Total Synthesis of γ-Alkylidenebutenolides, Potent Melanogenesis

Article Cite This: J. Org. Chem. 2018, 83, 8250−8264

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Total Synthesis of γ‑Alkylidenebutenolides, Potent Melanogenesis Inhibitors from Thai Medicinal Plant Melodorum f ruticosum Genzoh Tanabe,*,†,‡,∥ Yoshiaki Manse,‡,∥ Teppei Ogawa,† Naoki Sonoda,† Shinsuke Marumoto,§ Fumihiro Ishikawa,† Kiyofumi Ninomiya,‡ Saowanee Chaipech,⊥ Yutana Pongpiriyadacha,¶ Osamu Muraoka,‡ and Toshio Morikawa*,‡

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†

Faculty of Pharmacy, ‡Pharmaceutical Research and Technology Institute, and §Joint Research Center, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan ⊥ Faculty of Agro-Industry and ¶Faculty of Science and Technology, Rajamangala University of Technology Srivijaya, Thungyai, Nakhon Si Thammarat 80240, Thailand S Supporting Information *

ABSTRACT: A hitherto unreported member of γ-alkylidenebutenolides in Melodorum f ruticosum (Annonaceae), (4E)6-benzoyloxy-7-hydroxy-2,4-heptadiene-4-olide, named as isofruticosinol (4) was isolated from the methanol extract of flowers, along with the known related butenolides, namely, the (4Z)-isomer (3) of 4, melodrinol (1), and its (4E)-isomer (2). To unambiguously determine the absolute configuration at the C-6 position in these butenolides, the first total syntheses of both enantiomers of 2−4 were achieved over 6− 7 steps from commercially available D- or L-ribose (D- and L5). Using the same protocol, both enantiomers of 1 were also synthesized. Based on chiral HPLC analysis of all synthetic compounds (S- and R-1−4), all naturally occurring butenolides were assigned as partial racemic mixtures with respect to the chiral center at C-6 (enantiomeric ratio, 6S/6R = ∼83/17). Furthermore, the melanogenesis inhibitory activities of S- and R-1−4 were evaluated, with all shown to be potent inhibitors with IC50 values in the range 0.29−2.9 μM, regardless of differences in the stereochemistry at C-6. In particular, S-4 (IC50 = 0.29 μM) and R-4 (0.39 μM) showed potent inhibitory activities compared with that of reference standard arbutin (174 μM).

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stimulant, antipyretic, and hematinic.10 The essential oil of M. f ruticosum flowers is also a traditional medicine used in aromatherapy in Thailand.10 In the early 1990s, three γalkylidenebutenolides (1−3) were isolated from M. fruticosum as cytotoxic constituents against several tumor cell lines (Figure 2).11 Melodorinol (1) was first identified by Jung et al. as a partially racemic mixture ([α]D = −4011a and −37,11b both in CHCl3) predominantly containing the S-isomer (S-1) by applying Horeau’s method for the determination of absolute configuration.11b Thereafter, Lu et al.12b successfully achieved the syntheses of both enantiomers of 1 and concluded, contrary to Jung’s report,11b that R-1 should be the main constituent of natural melodorinol (1) with an enantiomeric excess (ee) of approximately 45% by comparing the specific rotations of the synthetic enantiomers (S-1: [α]D = +86.4 and R-1: [α] D = −88.0, both in CHCl 3 ). Furthermore, phytochemical analysis of M. f ruticosum10a,13 and other Annonaceae family plants (Cleistochlamys kirkii,14c Xylopia pierrei,14d and Artabotrys madagascariensis)14e was subsequently performed, which identified 1 as a constituent of these plants.

INTRODUCTION Δα,β-Butenolides [furan-2(5H)-ones] are important heterocyclic compounds that constitute the structural core of numerous natural products and related bioactive compounds.1Natural and synthetic compounds containing the butenolide ring system have received considerable attention because they exhibit a diverse range of biological activities,2which depend on the substitution pattern on the furanone ring. In past decades, a large number of Δα,β-butenolides, commonly bearing an alkylidene appendage at the γ-position, have been isolated from natural sources.3 Examples of γalkylidenebutenolides, namely asruncin B,4 aspergon B,5 cryptoconcatone,6 goniobutenolide B,7 hygrophorone F,8 and tetrenolin,9 which exhibit various biological activities, such as cytotoxic,4,7 α-glucosidase inhibitory,5 nitric oxide production inhibitory,6 fungicidal,8and antibacterial9 activities, are shown in Figure 1. Melodorum f ruticosum (Annonaceae) is a shrub found in Southeast Asia and commonly known as “Devil Tree” or “White Cheesewood” and locally known as “Lamduan” in Thailand. The flower of M. f ruticosum is used as an ingredient in Thai traditional medicine recipe “Geasorn Thung Gao”, which is traditionally prescribed as a tonic, mild cardiotonic © 2018 American Chemical Society

Received: April 18, 2018 Published: July 4, 2018 8250

DOI: 10.1021/acs.joc.8b00986 J. Org. Chem. 2018, 83, 8250−8264

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Figure 1. Representative natural γ-alkylidenebutenolides.

Figure 2. Chemical structures of butenolides (S- and R-1−4).

The (R)-isomer (R-1) was isolated from C. kirkii and X. pierrei and showed an even smaller specific rotation ([α]D = −514c in MeOH and [α]D = −1114d in CHCl3) than that of early reports by Tuchinda et al.11a and Jung et al.11b Meanwhile, Hongnak et al.13a and Murphy et al.14e isolated the counterpart isomer (S-1) from M. f ruticosum and A. madagascariensis, respectively. However, their assignment was not reliable because no [α]D values were reported, even though 1 had been isolated as a dextrorotatory compound for the first time. Therefore, the stereochemistry of natural melodorinol (1) remains ambiguous. Furthermore, the absolute configurations of other related natural butenolides (2 and 3) have yet to be determined, despite approximately 30 years having passed since the first isolation report.11 Therefore, there is strong demand for an unambiguous determination of the absolute configuration of all butenolides isolated from the Annonaceae family. In the course of our characterization studies on bioactive constituents from Thai medicinal plants,15 the methanol extract of flowers of M. fruticosum was found to inhibit theophylline-stimulated melanogenesis in murine B16 melanoma 4A5 cells. Through bioassay-guided separation, a hitherto missing member of the γ-alkylidenebutenolides in M. f ruticosum, isofruticosinol [(4E)-6-benzoyloxy-7-hydroxy2,4-heptadiene-4-olide (4)], was isolated, together with related butenolides 1−3. Herein, full details of the isolation and identification of new butenolide 4 are described, in addition to the first total syntheses of the S- and R-enantiomers of 2−4, starting from D- and L-ribose (D- and L-5), respectively. The protocol also successfully yielded enantiomers S-1 and R-1 in good yield. Using chiral HPLC analysis of the synthetic butenolides, all isolates (1−4) were unambiguously identified as partially racemic mixtures predominantly containing S-

isomers (∼66% ee). Furthermore, it is of interest to identify why the value of the specific rotation varied for each isolation of melodorinol (1). If variations in the specific rotation were not caused by experimental error or contamination, then epimerization at the allylic chiral center (C-6) during the extraction process should be accounted for. Therefore, attempts to explore the cause of epimerization of 1 are also described. Furthermore, structure−activity relationship (SAR) studies on the melanogenesis inhibitory activities of S- and Renantiomers of synthetic butenolides (1−4) showed that all enantiomers are potent melanogenesis inhibitors (IC50 = 0.29−2.9 μM). Among them, S-2 and R-2 were over 400-fold more potent than arbutin, which was used as a positive control.

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RESULTS AND DISCUSSION Dried flowers (1.86 kg) of M. fruticosum collected in Thailand were extracted with MeOH to afford an extract (11.31% from the dried materials). The MeOH extract inhibited melanogenesis (IC50 = 5.8 μg/mL) in theophylline-stimulated murine B16 melanoma 4A5 cells without inducing cytotoxicity at the effective concentration (see Supporting Information, Table S1). The extract was then partitioned with a mixture of EtOAc and H2O (1/1, v/v) and divided into two fractions. The EtOAc-soluble fraction (6.31%) was active for melanogenesis inhibition (IC50 = 2.1 μg/mL) without notable cytotoxicity at the effective concentration. Meanwhile, the aqueous fraction was separated by Diaion HP-20 column chromatography (H2O → MeOH) into H2O and MeOH-eluted fractions (2.98% and 1.67%, respectively), both of which showed no notable activity. The EtOAc-soluble fraction was then subjected to a combination of normal-phase silica gel and reversed-phase octadecylsilyl (ODS) column chromatography and ODS HPLC to isolate a new butenolide, isofruticosinol (4, 8251

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configuration. Therefore, the planar structure of 4 was clarified. Although isolate 4 was a levorotatory compound ([α]D25 = −53.4 in CHCl3), as shown in Table 2, the absolute

0.0054%), along with melodorinol (1, 0.0910%), 2 (0.0010%), and 3 (0.0237%) as shown in Figure 2. The 1H and 13C NMR spectral properties of isolates 1−3 were assigned by comparison with those reported.11a High-resolution mass spectroscopy (HRMS) of 4 recorded in positive ion mode (ESI) showed a peak at m/z 283.0574 due to the sodium adduct ion, which indicated that the molecular formula was C14H12O5. The infrared (IR) spectrum of 4 showed two absorptions at 1786 and 1759 cm−1, which were characteristic of carbonyl stretching at the α-position of the unsaturated Δα,β-butenolide system16 and an absorption at 1716 cm−1 that was attributed to carbonyl stretching of the benzoate moiety. Furthermore, a broad absorption at 3460 cm−1 indicated the presence of a hydroxy group. As shown in Table 1, the 1H NMR spectrum of 4 had four signals at δH‑7a

Table 2. Specific Rotations of Natural and Synthetic Butenolides (1−4) naturals

1

2

present study reported

[α]D26 +66.9 (c 0.52) [α]D20 −40 (c 0.26)11a [α]D −37 (c 1.0)11b [α]D20 −5.0 (c 0.01a)14c [α]D29 −11 (c 0.30)14d S-1

[α]D26 −29.3 (c 0.48) [α]D20 +21.9 (c 0.35)11a

synthetics present study reported

Table 1. 1H and 13C NMR Data (in CDCl3) of Isofruticosinol (4) δH (800 MHz)

position 1 2 3 4 5 6 7 7-OH OCOPh 1′ 2′,6′ 3′,5′ 4′

6.33 (dd, 5.6, 1.8) 7.97 (dd, 5.6, 0.8) 5.80 5.90 3.93 3.98 2.29

(ddd, 10.0, 1.8, 0.8) (ddd, 10.0, 6.3, 4.3) (dd, 12.0, 4.4) (dd, 12.0, 6.3) (br s)

8.03 (2H, dd, 8.3, 1.4) 7.45 (2H, dd, 8.3, 7.5) 7.59 (tt, 7.5, 1.4)

δC (200 MHz)

+94.2 (c 1.32) +72 (c 1)12a +86.4 (c 0.95)12b +92.5 (c 0.08)12c +91.0 (c 0.98)12d R-1

[α]D25 −42.7 (c 1.00)

[α]D24 [α]D20

−94.0 (c 1.25) −88.0 (c 1.36)12b 3

[α]D25

synthetics

168.8 122.4 140.5 153.6 108.6 70.3 64.4

present study reported naturals

165.9 129.4 129.7 128.5 133.5

a

S-2

[α]D24 [α]D22 [α]D22 [α]D25 [α]D26

R-2 +41.6 (c 1.10) 4

present study reported synthetics

[α]D25 −146 (c 1.51) [α]D20 +130 (c 0.7)11a S-3

[α]D25 −53.4 (c 1.31)

pesent study synthetics

[α]D25 −219 (c 1.08) R-3

[α]D24 −80.6 (c 1.10) R-4

present study

[α]D25 +221 (c 0.95)

[α]D25 +80.5 (c 1.05)

S-4

Measured in methanol; others, measured in CHCl3.

