Scopranones with Two Atypical Scooplike Moieties Produced by

Oct 24, 2017 - Scopranone A (1): compound 1 showed a molecular ion peak at m/z 303 [M + Na]+ in ESI-MS, and the molecular formula C17H28O3 was assigne...
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Letter Cite This: Org. Lett. 2017, 19, 5980-5983

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Scopranones with Two Atypical Scooplike Moieties Produced by Streptomyces sp. BYK-11038 Ryuji Uchida,†,§ Daiki Lee,†,§ Ibuki Suwa,† Masaki Ohtawa,† Nozomu Watanabe,† Ayumu Demachi,† Satoshi Ohte,† Takenobu Katagiri,‡ Tohru Nagamitsu,† and Hiroshi Tomoda*,† †

Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka-shi, Saitama 350-1298, Japan

‡

S Supporting Information *

ABSTRACT: Three new compounds, designated scopranones A−C, were isolated from the culture broth of a soil isolate, Streptomyces sp. BYK-11038, and shown to be inhibitors of bone morphogenetic protein (BMP) induced alkaline phosphatase activity in a BMP receptor mutant cell line. The structures were elucidated using NMR and other spectral data. The scopranones have an unusual structure with two atypical scooplike moieties linked at the tails to form part of a unique 3-furanone ring. ince the discovery of the first antibiotic, penicillin, in 1928, microorganisms such as actinomycetes and fungi have been extensively utilized as a source of new drugs and, subsequently, have provided numerous drugs or drug leads in clinical use, including penicillin, streptomycin, amphotericin B, compactin, tacrolimus, mitomycin C, and avermectin. Thus, microbial secondary metabolites exhibit diversity in chemical structures. Additionally, microorganisms have great potential for producing unusual secondary metabolites beyond our comprehension. Natural product chemistry has furthered not only basic research through its contributions regarding structural elucidation, total synthesis, chemical biology, and biosynthesis but also drug development for human health. Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder of progressive and widespread postnatal heterotopic bone formation in soft tissues.1 A recurrent mutation (R206H) within ACVR1/ALK2, one of the bone morphogenetic protein (BMP) type I receptors, was identified in FOP patients.2 Because this mutation results in the activation of intracellular BMP signaling to induce heterotopic bone formation, BMP signaling inhibitors might offer therapeutic benefits for FOP treatment.3 Accordingly, we have screened microbial metabolites for inhibitors of BMP-induced alkaline phosphatase (ALP) activity (BMP signaling inhibitors) using stable ALK2(R206H)-expressing C2C12 cells (abbreviated as C2C12(R206H) cells)4 and discovered a variety of BMP signaling inhibitors of microbial origin, such as trichocyalides, lucilactaene, hydroxylucilactaene, NG-391, NG-393, and 5prenyltriptophol.5 During our continuous screening for microbial BMP-signaling inhibitors, three novel compounds, designated scopranones A (1), B (2), and C (3), were isolated from the culture broth of Streptomyces sp. BYK-11038 (Figure 1). Scopranones have an unusual common structure with two atypical scooplike moieties linked at the tails to form part of a unique 3-furanone ring. In the present study, the isolation,

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© 2017 American Chemical Society

Figure 1. Structures of scopranones A (1), B (2), and C (3).

structural elucidation, total synthesis, and BMP-induced ALP inhibitory activity of the scopranones are described. Scopranones A−C (1−3) were obtained as yellow oils. In their UV spectra obtained in MeOH, 1 and 3 showed similar absorption maxima at approximately 226, 243, and 306 nm, whereas 2 showed maxima at 223, 245, and 286 nm. In the IR spectra of the compounds, broad OH absorption at approximately 3400 cm−1, three typical C−H (CH2) stretching absorptions near 2964, 2934, and 2876 cm−1, CO stretching absorption at 1685−1691 cm−1, and C−H bending absorption at 1459 cm−1 were observed. Scopranone A (1): compound 1 showed a molecular ion peak at m/z 303 [M + Na]+ in ESI-MS, and the molecular formula C17H28O3 was assigned on the basis of high-resolution electrospray ionization mass spectrometry (HRESI-MS) [m/z 303.1919 [M + Na]+, Δ −1.7 mmu], indicating four degrees of unsaturation. The 1H and 13C NMR spectra of 1 in CDCl3 (Table S1) showed 27 proton and 17 carbon signals, which were confirmed via analysis of the 2D NMR correlations. The multiplicity of the carbon signals was categorized into five methyl carbons, five sp3 methylene carbons, one sp3 methine carbon, one sp3 hemiketal quaternary carbon, one sp2 methine carbons, two sp2 quaternary carbons, one sp2 oxygenated Received: September 25, 2017 Published: October 24, 2017 5980

