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Oct 4, 2016 - Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. ‡. Alps-Pharmaceu...
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Semisynthesis and Structure−Activity Studies of Uncarinic Acid C Isolated from Uncaria rhynchophylla as a Specific Inhibitor of the Nucleation Phase in Amyloid β42 Aggregation Takuya Yoshioka,† Kazuma Murakami,† Kyohei Ido,† Mizuho Hanaki,† Kanoko Yamaguchi,† Satohiro Midorikawa,‡ Shinji Taniwaki,‡ Hiroki Gunji,‡ and Kazuhiro Irie*,† †

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan Alps-Pharmaceutical Industry Co., Ltd., Gifu 509-4241, Japan

‡

S Supporting Information *

ABSTRACT: Oligomers of the 42-mer amyloid-β protein (Aβ42), rather than fibrils, cause synaptic dysfunction in the pathology of Alzheimer’s disease (AD). The nucleation phase in a nucleationdependent aggregation model of Aβ42 is related to the formation of oligomers. Uncaria rhynchophylla is one component of “Yokukansan”, a Kampo medicine, which is widely used for treating AD symptoms. Previously, an extract of U. rhynchophylla was found to reduce the aggregation of Aβ42, but its active principles have yet to be identified. In the present work, uncarinic acid C (3) was identified as an inhibitor of Aβ42 aggregation that is present in U. rhynchophylla. Moreover, compound 3 acted as a specific inhibitor of the nucleation phase of Aβ42 aggregation. Compound 3 was synthesized from saponin A (10), an abundant byproduct of rutin purified from Uncaria elliptica. Comprehensive structure−activity studies on 3 suggest that both a C-27 ferulate and a C-28 carboxylic acid group are required for its inhibitory activity. These findings may aid the development of oligomer-specific inhibitors for AD therapy.

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during oligomerization and that the oligomers can serve as nuclei to accelerate the Aβ42 fibrillization. Therefore, selective inhibition of the nucleation phase for the purpose of removing toxic Aβ42 oligomers could be an effective strategy for preventing AD progression during its early stages. Kampo medicines have been widely used as symptomatic treatments for many diseases. “Yokukansan”, a traditional medicine of Kampo, was found to delay the onset of impaired behavioral and psychological symptoms of AD patients.10 Terasawa et al. reported that the dried hooks and stems of Uncaria rhynchophylla Miq. (Rubiaceae) (Japanese “chotoko”), which is an ingredient of “Yokukansan”, improved cognitive impairment in patients with vascular dementia when used as a “Shoyaku” or herbal remedy.11 In addition, Fujiwara et al. demonstrated that U. rhynchophylla extract not only prevents the aggregation of Aβ42 but also destabilizes preformed fibrils.12 Analysis by HPLC indicated that U. rhynchophylla extract contains indole alkaloids such as isorhynchophylline and hirsutine.12 According to preliminary experiments performed, isorynchophylline did not inhibit Aβ42 aggregation measurably (data not shown), which prompted the present study to identify the active components in U. rhynchophylla. Reported herein are the isolation of uncarinic acids A−D (1−4, Figure

lzheimer’s disease (AD) is characterized by senile plaques, which are composed mainly of 40- and 42-mer amyloid-β proteins (Aβ40 and Aβ42, respectively), generated from Aβ precursor protein (APP).1 Aβ42 aggregates rapidly and shows potent neurotoxicity compared with Aβ40. The aggregation mechanism of Aβ42 is well explained by the nucleationdependent polymerization model, which consists of nucleation and elongation phases.2 During the nucleation phase, Aβ42 monomer gradually forms low-molecular-weight intermediates, called “nuclei”. In the subsequent elongation phase, each nucleus acts as an aggregation template that reacts with monomers to further polymerize, resulting in the formation of amyloid fibrils. The nucleus may be closely related to the formation of toxic Aβ42 oligomers.3,4 Mounting evidence shows that soluble oligomers of Aβ42, rather than insoluble fibrils, induce cognitive impairment and synaptic loss; the term “aggregation” in the present study refers to the change from Aβ monomers into amyloid fibrils through oligomerization.5,6 The minimum unit of these oligomers, which are divided into two groups with either low (2−12-mer) or high (24−100-mer) molecular weight, is thought to be either a dimer or trimer (2 or 3 × n-mer).7,8 Sciarretta et al. demonstrated that Aβ40-lactam(Asp23/Lys28), a conformationally favorable model to initiate the nucleation, has shown a greater ability to generate oligomers than wild-type Aβ40.9 These findings indicate the significance of the nucleation phase © 2016 American Chemical Society and American Society of Pharmacognosy

Received: May 2, 2016 Published: October 4, 2016 2521

DOI: 10.1021/acs.jnatprod.6b00392 J. Nat. Prod. 2016, 79, 2521−2529

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Figure 1. (A) Structure of uncarinic acids (1−4). (B) Structure of C-27 derivatives (5−7) and C-28 derivatives (8, 9) of 3.

Figure 2. Effects of 1−4 on Aβ42 aggregation. (A) Th-T assay. HFIP-treated Aβ42 (25 μM) and 1−4 (50 μM) were incubated in PBS (pH 7.4) at 37 °C. Data are presented as means ± SD (n = 8). (B) Sedimentation assay. HFIP-treated Aβ42 (25 μM) and 3 (50 μM) were incubated in PBS (pH 7.4) at 37 °C. Data are presented as means ± SE (n = 2).

1A) from an acetone extract of U. rhynchophylla as specific inhibitors of the nucleation phase in Aβ42 aggregation and the first semisynthesis of uncarinic acid C (3), performed in order to examine its structure−activity relationships (SAR) using its derivatives (5−9, Figure 1B).

physical and spectroscopic parameters with literature values.16,17 Given the inhibition of the early stages (8 h incubation) of Aβ42 aggregation by 1−4 (data not shown), the effects of 1−4 on the nucleation phase were investigated using Aβ42 treated with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to dissociate aggregation seeds (nuclei) of Aβ42 that formed during purification or storage. As previously reported,14 synthetic Aβ42 required ca. 1 h for nucleation, but HFIP treatment extended the nucleation phase from ca. 1 h to ca. 4 h. Such an extension was also observed in this study (Figure 2A). The addition of 1−4 further extended the time required for nucleation of Aβ42 (ca. 10 h, Figure 2A), and 3 was the most preventive among the four uncarinic acids (ca. 12 h). In contrast, the maximum fluorescence intensity of Aβ42 treated with 1−4 was not significantly reduced (Figure 2A). The molecular ratio (1:2) of Aβ42 (25 μM) to compounds (50 μM) was selected based on the previous titration studies in the drug screen using natural products.13,14 All the compounds at the concentration of 50 μM tested in the following experiments

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RESULTS AND DISCUSSION An acetone extract of U. rhynchophylla branches was partitioned between ethyl acetate (EtOAc) and water and was fractionated on the basis of an ability to inhibit Aβ42 aggregation after an 8 h incubation, which falls within the nucleation phase measured in previous studies,13,14 using a thioflavin-T (Th-T) fluorescence assay. Th-T fluoresces when bound to Aβ aggregates with a β-sheet structure.15 Normal-phase chromatography of the EtOAc extract, followed by reversed-phase HPLC separation, gave four compounds with antiaggregative ability against Aβ42 (1−4), 1 (1.9 mg, 0.02%), 2 (2.7 mg, 0.03%), 3 (4.8 mg, 0.06%), and 4 (4.4 mg, 0.05%), from 8.2 g of EtOAc extract. Compounds 1−4 (Figure 1A) were identified as uncarinic acids A−D, respectively, by comparison of their 2522

DOI: 10.1021/acs.jnatprod.6b00392 J. Nat. Prod. 2016, 79, 2521−2529

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Scheme 1

Reagents and conditions: (a) HCl−MeOH, 50 °C, 4 h (50%); (b) K2CO3, MeI, rt, 16 h (97%); (c) DIBAL, 0 °C, 20 min (90%); (d) TEMPO, NaClO, rt, 80 min (46%); (e) 17, EDCI, 4-DMAP, 40 °C, 48 h (26%); (f) TBAF, rt, 3 h (65%); (g) NaClO2, amylene, NaH2PO4, rt, 5 h (76%).

a

Scheme 2

a Reagents and conditions: (a) NaClO2, amylene, NaH2PO4, rt, 5 h (79%); (b) Ac2O, rt, 27 h (37%); (c) NaClO2, amylene, NaH2PO4, rt, 5 h (98%); (d) TBSCl, imidazole, rt, 24 h, 49%; (e) 17, EDCI, DMAP, 40 °C, 48 h (14%); (f) TBAF, rt, 3 h (80%); (g) TMSCHN2, rt, 12 h (57%); (h) 17, EDCI, DMAP, 40 °C, 48 h (28%); (i) TBAF, rt, 3 h (81%).