configuration of 4 could not be unambiguously determined at this stage because both enantiomers were not in hand. In contrast, the optical rotation of melodorinol (1) was positive ([α]D26 = +66.9 in CHCl3), which was the opposite sign to that reported previously, as summarized in Table 2.11,14c,d Therefore, we concluded that natural product 1 itself must contain the S-isomer as the main component. Other isolates 2 and 3 showed negative optical rotations (2: [α]D26 = −29.3 and 3: [α]D25 −146; both in CHCl3). Interestingly, their specific rotations were also the opposite sign to those previously reported for 2 and 3 by Tuchinda et al.11a Therefore, to determine the absolute configurations and optical purities of isolated butenolides 1−4 unambiguously, all chiral butenolides (S- and R-1−4) were synthesized. Retrosynthetically, construction of the γ-alkylidenebutenolide core structures (C and D) for targets S-1−4 was envisioned from the E1cb elimination of the C-5 hydroxyl group of 5-hydroxybytenolide (B), which could be formed through the hydrolysis and intramolecular cyclization of acetonide-protected (Z)-ester A. Compound A would be prepared by Wittig reaction of commercially available D-ribose (D-5) with a stable ylide (Ph3PCHCO2CH3). Using the strategy designed for S-1−4, opposite enantiomers R-1−4 were synthesized by starting from L-ribose (L-5) (Scheme 1). First, the syntheses of (S)-melodorinol (S-1) and its geometric isomer (S-2) began with selective acetalization of hydroxyls at the C-2 and C-3 position of D-ribose (D-5) by treatment with acidic acetone, with the corresponding acetonide18 (D-6) obtained in 88% yield. After selective protection of the primary hydroxyl of D-6 by a benzoyl moiety using a slight excess of benzoyl chloride (1.2 equiv) at −15 °C,

3.93, δH‑7b 3.98, δH‑5 5.80, and δH‑6 5.90 forming an ABXM pattern. Furthermore, two signals at δH 6.33 and δH 7.97 resonated as doublets of doublets, corresponding to olefinic protons H-2 and H-3, respectively. This indicated the presence of the Δα,β-unsaturated enone system of butenolide. The small coupling constants (J2−5 = 1.8 Hz) and (J3−5 = 0.8 Hz) of these signals were clearly attributed to a long-range coupling with olefinic proton H-5. The value of the former coupling constants (J2−5) was typical of a trans/trans-configuration between H-2 and H-5 protons in the conjugated diene system.17 The stereochemistry of the exo-double bond was further supported by differential nuclear Overhauser effect (NOE) experiments. Meanwhile, the connectivity of seven carbons (C-1−C-7) and the position of the benzoyl (Bz) moiety were confirmed using heteronuclear multiple bond correlation (HMBC) experiments, as shown in Figure 3. Furthermore, a marked NOE enhancement was observed between H-3 and H-6 but not between H-3 and H-5, supporting that the exo-double bond at C-4 had a Z-

Figure 3. HMBC and NOE correlations of 4. 8252

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The Journal of Organic Chemistry Scheme 1. Retrosynthetic Analysis of Butenolides (1−4)

Scheme 2. Syntheses of Butenolides S-1, S-2, R-1, and R-2a

Reagents and conditions: i. acetone, conc. H2SO4, rt (D-6: 88%; L-6: 86%); ii. BzCl, pyridine, −15 °C (D-7: 73%; L-7: 71%); iii. Ph3P = CHCO2CH3, CH2Cl2, reflux; iv. TBDPSCl, AgNO3, Py, CH3CN, 0 °C−rt; v. AcOH/H2O (6/1, v/v), 100 °C (10: 56%, 11: 15% from D-7; ent10: 53%, ent-11: 14% from L-7); vi. Ac2O, Et3N, DMAP, CH2Cl2, 0 °C−rt (E-12: 46%, Z-12: 45%, ent-E-12: 47%, ent-Z-12: 42%; vii. TBAF/ AcOH (1/3.3), THF, 0 °C (S-1: 84%, S-2: 86%, R-1: 83%, R-2: 82%). a

the resultant monobenzoate19 (D-7) was subjected to Wittig reaction with phosphorane Ph3PCHCO2CH3 to give a mixture of geometric isomers Z- and E-8 (E/Z = approximately 3.7/1) that was difficult to separate. The isomeric mixture was silylated with TBDPSCl in pyridine in the presence of silver nitrate and to give a mixture of silyl ethers Z- and E-9 that was also difficult to separate. The mixture of Z- and E-9 was directly heated in aqueous acetic acid at 100 °C to achieve deacetalization followed by lactonization of the major E-9 isomer to afford corresponding butenolide 10 in 56% yield from D-7 along with the formation of small amount of (E)-ester 11. Major product 10 showed IR absorptions at 1786, 1755, and 1724 cm−1, with the first two characteristic of α-unsubstituted butenolide carbonyl stretching and the last peak attributed to ester carbonyl stretching. Finally, Δα,β-butenolide formation was confirmed by the HMBC correlation observed between the H-4 and the lactone carbonyl carbon (C-1), as shown in Scheme 2. Furthermore, the 1H NMR spectrum of 10 showed two signals at δH 6.10 and 7.18 attributed to α and β protons of the

butenolide ring, respectively, with a J2−3 value of 5.8 Hz supporting a cis-configuration. In contrast, minor product 11 showed two carbonyl absorptions at 1724 and 1712 cm−1 attributed to C6H5(CO)− and −(CO)OCH3 moieties, respectively. In the 1H NMR spectrum of 11, signals at δH 6.78 and 6.96 corresponded to protons α and β of the ester carbonyl, respectively, with a large J value of 15.8 Hz indicating that these protons had a trans-configuration. Subsequently, major butenolide 10 was subjected to E1cb elimination by treatment with acetic anhydride to give E-12 and Z-12 both in good yields. The stereochemistry of the newly introduced exodouble bond of E-12 was determined to be trans based on differential NOE experiments, as shown in Scheme 2. A marked NOE enhancement was observed between H-3 and H-6 but not between H-3 and H-5, supporting that the exo-double bond had a Z-configuration. Furthermore, the geometry of the double bond in counterpart Z-12 was not characterized by NOE experiments because the signals of H-5 and H-6 overlapped in the range 5.18−5.22 ppm. Fortunately, single crystals of Z-12 were obtained, and structural 8253

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of D-6 was selectively protected with a TBDPS group to afford silyl ether D-1320 in 97% yield. Compound D-13 was then subjected to Wittig reaction with Ph3PCHCO2CH3 to give an approximately 4:1 mixture of α,β-unsaturated (Z)-ester (Z14) and its E-isomer (E-14) in good yield. Major ester Z-14 was then heated in aqueous acetic acid at 100 °C to afford the corresponding butenolide (15) in 82% yield. The IR spectrum of 15 showed characteristic bands at 1786 and 1751 cm−1, which indicated Δα,β-butenolide formation, which was confirmed by HMBC correlation between H-4 and C-1 depicted in Scheme 3. Upon treating 15 with benzoic anhydride in the presence of NEt3 and DMAP, benzoylation of the two hydroxyl groups at C-5 and C-6 and subsequent E1cb elimination of the benzoyloxy moiety at C-5 smoothly proceeded to give (E)-γ-alkylidenebutenolide (E-16) and its Zisomer (Z-16) in 38% and 51% yields, respectively. The stereochemistry of the newly introduced exo-double bond in E16 and Z-16 was confirmed by NOE experiments, as shown in Scheme 3. Finally, deprotection of the TBDPS moiety of Z-16 and E-16 was attempted using the TBAF conditions that desilylated Z-12 and E-12, which succeeded in removing the TBDPS moiety. However, in both cases, gradual 1,2-migration of the benzyl moiety from the oxygen on the secondary carbon to the primary carbinol oxygen was unavoidable, even though excess acetic acid (5 equiv) was present to quench the alkoxide anion. Therefore, products S-3 and S-4 were contaminated by small amounts of the undesired 1,2-migration products S-1 and S-2, respectively. This undesired migration was significantly suppressed by treating Z-16 and E-16 with pyridinium poly(hydrogen fluoride) in acetonitrile at −50 °C, with targets S-3 and S-4 obtained in 85% and 89% yields, respectively. Synthetic S-3 and S-4 were given the common names (S)fruticosinol and (S)-isofruticosinol, respectively. Counterparts R-3 and R-4 were also synthesized using the same pathway as S-3 and S-4. The 1H and 13C NMR data of synthetic S-isomers S-3 and S-4 and R-isomers R-3 and R-4 were in good agreement with those of the natural products. The specific rotation values of both enantiomers showed good correlation, as shown in Table 2. With S- and R-enantiomers of synthetic 1−4 in hand, the enantiomeric purities of natural butenolides were determined

determination, including confirmation of the (Z)-geometry of the double bond, was established by X-ray crystallographic analysis (Figure 4).

Figure 4. Perspective view of Z-12.

Finally, desilylation of Z-12 and E-12 using tetrabutylammonium fluoride (TBAF; 2 equiv) in the presence of excess acetic acid (5 equiv) in THF gave targets S-1 and S-2 in 84% and 86% yields, respectively. 1H and 13C NMR data for synthetic S-1 and S-2 were in good agreement with those of naturally occurring butenolides 1 and 2. The systematic name of the E-isomer (S-2) of (S)-melodorinol (S-1) was (4E,6S)benzyloxy-6-hydroxy-2,4-heptadien-4-olide, to which we gave the common name (S)-isomelodorinol. The specific rotations of S-1 and S-2 were [α]D24 +94.2 (c 1.32, CHCl3) and [α]D24 −42.7 (c 1.00, CHCl3), respectively. Analogously, R-1 and R-2 were synthesized starting from L-ribose (L-5). The spectral properties of synthesized R-1 and R-2 were in good agreement with those of the corresponding S-isomers S-1 and S-2, and the absolute values of specific rotation (R-1: −94.0 (c 1.25 CHCl3) and R-2: [α]D25 +41.6 (c 1.10, CHCl3)) were in good agreement with those of S-1 and S-2 (Scheme 2). Compounds S-3 and S-4 were synthesized following the sequence starting from D-ribose derivative D-6 used for the preparation of S-1 and S-2 (Scheme 3). The primary hydroxyl Scheme 3. Syntheses of Butenolides S-3, S-4, R-3, and R-4a

a Reagents and conditions: i. TBDPSCl, imidazole, DMF, 0 °C (D-13: 97%; L-13: 96%); ii. Ph3PCHCO2CH3, CH2Cl2, reflux (Z-14: 72%, E-14: 18%; ent-Z-14: 70%, ent-E-14: 19%); iii. AcOH/H2O (6/1, v/v), 100 °C (15: 82%; ent-15: 88%); iv. Bz2O, Et3N, DMAP, CH2Cl2, 0 °C−rt (Z-16: 51%, E-16: 38%; ent-Z-16: 49%, ent-E-16: 42%); v. C6H5N·(HF)X, CH3CN, −50 °C (S-3: 86%, S-4: 88%; R-3: 89%, R-4: 89%).

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The Journal of Organic Chemistry by chiral HPLC (Ceramospher Chiral RU-1) using MeOH as the mobile phase. As shown in Figure 5, HPLC chromato-

grams of 1−4 supported their good optical purity. Natural butenolides 1−4 showed two peaks in chiral HPLC analysis. In all cases, the first eluted large peak and the subsequent small peak corresponded to the S-isomer and R-isomer, respectively. Therefore, S-1−4 were found to be the dominant enantiomers of natural butenolides 1−4. The S/R ratio, estimated from the peak area, ranged from 82/18 (64% ee) to 83/17 (66% ee). The specific rotations ([α]D: 1 = +66.9, 2 = −29.3, 3 = −146, and 4 = −53.4, all in CHCl3) reflected their ee values, taking into account the specific rotation of the synthetic compounds (Table 2). Interestingly, the signs of their specific rotations were opposite to those previously reported for 1−3, which were earlier isolated from M. f ruticosum and characterized as mixtures containing R-isomers as the main components.11Therefore, it is notable that natural butenolides 1−3 were isolated with opposite enantiomeric preferences from the same species, although it has been reported that the enantiomeric preference of natural products can vary with respect to each plant from which it is extracted.21 Owing to the [α]D values reported in previous studies for (R)-melodorinol (R-1) varying in the range −40 and −5 (in CHCl3), we speculated that the isolated R-1 might partially racemize at the allylic C-6 position or gradually transform into other compounds. Therefore, the specific rotation of synthetic R-1 was measured again in CHCl3 at room temperature. The value of the specific rotation of R-1 was nearly unchanged after 12 h in the solvent, suggesting that R-1 was stable at room temperature in CHCl3. This was well supported by the 1H NMR spectrum of R-1 recovered from the cell. Next, assuming that the stereochemical configuration of R-1 changed during the extraction process from M. f ruticosum, we examined the change in configuration by heating R-1 in refluxing aqueous methanol in the presence of benzoic acid or sodium benzoate,

Figure 5. HPLC chromatograms of 1−4: (A) natural 1 (S/R = ca. 82/18) and synthetics (S-1 and R-1); (B) natural 2 (S/R = ca. 83/17) and synthetics (S-2 and R-2); (C) natural 3 (S/R = ca. 82/18) and synthetics (S-3 and R-3); (D) natural 4 (S/R = ca. 83/17) and synthetics (S-4 and R-4). Column: Ceramospher Chiral RU-1, detection: UV (254 nm), mobile phase: MeOH; flow rate: 0.7 mL/ min, column temp.: 30 °C.

