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Apr 19, 2017 - disease that is caused by the human T-cell lymphotropic type I virus. ... reported that hyptinin (1) and hyptoside (2),5 obtained from ...
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Total Synthesis of the Claimed Structure of (±)-Hyptinin and Structural Revision of Natural Hyptinin Kazuto Maeda, Toshiyuki Hamada, Satoaki Onitsuka, and Hiroaki Okamura* Department of Chemistry and Bioscience, Graduate School of Science and Engineering, Kagoshima University, Korimoto 1-21-35, Kagoshima 890-0065, Japan S Supporting Information *

ABSTRACT: A total synthesis of (±)-hyptinin was achieved via a convergent route using the key phosphonate, cyclic ketone, and aryl Grignard components. The 1H and 13C NMR spectra of natural hyptinin did not agree with those of the synthesized compound. In particular, there were considerable differences between the signals assigned to the protons and carbons surrounding the lactone carbonyl group for the natural and synthesized compounds. The NMR data strongly suggested that the naturally occurring compound, hyptinin, was a structural isomer of the synthesized compound. The structure of the natural compound was eventually established as (+)-β-apopicropodophyllin, based on the synthesis results.

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dult T-cell leukemia (ATL) is an aggressive malignant disease that is caused by the human T-cell lymphotropic type I virus. It is recognized to be a rare form of leukemia, and an effective treatment is still under development.1 Recently, mogamulizumab, a monoclonal antibody drug, has been approved for the treatment of ATL in Japan.2−4 Despite this, the development of effective drugs with lower molecular weight is still a high-priority objective. Our research group has reported that hyptinin (1) and hyptoside (2),5 obtained from an extract of the Jamaican herb, Hyptis verticillata Jacq., exhibited potent activity as growth inhibitors against S1T cells from an ATL patient (IC50 5.1 nM and 7.0 nM, respectively). As such, 1 and 2 may be considered as potential lead compounds for the development of new anti-ATL drugs.5,6 In this article, the first total synthesis of (±)-1 via a convergent pathway starting from three simple components is described. In addition, the structural correction of naturally occurring hyptinin is reported on the basis of detailed spectroscopic and synthesis studies. Compound 1 was first reported in 1994 as one of the seven lignans from Hyptis verticillata Jacq., and its structure was established by 1H and 13C NMR analyses (Figure 1).7 Compound 1 is an uncommon 1,4-dihydronaphthalene lignan lactone with a C−8−C−8′ double bond and a C−9 lactone carbonyl group (according to the lignan lactone numbering system).5,7,8 Three other compounds 2, 3,9 and 410 have been reported to be natural products related to 1, but their syntheses have not yet been reported. Retrosynthesis analysis for the preparation of 1 is shown in Scheme 1. Since the C-ring seems to be readily aromatized, it is planned to be formed during the final step of the synthesis by cationic cyclization of I that is available via the reaction of II and III. The characteristic C−8−C−8′ double bond and the C−9 lactone carbonyl may be introduced by a Horner− © 2017 American Chemical Society and American Society of Pharmacognosy

Figure 1. Originally proposed structures of hyptinin and related compounds.

Wadsworth−Emmons (HWE) reaction of phosphonate IV and 1,3-dioxy-2-propanone (V). Compound (±)-1 was synthesized as shown in Scheme 2. Piperonyl alcohol 5 was converted to phosphonate 6 by tosylation and subsequent treatment with the carbanion of triethyl phosphonoacetate. The HWE reaction of 6 and the ketoacetal 711 followed by acid hydrolysis afforded unsaturated lactone 8. The HWE reaction is sensitive to steric hindrance of the substrate and is mainly applied to the preparation of mono-, di-, or trisubstituted olefins.12 However, the reaction with 7, that is known as a less sterically hindered and reactive ketone,11 gave tetrasubstituted olefin 8, although in low yield. All attempts to improve the yield (changing the solvent, temperature, and base) were unsuccessful, and a highly polar byproduct was obtained. After oxidation of the resulting primary alcohol 8 to aldehyde 9, the aromatic group corresponding to the E-ring was introduced by the reaction Received: December 4, 2016 Published: April 19, 2017 1446