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DOI: 10.1021/acs.jnatprod.6b00392 J. Nat. Prod. 2016, 79, 2521−2529

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Figure 3. Effects of 5−7 and natural 3 on Aβ42 aggregation as determined by a Th-T assay. HFIP-treated Aβ42 (25 μM) and (A) 5−7 or (B) 8 and 9 (50 μM) were incubated in PBS (pH 7.4) at 37 °C. Data are presented as means ± SD (n = 8).

periodinane,22 tetrapropylammonium perruthenate (TPAP),23 and 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO).24 DessMartin oxidation and TPAP oxidations were unsuccessful because of the low solubility of 13 in CH2Cl2. Surprisingly, a two-phase system between H2O and CH2Cl2 dissolved 13 completely, and then TEMPO oxidation gave 14 in moderate yield (46%). Coupling of 14 with 4-O-tert-butyldimethylsilyl (TBS) ferulic acid (17) by Steglich esterification,25 followed by deprotection and Pinnick oxidation,26 gave 3 in 19% yield (three steps). The structure of 3 was confirmed by its spectroscopic data (1H NMR, 13C NMR, HRESITOFMS, and optical rotation) and by cochromatography with natural 3. Synthetic 3 as well as natural 3 prevented the nucleation phase (ca. 12 h) in Aβ42 aggregation (Figure S1, Supporting Information). For SAR studies, four derivatives of 3 in Figure 1B (6−9) were prepared to examine the contribution of the C-27 ferulate and C-28 carboxylic acid groups toward their antiaggregative activity. These derivatives were prepared from 13 and 14 by conventional methods as shown in Scheme 2. In contrast to 3, all C-27 derivatives (5−7) failed to inhibit the nucleation phase as well as the elongation phase in Aβ42 aggregation (Figure 3A), suggesting that the C-27 ferulate group in 3 is important in the specific inhibition of nucleation. In addition, both C-28 derivatives (8 and 9) lacked any inhibitory activities, indicating that the C-28 carboxylic acid group is also required for the inhibitory activity. There have been a number of studies on the reduction of Aβ42 aggregation by natural products, which include flavonoids, polyketides, alkaloids, and terpenoids,27 but almost all of them either target only the elongation phase (e.g., myricetin and (+)-taxifolin)13,28 or inhibit both the nucleation and elongation phases (e.g., morin and datiscetin).14 Compounds 1−4 are the first inhibitors specific for the nucleation phase of Aβ42 aggregation and possess a triterpenoid skeleton without a catechol moiety.13,14 A sugar derivative with a carboxyl group, α-D-mannosylglycerate, inhibited both Aβ42induced aggregation and neurotoxicity by forming electrostatic interactions with Lys residues in Aβ42.29 Intriguingly, Klabunde et al. showed that the aggregation of transthyretin was inhibited by flurbiprofen due to formation of a salt bridge between its carboxylic acid group and the Arg residue in transthyretin.30 Given the importance of the C-28 carboxylic acid group to the antiaggregative activity of 3, similar inhibitory mechanisms may exist. At the same time, the C-27 ferulate group can participate in the interference of π−π stacking with aromatic residues such as His13,14 and Phe19,20 in Aβ42, like non-catechol-type flavonoids possessing planarity,14 because the inhibitory activity

showed a good solubility in the assay buffer. In fact, 3 did not completely inhibit the aggregation of Aβ42 at the maximum concentration tested (data not shown). These results suggest that 1−4 could preferentially inhibit nucleation compared with elongation. The inhibitory activities of 1, 2, and 4 were comparable to that of morin (ca. 10 h), a noncatechol-type flavonoid.14 Since 3 was more potent than morin, uncarinic acid C was focused on in subsequent experiments. Although the Th-T assay is one of the most conventional and useful methods for quantifying Aβ aggregates, caution should be exercised when evaluating the antiamyloidogenic properties of compounds, because of competitive binding with Th-T, as exemplified by curcumin and resveratrol.18 To validate whether the nucleation phase was inhibited by 3, a sedimentation assay19 using HPLC after centrifugation was carried out. After incubating for 2 h, Aβ42 started to aggregate, before reaching a plateau after 8 h (Figure 2B). In contrast, the aggregation velocity of Aβ42 was delayed at least by 8 h in the presence of 3, and this tendency was in good agreement with the results of the Th-T assay (Figure 2A). The inhibition of Aβ42 aggregation by natural products has been well explained by mechanisms that focus on either the nucleation or elongation phase.13,14 Catechol-type flavonoids [e.g., (+)-taxifolin] prevent the elongation phase by forming an o-quinone structure derived from autoxidation to target Lys16 and/or Lys28 in Aβ42 via a Michael addition.13 On the other hand, non-catechol-type flavonoids such as morin and datiscetin inhibit nucleation as well as elongation by interacting with the intermolecular β-sheet regions in Aβ42.14 Furthermore, Ono et al. implicated the carboxyl group of rosmarinic acid in its antiaggregative activity through the formation of salt bridges with either Lys or His residues in Aβ42.20 To identify the structural factors involved in the specific inhibition of the nucleation phase, the SAR of 3 was examined. Due to the low isolation yield for 3 (0.06%), this compound was synthesized from saponin A (10)21 as a starting material, which is an abundant byproduct of the purification of rutin from Uncaria elliptica (Scheme 1). Acidic cleavage of 10 generated quinovic acid (11, Scheme 1) in 50% yield. Since direct reduction of both the C-27 and C-28 carboxylic acid groups of quinovic acid to the corresponding primary hydroxy groups using BH3 or LiAlH4 was not successful, it was instead converted to the 27,28-dimethyl ester by iodomethane through an SN2 reaction. Subsequent reduction by diisobutylaluminum hydride (DIBAL) gave 13 in 87% yield (two steps). LiAlH4 or LiBH4, instead of DIBAL, did not reduce the methyl esters. Regioselective oxidation of the C-28 hydroxy group of 13 was attempted using bulky oxidizing agents, including Dess-Martin 2524

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of ferulic acid itself was detectable toward Aβ42 aggregation.31 These findings may be helpful to the development of oligomerspecific inhibitors for AD therapy. Further study is necessary to clarify how large the oligomers that 1−4 target are at a molecular level and the nature of the residues with which they interact.