Scheme 4. Degradation Study of R-1 and R-3

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The Journal of Organic Chemistry Table 3. Inhibitory Effects of S- and R-1−4 on Melanogenesis and Viability in B16 4A5 Cellsa inhibition (%) S-1 R-1 S-2 R-2 S-3 R-3 S-4 R-4

arbutine

0 μM

0.3 μM

0.0 ± 9.1 (100.0 ± 5.3) 0.0 ± 11.3 (100.0 ± 4.4) 0.0 ± 1.5 (100.0 ± 3.6) 0.0 ± 8.3 (100.0 ± 3.7) 0.0 ± 8.8 (100.0 ± 5.0) 0.0 ± 9.5 (100.0 ± 1.5) 0.0 ± 9.2 (100.0 ± 2.9) 0.0 ± 5.1 (100.0 ± 2.8)

25.1 ± 3.0 (100.6 ± 3.6) 23.1 ± 2.6b (105.5 ± 4.1) 52.6 ± 5.7c (109.3 ± 3.2) 44.2 ± 1.8c (103.4 ± 2.7) 26.5 ± 4.3c (104.5 ± 0.7) 11.8 ± 9.9 (93.8 ± 3.2) 29.1 ± 8.3b (101.3 ± 1.8) 18.6 ± 6.8b (100.5 ± 4.7) b

1 μM

3 μM

32.6 ± 6.2 (100.0 ± 4.2) 34.4 ± 6.8c (102.8 ± 2.2) 61.8 ± 2.9c (111.4 ± 3.2) 65.9 ± 2.6c (105.4 ± 3.9) 36.4 ± 5.1c (106.6 ± 3.8) 43.1 ± 4.2c (106.3 ± 2.2) 58.4 ± 5.5c (111.3 ± 4.6) 56.4 ± 3.1c (106.8 ± 4.1) inhibition (%) c

10 μM

56.3 ± 6.0 (104.5 ± 3.7) 66.0 ± 3.1c (111.2 ± 2.3) 89.4 ± 2.6c (105.7 ± 5.7) 89.8 ± 1.9c (102.5 ± 1.8) 63.3 ± 3.8c (115.2 ± 4.5) 64.1 ± 2.8c (109.3 ± 2.6) 75.9 ± 4.7c (113.8 ± 4.8) 74.3 ± 1.3c (109.6 ± 3.4) c

IC50 (μM)

95.2 ± 5.7 (107.2 ± 5.4) 95.1 ± 2.6c (110.2 ± 3.1) 100.7 ± 2.6c (78.4 ± 3.1)d 104.2 ± 2.4c (79.2 ± 1.9)d 86.4 ± 1.6c (108.6 ± 1.4) 93.5 ± 1.9c (106.2 ± 2.6) 94.6 ± 4.0c (96.8 ± 1.7) 98.8 ± 3.6c (95.4 ± 3.2)

2.5

c

2.9 0.29 0.39 1.5 1.5 0.80 1.0

0 μM

30 μM

100 μM

300 μM

1000 μM

IC50 (μM)

0.0 ± 1.4 (100.0 ± 2.1)

20.4 ± 0.5 (82.4 ± 3.0)

38.1 ± 0.9c (78.1 ± 1.9)

61.5 ± 0.6c (79.8 ± 2.2)

83.7 ± 0.5c (53.1 ± 1.8d)

174

Each value represents mean ± SEM (N = 4). bSignificantly different from control. p < 0.05. cSignificantly different from control. p < 0.01. Cytotoxic effects were observed, and values in parentheses indicate cell viability (%) in MTT assay. eCommercial arubutin was purchased from Nakalai Tesque Inc., (Kyoto, Japan). Lit.15a, 15b, 15c, 15d, 22.

a

d

the presence of which would be expected in the plant. The reaction of R-1 with benzoic acid was monitored by ODSHPLC and chiral HPLC analysis for 9 h, but R-1 was shown to remain nearly unchanged under these acidic conditions. However, upon treatment with sodium benzoate, R-1 gradually transformed into the corresponding 1,2-benzoyl migration product (R-3) accompanied by slight decomposition with approximately 20% of R-1 converted to R-3 (R-1/R-3 = 3.6:1) after 9 h. After purification of the crude mixture by preparative ODS-HPLC, each pure sample of R-1 and R-3 was analyzed by chiral HPLC to determine the enantiomeric purity. No peak supporting the formation of corresponding opposite enantiomers S-1 and S-3 was observed, suggesting that stereoinversion at the allylic C-6 position caused by benzoate anions did not occur during extraction from the plants. However, it was phytochemically noteworthy that 1,2-benzoyl migration of R-1 took place during the above experiment to give R-3. Even without sodium benzoate, the conversion efficiency of benzoyl migration in R-1 was nearly unchanged, giving an approximately 3.7/1 mixture of R-1/R-3 after 9 h. Meanwhile, the 1,2-benzoyl migration of R-3 proceeded faster than that of R-1 to R-3, with the conversion rate reaching approximately 60% (R-1/R-3 = 1.5/1) after 9 h. Therefore, although the examination showed that 1 and 3 could interconvert during the extraction process, it was difficult to strictly determine which compounds were genuine or artifacts. Interestingly, these results were in agreement with the fact that the isolated amount of 1 always exceeds that of 3 from Annonaceae plants (Scheme 4). Additionally, E-isomers R-2 and R-4 were also individually heated under the refluxing conditions in aqueous methanol. As shown in Scheme 4, reactions of R-2 and R-4 gave an approximately 3.7/1 and 2.5/1 mixture of R-2/R-4, respectively, after 9 h, suggesting that the 1,2-benzoyl

migration between 2 and 4 is also likely to occur during the isolation process. Melanin is a term for referring to a group of natural pigments found in most organisms such as bacteria, fungi, plants, and animals. The structure is complex comprised of a heterogeneous polyphenol-like biopolymer. The color varies from yellow to black through development, and the difference of the degree and distribution of melanin pigmentation affects mammalian skin, eyes, and hair color. Melanin plays important roles in protecting the skin by absorbing harmful ultraviolet (UV) light and in scavenging reactive oxygen species (ROS) generated in vivo. However, melanin overproduction resulting from prolonged exposure to UV light causes dermatological disorders, such as freckles, melisma, postinflammatory melanoderma, and solar lentigines. Melanogenesis takes place in melanocytes, distributed in the basal layer of the epidermis. Melanocytes are known to respond to various exogenous stimulation involving UV radiation, α-melanocyte-stimulating hormone (α-MSH), and theophylline (a phosphodiesterase inhibitor) to produce melamine.15b,c,22 As part of our ongoing investigation of bioactive natural products found in folk medicinal plants, we revealed that several alkaloids,15b,22 phenylethanoid glycosides,22b methoxyflavones,15a phenylpropanoids,15c neolignans,15c and diterpenes15d exhibited potent melanogenesis inhibitory activity against theophylline-stimulated melanogenesis in B16 melanoma 4A5 cells. Therefore, as a continuous research project synthetic S-butenolides S-1−4 and their opposite enantiomers (R-1−4) were evaluated as melanogenesis inhibitors. As summarized in Table 3, the eight butenolides showed potent inhibitory activities with IC50 values ranging from 0.29 to 2.9 μM, which were better than that of the positive control, arbutin (IC50 = 174 μM)15a−d,22 Among the butenolides, S-2 and R-2 were especially active, giving inhibitory activities (0.29−0.39 μM) that were several 8256

DOI: 10.1021/acs.joc.8b00986 J. Org. Chem. 2018, 83, 8250−8264

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The Journal of Organic Chemistry

enantiomers of 2 and 3 and the synthesis of melodorinol (1) were also achieved. All natural compounds 1−4 were shown to be partially racemic mixtures, predominantly comprising the Sisomer (∼66% ee) by chiral HPLC analysis in comparison with the synthetic compounds. In the three decades since melodorinol (1) was first isolated from M. f ruticosum, phytochemical studies on plants of this family have been continued by many different research groups. Compound 1 has been also isolated from several other Annonaceae genera. Since publication of the synthesis of Rand S-enantiomers of 1 by Lu et al. in 1997, it has been assumed that levorotatory R-1 was isolated from Annonaceae plants as the major component.12b In this study, we unambiguously showed that dextrorotatory S-1 was isolated as the major component from the same family. Although the longstanding issue regarding contradictory optical rotations of historical samples of 1 was not completely solved, we assumed that the enantiomeric excess of 1 varied among plants because epimerization between S-1 and R-1 was not observed at the allylic position. Otherwise, variation in the specific rotation could be attributed to contamination by impurities or a mere experimental error. However, it is not possible to know whether impurities were present in historical samples from other researchers. Thermodynamic isomerization between 1 and 3 by heating in aqueous methanol showed that the phytochemically important features of the butenolide structure were the following: (i) The molar ratio of 1 and 3 in the extracts might be artificially changed during extraction because 1 and 3 are interconvertible with through 1,2-migration of the benzoyl moiety; (ii) the conversion of 3 to 1 was easier than that of 1 to 3. This indicated that 1 has been regarded as the main Ztype butenolide in this plant to date. Evaluation of the melanogenesis inhibitory activities of 1−4 showed that all candidates were potent inhibitors, regardless of differences in the C-6 configuration. In particular, S-2 and R-2 were approximately 400-fold more potent than arbutin, the reference standard. Furthermore, the mechanism of action of 1−4 for the onset of inhibitory activities was shown to result from the inhibition of mRNA expression of the tyrosinase family. Further SAR studies on this series of compounds to obtain stronger melanogenesis inhibitory activities are in progress.

hundred times more potent than arbutin. Therefore, C-6 stereochemistry was found not to be an important determinant for the onset of potent inhibitory activity. Furthermore, slight cytotoxicity was observed when E-isomers S-2 and R-2 were used at a concentration of 10 μM, while all tested compounds showed no notable cytotoxicity at the effective concentrations. Tyrosinase (TYR) is a key enzyme in melanin biosynthesis and associated with skin, eyes, and hair color.23 TYR catalyzes the oxidation of L-tyrosine and 3-hydroxy-L-tyrosine (L-DOPA) to dopaquinone, which in turn is oxidized and polymerized to melanin. To characterize the mechanism of action of these butenolides, we examined the inhibitory activity of representative compounds S-1−4 against tyrosinase using L-tyrosine or LDOPA. As a result, no inhibition of the melanin synthesis was observed in the presence of candidates, suggesting that the candidates did not directly inhibit TYR (see Supporting Information, Table S2). Next, we examined mRNA expression of TYR and tyrosinase-related protein-1 (TYRP-1) and TYRP2 using S-1−4 because these enzymes catalyze the transformation of L -tyrosine into dopachrome in melanin synthesis.24As summarized in Table 4, S-1−4 effectively inhibited mRNA expression of TYR, TRP-1, and TRP-2 in the range 3−10 μM and contributed to mRNA expression of the tyrosinase family. Table 4. Effects of S-1−4 on Expression of TYR, TRP-1, and TRP-2 mRNA in B16 4A5 Cellsa Tyrosinase mRNA/β-Actin mRNA 0 μM