DOI: 10.1021/acs.jnatprod.6b01116 J. Nat. Prod. 2017, 80, 1446−1449

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Scheme 1. Retrosynthesis Analysis of 1

phyllin (12)13 has been obtained by the acid-catalyzed dehydration of podophyllotoxin (Scheme 3).14−16 Significantly, the 1H NMR data of synthesized compound 12 were highly similar to those of natural hyptinin (the assignments corresponding to H−9 and H−9′ were inverted). Also, the 13 C NMR data of 12 and natural hyptinin (Table 2) showed good agreement. (see Supporting Information for detailed discussion). In addition, the [α]D values of these two compounds are in good agreement {natural hyptinin: [α]D25 + 77.0 (CHCl3),7 12: [α]D24 + 63 (c 1.0, CHCl3)}. Therefore, we conclude that the compound isolated from the Jamaican herb is actually (+)-12. In conclusion, the first total synthesis of (±)-1 was achieved using a convergent pathway. There was a significant disagreement between the NMR data of the synthesized and naturally occurring compounds. By comparison with synthetic βapopicropodophyllin (12) and existing data, the naturally occurring compound has been identified to be (+)-12. Since 1,4-dihydronaphthalene lignan lactones have no vicinal protons between rings C and D, it is impossible to establish the position of their lactone carbonyl units by 3JH−H couplings in their 1H NMR spectra. In previous studies, the positions of the lactone units in compounds 1−4 were determined on the basis of long-range couplings in their NMR spectra. Therefore, a careful reexamination of the structures of 2, 3, and 4 is necessary. Further studies toward this aim are in progress in our laboratory.

Scheme 2. Total Synthesis of (±)-1

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were recorded on JEOL JNM-ECX 400, JEOL JNM-ECX 600, or Bruker AVANCE III 600 instruments, and chemical shifts are given in parts per million (ppm, δ). IR spectra were recorded on a PERKIN ELMER spectrum one spectrometer. Optical rotations were recorded on a HORIBA high sensitive polarimeter SEPA-300. Melting points were recorded on a Büchi M-560 melting point apparatus. Reagents and solvents were purchased from Wako Pure Chemical Industries. Ltd., Tokyo Chemical Industry Co. Ltd., and Kanto Chemical. Co. Inc. Podophyllotoxin was purchased from Sigma-Aldrich Co. Llc. Ethyl 2-(diethoxyphosphoryl)-3-[3,4-(methylenedioxy)phenyl]propanoate (6). A solution of TsCl (6.9 g, 36 mmol) and Et3N (5.4 mL, 39 mmol) in CH2Cl2 (10 mL) was added to a solution of 5 (5.0 g, 30 mmol) and DMAP (730 mg, 6.0 mmol) in dry CH2Cl2 (20 mL) under N2 at rt. The mixture was stirred for 12 h, and extracted with CH2Cl2 (3 × 50 mL). The combined organic layer was washed with H2O, dried over MgSO4, filtered, and concentrated. Since the

of 9 and Grignard reagent 10. The final ring-closing reaction of the resulting 11 proceeded smoothly, under acid catalysis, to give the desired (±)-1. The 1H NMR spectrum of synthesized (±)-1 was compared with that of natural hyptinin7 in order to confirm its structure. As shown in Table 1, the spectra of (±)-1 and that reported for hyptinin did not agree. In particular, the following significant chemical shift differences (Δδ = δsynth − δnat) between (±)-1 and natural hyptinin are observed: H−5 (Δδ = −0.11), H−7 (Δδ = −0.17), H−2′ and 6′ (Δδ = −0.10), and H−9′ (Δδ = −0.28 and −0.12). The obvious discrepancies at H−7 and H− 9′ strongly suggested that the positions of the lactone carbonyl unit in the natural and synthesized compounds are interchanged. A structural isomer of (±)-1, β-apopicropodo1447

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Table 1. 1H NMR Data of Synthesized and Natural 1,4-Dihydronaphthalene Lignan Lactones (in CDCl3) synthesized (±)-hyptinin (1)a 6.75 6.51 6.26 5.94 5.93 4.75 4.73 4.51 3.82 3.79 3.70 3.60 a