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NMR data (Table S2, Supporting Information); (−)-HRESITOFMS m/z 647.3948 [M − H]− (calcd for C40H55O7, 647.3948). Uncarinic acid C (3): [α]27D +87 (c 3.7 × 10−3, i-PrOH); FT-IR (KBr) νmax 3449, 1688, 1630, 1514, 1164, 1032 cm−1; 1H and 13C NMR data (Table S3, Supporting Information); (−)-HRESITOFMS m/z 647.4042 [M − H]− (calcd for C40H55O7, 647.3948). Uncarinic acid D (4): [α]18D + 47 (c 1.9 × 10−3, i-PrOH); FT-IR (KBr) νmax 3423, 1697, 1630, 1595, 1033 cm−1; 1H and 13C NMR data (Table S4, Supporting Information); (−)-HRESITOFMS m/z 647.3948 [M − H]− (calcd for C40H55O7, 647.3948). Synthesis of Uncarinic Acid C (3) and Its Derivatives. The synthetic procedure for 3 is summarized in Scheme 1, while the synthetic procedures for the derivatives (6−9) of 3 are summarized in Scheme 2. Saponin A (10). A suspension of solid rutin-containing extracts21 (651 g) in EtOAc (3 L) was stirred at 55 °C for 4 h and filtered. The filtrate was evaporated, and the residue was treated with H2O (100 mL), EtOAc (100 mL), and KHCO3 (3.0 g). After stirring at 50 °C for 30 min, the layers were separated. The organic layer was washed with 0.01 M aqueous KHCO3 solution (2 × 50 mL), and the combined aqueous layers were treated with 0.24 M HCl (180 mL). The resultant precipitate was collected by filtration and dried in vacuo to give 10 (3.5 g) as a gray solid: [α]19D +46 (c 6.13 × 10−3, MeOH); 1H NMR (pyridine-d5, 400 MHz) δ 0.79 (3H, d, J = 6.4 Hz, H3-30), 0.86 (3H, s, H3-25), 0.93 (3H, s, H3-23 or 24), 0.92−1.10 (3H, m, H-5, 20, and 22a), 1.09 (3H, s, H3-26), 1.14 (3H, s, H3-23 or 24), 1.21 (3H, d, J = 6.1 Hz, H3-29), 1.21−1.52 (5H, m, H-6a, 6b, 19, 21a, and 21b), 1.65 (3H, d, J = 6.0 Hz, H3-6′), 1.52−1.76 (2H, m, H-7a and 22b), 1.76− 2.02 (5H, m, H-1a, 1b, 2a, 7b, and 11a), 2.04−2.37 (4H, m, H-2b, 11b, 15a, and 16a), 2.50−2.63 (2H, m, H-15b and 16b), 2.71 (1H, dd, J = 11.4, 5.0 Hz, H-9), 2.79 (1H, d, J = 11.3 Hz, H-18), 3.18 (1H, dd, J = 11.7, 4.4 Hz, H-3), 3.69 (1H, dd, J = 8.9, 8.9 Hz, H-4′), 3.78 (1H, dq, J = 8.9, 6.0 Hz, H-5′), 4.09 (1H, dd, J = 8.8, 7.7 Hz, H-2′), 4.09 (1H, dd, J = 8.8, 8.8 Hz, H-3′), 4.68 (1H, d, J = 7.8 Hz, H-1′), 6.00 (1H, br d, J = 2.6 Hz, H-12); 13C NMR (pyridine-d5, 100 MHz) δ 17.0 (q, C-25), 17.5 (q, C-23 or 24), 18.7 (q, C-29), 19.1 (t, C-6), 19.3 (q, C-6′), 19.4 (q, C-26), 21.8 (q, C-30), 23.8 (t, C-11), 26.0 (t, C-15), 26.8 (t, C16), 27.3 (t, C-2), 28.5 (q, C-23 or 24), 31.0 (t, C-21), 37.5 (s, C-10), 37.6 (t, C-1), 38.0 (t, C-7), 38.2 (d, C-19), 39.5 (t, C-22), 39.8 (d, C20), 40.0 (s, C-4), 40.5 (s, C-8), 47.7 (d, C-9), 49.2 (s, C-17), 55.4 (d, C-18), 56.3 (d, C-5), 57.2 (s, C-14), 73.1 (d, C-5′), 76.4 (d, C-2′), 77.4 (d, C-4′), 78.9 (d, C-3′), 88.9 (d, C-3), 107.2 (d, C-1′), 129.4 (d, C-12), 134.6 (s, C-13), 178.5 (s, C-27 or 28), 180.6 (s, C-27 or 28); (−)-HRESITOFMS m/z 631.3832 [M − H] − (calcd for C36H55O9, 631.3846). Quinovic Acid (11). A solution of 10 (1.93 g, 3.1 mmol) in MeOH (46 mL) was treated with 5% HCl−MeOH (12 mL) at room temperature. After stirring for 4 h at 50 °C, the reaction mixture was cooled to room temperature and was evaporated. The residue was coevaporated several times with MeOH to remove HCl. The resulting oily material was treated with acetone (4 mL) to yield a white precipitate. After stirring at room temperature for 30 min, the mixture was filtered. The cake was washed several times with acetone and dried in vacuo to yield 11 (735 mg, 50%) as a white solid: [α]19D +86 (c 1.88 × 10−3, MeOH); 1H NMR (pyridine-d5, 400 MHz) δ 0.82 (3H, d, J = 6.4 Hz, H3-30), 0.94 (3H, s, H3-25), 1.00 (3H, s, H3-3 or 24), 1.02− 1.17 (3H, m, H-5, 20, and 22a), 1.08 (3H, s, H3-26), 1.15 (3H, s, H323 or 24), 1.24 (3H, d, J = 6.1 Hz, H3-29), 1.28−1.51 (5H, m, H-6a, 6b, 19, 21a, and 21b), 1.52−1.70 (2H, m, H-7a and 22b), 1.70−2.02 (5H, m, H-1a, 1b, 2a, 7b, and 11a), 2.04−2.24 (4H, m, H-2b, 11b, 15a, and 16a), 2.53−2.69 (2H, m, H-15b and 16b), 2.76 (1H, dd, J = 11.5, 5.2 Hz, H-9), 2.82 (1H, d, J = 11.3 Hz, H-18), 3.32 (1H, dd, J = 11.2, 4.8 Hz, H-3), 6.04 (1H, br d, J = 2.6 Hz, H-12); 13C NMR (pyridined5, 100 MHz) δ 17.0 (q, C-25), 17.1 (q, C-23 or 24), 18.7 (q, C-29), 19.4 (t, C-6), 19.4 (q, C-26), 21.8 (q, C-30), 23.8 (t, C-11), 26.0 (t, C15), 26.9 (t, C-16), 28.7 (t, C-2), 29.1 (q, C-23 or 24), 31.1 (t, C-21), 37.6 (t, C-1), 37.8 (s, C-10), 38.1 (t, C-7), 38.2 (d, C-19), 39.7 (t, C22), 39.8 (s, C-4), 39.9 (d, C-20), 40.5 (s, C-8), 47.8 (d, C-9), 49.2 (s, C-17), 55.4 (d, C-18), 56.2 (d, C-5), 57.3 (s, C-14), 78.4 (d, C-3), 129.5 (d, C-12), 134.6 (s, C-13), 178.5 (s, C-27 or 28), 180.6 (s, C-27