3 μM

10 μM

S-1 S-3

1.00 ± 0.08 1.00 ± 0.30 0 μM

0.19 ± 0.09b 0.22 ± 0.15b 1 μM

0.21 ± 0.01b 0.14 ± 0.05b 3 μM

S-2 S-4

1.00 ± 0.08 0.16 ± 0.08b 1.00 ± 0.15 0.10 ± 0.01b TRP-1 mRNA/β-Actin mRNA 0 μM

3 μM

10 μM

S-1 S-3

1.00 ± 0.13 1.00 ± 0.06 0 μM

0.62 ± 0.12 0.13 ± 0.06b 1 μM

0.33 ± 0.06b 0.11 ± 0.03b 3 μM

S-2 S-4

1.00 ± 0.13 0.59 ± 0.26 1.00 ± 0.06 0.42 ± 0.03b TRP-2 mRNA/β-Actin mRNA 0 μM

3 μM

S-1 S-3 S-2 S-4

0.08 ± 0.04b 0.06 ± 0.01b

0.30 ± 0.12b 0.35 ± 0.07b

■

10 μM

1.00 ± 0.03 1.00 ± 0.11 0 μM

b

0.56 ± 0.07 0.11 ± 0.05b 1 μM

0.34 ± 0.04 0.06 ± 0.02b 3 μM

1.00 ± 0.03 1.00 ± 0.10

0.72 ± 0.31 0.19 ± 0.01b

0.31 ± 0.13b 0.09 ± 0.02b

b

EXPERIMENTAL SECTION

General Experimental Procedures. IR spectra were measured on a FT-IR spectrophotometer. NMR spectra were recorded on a FTNMR spectrometer (1H, 500 or 800 MHz; 13C, 125 or 200 MHz). Chemical shifts (δ) and coupling constants (J) are given in ppm and Hz, respectively. Tetramethylsilane (TMS) was used as an internal standard for 1H NMR measurements in CDCl3, whereas 13C NMR measurements utilized the solvent signal (77.0 ppm) of CDCl3 for this purpose. 1D NMR peak assignments were confirmed by COSY and HSQC spectra. Low-resolution and high-resolution mass spectra were recorded on a double-focusing mass spectrometer (FAB) or an orbitrap mass spectrometer (ESI). Optical rotations were determined with a digital polarimeter. All the organic extracts were dried over anhydrous Na2SO4 prior to evaporation. Analytical and preparative HPLC were performed on a HPLC equipped with UV−vis detector (UV 230 or 254 nm) using Cosmosil 5C18-MS-II or Ceramospher Chiral RU-1 (4.6 mm I.D. × 250 mm or 20 mm I.D. × 250 mm). For the purification of synthetic compounds, column chromatography was performed over normal-phase silica gel (45−106 μm). For the isolation of natural products, column chromatography (C.C.) was performed over highly porous synthetic resin (Diaion HP-20),

Each value represents mean ±SEM (N = 3). bSignificantly different from control. p < 0.01. a

In summary, through extensive exploratory studies of M. f ruticosum extracts, a hitherto unreported member of the γalkylidenebutenolides, isofruticosinol (4), was isolated along with related butenolides 1−3. The structure of 4 was unambiguously identified by comparing the spectral data with authentic specimens obtained from the first total synthesis of both enantiomers of 4, which was accomplished in six steps starting from commercially available D- and L-ribose (D- and L-5). Using this synthetic strategy, the first syntheses of both 8257

DOI: 10.1021/acs.joc.8b00986 J. Org. Chem. 2018, 83, 8250−8264

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dd, J = ca. 3.1, 2.3, H-4(β)], 4.59 [0.87H, d, J = 6.0, H-2(β)], 4.65 [0.13H, dd, J = 6.7, 4.3, H-2(α)], 4.74 [0.13H, dd, J = 6.7, 2.4, H3(α)], 4.83 [0.87H, br d, J = ca. 6.0, H-3(β)], 5.42 [0.87H, s, H1(β)], 5.43 [0.13H, d-like, J = ca. 4.3, H-1(α)]. 13C NMR (125 MHz, CDCl3) δ: 24.7/26.2 [CH3(α)], 24.7/26.3 [CH3(β)], 63.2/63.6 [C5(α)/C5(β)], 79.5/86.8 [C2(α)/C2(β)] 81.0/81.6 [C3(α)/ C3(β)], 81.4/87.8 [C4(α)/C4(β)], 96.9/102.9[C1(α)/C1(β)], 112.1/114.3 [(CH3)2C (β)/(α)]. HRMS (ESI) m/z: [M + Na]+ Calcd for C8H14O5Na 213.0733; Found 213.0722. In a similar manner, L-ribose (L-5, 25 g, 167 mmol) gave L-6 (27.2 g, 86%) as a colorless oil. 1H and 13C NMR spectral data of L-6 agreed well with those of D-6. 5-O-Benzoyl-2,3-O-isopropylidene-D- and L-Ribofuranose (D-7 and L-7). To a solution of D-6 (18.0 g, 94.7 mmol) in pyridine (76 mL) was added benzoyl chloride (13.2 mL, 114 mmol) at −15 °C, and the mixture was stirred at −15 °C for 1 h. The reaction mixture was diluted with ethyl acetate (200 mL), and the resulting mixture was successively washed with ice-cold 10% aqueous sulfuric acid, aqueous NaHCO3, and brine and condensed to give a pale yellow paste (28.8 g), which on column chromatography (nhexane/EtOAc, 10/1 → 5/1 → 3/1) gave an anomeric mixture of D719 (20.3 g, 73%, α/β = ca. 1/1.6) as a colorless viscous oil. 1H NMR (800 MHz, CDCl3) δ: 1.34/1.50 [each 1.86H, s, CH3(β)], 1.40/1.59 [each 1.14H s, CH3(α)], 3.32 [0.62H, d, J = 1.2, OH(β)], 3.99 [0.38H, d, J = 10.5, OH(α)], 4.36 [0.62H, dd, J = 11.2, 6.4, H-5a(β)], 4.39 [0.38H, dd, J = 10.9, 2.7, H-5a(α)], 4.45 [0.38H, ddd, J = 3.8, 2.7, 1.6, H-4(α)], 4.46 [0.38H, dd, J = 10.9, 3.8, H-5b(α)], 4.53 [0.62H, ddd, J = 6.9, 6.4, 0.9, H-4(β)], 4.58 [0.62H, dd, J = 11.2, 6.9, H-5b(β)], 4.69 [0.38H, dd, J = 6.4, 4.0, H-2(α)], 4.70 [0.62H, d, J = 6.0, H-2(β)], 4.77 [0.38H, dd, J = 6.4, 1.6, H-3(α)], 4.81 [0.62H, dd, J = 6.0, 0.9, H-3(β)], 5.49 [0.38H, dd, J = 10.5, 4.0, H-1(α)], 5.51 [0.62H, d, J = 1.2, H-1(β)], 7.43−7.48 (2H, m, arom.), 7.56−7.61 (1H, m, arom.), 7.98−8.08 (2H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 24.8/26.2 [CH3(α)], 25.0/26.5 [CH3(β)], 65.5/65.8 [C5(α)/C5(β)), 78.4/84.9 [C4(α)/C4(β)], 79.3/86.0 [C2(α)/ C2(β)], 81.5/82.0 [C3(α)/C3(β)], 97.5/103.2 [C1(α)/C1(β)], 112.7/114.1 [(CH3)2C (β)/(α)], [128.4/129.7/133.2(d)/129.7(s), arom.(β)], [128.6/129.6/133.4(d)/129.4(s), arom.(α)], 166.1/166.5 [CO, (α)/(β)]. HRMS (ESI) m/z: [M + Na]+ Calcd for C15H18O6Na 317.0996; Found 317.0994. In a similar manner, L-6 (16.8 g, 88.3 mmol) gave (L-7, 18.5 g, 71%). 1H and 13C NMR spectral data of L-7 agreed well with those of the corresponding antipode (D-7). Wittig Reaction of D-7 and L-7. A mixture of D-7 (9.15 g, 31.1 mmol), Ph3PCHCO2CH3 (12.5 g, 37.4 mmol), and dichloromethane (60 mL) was heated under reflux for 1 h. After removal of the solvent, the residue was triturated with diethyl ether. The solidified material was filtered off and washed with diethyl ether. The combined filtrate and washings were condensed to give a pale orange oil (16.2 g), which on column chromatography (n-hexane/EtOAc, 5/ 1 → 2/1, v/v) gave methyl (2Z)- and (2E)-7-O-benzoyl-2,3-dideoxy4,5-O-isopropylidene-D-ribo-hept-2-enoate (Z-8 and E-8, Z/E = ca. 3.7/1, 9.0 g, 83%) as an inseparable mixture. NMR data for major isomer (Z-8) extracted from the spectrum of a mixture (Z/E = ca. 3.7/1): 1H NMR (800 MHz, CDCl3) δ: 1.390/ 1.522 [each 3H, s, C(CH3)2], 3.20 (1H, br s, OH), 3.75 (3H, s, CO2CH3), 3.95 (1H, ddd, J = 8.1, 6.5, 2.5, H-6), 4.380 (1H, dd, J = 11.6, 6.5, H-7a), 4.43 (1H, dd, J = 8.1, 6.3, H-5), 4.62 (1H, J = dd, 11.6, 2.5, H-7b), 5.65 (1H, ddd, J = 8.4, 6.3, 1.4, H-4), 6.05 (1H, dd, J = 11.7, 1.4, H-2), 6.35 (1H, dd, J = 11.7, 8.4, H-3), 7.41−8.08 (5H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 25.3/27.8 [C(CH3)2], 52.0, (CO2CH3), 66.7 (C-7), 69.0 (C-6), 74.6 (C-4), 78.8 (C-5), 109.67 [C(CH3)2], 122.1 (C-2), 128.3/129.68/133.0 (d, arom.), 130.0 (s, arom.), 145.8 (C-3), 166.8/167.2 (COPh, CO2CH3). NMR data for minor isomer (E-8) extracted from the spectrum of a mixture (Z/E = ca. 3.7/1): 1H NMR (800 MHz, CDCl3) δ: 1.394/ 1.519 [each 3H, s, C(CH3)2], 3.20 (1H, br s, OH), 3.74 (3H, s, CO2CH3), 3.89 (1H, ddd, J = 9.2, 6.6, 2.4, H-6), 4.24 (1H, dd, J = 9.2, 6.6, H-5), 4.377 (1H, dd, J = 11.6, 6.6, H-7a), 4.65 (1H, J = dd, 11.6, 2.4, H-7b), 4.91 (1H, ddd, J = 6.6, 4.8, 1.7, H-4), 6.20 (1H, dd, J =

normal-phase silica gel (63-210 mesh), or reversed-phase ODS (100− 200 mesh). TLC analysis was performed using precoated TLC plates with silica gel 60F254 (normal-phase) or silica gel RP-18 WF254S (reversed-phase). Phosphomolybdic acid or ceric sulfate was used as coloring reagent. Plant Material. The flowers of M. f ruticosum were collected at Nakhonsithammarat Province, Thailand in September 2011. The plant material was identified by one of the authors (Y.P.). A voucher specimen (2011.06. Raj-06) of this plant material is on file in our laboratory. Extraction and Isolation. Dried flowers of M. fruticosum (1.86 kg) were extracted three times with MeOH under reflux for 3 h. Evaporation of the combined extracts under reduced pressure yielded the MeOH extract (210.4 g, 11.31%). An aliquot (183.8 g) was partitioned in an EtOAc/H2O (1/1, v/v) mixture to furnish an EtOAc-soluble fraction (102.6 g, 6.31%) and an aqueous phase. The aqueous phase was subjected to Diaion HP-20 C.C. (3.0 kg, H2O → MeOH) to yield H2O-eluted (47.9 g, 2.98%) and MeOH-eluted (27.2 g, 1.67%) fractions. An aliquot (82.6 g) of the EtOAc-soluble fraction was subjected to normal-phase silica gel C.C. [n-hexane/EtOAc, 20/1 → 10/1 → 2/1 → 1/1, v/v] → EtOAc → MeOH] to give 10 fractions [Fr. One (259.0 mg), Fr. Two (2.55 g), Fr. Three (3.71 g), Fr. Four (5.41 g), Fr. Five (4.42 g), Fr. Six (18.20 g), Fr. Seven (2.22 g), Fr. Eight (4.97 g), Fr. Nine (12.30 g), and Fr. Ten (18.70 g)]. The fraction 6 (18.20 g) was subjected to reversed-phase ODS C.C. [MeOH/H2O, 60/40 → 70/30 → 90/10, v/v] → MeOH → acetone] to afford six fractions {Fr. 6−1 [benzoic acid, 3.14 g (0.2982%)], Fr. 6−2 (4.62 g), Fr. 6−3 (1.60 g), Fr. 6−4 (20.0 mg), Fr. 6−5 [chrysin, 6.73 g (0.6391%)], and Fr. 6−6 (1.78 g)}. The fraction 6−2 (535.5 mg) was purified by HPLC [Cosmosil 5C18-MSII, UV (230 nm), MeOH−1% aqueous AcOH (60/40, v/v)] to give three fractions {Fr. 6−2−1 [benzoic acid (19.5 mg, 0.0016%)], Fr. 6− 2−2 [52.5 mg], and Fr. 6−2−3 (453.3 mg)}. The fraction 7 (2.21 g) was subjected to reversed-phase ODS C.C. [80 g, H2O → (MeOH/ H2O, 40/60 → 70/30, v/v) → MeOH → acetone] to afford 10 fractions [Fr. 7−1 (4.0 mg), Fr. 7−2 (85.7 mg), Fr. 7−3 (72.2 mg), Fr. 7−4 (319.1 mg), Fr. 7−5 (160.0 mg), Fr. 7−6 (154.9 mg), Fr. 7− 7 (104.5 mg), Fr. 7−8 (172.3 mg), Fr. 7−9 (295.1 mg), and Fr. 7−10 (448.8 mg)]. The fraction 7−4 (319.1 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (230 nm), MeOH/1% aqueous AcOH (50/50, v/v)] to give six fractions {Fr. 7−4−1 (2.8 mg), Fr. 7−4−2 (122.6 mg), Fr. 7−4−3 (22.0 mg), Fr. 7−4−4 [isofruticosinol (4), 56.9 mg (0.0054%)], Fr. 7−4−5 [isomelodorinol (2),11b 10.9 mg (0.0010%)], and Fr. 7−4−6 (94.0 mg)}. The fraction 8 (4.97 g) was subjected to reversed-phase ODS C.C. [190 g, MeOH/H2O (30/70 → 50/50 → 70/30, v/v) → MeOH → acetone] to afford seven fractions [Fr. 8−1 (283.5 mg), Fr. 8−2 (225.3 mg), Fr. 8−3 (2.07 g), Fr. 8−4 (266.5 mg), Fr. 8−5 (105.2 mg), Fr. 8−6 (1.09 g), and Fr. 8−7 (720.3 mg)]. The fraction 8−3 (1.67 g) was purified by HPLC [Cosmosil 5C18-MS-II, UV (230 nm), MeOH/1% aqueous AcOH (50/50, v/v)] to give three fractions {Fr. 8−3−1 [fruticosinol (3),11a 239.4 mg, (0.0237%)], Fr. 8−3−2 [melodorinol (1),11a 877.0 mg (0.0910%)], and Fr. 8−3−3 (530.1 mg)}. Benzoic acid and chrysin were identified by comparison of their physical and spectral data with those of commercially available samples. 2,3-Isopropylidene-D- and L-Ribofuranose (D-6 and L-6). A mixture of D-ribose (D-5, 25 g, 167 mmol), acetone (500 mL), and conc. H2SO4 (2 mL) was stirred at room temperature for 1 h, and excess Na2CO3 was added to the reaction mixture. The resulting mixture was stirred at room temperature until quenching of the reaction was complete. The mixture was filtered by suction, and the filter cake was washed with acetone. The combined filtrate and washings were condensed in vacuo to give a colorless oil (33.8 g), which on column chromatography (n-hexane/AcOEt, 1/1 → 1/2) gave an anomeric mixture of D-618 (28.0 g, 88%, α/β = ca. 1/7) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ: 1.32/1.49 [each 2.63H, s, CH3(β)], 1.40/1.58 [each 0.37H s, CH3(α)], 3.66 [0.13H, dd, J = 11.8, 3.1, H-5a(α)], 3.71 [0.87H, J = 11.9, 3.1, H-5a(β)], 3.76 [0.87H, dd, J = 11.9, 2.3, H-5b(β)], 3.79 [0.13H, dd, J = 11.8, 3.1, H5b(α)], 4.19 [0.13H, ddd, J = 4.0, 3.1, 2.4, H-4(α)], 4.41 [0.87H, br 8258