(1H, (1H, (2H, (1H, (1H, (1H, (1H, (1H, (3H, (6H, (1H, (1H,

s, H−2) s, H−5) s, H−2′ & H−6′) brs, O-CH2-O) brs, O-CH2-O) brd, J = 10.0 Hz, H−9′) m, H−7′) brd, J = 11.6 Hz, H−9′) s, H−4′-OMe) s, H−3′-OMe & H−5′-OMe) dd, J = 14.4, 1.6 Hz, H−7) dd, J = 14.4, 2.2 Hz, H−7)

natural hyptinin7,b 6.70 6.62 6.36 5.94 5.93 4.87 4.79 4.79 3.87 3.78 3.76 3.63

(s, H−2) (s, H−5) (s, H−2′ & H−6′) (d, J = 1.5 Hz, O-CH2-O) (d, J = 1.5 Hz, O-CH2-O) (d, J = 17.2 Hz, H−9′) (m, H−7′) (dd, J = 17.2, 2.3 Hz, H−9′) (dd, J = 22.5, 4.5 Hz, H−7) (s, H−4′-OMe) (s, H−3′-OMe & H−5′-OMe) (dd, J = 22.5, 4.1 Hz, H−7)

synthesized β-apopicropodophyllin (12)b 6.72 6.63 6.37 5.95 5.94 4.90 4.81 4.81 3.85 3.79 3.78 3.65

(1H, (1H, (2H, (1H, (1H, (1H, (1H, (1H, (1H, (3H, (6H, (1H,

s, H−2) s, H−5) s, H−2′ & H−6′) brs, O-CH2-O) brs, O-CH2-O) d, J = 17.6 Hz, H−9) d, J = 17.6 Hz, H−9) m, H−7′) dd, J = 22.0, 4.6 Hz, H−7) s, H−4′-OMe) s, H−3′-OMe & H−5′-OMe) dd, J = 22.2, 3.9 Hz, H−7)

Measured at 600 MHz 1H NMR. bMeasured at 400 MHz 1H NMR. by stirring for an additional 3 h. The reaction was quenched with H2O, and the mixture was extracted with EtOAc (3 × 50 mL). The combined organic layer was washed with H2O and a saturated aqueous solution of NaCl, dried over MgSO4, filtered, and concentrated. The resulting crude product was purified by silica gel column chromatography (gradient n-hexane:EtOAc = 1:1 to 3:7). The title compound 6 (5.1 g, 15 mmol, 48% over two steps) was obtained as a yellow oil. IR (thin film) 2097, 1731, 1648, 1491, 1444, 1249, 1039, 969, 810 cm−1; 1H NMR (CDCl3, 400 MHz) δ 6.70 (3H, m), 5.95 (2H, s), 4.20 (7H, m), 3.15 (3H, m), 1.40 (2H, t, J = 6.9 Hz), 1.20 (6H, t, J = 7.1 Hz); 13C NMR (CDCl3, 100 MHz) δ 168.6, 147.7, 146.4, 132.4, 121.7, 109.1, 108.3, 101.0, 62.9, 61.5, 48.7, 47.5, 32.6, 16.5, 14.3 (2C); HRFABMS m/z 358.1180 (calcd. for C16H23O7P, 358.1181). 4-(Hydroxymethyl)-3-[3,4-(methylendioxy)benzyl]furan-2(5H)one (8). Ethyl 2-(diethoxyphosphoryl)-3-[3,4-(methylenedioxy)phenyl]propanoate (6, 1.8 g, 5.0 mmol) was slowly added to a suspension of NaH (60% in oil, 240 mg, 6.0 mmol) in dry THF (50 mL) under N2 at 0 °C, followed by stirring for 1 h. Cyclic ketone 7 (1.0 g, 7.5 mmol) was added dropwise with further stirring for 36 h at 60 °C. The reaction was quenched with H2O, and the mixture was extracted with EtOAc (3 × 100 mL). The combined organic layer was