EXPERIMENTAL SECTION

General Experimental Procedures. The following spectroscopic and analytical instruments were used for the isolation of compounds 1−4: Digital polarimeter, model P-2200 (JASCO); IR, FT/IR-470 Plus (JASCO); NMR spectrometers, Avance III 400, Avance III 500, and Avance II 800 (Bruker), tetramethylsilane was used as reference; HRESITOFMS, Xevo G2-S QTof (Waters); HPLC, Waters model 600E with a model 2487 UV detector. HPLC was carried out on YMC-packed ODS-AL (20 mm i.d. × 150 mm) and ODS-AM (10 mm i.d. × 250 mm) (Yamamura Chemical Laboratory, Kyoto, Japan). Wakogel C-200 (silica gel, Wako Pure Chemical Laboratory, Osaka, Japan) and Kieselgel 60 (silica gel, Merck, Darmstadt, Germany) were used for column chromatography of natural products. In the synthetic section, NMR spectra were recorded on a Bruker AV400 M spectrometer (Bruker, Germany), operating at 400 MHz for 1 H and 100 MHz for 13C NMR in CDCl3 unless otherwise noted. All the chemical shifts are reported in the scale relative to tetramethylsilane (0.00 ppm) as an internal standard. Reaction solvents used were the super-dehydrated grade, purchased from Wako Pure Chemical Industries, Ltd. Flash column chromatography was performed with silica gel 60, spherical (40−100 μm), purchased from Kanto Chemical Co., Inc. Abbreviations used in this synthetic section are as follows; Ac = acetyl, Ac2O = acetic anhydride, Bu = butyl, BuOH = butanol, DIBAL = diisobutylaluminum hydride, 4-DMAP = 4-(dimethylamino)pyridine, DMF = N,N-dimethylformamide, EDCI·HCl = 1-(3(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride, Et = ethyl, Et2O = diethyl ether, EtOAc = ethyl acetate, Me = methyl, MeI = iodomethane, MeOH = methanol, OMe = methoxy, TBAF· 3H2O = tetrabutylammonium fluoride trihydrate, TBS = tertbutyldimethylsilyl, TBSCl = tert-butyldimethylchlorosilane, TEMPO = 2,2,6,6-tetramethylpiperidin-1-oxyl, THF = tetrahydrofuran, TMS = trimethysilyl, and TMSCHN2 = trimethysilyldiazomethane. Plant Material. The dried branches (1 kg) of U. rhynchophylla were obtained from a distributor of Kampo medicine, Tochimoto Tenkaido (Osaka, Japan). A voucher specimen is deposited at the Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University. Extraction and Isolation. U. rhynchophylla (dried branches, 1 kg) was extracted with acetone, and the concentrate after evaporation was partitioned between EtOAc and water. The EtOAc extract (8.2 g) was chromatographed on Wako gel C-200, eluted with toluene containing increasing amounts of EtOAc (10, 20, 50, and 100%) and subsequently with 20% MeOH−CHCl3. The eluate with 50% EtOAc in toluene (1.1 g) was subjected to further chromatography on Kieselgel 60, eluted with CHCl3 containing increasing amounts of EtOAc (5, 10, 20, 50, and 100% EtOAc) and subsequently with 20% MeOH−CHCl3. The eluates obtained with 5% and 10% EtOAc in CHCl3 had potent inhibitory activity against Aβ42 aggregation and were purified by reversed-phase HPLC on YMC Pack ODS-AL using 70% acetonitrile in water, resulting in the isolation of two active fractions (Fr.1, 8.1 mg, and Fr.2, 8.8 mg). HPLC separation of Fr.1 on YMC Pack ODS-AM using 82% methanol in water led to the isolation of compounds 1 (1.9 mg) and 3 (4.8 mg). Further HPLC separation of Fr.2 on YMC Pack ODS-AM using 82% methanol in water gave compounds 2 (2.7 mg) and 4 (4.4 mg). Uncarinic acid A (1): [α]27D +64 (c 1.3 × 10−3, i-PrOH); FT-IR (KBr) νmax 3412, 1693, 1630, 1597, 1180 cm−1; 1H and 13C NMR data (Table S1, Supporting Information); (−)-HRESITOFMS m/z 647.3948 [M − H]− (calcd for C40H55O7, 647.3948). Uncarinic acid B (2): [α]18D +121 (c 5.1 × 10−4, i-PrOH); FT-IR (KBr) νmax 3404, 1696, 1626, 1594, 1260, 1161 cm−1; 1H and 13C 2525

DOI: 10.1021/acs.jnatprod.6b00392 J. Nat. Prod. 2016, 79, 2521−2529

Journal of Natural Products

Article

or 28); (−)-HRESITOFMS m/z 485.3260 [M − H]− (calcd for C30H45O5, 485.3267). Dimethyl 3β-hydroxyurs-12-en-27,28-oate (12). A solution of 11 (1.05 g, 2.2 mmol) in DMF (21 mL) was treated with K2CO3 (0.896 g, 6.5 mmol) and MeI (0.40 mL, 6.5 mmol) at room temperature. After stirring for 16 h, the reaction mixture was poured into H2O (100 mL) and was extracted with EtOAc−Et2O (1:1) (3 × 20 mL). The combined organic layer was washed with H2O (15 mL), dried over Na2SO4, and evaporated in vacuo to dryness. The residue was purified by flash column chromatography (50 g of silica gel; hexane−EtOAc, 9:4) to yield 12 (1.08 g, 97%) as a white solid: 1H NMR (CDCl3, 400 MHz) δ 0.56−0.65 (2H, m), 0.76 (3H, s, H3-25), 0.83 (3H, s, H3-23 or 24), 0.85 (3H, d, J = 6.2 Hz, H3-30), 0.87 (3H, d, J = 6.2 Hz, H329), 0.90 (3H, s, H3-26), 0.93 (3H, s, H3-23 or 24), 0.93−1.05 (3H, m), 1.16−1.35 (4H, m), 1.37−1.74 (9H, m), 1.87−2.14 (5H, m), 2.27 (1H, d, J = 11.4 Hz, H-18), 3.19 (1H, t, J = 5.5 Hz, H-3), 3.62 (3H, s, OMe), 3.63 (3H, s, OMe), 5.64 (1H, dd, J = 4.4, 2.2 Hz, H-12); (+)-HRESITOFMS m/z 515.3751 [M + H]+ (calcd for C32H51O5, 515.3736). 3β,27,28-Trihydroxyurs-12-ene (13). A solution of 12 (1.08 g, 2.1 mmol) in CH2Cl2 (22 mL) was cooled at 0 °C and was treated dropwise over 10 min with a 1.0 M DIBAL−hexane solution (18 mL, 18.0 mmol). After stirring for 20 min, the reaction mixture was warmed to room temperature and was additionally stirred for 3 h. The reaction mixture was treated carefully with EtOAc (15 mL) and 0.5 N HCl (50 mL) at 0 °C to quench the excess reagent and was stirred for 5 min. After separation of the organic layer, the aqueous layer was extracted with EtOAc (2 × 10 mL) and toluene (2 × 10 mL). The combined organic layers were washed with brine (15 mL), dried over Na2SO4, and evaporated in vacuo to dryness. The residue was purified by flash column chromatography (50 g of silica gel, hexane−EtOAc, 1:1) to yield 13 (0.87 g, 90%) as a white solid: 1H NMR (CDCl3, 400 MHz) δ 0.79 (3H, s, H3-25), 0.93 (3H, d, J = 6.1 Hz, H3-29 or 30), 0.95 (3H, d, J = 6.1 Hz, H3-29 or 30), 0.83−0.90 (2H, m), 0.96 (3H s, H3-23 or 24), 0.96 (3H, s, H3-26), 1.00 (3H, s, H3-23 or 24), 1.01− 1.28 (6H, m), 1.28−1.66 (13H, m), 1.67−1.86 (2H, m), 1.90−2.11 (3H, m), 3.24 (1H, dd, J = 10.8, 5.0 Hz, H-3), 3.26 and 3.58 (2H, ABq, J = 10.9 Hz, H2-27 or 28), 3.62 and 3.46 (2H, ABq, J = 12.2 Hz, H2-27 or 28), 5.63 (1H, t, J = 3.4 Hz, H-12); 13C NMR (CDCl3, 100 MHz) δ 15.6 (q, C-25), 16.2 (q, C-23 or 24), 18.0 (q, C-29), 18.3 (t, C-6), 18.9 (q, C-26), 21.4 (q, C-30), 22.3 (t, C-11), 23.2 (t, C-15), 23.9 (t, C-16), 27.2 (t, C-2), 28.0 (q, C-23 or 24), 29.8 (t, C-21), 33.0 (t, C-1), 35.1 (t, C-7), 37.0 (s, C-17), 37.7 (s, C-10), 38.5 (d, C-19), 38.6 (t, C-22), 38.8 (s, C-4), 39.9 (d, C-20), 40.6 (s, C-8), 47.6 (s, C14), 48.2 (d, C-9), 52.8 (d, C-18), 54.9 (d, C-5), 64.2 (t, C-27 or 28), 69.8 (t, C-27 or 28), 78.8 (d, C-3), 132.6 (d, C-12), 133.8 (s, C-13); (+)-HRESITOFMS m/z 441.3722 [M + H − H2O]+ (calcd for C30H49O2, 441.3733). 3β,27-Dihydroxyurs-12-en-28-aldehyde (14). A suspension of 13 (37.7 mg, 82 μmol) in CH2Cl2 (1.1 mL) was treated with H2O (0.57 mL) at room temperature and was stirred until the white solid was dissolved completely. The resulting two-phase solution was treated with KBr (20.3 mg, 0.17 mmol), NaHCO3 (21.1 mg, 0.25 mmol), 12% aqueous NaClO solution (46 μL, 90 μmol), and TEMPO24 (1.2 mg, 7.7 μmol). After stirring for 80 min, the layers were separated. The aqueous layer was extracted with CH2Cl2 (2 × 1.0 mL). The combined organic layers were washed with brine (1.0 mL), dried over Na2SO4, and evaporated in vacuo to dryness. The residue was purified by flash column chromatography (1.8 g of silica gel, hexane−EtOAc, 7:5) to yield 14 (17.4 mg, 46%) as a white solid: 1H NMR (CD3OD, 400 MHz) δ 0.73 (3H, s, H3-26), 0.77 (3H, s, H3-23 or 24), 0.80−0.88 (1H, m, H-5), 0.93 (3H, d, J = 6.6 Hz, H3-29 or 30), 0.94 (3H, s, H325), 0.97 (3H, s, H3-23 or 24), 0.97 (3H, d, J = 6.6 Hz, H3-29 or 30), 1.01−1.12 (2H, m, H-20a, and 22a), 1.18−1.47 (6H, m, H-1a, 1b, 6a, 7a, 19, and 21a), 1.47−1.70 (8H, m, H-2a, 2b, 6b, 15a, 15b, 16a, 21b, and 22b), 1.70−1.81 (2H, m, H-7b and 9), 1.86−2.04 (3H, m, H-11a, 11b, and 16b), 2.08 (1H, d, J = 11.3 Hz, H-18), 3.16 (1H, dd, J = 11.3, 5.0 Hz, H-3), 3.62 and 3.70 (2H, ABq, J = 12.5 Hz, H2-27), 5.53 (1H, dd, J = 3.8, 3.4 Hz, H-12), 9.32 (1H, d, J = 1.2 Hz, H-28); 13C NMR (CD3OD, 100 MHz) δ 16.4 (q, C-23 or 24), 16.5 (q, C-25), 17.6 (q,

