection of mono MOM ether 41 using aqueous HCl resulted in a formation of complex mixture. However, application of BCl3 to 41 at −78 °C afforded DDHK (3) in 69% yield (Scheme 6).

Semisynthesis of DDHK (3) and Its Epimer 7 from (S)-DNPA (6)
To confirm the structures of the synthetic DDHK (3) and its epimer 7, (S)-DNPA (6) was isolated from a transformant of S. coelicolor [34] and then subjected to NaBH4 reduction in methanol (Scheme 7). The crude product consisting of an approximately 1:1 mixture of diastereoisomers was purified by reverse-phase HPLC to afford DDHK (3) and its epimer 7. The semisynthetic products were identified by comparison of their spectral data with those of synthetic 3 and 7 (Figure 1). DDHK (3) was observed to be more polar than epi-DDHK (7).

Semisynthesis of DDHK (3) and Its Epimer 7 from (S)-DNPA (6)
To confirm the structures of the synthetic DDHK (3) and its epimer 7, (S)-DNPA (6) was isolated from a transformant of S. coelicolor [34] and then subjected to NaBH 4 reduction in methanol (Scheme 7). The crude product consisting of an approximately 1:1 mixture of diastereoisomers was purified by reverse-phase HPLC to afford DDHK (3) and its epimer 7. The semisynthetic products were identified by comparison of their spectral data with those of synthetic 3 and 7 (Figure 1). DDHK (3) was observed to be more polar than epi-DDHK (7). was isolated from a transformant of S. coelicolor [34] and then subjected to NaBH4 reduction in methanol (Scheme 7). The crude product consisting of an approximately 1:1 mixture of diastereoisomers was purified by reverse-phase HPLC to afford DDHK (3) and its epimer 7. The semisynthetic products were identified by comparison of their spectral data with those of synthetic 3 and 7 (Figure 1). DDHK (3) was observed to be more polar than epi-DDHK (7).
IR spectra were recorded on JASCO FT/IR-4100 and FT/IR-4600 spectrophotometers (Tokyo, Japan) with NaCl plate or Attenuated Total Reflectance Unit ATR PRO450-S. EI-MS was recorded on a JEOL GC-Mate II (Tokyo, Japan). ESIMS was recorded on a JEOL JMS-T100LP in positive ion mode and Thermo Fisher Scientific LTQ Orbitrap XL (Waltham, MA, USA) in positive mode. Optical rotation was measured by JASCO P-1020 and DIP-1000 polarimeters (Tokyo, Japan) 1 H and 13 C-NMR spectra were recorded on JEOL ECX 400 (400 MHz for 1 H-NMR and 100 MHz for 13 C-NMR) and a JEOL LA 500 spectrometers (125 MHz for 13 C-NMR)). Chemical shifts were reported in ppm and J in Hz. Abbreviations were used for multipilicity: s = singlet, d = doublet, dd = doublets of doublet, ddd = doublets of doublets of doublet, dddd = doublets of doublets of doublets of doublet, t = triplet, quint = quintet, m = multiplet.
IR spectra were recorded on JASCO FT/IR-4100 and FT/IR-4600 spectrophotometers (Tokyo, Japan) with NaCl plate or Attenuated Total Reflectance Unit ATR PRO450-S. EI-MS was recorded on a JEOL GC-Mate II (Tokyo, Japan). ESIMS was recorded on a JEOL JMS-T100LP in positive ion mode and Thermo Fisher Scientific LTQ Orbitrap XL (Waltham, MA, USA) in positive mode. Optical rotation was measured by JASCO P-1020 and DIP-1000 polarimeters (Tokyo, Japan) 1 H and 13 C-NMR spectra were recorded on JEOL ECX 400 (400 MHz for 1 H-NMR and 100 MHz for 13 C-NMR) and a JEOL LA 500 spectrometers (125 MHz for 13 C-NMR)). Chemical shifts were reported in ppm and J in Hz. Abbreviations were used for multipilicity: s = singlet, d = doublet, dd = doublets of doublet, ddd = doublets of doublets of doublet, dddd = doublets of doublets of doublets of doublet, t = triplet, quint = quintet, m = multiplet.