Scheme 3. Preparation of (+)-β-Apopicropodophyllin (12) from (−)-Podophyllotoxin

resulting tosylate was unstable on silica gel, it was used in the next step without further purification. Triethyl phosphonoacetate (7.4 g, 33 mmol) was added slowly to a suspension of NaH (60% in oil, 1.3 g, 33 mmol) in dry THF (40 mL) under N2 at 0 °C, followed by stirring for 1 h. The mixture was added dropwise to a solution of the tosylate in dry THF (2.0 mL), followed

Table 2. 13C NMR Data of Synthesized and Natural 1,4-Dihydronaphthalene Lignan Lactones (in CDCl3) synthesized (±)-hyptinin (1)a 173.3 160.7 153.8 153.8 147.1 147.1 137.7 137.5 127.9 124.8 123.9 108.8 108.3 105.3 105.3 101.3 71.0 60.6 56.0 46.3 25.6 a

(C−9) (C−8′) (C−3′) (C−5′) (C−3) (C−4) (C−1′) (C−4′) (C−8) (C−6) (C−1) (C−5) (C−2) (C−2′) (C−6′) (O-CH2-O) (C−9′) (C−4′-OMe) (C−3′-OMe & C−5′-OMe) (C−7′) (C−7)

natural hyptinin7,b 172.3 157.9 157.9c 153.3 147.3 147.1 138.3 129.7 128.2 123.9c 123.7 109.6 107.8 105.8 105.8 101.4 71.1 60.8 56.3 42.8 29.3

synthesized β-apopicropodophyllin (12)b 172.4 157.5 153.2 153.2 147.3 147.1 138.4 137.0 129.7 128.1 123.8 109.6 107.8 105.6 105.6 101.4 71.1 60.8 56.2 42.8 29.3

(C−9′) (C−8) (C−3′) (C−5′) (C−4′) (C−3) (C−4) (C−1′) (C−1) (C−8′) (C−5) (C−6) (C−2) (C−2′) (C−6′) (O-CH2-O) (C−9) (C−4′-OMe) (C−3′-OMe & C−5′-OMe) (C−7′) (C−7)

Measured at 150 MHz 13C NMR. bMeasured at 100 MHz 13C NMR. cErroneously assigned. 1448