C-29), 19.0 (q, C-26), 19.4 (t, C-6), 21.4 (q, C-30), 22.0 (t, C-15), 24.4 (t, C-11 or 16), 24.6 (t, C-11 or 16), 27.9 (t, C-2), 28.7 (q, C-23 or 24), 31.1 (t, C-21), 33.0 (t, C-1), 35.3 (t, C-7), 38.2 (s, C-8), 39.6 (d, C-19), 39.9 (s, C-10), 40.0 (t, C-22), 40.5 (d, C-20), 41.9 (s, C14), 49.3 (d, C-9), 49.9 (s, C-4), 51.3 (s, C-17), 54.0 (d, C-18), 56.5 (d, C-5), 64.5 (t, C-27), 79.7 (d, C-3), 131.3 (d, C-12), 136.1 (s, C13), 209.3 (d, C-28); (+)-HRESITOFMS m/z 441.3722 [M + H − H2O]+ (calcd for C30H49O2, 441.3733). 3β-Hydroxy-27-[(E)-4′-(O-tert-butyldimethylsilyl)feruloyloxy]urs12-en-28-aldehyde (15). A solution of 14 (17.0 mg, 37 μmol) and 4O-tert-butyldimethylsilylferulic acid (17)25 (41.6 mg, 0.13 mmol) in ClCH2CH2Cl (1.7 mL) was treated at room temperature with EDCI· HCl (27.8 mg, 0.15 mmol) and DMAP (17.2 mg, 0.14 mmol) and was warmed to 40 °C. After stirring for 48 h, the reaction mixture was cooled to room temperature and diluted with CHCl3 (20 mL). The solution was washed with 10% aqueous citric acid solution (8 mL) and brine (8 mL), dried over Na2SO4, and evaporated in vacuo to dryness. The residue was purified by flash column chromatography (25 g of silica gel; hexane−EtOAc, 3:1) to yield 15 (7.3 mg, 26%) as a white solid: 1H NMR (CDCl3, 400 MHz) δ 0.17 (6H, s, Me2Si), 0.68−0.85 (2H, m), 0.77 (3H, s, H3-25), 0.78 (3H, s, H3-23 or 24), 0.85−1.00 (15H, m), 1.00 (9H, s, t-Bu), 1.11−1.45 (7H, m), 1.45−1.71 (6H, m), 1.71−1.85 (2H, m), 1.90−2.08 (2H, m), 2.08 (1H, d, J = 11.1 Hz, H18), 3.17 (1H, t, J = 7.8 Hz, H-3), 3.85 (3H, s, OMe), 4.22 and 4.34 (2H, ABq, J = 12.8 Hz, H2-27), 5.63 (1H, t, J = 3.3 Hz, H-12), 6.23 (1H, d, J = 15.9 Hz, CHCHAr), 6.85 (1H, d, J = 7.8 Hz, H-5′), 6.97−7.02 (2H, m, H-2′ and 6′), 7.55 (1H, d, J = 15.9 Hz, CH CHAr), 9.35 (1H, s, H-28). 3β-Hydroxy-27-[(E)-feruloyloxy]urs-12-en-28-aldehyde (16). A solution of 15 (7.3 mg, 10 μmol) in THF (0.22 mL) was treated with TBAF·3H2O (5.3 mg, 17 μmol) at room temperature. After stirring for 3 h, the reaction mixture was evaporated in vacuo to dryness. The residue was purified by flash column chromatography (2.0 g of silica gel; hexane−EtOAc, 4:7) to yield 16 (4.0 mg, 65%) as a white solid: 1H NMR (CDCl3, 400 MHz) δ 0.71−0.89 (3H, m), 0.77 (3H, s, H3-25), 0.78 (3H, s, H3-23 or 24), 0.89 (3H, d, J = 6.4 Hz, H330), 0.91−1.01 (10H, m), 1.20−1.32 (6H, m), 1.32−1.43 (3H, m), 1.46−1.62 (6H, m), 1.62−1.85 (2H, m), 1.90−2.05 (2H, m), 2.08 (1H, d, J = 11.3 Hz, H-18), 3.16 (1H, t, J = 8.2 Hz, H-3), 3.94 (3H, s, OMe), 4.21 and 4.35 (2H, ABq, J = 12.8 Hz, H2-27), 5.63 (1H, t, J = 3.4 Hz, H-12), 5.83 (1H, br s, phenol), 6.23 (1H, d, J = 15.9 Hz, CHCHAr), 6.93 (1H, d, J = 8.2 Hz, H-5′), 7.01 (1H, d, J = 1.7 Hz, H-2′), 7.06 (1H, dd, J = 8.2, 1.7 Hz, H-6′), 7.55 (1H, d, J = 15.9 Hz, CHCHAr), 9.35 (1H, s, H-28); 13C NMR (CDCl3, 100 MHz) δ 15.7 (q, C-25), 15.9 (q, C-23 or 24), 17.2 (q, C-29), 18.3 (t, C-6), 18.3 (q, C-26), 21.0 (q, C-30), 22.3 (t, C-11), 23.2 (t, C-15), 23.6 (t, C16), 27.2 (t, C-2), 28.1 (q, C-23 or 24), 29.7 (t, C-21), 31.8 (t, C-1), 33.8 (t, C-7), 37.2 (s, C-10), 38.3 (d, C-19), 38.7 (t, C-22), 38.8 (s, C4), 39.2 (d, C-20), 40.5 (s, C-8), 45.7 (s, C-14), 48.4 (d, C-9), 50.0 (s, C-17), 52.3 (d, C-5), 55.3 (d, C-18), 56.0 (q, OMe), 65.7 (t, C-27), 78.8 (d, C-3), 109.4 (d, C-2′), 114.8 (d, C-5′), 115.7 (d, CHCHAr), 122.9 (d, C-6′), 126.9 (s, C-3′), 130.7 (d, C-12), 133.0 (s, C-13), 144.8 (d, CHCHAr), 146.8 (s, C-4′), 148.1 (s, C-1′), 166.9 (s, C O), 207.1 (C-28). Uncarinic Acid C (3). A solution of 16 (4.0 mg, 6.3 μmol) in tBuOH (0.12 mL) was treated at room temperature with H2O (24 μL), 2-methyl-2-butene (24 μL), NaH2PO4 (4.9 mg, 41 μmol), and NaClO2 (1.9 mg, 21 μmol). After stirring for 5 h, the reaction mixture was diluted with CHCl3 (1.0 mL). The solution was washed with H2O (0.5 mL) and brine (0.5 mL), dried over Na2SO4, and evaporated in vacuo to dryness to yield 3 (3.1 mg, 76%) as a white solid: [α]20D +93 (c 4.3 × 10−3, i-PrOH); 1H NMR (CDCl3, 400 MHz) δ 0.71−0.76 (1H, m, H-5), 0.76 (3H, s, H3-23 or 24), 0.78 (3H, s, H3-26), 0.86 (3H, d, J = 6.2 Hz, H3-29), 0.91 (3H, d, J = 6.1 Hz, H3-30), 0.95 (3H, s, H3-25), 0.97 (3H, s, H3-23 or 24), 0.96−1.05 (2H, m, H-20 and 22a), 1.05−1.20 (1H, m, H-19), 1.20−1.45 (4H, m, H-1a, 6a, 7a and 15a), 1.45−1.64 (5H, m, H-1b, 2a, 2b, 6b and 7b), 1.64−1.78 (5H, m, H-9, 16a, 21a, 21b and 22b), 1.78−1.92 (1H, m, H-15b), 1.92−2.10 (3H, m, H-11a, 11b and 16b), 2.29 (1H, d, J = 11.2 Hz, H-18), 3.18 (1H, t, J = 8.4 Hz, H-3), 3.94 (3H, s, OMe), 4.19 and 4.33 (2H, ABq, J 2526