DOI: 10.1021/acs.orglett.7b03003 Org. Lett. 2017, 19, 5980−5983

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Organic Letters

chemical shifts of the four carbons C-2 to C-5 in 1 were comparable to those of the corresponding carbons in actinofuranone A,6 which possesses the same 2-hydroxy-3furanone ring. Compound 1 has one chiral carbon at C-2 in the hemiacetal moiety. Considering the small specific rotation (see the Supporting Information), 1 might exist as a racemic mixture. Thus, the full planar structure of 1 was elucidated as shown in Figure 1 and featured two atypical scooplike (2ethylbutyl and 1-ethyl-1-propenyl) moieties linked at the tails to form part of a unique 3-furanone ring. Scopranone B (2): the molecular formula of 2 is C17H28O2 on the basis of the HRESI-MS [m/z 265.2170 [M + Na]+, Δ +0.2 mmu], indicating that 2 contains one oxygen atom less than 1. Comparison of the 1H and 13C NMR spectra (Table S1) of 1 and 2 indicated that the sp3 dioxygenated quaternary carbon at C-2 (δC 102.3) in 1 is replaced by an sp3 oxygenated methine at C-2 (δC 85.6, δH 4.44) in 2. As shown by the bold lines for 2 in Figure 2B, the 1H−1H COSY correlations between H-2 and the sp3 methylene protons H2-6 (δ 1.97, 1.72) and between H2-6 and the methyl protons H3-7 (δ 0.95) yielded the structure of 2 shown in Figure 1, which satisfied the molecular formula and the degrees of unsaturation. Compound 2 might exist as a racemic mixture as well as compound 1. Scopranone C (3): the molecular formula of 3 is C17H28O4 on the basis of HRESI-MS [m/z 297.2053 [M + Na]+, Δ +1.3 mmu], indicating that 3 contains one more oxygen atom than 1. Comparison of the 1H and 13C NMR spectra (Table S1) of 1 and 3 indicated that the sp3 methylene carbon at C-6 (δC 29.2, δH 1.90) in 1 is replaced by an sp3 oxygenated methine at C-6 (δC 69.6, δH 3.96) in 3. As shown by the bold lines for 3 in Figure 2C, partial structure I (−CH(O)CH3) was obtained from the 1H−1H COSY spectrum. In the HMBC experiments, the cross peaks from H-6 and the methyl protons H3-7 (δ 1.17) to the sp3 hemiketal quaternary carbon C-2 (δ 100.3) and from H-6 to the ketone carbon C-3 (δ 202.1) indicated that partial structure I is attached to C-2 (Figure 2C). Furthermore, the chemical shifts and the molecular formula (C17H28O4) of 3 indicated that a hydroxyl group was attached to C-6. Furthermore, the oxymethine proton H-6 was observed as two pairs of quartets (δ H3.96 and 3.98) in 1H NMR spectrum, and some 13C NMR signals were observed as doublets (see the Supporting Information), indicating that 3 exists as a diastereomer. Thus, the structure of 3 was elucidated as

quaternary carbon, and one ketone carbon based on the DEPT and HMQC data. The connectivity of the proton and carbon atoms was established using HMQC. As shown in Figure 2A,

Figure 2. Key correlations in the 1H−1H COSY (bold line), HMBC (solid arrow), and ROESY (dashed arrow) spectra of the scopranones: (A) scopranone A (1); (B) scopranone B (2); and (C) scopranone C (3).