DOI: 10.1021/acs.joc.8b00986 J. Org. Chem. 2018, 83, 8250−8264

Article

The Journal of Organic Chemistry

yellow oil (16.4 g), which on column chromatography (CHCl3 → CHCl3/MeOH, 50/1, v/v) gave (4S,5S,6R)-7-benzoyloxy-6-(tertbutyldiphenylsilyloxy)-5-hydroxy-2-hepten-4-olide (10, 8.23 g, 56% from D-7) and methyl (2E,4S,5S,6R)-7-benzoyloxy-6-(tert-butyldiphenylsilyloxy)-4,5-dihydroxy-2-heptenoate (11, 2.34 g, 15% from D7). Lactone (10): Colorless viscous oil. [α]D26 −62.5 (c 3.00, CHCl3). IR (neat): 3460, 2931, 2859, 1786, 1755, 1724, 1600, 1454, 1427, 1273, 1111 cm−1. 1H NMR (800 MHz, CDCl3) δ: 1.09 [9H, s, C(CH3)3], 2.69 (1H, d, J = 4.9, OH), 3.95 (1H, ddd, J = 6.0, 5.3, 4.9, H-5), 4.10 (1H, ddd, J = 6.0, 4.2, 3.8, H-6), 4.40 (1H, dd, J = 12.0, 3.8, H-7a), 4.54 (1H, J = dd, 12.0, 4.2, H-7b), 5.28 (1H, ddd, J = 5.3, 2.0, 1.5, H-4), 6.10 (1H, dd, J = 5.8, 2.0, H-2), 7.18 (1H, dd, J = 5.8, 1.5, H-3), 7.31−7.89 (15H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 19.2 [C(CH3)3], 27.0 [C(CH3)3], 64.7 (C-7), 72.1 (C-5), 72.5 (C6), 82.5 (C-4), 122.8 (C-2), 127.8/128.2/128.3/129.7/130.2/130.4/ 133.2/135.8/135.9 (d, arom.), 129.6/132.1/132.7 (s, arom.), 153.5 (C-3), 166.5 (COPh), 172.6 (C-1). HRMS (ESI) m/z: [M + Na]+ Calcd for C30H32O6SiNa 539.1860; Found 539.1863. Ester (11): Colorless viscous oil. [α]D27 −34.9 (c 3.10, CHCl3). IR (neat): 3460, 2931, 2859, 1724, 1712, 1662, 1600, 1454, 1427, 1392, 1277, 1177, 1111 cm−1. 1H NMR (800 MHz, CDCl3) δ: 1.07 [9H, s, C(CH3)3], 2.68 (1H, d, J = 6.2, OH), 2.9. (1H, d, J = 4.4, OH), 3.73 (3H, s, CO2CH3), 3.80 (1H, ddd-like, J = 5.7, 5.7, 4.4, H-5), 4.04 (1H, ddd, J = 5.7, 4.8, 3.0, H-6), 4.34 (1H, dd, J = 12.1, 3.0, H-7a), 4.49 (1H, J = dd, 12.1, 4.8, H-7b), 4.57 (1H, dddd-like, J = 6.2, 5.7, 4.8, 1.8, H-4), 6.08 (1H, dd, J = 15.8, 1.8, H-2), 6.96 (1H, dd, J = 15.8, 4.8, H-3), 7.28−7.86 (15H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 19.3 [C(CH3)3], 26.9 [C(CH3)3], 51.6 (CO2CH3), 65.6 (C-7), 71.0 (C-4), 73.0 (C-6), 74.6 (C-5), 122.3 (C-2), 127.7/128.0/ 128.3/129.7/129.9/130.2/133.2/135.78/135.83 (d, arom.), 129.5/ 132.3/133.0 (s, arom.), 145.7 (C-3), 166.4/167.0 (COPh, C-1). LRMS (FAB) m/z: 549 [M + H]+. HRMS (FAB) m/z: [M + H]+ Calcd for C31H37O7Si 549.2308; Found 549.2328. In a similar manner, a mixture of ent-Z-9 and ent-E-9 (8.2 g, 23.4 mmol) gave ent-10 (7.6 g, 53%) and ent-11 (2.2 g, 14%). 1H and 13C NMR spectral data of ent-10 and ent-11 agreed well with those of the corresponding antipodes (10 and 11). Ent-10 and ent-11 exhibited the specific rotation [α]D22 +62.4 (c 0.99, CHCl3) and [α]D27 +34.8 (c 3.09, CHCl3), respectively. E1cb Reaction of Butenolides (10 and ent-10). To a mixture of 10 (7.28 g, 14.1 mmol), triethylamine (4.8 mL, 34.7 mmol), dimethylaminopyridine (DMAP, 1.75 g, 14.3 mmol), and dichloromethane (20 mL) was added acetic anhydride (1.62 mL, 17.2 mmol) at 0 °C. The reaction mixture was allowed to reach room temperature and was stirred for 1 h. The reaction mixture was poured into cold water (100 mL) and extracted with dichloromethane. The extract was washed with brine and condensed to give a brown solid (8.65 g), which which on column chromatography (n-hexane/EtOAc, 30/1 → 10/1 → 5/1, v/v) gave (4Z,6S)-7-benzoyloxy-6-(tert-butyldiphenylsilyloxy)-2,4-heptadien-4-olide (Z-12, 3.16 g, 45%) and its E-isomer (E12, 3.23 g, 46%). Z-isomer (Z-12): Colorless prisms (from MeOH/AcOEt). Mp 145−146 °C. [α]D26 −73.8 (c 1.33, CHCl3). IR (KBr): 2963, 2855, 1786, 1705, 1674, 1598, 1450, 1427, 1381, 1315, 1276, 1107, 1083, 1015 cm−1. 1H NMR (800 MHz, CDCl3) δ: 1.05 [9H, s, C(CH3)3], 4.36−4.44 (2H, m, H-7a and H-7b), 5.18−5.22 (2H, m, H-5 and H6), 6.05 (1H, dd, J = 5.5, H-2), 7.11 (1H, dd, J = 5.5, H-3), 7.31 (4H, m, arom.), 7.36 (1H, m, arom.), 7.39 (1H, m, arom.), 7.41 (2H, m, arom.), 7.55 (1H, m, arom.), 7.63 (2H, m, arom.), 7.65 (2H, m, arom), 7.90 (2H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 19.2 [C(CH3)3], 26.8 [C(CH3)3], 66.9 (C-6), 67.4 (C-7), 114.2 (C-5), 120.6 (C-2), 127.5/127.6/128.3/129.7/129.90/129.92/133.0/135.7/ 135.8 (d, arom.), 132.9/133.3 (s, arom.), 143.2 (C-3), 149.1 (C-4), 166.2 (COPh), 168.7 (C-1). LRMS (FAB) m/z: 521 [M + Na]+. HRMS (FAB) m/z: [M + Na]+ Calcd for C30H30O5SiNa 521.1760; Found 521.1748. E-isomer (E-12): Colorless viscous oil. [α]D26 +46.0 (c 1.32, CHCl3). IR (neat): 2931, 2859, 1790, 1762, 1724, 1670, 1600, 1469, 1454, 1427, 1389, 1269, 1192, 1177, 1111, 1069 cm−1. 1H NMR (800