DOI: 10.1021/acs.jnatprod.6b00392 J. Nat. Prod. 2016, 79, 2521−2529

Journal of Natural Products

Article

3β,27-Dihydroxy-28-tert-butyldimethylsilyloxyurs-12-ene (19). A solution of 13 (324.6 mg, 0.71 mmol) in DMF (6.5 mL) was treated with TBSCl (26.7 mg, 1.06 mmol) and imidazole (73.7 mg, 1.08 mmol) at room temperature. After stirring for 24 h, the reaction mixture was diluted with Et2O (65 mL). The solution was washed with H2O (3 × 10 mL), dried over Na2SO4, and evaporated in vacuo to dryness. The residue was purified by flash column chromatography (20 g of silica gel; hexane−EtOAc, 2:1) to yield 19 (198.9 mg, 49%) as a white solid: 1H NMR (CDCl3, 400 MHz) δ −0.01 (6H, s, Me2Si), 0.78 (3H, s, H3-25), 0.81−1.00 (29H, m), 1.00−1.49 (10H, m), 1.49− 1.76 (6H, m), 1.84−2.03 (3H, m), 2.07 (1H, dd, J = 10.8, 6.4 Hz, H9), 3.08 and 3.13 (2H, ABq, J = 9.6 Hz, H2-28), 3.24 (1H, dd, J = 10.6, 5.0 Hz, H-3), 3.45 and 3.81 (2H, ABq, J = 12.2 Hz, H2-27), 5.58 (1H, t, J = 3.3 Hz, H-12); (+)-HRESITOFMS m/z 555.4631 [M + H − H2O]+ (calcd for C36H63O2Si, 555.4597). 3β-Hydroxy-28-(tert-butyldimethylsilyl)oxy-27-[(E)-4′-(O-tertbutyldimethylsilyl)feruloyloxy]urs-12-ene (20). A procedure similar to that used in the synthesis of 15 gave 20 (6.6 mg, 14%), using 19 (30.6 mg, 53 μmol), 4-O-tert-butyldimethylsilylferulic acid (33.8 mg, 0.11 mmol), EDCI·HCl (31.6 mg, 0.16 mmol), DMAP (20.3 mg, 0.17 mmol), and ClCH2CH2Cl (3.2 mL): 1H NMR (CDCl3, 400 MHz) δ 0.17 (6H, s, Me2Si), 0.74 (1H, d, J = 11.3 Hz, H-5), 0.78 (3H, s, H325), 0.78−1.08 (38H, m), 1.08−1.48 (9H, m), 1.48−1.65 (4H, m), 1.65−1.90 (3H, m), 1.90−2.08 (2H, m), 3.07 and 3.56 (2H, ABq, J = 9.6 Hz, H2-28), 3.16 (1H, br t, J = 5.0 Hz, H-3), 3.84 (3H, s, OMe), 4.26 and 4.36 (2H, ABq, J = 12.8 Hz, H2-27), 5.42 (1H, br t, J = 3.0 Hz, H-12), 6.23 (1H, d, J = 15.9 Hz, CHCHAr), 6.84 (1H, d, J = 8.6 Hz, H-5′), 6.98−7.01 (2H, m, H-2′ and 6′), 7.54 (1H, d, J = 15.9 Hz, CHCHAr). 3β,28-Dihydroxy-27-[(E)-feruloyloxy]urs-12-ene (8). A procedure similar to that in the synthesis of 16 gave 8 (3.9 mg, 80%), using 20 (6.6 mg, 7.6 μmol), TBAF·3H2O (11.2 mg, 35 μmol), and THF (0.20 mL): 1H NMR (CDCl3, 400 MHz) δ 0.74 (1H, d, J = 11.3 Hz, H-5), 0.78 (3H, s, H3-25), 0.78−1.00 (17H, m), 1.00 (3H, s, H3-23 or 24), 1.00−1.22 (3H, m), 1.22−1.34 (2H, m), 1.34−1.45 (4H, m), 1.45− 1.65 (3H, m), 1.65−1.86 (4H, m), 1.92−2.11 (2H, m), 3.07 and 3.56 (2H, ABq, J = 9.6 Hz, H2-28), 3.16 (1H, t, J = 7.7 Hz, H-3), 3.94 (3H, s, OMe), 4.25 and 4.38 (2H, ABq, J = 12.8 Hz, H2-27), 5.43 (1H, br t, J = 3.0 Hz, H-12), 5.85 (1H, s, phenol), 6.22 (1H, d, J = 15.9 Hz, CHCHAr), 6.92 (1H, d, J = 8.2 Hz, H-5′), 7.00 (1H, d, J = 1.8 Hz, H-2′), 7.05 (1H, dd, J = 8.2, 1.8 Hz, H-6′), 7.54 (1H, d, J = 15.9 Hz, CHCHAr); (−)-HRESITOFMS m/z 633.4141 [M − H]− (calcd for C40H57O6, 633.4155). 3β,27-Dihydroxy-28-methoxycarbonylurs-12-ene (21). A solution of 6 (85.0 mg, 0.18 mmol) in THF (0.85 mL) was treated with MeOH (0.85 mL) and 0.6 M TMSCHN2−hexane solution (0.60 mL, 0.36 mmol) at room temperature. After stirring for 12 h, the reaction mixture was evaporated in vacuo to dryness. The residue was purified by flash column chromatography (4.0 g of silica gel; hexane−EtOAc, 3:2) to yield 21 (50.2 mg, 57%) as a white solid: 1H NMR (CDCl3, 400 MHz) δ 0.69 (3H, s, H3-25), 0.78 (3H, s, H3-23 or 24), 0.82−0.93 (2H, m), 0.89 (3H, s, H3-26), 0.94 (3H, d, J = 5.8 Hz, H3-30), 0.99 (3H, s, H3-23 or 24), 0.99 (3H, d, J = 5.8 Hz, H3-29), 1.01−1.24 (4H, m), 1.24−1.42 (4H, m), 1.42−1.51 (2H, m), 1.51−1.62 (5H, m), 1.62−1.78 (3H, m), 1.83−2.02 (4H, m), 2.36 (1H, d, J = 10.7 Hz, H18), 3.17−3.27 (1H, m, H-3), 3.34 (1H, d, J = 11.5 Hz, H-27a), 3.63 (3H, s, OMe), 3.79 (1H, dd, J = 11.5, 10.4 Hz, H-27b), 5.74 (1H, t, J = 3.1 Hz, H-12); 13C NMR (CDCl3, 100 MHz) δ 15.6 (q, C-23 or 24), 15.9 (q, C-25), 18.0 (q, C-29), 18.3 (t, C-6), 18.6 (q, C-26), 21.2 (q, C-30), 23.9 (t, C-15), 24.0 (t, C-11 or 16), 24.2 (t, C-11 or 16), 27.2 (t, C-2), 28.0 (q, C-23 or 24), 29.7 (t, C-21), 33.0 (t, C-1), 36.6 (t, C7), 37.1 (s, C-8), 38.0 (d, C-19), 38.3 (t, C-22), 38.7 (s, C-10), 39.2 (d, C-20), 40.1 (s, C-14), 47.5 (s, C-17), 47.7 (s, C-4), 48.2 (d, C-9), 51.7 (q, OMe), 51.7 (d, C-18), 54.9 (d, C-5), 64.4 (t, C-27), 78.8 (d, C-3), 133.0 (d, C-12), 133.4 (s, C-13), 177.8 (s, C-28). 3β,27-Dihydroxy-27-[(E)-4′-(O-tert-butyldimethylsilyl)feruloyloxy]-28-methoxycarbonylurs-12-ene (22). A procedure similar to that in the synthesis of 15 gave 22 (22.7 mg, 28%), using 21 (50.2 mg, 0.10 mmol), 4-O-tert-butyldimethylsilylferulic acid (41.6 mg, 0.13 mmol), EDCI·HCl (70.2 mg, 0.37 mmol), DMAP (44.2 mg, 0.36