the partial structures I−IV, including a scooplike 2-ethylbutyl residue (partial structure II), were elucidated by 1H−1H COSY spectra. The 13C−1H long-range couplings of 2J and 3J in the HMBC spectrum (Figure 2A) confirmed the structural linkages in 1 as follows: 1) The cross peaks from the sp2 methine proton H-15 (δ 6.28) and the methyl protons H3-16 (δ 1.88) in partial structure III to the sp2 quaternary carbons C-14 (δ 134.4), from the sp3 methylene protons H2-17 (δ 2.42) and the methyl protons H3-18 (δ 1.03) in partial structure IV to C-14, from H15 to the sp3 methylene carbon C-17 (δ 20.3), from H-17 to the sp2 methine carbon C-15 (δ 133.7), and from H-15 and H217 to the sp2 oxygenated quaternary carbon C-5 (δ183.4) yielded the expanded partial structure V containing a second scooplike 1-ethyl-1-propenyl residue. Furthermore, NOE correlation was observed between H-8 and H-15 in the ROESY experiment, indicating that the configuration of the double bond in partial structure V was 14E. 2) The long-range couplings from the sp3 methylene protons H2-6 (δ 1.90) and methyl protons H3-7 (δ 0.92) to the sp3 hemiketal quaternary carbon C-2 (δ 102.3), from H2-6 and the sp3 methylene protons H2-8 (δ 2.16) to the ketone carbon C-3 (δ 203.6), and from H2-8 to the sp2 quaternary carbons C-4 (δ 112.1) and C-5 were observed. Thus, to account for the four degrees of unsaturation, the structure should establish the presence of a 3furanone ring in 1 (Figure 2A). Furthermore, the chemical shifts and the molecular formula (C17H28O3) of 1 indicated that a hydroxyl group was attached to C-2, forming a hemiacetal moiety in the 3-furanone ring. Additionally, the 13C NMR Scheme 1. Total Synthesis of Scopranone A (1)

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DOI: 10.1021/acs.orglett.7b03003 Org. Lett. 2017, 19, 5980−5983

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Organic Letters shown in Figure 1, which satisfied the molecular formula and the degrees of unsaturation. Structure elucidation of the scopranones revealed that these compounds are unusual microbial metabolites because of the two rare scooplike moieties connected at the tails to form a part of their unique 3-furanone skeleton. Natural products bearing a scooplike moiety have been isolated from actinomycetes (K259-2, TAN-1518B, BOS-013-II, and bis(5-ethylheptyl)phosphate),7 plants (rhaponticum and quinolinone alkaloid),8 sponges (cladocrocin A),9 and soft coral (xestosterol esters).10 Most of these natural products have one scooplike moiety in the structures, whereas bis(5-ethylheptyl)phosphate has two scooplike moieties linked via a phosphate group. Therefore, scopranones are the first microbial metabolites with two scooplike moieties directly linked at the tails. Furthermore, only two natural products have a 3-furanone ring; aurafurones isolated from a myxobacterium11 and an actinofuranone from an actinomycete.6 Thus, from a structural perspective, the scopranones have a simple but unusual skeleton. Next, we embarked on the total synthesis of scopranone A to confirm the proposed structure. The Appel reaction12 of commercially available 2-ethylbutan-1-ol gave iodide 413 in 90% yield (Scheme 1). Alkylation of 4 with the corresponding enolate derived from commercially available ethyl trans-2hexenoate treated with LDA and HMPA afforded 5 in 66% yield as an E/Z mixture (E/Z = 1:5.7).13 Subsequent dihydroxylation of 5 with OsO4/N-methylmorpholine oxide (NMO) effected an intramolecular cyclization to give lactone 6 in 72% yield as a mixture of diastereomers. DIBAL reduction of 6 gave lactol 7 in 91% yield. The Shapiro reaction14 of 7 with 815 afforded an unstable triol (9) possessing the desired (E)-1ethyl-1-propenyl substituent at C5, which was immediately subjected to Swern oxidation conditions16 to furnish the desired scopranone A (1) in 41% yield over two steps. The synthetic scopranone A (1) was completely identical to natural scopranone A in all aspects (1H NMR, 13C NMR, IR, MS, and biological evaluation). Thus, the structure of 1 was confirmed as elucidated. When 1 was stored in 80% CH3CN aq at −20 °C for 3 weeks, we observed the E/Z isomerization of the (E)trisubstituted olefin in 1. To investigate the mechanism, 1 was dissolved in a DMSO-d6−D2O mixture (4:1) and analyzed using 1H NMR (400 MHz). Figure 3 shows that the E/Z isomerization was observed after several hours, at which point the 1H NMR peaks originating the from (Z)-trisubstituted olefin were detected, and then proceeded very slowly. The E/Z ratio was 1:1 after 7 days and reached 1:6 after 23 days. This 1H NMR experiments revealed that the E/Z isomerization in the presence of water proceeded very slowly via the 1,6-addition of water to 1 to produce 1′, which possesses the thermodynamically favored (Z)-trisubstituted olefin at C-15 as a major isomer (Scheme 2). The BMP-induced ALP inhibitory activity and cytotoxicity of 1−3 and dorsomorphin in C2C12(R206H) cells were measured according to a previously reported method.4 Dorsomorphin, a potent inhibitor of BMP signaling, functions via the inhibition of the BMP type I receptors ALK2, ALK3, and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation.17 ALP activity is one of the key markers in osteoblast differentiation of C2C12(R206H) cells. Accordingly, compounds 1−3 and dorsomorphin exhibited ALP inhibitory activity in a dose-dependent manner with IC50 values of 2.70, 28.9, 11.9, and 0.00610 μM and showed weak cytotoxicity to