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washed with H2O followed by saturated aqueous solution of NaCl, dried over MgSO4, filtered, and concentrated. PPTS (130 mg, 0.50 mmol) was added at 60 °C to a solution of the crude extract in MeOH (20 mL), and the reaction mixture was stirred at this temperature for 48 h. The solution was concentrated, and the resulting crude product was purified by silica gel column chromatography (n-hexane:EtOAc = 1:1). The title compound 8 (100 mg, 0.41 mmol, 13% over two steps) was obtained as a colorless oil. IR (thin film) 2916, 2840, 1650, 1440, 1260, 799 cm−1; 1H NMR (CDCl3, 400 MHz) δ 6.83 (3H, m), 5.92 (2H, s), 4.90 (2H, s), 4.55 (2H, s), 3.8 (2H, s); 13C NMR (CDCl3, 100 MHz) δ 175.0, 160.3, 148.0, 146.4, 131.4, 126.1, 121.5, 109.1, 108.5, 101.1, 70.7, 57.9, 29.5; HRFABMS m/z 248.0684 (calcd. for C13H12O5, 248.0685). 4-Formyl-3-(3,4-methylendioxybenzyl)furan-2(5H)-one (9). 4(Hydroxymethyl)-3-[3,4-(methylendioxy)benzyl]furan-2(5H)-one (8, 90 mg, 0.37 mmol) in CH2Cl2 (5 mL) was added to a suspension of PDC (190 mg, 0.50 mmol) and powdered 4 Å molecular sieve (380 mg) in dry CH2Cl2 (20 mL), under N2. After stirring for 6 h, Celite was added to the reaction mixture. The mixture was filtered, and the filtrate was concentrated to give the crude product, which was purified by silica gel column chromatography (n-hexane:EtOAc = 8:2). The title compound 9 (78 mg, 0.32 mmol, 86%) was obtained as a colorless oil. 1H NMR (CDCl3, 400 MHz) δ 10.00 (1H, s), 6.80 (3H, m), 5.90 (2H, s), 4.93 (2H, s), 3.95 (2H, s); 13C NMR (CDCl3, 100 MHz) δ 186.3, 173.4, 149.9, 148.4, 147.2, 141.5, 129.5, 122.0, 109.2, 108.9, 101.4, 87.2, 68.8, 30.5. Since the product was unstable in the air, it was used in the next step without further spectroscopic analysis. 4-[Hydroxy-(3,4,5-trimethoxyphenyl)methyl]-3-(3,4methylendioxybenzyl)furan-2(5H)-one (11). A solution of 3,4,5trimethoxyphenylmagnesium bromide (10, ca. 0.10 M in THF, 1.0 mL) was added dropwise to a solution of 4-formyl-3-(3,4methylendioxybenzyl)furan-2(5H)-one (9, 13 mg, 0.050 mmol) in dry THF (10 mL), under N2, and the reaction mixture was stirred for 4 h at 0 °C with warming to rt. The mixture was quenched with H2O, and the product was extracted with EtOAc (3 × 30 mL). The combined organic layer was washed with H2O, dried over MgSO4, filtered, and concentrated. The product was purified by silica gel column chromatography (gradient, n-hexane:EtOAc = 7:3 to 1:1). Compound 11 (14 mg, 0.033 mmol, 66%) was obtained as a pale yellow oil. IR (thin film) 2938, 1752, 1593, 1503, 1243, 1126, 923 cm−1; 1H NMR (CDCl3, 400 MHz) δ 6.83 (3H, m), 6.50 (2H, s), 5.90 (2H, s), 5.71 (1H, brs), 4.83 (1H, d, J = 17.9 Hz), 4.67 (1H, d, J = 17.9 Hz), 3.85 (3H, s), 3.75 (6H, s), 3.61 (2H, brs); 13C NMR (CDCl3, 100 MHz): δ 174.9, 161.1, 153.7, 147.9, 146.4, 138.1, 135.7, 131.7, 126.7, 121.5, 109.2, 108.4, 104.7, 103.0, 101.1, 87.2, 70.4, 69.6, 61.0, 56.4, 56.2, 29.4; HRFABMS m/z 414.1313 (calcd. for C22H22O8, 414.1315). (±)-Hyptinin (1). To a solution of 11 (10 mg, 0.025 mmol) in dry CH2Cl2 (1.0 mL) was added BF3·Et2O (1 drop) at 0 °C. After stirring for 2 h at 0 °C, the mixture was concentrated and purified by preparative TLC using n-hexane:EtOAc = 1:1 as solvent. (±)-Hyptinin (1, 5.1 mg, 0.013 mmol, 51%) was obtained as a white solid. Mp. 177− 178 °C; IR (thin film) 2917, 1758, 1590, 1505, 1487, 1421, 1329, 1233, 1128, 1018 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) are provided in Tables 1 and 2 ; HRFABMS m/z 396.1210 (calcd. for C22H20O7, 396.1209). (+)-β-Apopicropodophyllin (12). Preparation of (+)-β-apopicropodophyllin (12) was carried out according to a literature procedure.13 BF3·Et2O (300 μL, 2.4 mmol) was added to a solution of (−)-podophyllotoxin (50 mg, 0.12 mmol) in 1,4-dioxane (2.0 mL), and the mixture was stirred at rt for 4 h under N2. After adding ice cold water (3.0 mL) to the mixture, it was extracted with Et2O (3 × 10 mL) and dried over Na2SO4. After evaporation, the product was purified by silica gel column chromatography (CHCl3), and further purified by recrystallization from petroleum ether. (+)-β-Apopicropodophyllin (12, 16.7 mg, 0.042 mmol, 35%) was obtained as a white solid. Mp. 215−217 °C (lit. 212−214 °C14, 214−215 °C16); 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) are provided in Tables 1 and 2.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01116. 1 H and 13C NMR, COSY, HSQC, HMBC, and NOESY spectra of selected compounds (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81 (0)99 285 8113. Fax: +81 (0)99 285 8117. E-mail: [email protected]. ORCID

Hiroaki Okamura: 0000-0003-0212-1829 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grants-in-Aid for Scientific Research (No. 23550160 and No. 26410096) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by the Cooperative Research Program of the “Network Joint Research Centre for Materials and Devices” (No. 2011287 and 2014440).

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