= 12.7 Hz, H2-27), 5.58 (1H, t, J = 3.2 Hz, H-12), 5.88 (1H, br s, phenol), 6.22 (1H, d, J = 15.9 Hz, CHCHAr), 6.93 (1H, d, J = 8.2 Hz, H-5′), 7.01 (1H, d, J = 1.7 Hz, H-2′), 7.06 (1H, dd, J = 8.2, 1.7 Hz, H-6′), 7.55 (1H, d, J = 15.9 Hz, CHCHAr); 13C NMR (CDCl3, 100 MHz) δ 15.7 (q, C-23 or 24), 15.9 (q, C-25), 17.5 (q, C-29), 18.1 (q, C-26), 18.2 (t, C-6), 21.1 (q, C-30), 23.5 (t, C-15), 23.7 (t, C-11), 24.1 (t, C-16), 27.1 (t, C-2), 28.0 (q, C-23 or 24), 30.2 (t, C-1), 33.4 (t, C-7), 36.7 (t, C-21), 37.3 (s, C-10), 38.4 (d, C-19), 38.6 (t, C-22), 38.7 (s, C-4), 39.0 (d, C-20), 40.5 (s, C-8), 45.4 (s, C-14), 47.7 (s, C17), 48.4 (d, C-9), 52.2 (d, C-18), 55.2 (d, C-5), 56.0 (q, OMe), 66.1 (t, C-27), 78.8 (d, C-3), 109.5 (d, C-2′), 114.8 (d, C-5′), 115.7 (d, CHCHAr), 122.9 (d, C-6′), 126.9 (s, C-3′), 130.5 (d, C-12), 133.0 (s, C-13), 144.8 (d, CHCHAr), 146.8 (s, C-4′), 148.1 (s, C-1′), 167.0 (s, CO), 183.6 (C-28); (−)-HRESITOFMS m/z 647.3948 [M − H]− (calcd for C40H55O7, 647.3948). 3β,27-Dihydroxyurs-12-en-28-oic acid (6). A procedure similar to that used in the synthesis of 3 gave 6 (12.4 mg, 79%), using 14 (15.1 mg, 3.3 μmol), H2O (60 μL), 2-methyl-2-butene (60 μL), NaH2PO4 (16.4 mg, 0.14 mmol), NaClO2 (6.5 mg, 72 μmol), and tert-BuOH (0.30 mL): 1H NMR (CDCl3, 400 MHz) δ 0.75 (3H, s, H3-25), 0.76 (3H, s, H3-23 or 24), 0.76−0.99 (16H, m), 0.90−1.25 (4H, m), 1.25− 1.46 (4H, m), 1.46−1.78 (8H, m), 1.78−2.06 (4H, m), 2.31 (1H, d, J = 10.5 Hz, H-18), 3.23 (1H, dd, J = 10.7, 4.8 Hz, H-3), 3.34 and 3.79 (2H, ABq, J = 12.0 Hz, H2-27), 5.73 (1H, br s, H-12); 13C NMR (CDCl3, 100 MHz) δ 15.6 (q, C-23 or 24), 15.9 (q, C-25), 18.2 (t, C6), 18.3 (q, C-29), 18.4 (q, C-26), 21.2 (q, C-30), 23.9 (t, C-15), 23.9 (t, C-11 or 16), 24.1 (t, C-11 or 16), 27.1 (t, C-2), 28.1 (q, C-23 or 24), 29.7 (t, C-21), 33.0 (t, C-1), 36.7 (t, C-7), 37.1 (s, C-8), 38.0 (d, C-19), 38.3 (t, C-22), 38.7 (s, C-10), 39.1 (d, C-20), 40.1 (s, C-14), 47.3 (s, C-17), 47.7 (s, C-4), 48.2 (d, C-9), 51.4 (d, C-18), 54.9 (d, C5), 64.3 (t, C-27), 78.8 (d, C-3), 133.1 (d, C-12), 132.7 (s, C-13), 182.6 (s, C-28); (−)-HRESITOFMS m/z 471.3445 [M − H]− (calcd for C30H47O4, 471.3474). 3β-Hydroxy-27-acetoxyurs-12-en-28-aldehyde (18). A solution of 14 (11.3 mg, 25 μmol) in pyridine (0.45 mL) was treated with Ac2O (2.8 μL, 30 μmol) at room temperature. After stirring for 3 h, the reaction mixture was treated with additional Ac2O (2.8 μL, 30 μmol) and was stirred at room temperature for 24 h. The reaction mixture was evaporated in vacuo to dryness, and the resultant residue was coevaporated with toluene until the pyridine smell disappeared and was purified by flash column chromatography (1.2 g of silica gel; hexane−EtOAc, 3:2) to yield 18 (4.5 mg, 37%) as a white solid: 1H NMR (CDCl3, 400 MHz) δ 0.66−0.80 (2H, m), 0.74 (3H, s, H3-25), 0.79 (3H, s, H3-23 or 24), 0.86−0.95 (3H, m), 0.90 (3H, d, J = 6.6 Hz, H3-30), 0.92 (3H, d, J = 6.6 Hz, H3-29), 0.95 (3H, s, H3-26), 0.98 (3H, s, H3-23 or 24), 1.11−1.45 (10H, m), 1.50−1.82 (5H, m), 1.86−1.98 (3H, m), 2.05 (3H, s, Ac), 2.06 (1H, d, J = 10.9 Hz, H-18), 3.17−3.22 (1H, m, H-3), 4.04 and 4.24 (2H, ABq, J = 12.7 Hz, H2-27), 5.60 (1H, t, J = 3.6 Hz, H-12), 9.33 (1H, d, J = 1.0 Hz, H-28). 3β-Hydroxy-27-acetoxyurs-12-en-28-oic acid (7). A procedure similar to that used in the synthesis of 3 gave 7 (4.5 mg, 98%), using 18 (4.5 mg, 9.0 μmol), H2O (18 μL), 2-methyl-2-butene (18 μL), NaH2PO4 (5.4 mg, 45 μmol), NaClO2 (2.6 mg, 29 μmol), and tBuOH (90 μL): 1H NMR (CDCl3, 400 MHz) δ 0.66−0.80 (2H, m, H-5 and 19), 0.75 (3H, s, H3-25), 0.76 (3H, s, H3-23 or 24), 0.87− 0.95 (2H, m, H-20 and 22a), 0.88 (3H, d, J = 6.2 Hz, H3-30), 0.92 (3H, s, H3-26), 0.94 (3H, d, J = 6.2 Hz, H3-29), 0.98 (3H, s, H3-23 or 24), 0.98−1.22 (3H, m), 1.23−1.76 (11H, m), 1.76−1.98 (4H, m), 2.03 (3H, s, Ac), 2.27 (1H, d, J = 10.9 Hz, H-18), 3.20 (1H, dd, J = 10.6, 5.0 Hz, H-3), 4.02 and 4.23 (2H, ABq, J = 12.7 Hz, H2-27), 5.54 (1H, t, J = 3.4 Hz, H-12); 13C NMR (CDCl3, 100 MHz) δ 15.6 (q, C23 or 24), 15.8 (q, C-25), 17.3 (q, C-29), 18.1 (q, C-26), 18.2 (t, C-6), 21.1 (q, C-30), 21.3 (q, Ac), 23.5 (t, C-15), 23.6 (t, C-11 or 16), 24.0 (t, C-11 or 16), 27.1 (t, C-2), 28.1 (q, C-23 or 24), 30.1 (t, C-21), 33.3 (t, C-1), 36.7 (t, C-7), 37.2 (s, C-8), 38.5 (d, C-19), 38.7 (s, C-10), 38.7 (t, C-22), 38.9 (d, C-20), 40.2 (s, C-14), 45.1 (s, C-17), 48.5 (s, C-4), 48.5 (d, C-9), 52.7 (d, C-18), 55.3 (d, C-5), 66.2 (t, C-27), 78.9 (d, C-3), 130.7 (d, C-12), 132.7 (s, C-13), 170.9 (s, CO of Ac), 182.4 (s, C-28); (−)-HRESITOFMS m/z 513.3569 [M − H]− (calcd for C32H49O5, 513.3580). 2527