15.6, 1.7, H-2), 7.14 (1H, dd, J = 15.6, 4.8, H-3), 7.41−8.08 (5H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 25.2/27.6 [C(CH3)2], 51.7, (CO2CH3), 67.4 (C-7), 68.9 (C-6), 76.6 (C-4), 77.6 (C-5), 109.74 [C(CH3)2], 122.0 (C-2), 128.4/129.70/133.3 (d, arom.), 130.0 (s, arom.), 143.6 (C-3), 166.7/167.1 (COPh, CO2CH3). In a similar manner, L-7 (13.6 g, 45.9 mmol) gave a mixture of entZ-8 and ent-E-8 (13.5 g, 84%). 1H and 13C NMR spectral data of entZ-8 and ent-E-8 agreed well with those of the corresponding antipodes (Z-8 and E-8). Silylation of a Mixtures of (Z-8 and E-8) and (ent-Z-8 and ent-E-8). To a mixture of a ca. 3.7/1 mixture of Z-8 and E-8 (8.3 g, 23.7 mmol), silver nitrate (6.0 g, 35.3 mmol), pyridine (9.5 mL), and acetonitrile (30 mL) was added a solution of tert-butyldiphenylchlorosilane (9.25 mL, 35.6 mmol) in acetonitrile (20 mL) at 0 °C, and the mixture was stirred in the dark at room temperature for 3 h. After the reaction mixture was diluted with ethyl acetate (100 mL), the resulting suspension was filtered through Celite, and the filter cake was washed with ethyl acetate. The combined filtrate and washings were subsequently washed with ice-cold 10% sulfuric acid and brine and concentrated to give (2Z)- and (2E)-7-O-benzoyl-6-O-(tertbutyldiphenylsilyl)-2,3-dideoxy-4,5-O-isopropylidene-D-ribohept-2enoate (Z-9 and E-9, 17.7 g) as a poorly separable ca. 3.7/1 mixture, which was used for the next step without further purification. For analytical purpose a small portion of the crude mixture was purified by column chromatography (n-hexane/EtOAc, 50/1 → 20/1, v/v). Major Z-ester (Z-9): Colorless viscous oil: [α]D27 +88.6 (c 1.29, CHCl3). IR (neat): 2934, 2856, 1728, 1712, 1647, 1600, 1454, 1431, 1381, 1269, 1226, 1200, 1111, 1069, 1002 cm−1. 1H NMR (800 MHz, CDCl3) δ: 1.02 [9H, s, C(CH3)3], 1.38/1.55 [each 3H, s, C(CH3)2], 3.67 (3H, s, CO2CH3), 4.03 (1H, ddd, J = 7.2, 3.0, 2.3, H-6), 4.27 (1H, dd, J = 11.9, 7.2, H-7a), 4.39 (1H, J = dd, 11.9, 2.3, H-7b), 4.68 (1H, dd, J = 7.4, 3.0, H-5), 5.69 (1H, ddd, J = 8.0, 7.4, 1.6, H-4), 5.80 (1H, dd, J = 11.6, 1.6, H-2), 6.36 (1H, dd, J = 11.6, 8.0, H-3), 7.21− 7.37 (15H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 19.3 [C(CH3)3], 24.6/26.8 [C(CH3)2], 26.9 [C(CH3)3], 51.4, (CO2CH3), 66.8 (C-7), 71.4 (C-6), 73.6 (C-4), 80.5 (C-5), 109.0 [C(CH3)2], 122.5 (C-2), 127.36/127.43/128.0/129.47/129.58/129.64/132.6/ 135.8/136.0 (d, arom.), 129.9/132.8/133.7 (s, arom.), 145.3 (C-3), 165.7/166.1 (COPh, CO2CH3). HRMS (ESI) m/z: [M + Na]+ Calcd for C34H40O7SiNa 611.2436; Found 611.2440. Minor E-ester (E-9): Colorless viscous oil: [α]D26 −4.22 (c 1.25, CHCl3). IR (neat): 2931, 2859, 1728, 1662, 1600, 1454, 1431, 1381, 1273, 1215, 1165, 1111, 1072, 1026 cm−1. 1H NMR (800 MHz, CDCl3) δ: 1.02 [9H, s, C(CH3)3], 1.35/1.47 [each 3H, s, C(CH3)2], 3.70 (3H, s, CO2CH3), 4.16 (1H, ddd, J = 7.4, 5.3, 2.1, H-6), 4.18 (1H, dd, J = 11.7, 5.3, H-7a), 4.25 (1H, J = dd, 11.7, 2.1, H-7b), 4.49 (1H, dd, J = 7.4, 6.4, H-5), 4.84 (1H, ddd, J = 6.4, 5.7, 1.5, H-4), 6.04 (1H, dd, J = 15.6, 1.5, H-2), 7.04 (1H, dd, J = 15.6, 5.7, H-3), 7.18− 7.74 (15H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 19.3 [C(CH3)3], 25.2/27.5 [C(CH3)2], 26.9 [C(CH3)3], 51.5, (CO2CH3), 66.4 (C-7), 70.8 (C-6), 76.6 (C-4), 78.6 (C-5), 109.4 [C(CH3)2], 123.3 (C-2), 127.5/127.7/128.1/129.5/129.8/129.9/132.7/135.6/ 135.8 (d, arom.), 130.0/132.8/133.4 (s, arom.), 143.7 (C-3), 166.0/166.1 (COPh, CO2CH3). HRMS (ESI) m/z: [M + Na]+ Calcd for C34H40O7SiNa 611.2436; Found 611.2435. In a similar manner, a mixture of ent-Z-8 and ent-E-8(8.2 g, 23.4 mmol) gave a mixture of ent-Z-9 and ent-E-9 (17.2 g), which was directly used for the next step. The 1H and 13C NMR spectral data of analytical samples of ent-Z-9 and ent-E-9, obtained by column chromatography, agreed well with those of the corresponding antipodes (Z-9 and E-9). Ent-Z-9 and ent-E-9 exhibited the specific rotation [α]D27 −88.4 (c 1.20, CHCl3) and [α]D27 +4.10 (c 1.40, CHCl3), respectively. Treatment of a Mixtures of (Z-9 and E-9) and (ent-Z-9 and ent-E-9) with Aqueous Acetic Acid. The crude mixture of Z-9 and E-9 (17.6 g), acetic acid (60 mL), and water (10 mL) was heated at 100 °C for 2 h. After being cooled, the reaction mixture was poured into ice-cold water (600 mL). The resulting mixture was neutralized with sodium hydrogen carbonate and extracted with ethyl acetate. The extract was washed with brine and condensed to give a pale 8259

DOI: 10.1021/acs.joc.8b00986 J. Org. Chem. 2018, 83, 8250−8264

Article

The Journal of Organic Chemistry MHz, CDCl3) δ: 1.05 [9H, s, C(CH3)3], 4.37 (1H, dd, J = 11.1, 5.3, H-7a), 4.41 (1H, dd, J = 11.1, 6.3, H-7b), 4.76 (1H, ddd, J = 9.5, 6.3, 5.3, H-6), 5.73 (1H, ddd, J = 9.5, 1.8, 0.7, H-5), 5.97 (1H, dd, J = 5.6, 1.8, H-2), 6.92 (1H, dd, J = 5.6, 0.7, H-3), 7.31 (4H, m, arom.), 7.41 (4H, m, arom.), 7.56 (1H, m, arom.), 7.58 (2H, m, arom.), 7.64 (2H, m, arom.), 7.90 (2H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 19.2 [C(CH3)3], 26.8 [C(CH3)3], 67.6 (C-6), 68.0 (C-7), 113.2 (C5), 121.1 (C-2), 127.8/128.4/129.6/130.0/130.1/133.2/135.7/ 135.9/139.4 (d, arom.), 132.6/132.9 (s, arom.), 139.4 (C-3), 151.0 (C-4), 166.2 (COPh), 168.9 (C-1). LRMS (FAB) m/z: 521 [M + Na]+. HRMS (FAB) m/z: [M + Na]+ Calcd for C30H30O5SiNa 521.1760; Found 521.1742. In a similar manner, ent-10 (8.98 g, 17.4 mmol) gave ent-Z-12 (3.65 g, 42%) and ent-E-12 (4.08 g, 47%). Recrystallization from methanol and ethyl acetate gave ent-Z-12 as colorless needles, mp 145−146 °C. 1H and 13C NMR spectral data of ent-Z-12 and ent-E12 agreed well with those of the corresponding antipodes (Z-12 and E-12). Ent-Z-12 and ent-E-12 exhibited the specific rotation [α]D26 −73.5 (c 1.04, CHCl3) and [α]D25 −45.5 (c 0.94, CHCl3), respectively. Desilylation of Z-12, E-12, ent-Z-12, and ent-E-12. (4Z,6S)and (4Z,6R)-7-Benzyloxy-6-hydroxy-2,4-heptadien-4-olide11a [(S)- and (R)-Melodorinol (S-1 and R-1)]. To a mixture of Z-12 (260 mg, 0.52 mmol), acetic acid (150 μL, 2.62 mmol), and THF (5 mL) was added 1 M solution of TBAF in THF (1.04 mL, 1.04 mmol) at 0 °C. The reaction mixture was allowed to reach room temperature and was stirred for 2 h. The reaction mixture was poured into cold water (200 mL) and extracted with ethyl acetate. The extract was washed with aqueous NaHCO3 and brine and condensed to give a pale yellow oil (270 mg), which on column chromatography (nhexane/EtOAc, 10/1 → 3/1, v/v) gave the title compounds (S-1, 114 mg, 84%) as a pale yellow viscous oil. IR (neat): 3460, 2954, 1778, 1748, 1712, 1681, 1600, 1558, 1454, 1273, 1115, 1069, 1026 cm−1. 1 H NMR (800 MHz, CDCl3) δ: 2.83 (1H, d, J = 4.4, OH), 4.46 (1H, dd, J = 11.5, 6.3, H-7a), 4.49 (1H, dd, J = 11.5, 4.1, H-7b), 5.18 (1H, dddd-like, J = 8.0, 6.3, 4.4, 4.1, H-6), 5.41 (1H, br d-like, J = 8.0, H5), 6.26 (1H, dd-like, J = 5.5, 0.7, H-2), 7.39 (1H, d, J = 5.6, H-3), 7.45 (2H, m, arom.), 7.58 (1H, m, arom.), 8.05 (2H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 66.0 (C-6), 67.5 (C-7), 113.0 (C-5), 121.1 (C-2), 128.5/129.7/133.4 (d, arom.), 129.5 (s, arom.), 143.6 (C-3), 150.1 (C-4), 166.7 (COPh), 168.8 (C-1). HRMS (ESI) m/z: [M + Na]+ Calcd for C14H12O5Na 283.0577; Found 283.0576. In a similar manner, ent-Z-12 (300 mg, 0.60 mmol) gave R-1 (130 mg, 83%). 1H and 13C NMR spectral data of R-1 agreed well with those of the corresponding antipode (S-1). (4E,6S)- and (4E,6R)-7-Benzyloxy-6-hydroxy-2,4-heptadien4-olide11b [(S)- and (R)-Isomelodorinol (S-2 and R-2)]. Following the procedure used for desilylation of Z-12, E-12 (300 mg, 0.6 mmol) was desilylated with 1 M solution of TBAF in THF (0.9 mL, 0.9 mmol) in THF (5 mL) in the presence of acetic acid (172 μL, 3.0 mmol). Workup and column chromatography gave S-2 (135 mg, 86%) as a colorless microcrystalline. Mp 109−110 °C11b mp 95−97 °C. IR (KBr): 3441, 2924, 2877, 1755, 1697, 1319, 1276, 1119, 1076, 1026 cm−1. 1H NMR (800 MHz, CDCl3) δ: 2.83 (1H, br s, OH), 4.39 (1H, dd, J = 11.6, 7.0, H-7a), 4.51 (1H, dd, J = 11.6, 3.9, H-7b), 4.94 (1H, ddd-like, J = 7.3, 7.0, 3.9, H-6), 5.81 (1H, ddd, J = 7.3, 1.8, 0.7, H-5), 6.27 (1H, dd, J = 5.6, 1.8, H-2), 7.46 (2H, m, arom.), 7.60 (1H, m, arom.), 7.91 (1H, dd, J = 5.6, 0.7, H-3), 8.03 (2H, m, arom.). 13 C NMR (200 MHz, CDCl3) δ: 67.0 (C-6), 68.2 (C-7), 111.9 (C-5), 121.6 (C-2), 128.5/129.6/133.5 (d, arom.), 129.6 (s, arom.), 140.8 (C-3), 151.8 (C-4), 166.6 (COPh), 168.8 (C-1). HRMS (ESI) m/z: [M + Na]+ Calcd for C14H12O5Na 283.0576; Found 283.0577. In a similar manner, ent-E-12 (300 mg, 0.6 mmol) gave R-2 (128 mg, 82%) as a colorless microcrystalline. Mp 109−110 °C. 1H and 13 C NMR spectral data of R-2 agreed well with those of the corresponding antipode (S-2). 5-O-(tert-Butyldiphenylsilyl)-2,3-O-isopropylidene-D- and L-Ribofuranose (D-13 and L-13). To a mixture of D-6 (12.0 g, 63 mmol), imidazole (9.7 g, 14 mmol), and DMF (80 mL) was added tert-butyldiphenylchlorosilane (18.6 mL, 71.5 mmol) at 0 °C, and the