Figure 3. Time course of the E/Z isomerization of the (E)trisubstituted olefin in the presence of water by 1H NMR (400 MHz NMR, DMSO-d6/D2O = 4:1): (A) day 0, (B) day 7, (C) day 23.

C2C12(R206H) cells in an MTT assay with IC50 values of 91.6, 119, 253, and 2.39 μM, respectively. Thus, the calculated selectivity indexes (the ratio of cytotoxicity/ALP inhibition) of 1−3 and dorsomorphin were 33.9, 4.12, 21.3, and 392, respectively. The activity of the natural isolated 1′ was almost equivalent to that of 1 (IC50 values for ALP and MTT were 8.21 μM and >35.7 μM, respectively). The induction of ALP activity in C2C12 cells is an indicator of the multiple intracellular events initiated by BMP signaling. BMP signaling is transduced via the transcriptional factor Smad1/5, which is phosphorylated and activated by BMP receptors.3b Therefore, to examine the direct effect of 1−3 and dorsomorphin on BMP signaling, a BMP−Smad-specific Id1WT4F-luciferase reporter assay in C2C12 cells was performed.18 Compound 2 and dorsomorphin showed inhibitory activity with IC50 values of 44.0 and 0.057 μM, respectively, whereas 1 and 3 showed no effect, even at 340 μM, indicating that despite the similar structures of the scopranones, only 2 might inhibit the early stage of BMP signaling. The detailed inhibitory mechanism of 1−3 remains to be elucidated. In conclusion, we isolated novel compounds, designated scopranones A−C, featuring two scooplike moieties linked at the tails to form part of a 3-furanone ring with a hemiketal from the culture broth of Streptomyces sp. BYK-11038. The chemical structures of the scopranones were elucidated by various instrumental analyses, including NMR and MS spectra, and confirmed via the total synthesis of scopranone A. Additionally, the E/Z isomerization mechanism of (E)-trisubstituted olefin of scopranone A was determined. Furthermore, the scopranones showed inhibitory activity of BMP-induced ALP activity in myoblasts with low cytotoxicity. Although they have similar structures, only scopranone B might exert a direct effect on the 5982

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Organic Letters Scheme 2. E/Z Isomerization of Scopranone A (1)

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early stage of BMP signaling via Smads. Hopefully, scopranone derivatives with more potent inhibitory activity of BMP signaling will be synthesized, leading to effective drugs for the treatment of FOP.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b03003. Experimental section, NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Hiroshi Tomoda: 0000-0002-1241-8901 Author Contributions §

R.U. and D.L. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We express our thanks to Dr. Kenichiro Nagai and Ms. Noriko Sato of the School of Pharmacy, Kitasato University, for measurements of NMR and mass spectra. This work was supported in part by JSPS KAKENHI Grant No. 16K15134 (to R.U.).

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REFERENCES

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DOI: 10.1021/acs.orglett.7b03003 Org. Lett. 2017, 19, 5980−5983