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mmol), and ClCH2CH2Cl (3.2 mL): 1H NMR (CDCl3, 400 MHz) δ 0.18 (6H, s, Me2Si), 0.75 (3H, s, H3-25), 0.78 (3H, s, H3-23 or 24), 0.86 (3H, d, J = 6.1 Hz, H3-29), 0.91 (3H, d, J = 6.1 Hz, H3-30), 0.89 (3H, s, H3-26), 0.94−1.03 (15H, m), 1.11−1.32 (4H, m), 1.32−1.51 (4H, m), 1.51−1.84 (9H, m), 1.90−2.11 (3H, m), 2.33 (1H, d, J = 11.3 Hz, H-18), 3.18 (1H, t, J = 8.0 Hz, H-3), 3.63 (3H, s, Me ether), 3.84 (3H, s, Me ester), 4.19 and 4.32 (2H, ABq, J = 12.6 Hz, H2-27), 5.57 (1H, br s, H-12), 6.23 (1H, d, J = 15.9 Hz, CHCHAr), 6.85 (1H, d, J = 8.0 Hz, H-5′), 6.99 (1H, s, H-2′), 7.00 (1H, d, J = 8.0 Hz, H-6′), 7.55 (1H, d, J = 15.9 Hz, CHCHAr); 13C NMR (CDCl3, 100 MHz) δ − 4.6 (q, Me2Si), 15.7 (q, C-25), 15.8 (q, C-23 or 24), 17.5 (q, C-29), 17.9 (q, C-26), 18.3 (t, C-6), 21.1 (q, C-30), 23.5 (t, C-11), 23.7 (t, C-15), 24.2 (t, C-16), 25.7 (q, t-Bu), 27.1 (t, C-2), 28.1 (q, C23 or 24), 30.2 (t, C-21), 33.5 (t, C-1), 36.6 (t, C-7), 37.2 (s, C-10), 38.5 (d, C-19), 38.7 (t, C-22), 38.7 (s, C-4), 39.1 (d, C-20), 40.2 (s, C8), 45.5 (s, C-14), 47.8 (s, C-17), 48.4 (d, C-9), 51.6 (q, Me ether), 52.5 (d, C-5), 55.2 (d, C-18), 55.5 (q, Me ester), 66.1 (t, C-27), 78.8 (d, C-3), 111.0 (d, C-2′), 116.1 (d, CHCHAr), 121.2 (d, C-5′), 122.1 (d, C-6′), 128.2 (s, C-3′), 130.4 (d, C-12), 133.3 (s, C-13), 144.7 (d, CHCHAr), 147.7 (s, C-4′), 151.2 (s, C-1′), 167.0 (s, C O), 178.0 (C-28). Uncarinic acid C methyl ester (9). A procedure similar to the synthesis of 16 gave 9 (15.2 mg, 81%), using 22 (15.5 mg, 20 μmol), TBAF·3H2O (10.1 mg, 32 μmol), and THF (0.47 mL): 1H NMR (CDCl3, 400 MHz) δ 0.75 (3H, s, H3-25), 0.77 (3H, s, H3-23 or 24), 0.86 (3H, d, J = 6.3 Hz, H3-29), 0.90 (3H, d, J = 6.2 Hz, H3-30), 0.93 (3H, s, H3-26), 0.97 (3H, s, H3-23 or 24), 0.94−1.04 (2H, m), 1.11− 1.32 (4H, m), 1.32−1.51 (4H, m), 1.51−1.82 (10H, m), 1.92−2.11 (3H, m), 2.33 (1H, d, J = 11.3 Hz, H-18), 3.17 (1H, t, J = 6.8 Hz, H3), 3.63 (3H, s, Me ether), 3.94 (3H, s, Me ester), 4.19 and 4.33 (2H, ABq, J = 12.7 Hz, H2-27), 5.57 (1H, t, J = 3.4 Hz, H-12), 5.90 (1H, br s, phenol), 6.22 (1H, d, J = 15.9 Hz, CHCHAr), 6.92 (1H, d, J = 8.2 Hz, H-5′), 7.00 (1H, d, J = 1.7 Hz, H-2′), 7.06 (1H, dd, J = 8.2, 1.7 Hz, H-6′), 7.55 (1H, d, J = 15.9 Hz, CHCHAr); (−)-HRESITOFMS m/z 661.4117 [M − H]− (calcd for C41H57O7, 661.4104). Thioflavin-T Assay. A fluorescence assay using Th-T (Sigma, St. Louis, MO, USA) was performed as described previously, with a slight modification.13 In brief, 445 μL of phosphate-buffered saline (PBS: 50 mM sodium phosphate and 100 mM NaCl, pH 7.4) was aliquoted into a 1.5 mL tube, followed by the addition of 5 μL of each test sample, which was dissolved with ethanol (5 mM). Then, a 50 μL solution of Aβ42 (250 μM in 0.1% NH4 OH), synthesized as reported previously,32 was added to the tube, so that the final concentration was 50 μM of each sample and 25 μM of Aβ42, respectively. Alternatively, Aβ42 dissolved in HFIP at 250 μM stood at room temperature for 30 min, as reported previously.14 After sonication for 5 min, the solution was air-dried and centrifuged for 10 min under reduced pressure to evacuate HFIP completely. The HFIP-treated Aβ42 was dissolved in 0.1% NH4OH and added to the reaction mixtures, as mentioned above. The resultant reaction mixture was incubated at 37 °C for the desired period, and then 2.5 μL was mixed periodically with 250 μL of Th-T (5.0 μM in 5.0 mM Gly-NaOH, pH 8.5). Fluorescence intensity was measured at 430 nm excitation and 485 nm emission using a microplate reader (Fluoroskan Ascent, Thermo Scientific). Eight points were measured at each time interval. Sedimentation Assay. A sedimentation assay using HPLC (Waters model 1525 with a model 2489 UV detector) was performed as described previously, with a slight modification.19 Preparation of the Aβ42 solution was the same as that described for the Th-T assay. After incubation at 37 °C for the desired period, each sample was centrifuged at 15 000 rpm at 4 °C for 10 min. Then, 25 μL of each supernatant was analyzed by reversed-phase HPLC on a Develosilpacked column (ODS-UG-5, 6.0 mm i.d. × 100 mm), with elution at 1.0 mL/min by a 30 min linear gradient of 10−50% CH3CN in 0.1% NH4OH. The absorption peak at 220 nm was integrated into the area proportional to the amount of substance. The value of the area at each period was subtracted from the highest value, and the percentage of the obtained to the highest was defined as “insoluble Aβ42”.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00392. NMR spectra for 1−4 and synthetic 3, cochromatography of natural and synthetic 3, and Th-T assay using synthetic 3 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-75-753-6281. Fax: +81-75-753-6284. E-mail: irie@ kais.kyoto-u.ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by JSPS Kakenhi grant 26221202 to K.I. and K.M., as well as grants 25850080 and 16H06194 to K.M. We thank Dr. K. Akagi at National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Japan, for NMR measurements using Bruker Avance II 800.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.6b00392 J. Nat. Prod. 2016, 79, 2521−2529

Journal of Natural Products

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DOI: 10.1021/acs.jnatprod.6b00392 J. Nat. Prod. 2016, 79, 2521−2529