mixture was stirred at 0 °C for 4 h and then stirred at room temperature for another 6 h. The reaction mixture was poured into cold water (400 mL) and extracted with ethyl acetate. The extract was washed with brine and condensed to give a colorless oil (32.4 g), which on column chromatography (n-hexane/EtOAc, 10/1 → 4/1, v/ v) gave an anomeric mixture of D-1320 (26.2 g, 97%, α/β = ca. 1:3.8) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ: 1.05 [1.89H, s, C(CH3)3 (α)], 1.09 [7.11H, s, C(CH3)3 (β)], 1.32/1.48 [each 2.37H, s, CH3(β)], 1.40/1.56 [each 0.63H s, CH3(α)], 3.63 [0.21H, dd, J = 11.1, 2.3, H-5a(α)], 3.66 [0.79H, J = 11.9, 3.1, H-5a(β)], 3.81 [0.21H, dd, J = 11.1, 2.6, H-5b(α)], 3.83 [0.79H, dd, J = 11.3, 2.9, H5b(β)], 3.97 [0.21H, d, J = 11.4, OH(α)], 4.15 [0.21H, br dd, J = ca. 2.6, 2.3, H-4(α)], 4.29 [0.79H, br dd, J = ca. 2.9, 2.7, H-4(β)], 4.56 [0.79H, d, J = 10.6, OH(α)], 4.62 [0.79H, d, J = 6.0, H-2(β)], 4.67 [0.21H, dd, J = 6.2, 4.0, H-2(α)], 4.72 [0.79H, dd, J = 6.0, 0.6, H3(β)], 4.79 [0.21H, bd, J = 6.0, 0.9, H-3(α)], 5.36 [0.79H, d, J = 10.6, H-1(β)], 5.53 [0.21H, dd, J = 11.4, 4.0, H-1(α)], 7.37−7.77 (10H, m, arom.). 13C NMR (125 MHz, CDCl3) δ: 19.0/19.1 [C(CH3)3 (α)/ (β)], 24.6/26.1 [CH3(α)], 24.9/26.5 [CH3(β)], 26.81/26.84 [C(CH3)3 (α)/(β)], 65.4/66.1 [C5(β)/C5(α)], 79.4/87.3 [C2(α)/ C2(β)] 81.2/87.6 [C4(α)/C4(β)], 81.6/87.9 [C3(β)/C3(α)], 98.0/ 103.4[C1(α)/C1(β)], 112.1/112.9 [(CH3)2C (β)/(α)], 127.9/ 128.0/128.1/129.9/130.0/130.2/130.4/135.5/135.7 (d, arom.), 131.5/131.6/132.3/132.6 (s, arom.). HRMS (ESI) m/z: [M + Na]+ Calcd for C24H32O5NaSi 451.1911; Found 451.1885. In a similar manner, L-6 (12.4 g, 65 mmol) gave L-13 (26.8 g, 96%) as a colorless oil. 1H and 13C NMR spectra data of L-13 agreed well with those of the corresponding antipode (D-13). Wittig Reaction of D-13 and L-13. A mixture of D-13 (26.2 g, 61.2 mmol), Ph3PCHCO2CH3 (25 g, 74.9 mmol), and dichloromethane (150 mL) was heated under reflux for 3 h. After removal of the solvent, the residue was triturated with diethyl ether. The solidified material was filtered off and washed with diethyl ether. The combined filtrate and washings were condensed to give a pale orange oil (38.1 g), which on column chromatography (n-hexane/EtOAc, 30/1 → 10/1 → 5/1, v/v) gave methyl (2Z)-7-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-4,5-O-isopropylidene-D-ribohept-2-enoate (Z14, 21.4 g, 72%) and its E-isomer (E-14, 5.4 g, 18%). Major Z-ester (Z-14): Colorless viscous oil. [α]D23 +83.8 (c 0.45, CHCl3). IR (neat): 2931, 2858, 1724, 1651, 1472, 1373, 1204, 1111, 1057 cm−1. 1H NMR (800 MHz, CDCl3) δ: 1.06 [9H, s, C(CH3)3], 1.35/1.38 [each 3H, s, C(CH3)2], 2.74 (1H, d, J = 4.8, OH), 3.65 (1H, dddd, J = 8.8, 5.6, 4.8, 3.2, H-6), 3.74 (3H, s, CO2CH3), 3.78 (1H, dd, J = 10.4, 5.6, H-7a), 3.80 (1H, J = dd, 10.4, 3.2, H-7b), 4.37 (1H, dd, J = 8.8, 6.4, H-5), 5.74 (1H, ddd, J = 8.8, 6.4, 1.6, H-4), 5.95 (1H, dd, J = 12.0, 1.6, H-2), 6.24 (1H, dd, J = 12.0, 8.8, H-3), 7.36− 7.71 (10H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 19.3 [C(CH3)3], 25.4/27.8 [C(CH3)2], 26.8 [C(CH3)3], 51.6, (CO2CH3), 65.2 (C-7), 70.3 (C-6), 73.9 (C-4), 78.1 (C-5), 109.1 [C(CH3)2], 121.8 (C-2), 127.70/127.73/129.7/129.8/135.55/135.61 (d, arom.), 133.1/133.2 (s, arom.), 144.8 (C-3), 166.5 (C-1). LSMS (FAB) m/z: 485 [M + H]+, 507 [M + Na]+. HRMS (FAB) m/z: [M + H]+ Calcd for C27H37O6Si 485.2359; Found 485.2364. Minor E-ester (E-14): Colorless viscous oil. [α]D24 +78.9 (c 0.93, CHCl3). IR (neat): 3502, 2931, 2859, 1728, 1659, 1462, 1431, 1372, 1311, 1258, 1215, 1165, 1111, 1065 cm−1. 1H NMR (800 MHz, CDCl3) δ: 1.07 [9H, s, C(CH3)3], 1.35/1.41 [each 3H, s, C(CH3)2], 2.59 (1H, d, J = 5.8, OH), 3.60 (1H, dddd, J = 9.5, 5.8, 5.4, 2.9, H-6), 3.758 (3H, s, CO2CH3) 3.760 (2H, dd, J = 10.3, 5.4, H-7a), 3.84 (2H, dd, J = 10.3, 2.9, H-7b), 4.21 (1H, dd, J = 9.5, 6.6, H-5), 4.86 (1H, ddd, J = 6.6, 4.9, 1.8, H-4), 6.16 (1H, dd, J = 15.8, 1.8, H-2), 7.14 (1H, dd, J = 15.8, 4.9, H-3), 7.34−7.69 (10H, m, arom.). 13C NMR (125 MHz, CDCl3) δ: 19.3 [C(CH3)3] 25.3/27.6 [C(CH3)2], 26.8 [C(CH3)3], 51.6 (CO2CH3), 65.2 (C-7), 69.8 (C-6), 76.8 (C-4), 77.4 (C-5), 109.5 [C(CH3)2], 121.8 (C-2), 127.75/127.81/129.85/ 129.89/135.5/135.6 (d, arom.), 132.8/132.3 (s, arom.), 144.1 (C3), 166.7 (C-1). LRMS (FAB) m/z: 485 [M + H]+, 507 [M + Na]+. HRMS (FAB) m/z: [M + H]+ Calcd for C27H37O6Si 485.2359; Found 485.2335. 8260

DOI: 10.1021/acs.joc.8b00986 J. Org. Chem. 2018, 83, 8250−8264

Article

The Journal of Organic Chemistry In a similar manner, L-13 (26.7 g, 65 mmol) gave ent-Z-14 (21.1 g, 70%) and ent-E-14 (5.74 g, 19%). 1H and 13C NMR spectral data of ent-Z-14 and ent-E-14 agreed well with those of the corresponding antipodes Z-14 and E-14. Ent-Z-14 and ent-E-14 exhibited the specific rotation [α]D26 −84.6 (c 1.10, CHCl3) and [α]D25 −77.8 (c 0.99, CHCl3), respectively. (4S,5R,6R)- and (4R,5S,6S)-7-(tert-Butyldiphenylsilyloxy)6,7-dihydroxy-2-hepten-4-olide (15 and ent-15). A mixture of Z-14 (10.0 g, 20.6 mmol), acetic acid (30 mL), and water (5 mL) was heated at 100 °C for 1 h. After being cooled, the reaction mixture was poured into cold water (20 mL). The resulting mixture was neutralized with sodium hydrogen carbonate and extracted with ethyl acetate. The extract was washed with brine and condensed to give a pale yellow solid (8.7 g), which on recrystallization from a mixture of n-hexane and ethyl acetate gave 15 (6.36 g, 75%) as colorless needles. The mother liquid was condensed, and the residue was purified by column chromatography (n-hexane/EtOAc, 30:1 → 10:1 → 5:1, v/v) to give 15 (631 mg, 7%). Mp 100−101 °C. [α]D21 −72.0 (c 1.06, CHCl3). IR (KBr): 3545, 3394, 2931, 2858, 1786 (sh), 1751 (s), 1600, 1473, 1427, 1396, 1315, 1180, 1115, 1037 cm−1. 1H NMR (800 MHz, CDCl3) δ: 1.09 [ 9H, s, C(CH3)3] 2.47 (1H, d, J = 5.6, OH), 2.70 (1H, d, J = 5.6, OH), 3.75 (1H, dddd, J = 7.2, 5.6, 4.8, 4.0, H-6), 3.88 (1H, dd, J = 11.2, 4.8, H-7a), 3.91 (1H, dd, J = 11.2, 4.0, H-7b), 4.00 (2H, ddd-like, J = 7.2, 5.6, 4.0, H-5), 5.28 (1H, ddd, J = 4.0, 1.6, 1.6, H-4), 6.18 (1H, dd, J = 5.6, 1.6, H-2),7.41 (4H, m, arom.), 7.46 (2H, m, arom.), 7.61 (2H, dd, J = 5.6, 1.6, H-3), 7.65 (4H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 19.2 [C(CH3)3], 26.9 [C(CH3)3], 64.6 (C-7), 71.6 (C-5 and C-6), 83.7 (C-4), 122.7 (C-2), 127.95/127.98/130.1/130.2/135.5 (d, arom.), 132.4/132.5 (s, arom.), 153.7 (C-3), 172.8 (C-1). LRMS (FAB) m/z: 435 [M + Na]+. HRMS (FAB) m/z: [M + Na]+ Calcd for C23H28O5SiNa 435.1604; Found 435.1588. In a similar manner, ent-Z-14 (10.0 g, 20.6 mmol) gave ent-15 (7.5 g, 88%). Recrystallization from n-hexane and ethyl acetate gave colorless needles, mp 100−101 °C. 1H and 13C NMR spectral data of ent-15 agreed well with those of the corresponding antipode (15). Ent-15 exhibited the specific rotation [α]D24 +73.1 (c 1.05, CHCl3). E1cb Reaction of Butenolides (15 and ent-15). To a mixture of 15 (4.12 g, 10 mmol), triethylamine (2.9 mL, 21 mmol), dimethylaminopyridine (DMAP, 1.22 g, 10 mmol), and dichloromethane (40 mL) was added benzoic anhydride (4.85 mg, 21.5 mmol) at 0 °C. The reaction mixture was allowed to reach room temperature and was stirred for 1 h. The reaction mixture was poured into cold water (100 mL) and extracted with dichloromethane. The extract was successively washed with ice-cold 5% aqueous sulfuric acid and brine and condensed to give a pale brown viscus oil (5.63 g), which on column chromatography (n-hexane/EtOAc, 50/1 → 30/1 → 20/1 → 5/1, v/v) gave (4Z,6S)-7-(tert-butyldiphenylsilyloxy)-6benzyloxy-2,4-heptadien-4-olide (Z-16, 2.53 g, 51%) and its E-isomer (E-16, 1.94 g, 38%). Major Z-lactone (Z-16): Colorless viscous oil. [α]D25 −52.0 (c 0.99, CHCl3). IR (neat): 2931, 2585, 1786, 1724, 1681, 1600, 1562, 1469, 1427, 1389, 1361, 1315, 1269, 1103, 1068, 1026 cm−1. 1H NMR (500 MHz, CDCl3) δ: 1.03 [9H, s, (CH3)3C], 4.00 (1H, dd, J = 11.0, 4.6, H-7a), 4.03 (1H, dd, J = 11.0, 4.6, H-7b), 5.44 (1H, d, J = 8.3, H-5), 6.14 (1H, dt, J = 8.3, 4.6, H-6), 6.25 (1H, d, J = 5.2, H-2), 7.35 (1H, d, J = 5.2, H-3), 7.29−7.46 (8H, m, arom.), 7.56 (1H, m, arom.), 7.62−7.67 (4H, m, arom.), 8.03−8.06 (2H, m, arom.). 13C NMR (125 MHz, CDCl3) δ: 19.2 [C(CH3)3], 26.7 [C(CH3)3], 65.0 (C-7), 70.6 (C-6), 110.7 (C-5), 121.2 (C-2), 127.7/128.4/129.7/ 129.8/133.1/135.5 (d, arom.), 129.9/132.98/133.01 (s, arom.), 143.4 (C-3), 150.3 (C-4), 165.5 (COPh), 168.8 (C-1). LRMS (FAB) m/z: 521 [M + Na]+. HRMS (FAB) m/z: [M + Na]+ Calcd for C30H30O5SiNa 521.1760; Found 521.1740. Minor E-lactone (E-16): Colorless viscous oil. [α]D25 −50.3 (c 1.00, CHCl3). IR (neat): 2931, 2585, 1790, 1763, 1724, 1670, 1558, 1469, 1427, 1393, 1265, 1111, 1068, 1026 cm−1. 1H NMR (500 MHz, CDCl3) δ: 1.03 [9H, s, C(CH3)3], 3.91 (1H, dd, J = 10.9, 4.6, H-7a), 4.01 (1H, dd, J = 10.9, 5.7, H-7b), 5.80 (1H, dd-like, J = 10.0, 1.7, H5), 5.90 (1H, ddd, J = 10.0, 5.7, 4.6, H-6), 6.29 (1H, dd, J = 5.7, 1.7,

H-2), 7.34−7.47 (8H, m, arom.), 7.59 (1H, m, arom.), 7.65 (4H, m, arom.), 7.83 (1H, d, J = 5.7, H-3), 7.99−8.01 (2H, m, arom.). 13C NMR (125 MHz, CDCl3) δ: 19.2 [C(CH3)3], 26.7 [C(CH3)3], 65.2 (C-7), 70.0 (C-6), 109.2 (C-5), 121.9 (C-2), 127.8/128.5/129.70/ 129.9/133.4/135.5/135.6 (d, arom.), 129.67/132.79/132.82 (s, arom.), 140.6 (C-3), 153.1 (C-4), 165.8(COPh), 169.0 (C-2). LRMS (FAB) m/z: 521 [M + Na]+. HRMS (FAB) m/z: [M + Na]+ Calcd for C30H30O5SiNa 521.1760; Found 521.1762. In a similar manner, ent-15 (3.0 g, 7.3 mmol) gave ent-Z-16 (1.77 g, 49%) and ent-E-16 (1.52 g, 42%). 1H and 13C NMR spectral data of ent-Z-16 and ent-E-16 agreed well with those of the corresponding antipodes (Z-16 and E-16). Ent-Z-16 and ent-E-16 exhibited the specific rotation [α]D26 +52.5 (c 1.04, CHCl3) and [α]D25 +50.4 (c 0.96, CHCl3), respectively. Desilylation of Z-16, E-16, ent-Z-16, and ent-E-16. (4Z,6S)and (4Z,6R)-6-Benzyloxy-7-hydroxy-2,4-heptadien-4-olide [(S)- and (R)-Fruticosinol11a (S-3 and R-3)]. To a solution of Z16 (300 mg, 0.6 mmol) in actonitrile (10 mL) was added pyridinium poly(hydrogen fluoride) (65% HF, 6 mL) at −50 °C, and the reaction mixture was stirred at −50 °C for 3 h. The reaction mixture was poured into cold aqueous NaHCO3 (300 mL) and extracted with ethyl acetate. The extract was successively washed with 5% sulfuric acid and brine and condensed to give a pale yellow oil (315 mg), which on column chromatography (n-hexane/EtOAc, 30/1 → 5/1 → 3/1, v/v) gave S-3 (135 mg, 86%) as a colorless viscous oil. IR (neat): 3479, 2927, 2878, 1778, 1746, 1713, 1601, 1558, 1450, 1315, 1261, 1107, 1069, 1026 cm−1. 1H NMR (800 MHz, CDCl3) δ: 2.32 (1H, br s, OH), 3.98 (1H, ddd, J = 12.1, 5.8, H-7a), 4.02 (1H, ddd, J = 12.1, 3.8, H-7b), 5.46 (1H, dd-like, J = 8.0, 0.5 H-5), 6.07 (1H, ddd, J = 8.0, 5.8, 3.8, H-6), 6.27 (1H, dd, J = 5.5, 0.5, H-2), 7.39 (1H, d, J = 5.5, H3), 7.45 (2H, m, arom.), 7.58 (1H, m, arom.), 8.06 (2H, m, arom.). 13 C NMR (200 MHz, CDCl3) δ: 64.2 (C-7), 71.3 (C-6), 109.9 (C-5), 121.3 (C-2), 128.5/129.8/133.4 (d, arom.), 129.6 (s, arom.), 143.5 (C-3), 150.4 (C-4), 165.8 (COPh), 168.7 (C-1). HRMS (ESI) m/z: [M + Na]+ Calcd for C14H12O5Na 283.0577; Found 283.0573. In a similar manner, ent-Z-16 (300 mg, 0.6 mmol) gave R-3 (139 mg, 89%). 1H and 13C NMR spectral data of R-3 agreed well with those of the corresponding antipode (S-3). (4E,6S)- and (4E,6R)-6-Benzyloxy-7-hydroxy-2,4-heptadien4-olide [(S)- and (R)-Isofruticosinol (S-4 and R-4)]. Following the procedure used for desilylation of Z-16, E-16 (231 mg, 1.0 mmol) was desilylated with pyridinium poly(hydrogen fluoride) (65% HF, 4.6 mL) at −50 °C. Workup and column chromatography gave S-4 (106 mg, 88%) as a colorless viscous oil. IR (neat): 3460, 2931, 2881, 1786, 1759, 1716, 1601, 1558, 1450, 1269, 1111, 1069, 1026 cm−1. 1 H NMR (800 MHz, CDCl3) δ: 2.29 (1H, br s, OH), 3.93 (1H, dd, J = 12.0, 4.4, H-7a), 3.98 (1H, dd, J = 12.0, 6.3, H-7b), 5.80 (1H, ddd, J = 10.0, 1.8, 0.8, H-5), 5.90 (1H, ddd, J = 10.0, 6.3, 4.4, H-6), 6.33 (1H, dd, J = 5.6, 1.8, H-2), 7.45 (2H, m, arom.), 7.59 (1H, m, arom.), 7.97 (1H, dd, J = 5.6, 0.8, H-3), 8.03 (2H, m, arom.). 13C NMR (200 MHz, CDCl3) δ: 64.3 (C-7), 70.4 (C-6), 108.3 (C-5), 122.3 (C-2), 128.5/129.7/133.5 (d, arom.), 129.4 (s, arom.), 140.6 (C-3), 153.5 (C-4), 165.9 (COPh) 168.9 (C-1). HRMS (ESI) m/z: [M + Na]+ Calcd for C14H12O5Na 283.0577; Found 283.0574. In a similar manner, ent-Z-16 (300 mg, 0.6 mmol) gave R-4 (139 mg, 89%). 1H and 13C NMR spectral data of R-4 agreed well with those of the antipode (S-1). X-ray Crystallographic Analysis. Data of butenolide Z-12 were taken on a Rigaku RAXIS RAPID imaging plate area detector with graphite monochromated Mo-Kα radiation. The structure of Z-12 was solved by direct methods with SIR97. Full-matrix least-squares refinement was employed with anisotropic thermal parameters for all non-hydrogen atoms. All calculations were performed using the Crystal Structure (ver. 3.8) crystallographic software package. ORTEP drawing of Z-12 is shown in Figure 4. The data of Z-12 have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number (CCDC 1824643). Crystal Data for Butenolide Z-12. Orthorhombic, space group P212121, a = 8.5428(4), b = 17.2873(9), c = 18.2419(8) Å, V = 2694.0(2) Å3, Z = 4, μ(Mo-Kα) = 1.24 cm−1, F(000) = 1056, Dc = 8261

DOI: 10.1021/acs.joc.8b00986 J. Org. Chem. 2018, 83, 8250−8264

Article

The Journal of Organic Chemistry 1.229 g/cm3, crystal dimensions: 0.35 × 0.30 × 0.25 mm. A total of 25946 reflections (6165 unique) were collected using the ω−2θ scan technique to a maximum 2θ value of 55°, and all reflections were used in the structure determination. Final R and Rw values were 0.045 and 0.093, respectively. The maximum and minimum peaks in the difference map were 1.52 and −1.74 e− Å−3, respectively. The absolute structure was deduced based on Flack parameter, 0.00(4), refined using 2690 Friedel pairs. HPLC Monitoring Experiments. Reaction of R-1 with Benzoic Acid. A mixture of R-1 (60.0 mg, 0.21 mmol), benzoic acid (51.2 mg, 0.42 mmol), methanol (3 mL), and water (3 mL) was heated under reflux. The reaction mixture, which was picked up (1 μL) at certain intervals (3, 6, and 9 h), was analyzed by ODS-HPLC. After 9 h, the reaction mixture was condensed at reduced pressure to give a mixture of R-1 and benzoic acid. Reaction of R-1 with Sodium Benzoate. A mixture of R-1 (60 mg, 0.21 mmol), sodium benzoate (77 mg, 0.63 mmol), methanol (3 mL), and water (3 mL) was heated under reflux. The reaction mixture, which was picked up (1 μL) at certain intervals (3, 6, and 9 h), was analyzed by ODS-HPLC. After 9 h, the reaction mixture was condensed at reduced pressure to give a pale brown solid, which on preparative ODS-HPLC [CH3CN/1% aqueous AcOH (30/70, v/v)] gave R-1 (19.6 mg), R-3 (5.4 mg). Reaction of R-1 without Sodium Benzoate. A mixture of R-1 (10 mg, 0.04 mmol), methanol (2 mL), and water (2 mL) was heated under reflux. The reaction mixture, which was picked up (1 μL) at certain intervals (3, 6, and 9 h), was analyzed by ODS-HPLC. After 9 h, ODS-HPLC analysis indicated the formation of an approximately 3.7/1 mixture of R-1/R-3. Reaction of R-3 without Sodium Benzoate. A mixture of R-3 (10 mg, 0.04 mmol), methanol (2 mL), and water (2 mL) was heated under reflux. The reaction mixture, which was picked up (1 μL) at certain intervals (3, 6, and 9 h), was analyzed by ODS-HPLC. After 9 h, ODS-HPLC analysis indicated the formation of an approximately 1.5/1 mixture of R-1/R-3. Reaction of R-2 without Sodium Benzoate. A mixture of R-2 (10 mg, 0.04 mmol), methanol (2 mL), and water (2 mL) was heated under reflux. The reaction mixture, which was picked up (1 μL) at certain intervals (3, 6, and 9 h), was analyzed by ODS-HPLC. After 9 h, ODS-HPLC analysis indicated the formation of an approximately 3.7/1 mixture of R-2/R-4. Reaction of R-4 without Sodium Benzoate. A mixture of R-4 (10 mg, 0.04 mmol), methanol (2 mL), and water (2 mL) was heated under reflux. The reaction mixture, which was picked up (1 μL) at certain intervals (3, 6, and 9 h), was analyzed by ODS-HPLC. After 9 h, ODS-HPLC analysis indicated the formation of an approximately 2.5/1 mixture of R-2 and R-4. Bioassays. Reagents. Dulbecco’s modified Eagle’s medium (DMEM, 4.5 g/L glucose) was purchased from Sigma-Aldrich (St. Louis, MO); fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco (Invitrogen, Carlsbad, CA); and other chemicals used in this study were purchased from Wako Pure Chemical Co., Ltd. (Osaka, Japan). The 48- and 96-well microplates (Sumilon) were purchased from Sumitomo Bakelite Co., Ltd. (Tokyo, Japan). Cell Culture. Murine B16 melanoma 4A5 cells (RCB0557)25 were obtained from Riken Cell Bank (Tsukuba, Japan). The cells were grown in DMEM (glucose; 4500 mg/L) supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37 °C in 5% CO2/air. Cells were harvested by incubation in phosphatebuffered saline (PBS) containing 0.05% ethylenediaminetetraacetic acid (EDTA) and 0.02% trypsin for ca. 5 min at 37 °C and used for the subsequent bioassays. Melanogenesis and Cell Viability. Effects on theophyllinestimulated melanogenesis and viability of B16 melanoma 4A5 cells were determined as described previously.15a−d,22a,b Mushroom Tyrosinase. Tyrosinase activities using L-tyrosine or 3,4-dihydroxyphenyl-L -alanine (L-DOPA) as a substrate were determined according to the protocol described previously.15a−d,22a,b

Expressions of Tyrosinase, TRP-1, and TRP-2 mRNA. The expressions of tyrosinase, TRP-1, and TRP-2 mRNA were assessed according to the previously reported method.15a−d,22a,b Statistical Analysis. Values are expressed as mean ± SEM. Oneway analysis of variance followed by Dunnett’s test was used for statistical analyses. Probability (p) values less than 0.05 were considered significant.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.8b00986. Tables of inhibitory effects of the methanol extract and its fractions from the flowers of M. f ruticosum on melanogenesis and viability in B16 4A5 cells and effects of S- and R-1−4 on activity of tyrosinase from mushroom, as well as 1H and 13C NMR spectra of synthesized compounds (PDF) X-ray data for Z-12 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +81 6 4307 4019. Fax: +81 6 6721 2505. E-mail: [email protected] (G.T.). *Tel.: +81 6 4307 4306. Fax: +81 6 6729 3577. E-mail: [email protected] (T.M.). ORCID

Genzoh Tanabe: 0000-0002-7954-8874 Fumihiro Ishikawa: 0000-0002-8681-9396 Toshio Morikawa: 0000-0003-2794-5365 Author Contributions ∥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science [JSPS (KAKENHI), 17K08377 (G.T.), 17H05438 (F.I.), 18K06726 (T.M.), and 18K06739 (K.N.)]. We are also thankful for the financial support extended by the MEXTSupported Program for the Strategic Research Foundation at Private Universities, 2014−2018 (S1411037, T.M.), Kobayashi International Scholarship Foundation, the Houansha Foundation, 2017−2020 (G.T.), and the Antiaging Project for Private Universities.

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REFERENCES

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DOI: 10.1021/acs.joc.8b00986 J. Org. Chem. 2018, 83, 8250−8264

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DOI: 10.1021/acs.joc.8b00986 J. Org. Chem. 2018, 83, 8250−8264