Miltiorins A-D, diterpenes from Radix Salviae miltiorrhizae

FITOTE-03111; No of Pages 7 Fitoterapia xxx (2015) xxx–xxx Contents lists available at ScienceDirect Fitoterapia R O O F journal homepage: www.elsevier.com/locate/fitote 1Q1 Miltiorins A–D, diterpenes from Radix Salviae miltiorrhizae 2Q2 Ai Hirata a, Sang-Yong Kim a,b, Natsuki Kobayakawa a, Naonobu Tanaka a, Yoshiki Kashiwada a,⁎ 3 4 a b Graduate School of Pharmaceutical Sciences, Tokushima University, Tokushima 770-8505, Japan Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Tobetsu 061-0293, Japan 5 a r t i c l e i n f o a b s t r a c t 8 9 10 11 12 Article history: Received 12 December 2014 Accepted in revised form 20 January 2015 Accepted 23 January 2015 Available online xxxx 21 22 20 23 24 25 Keywords: Radix Salviae miltiorrhizae Diterpene Anti-influenza Miltiorins A–D 26 1. Introduction 27 Radix Salviae miltiorrhizae, the dried root of S. miltiorrhiza Bge. (Laminaceae) is one of the most popular herbal traditional medicines in Asian countries, and has been used extensively for the treatment of coronary artery disease, angina pectoris, myocardial infarction, cerebrovascular diseases, chronic renal failure, dysmenorrhea, and various types of hepatitis [1]. In our continuing search for new natural templates of therapeutic agents [2–6], an influenza A neuraminidase (NA) inhibitory effect of several extracts from plants was evaluated. As a result, the extract from the dried roots of S. miltiorrhiza exhibited an anti-NA activity, which prompted us to investigate the constituents of the natural source. In this article, we describe the isolation and structure elucidation of four new diterpenes, miltiorins A–D (1–4), isolated from the roots of S. miltiorrhiza, as well as evaluation of an anti-NA activity of the isolated compounds. P 7 13 14 15 16 17 18 19 34 35 36 37 38 39 40 41 42 R R O C 32 33 N 30 31 U 28 29 E C T E D Constituents of the anti-influenza A neuraminidase (NA) active extract from the roots of Radix Salviae miltiorrhizae were investigated, resulting in the isolation of four new diterpenes, miltiorins A–D (1–4), together with eight known diterpenes. The structures of 1–4 were assigned by spectroscopic analysis. Miltiorins A–C (1–3) were abietane diterpenes possessing a 2α-acetoxy group and a 12-hydroxy group in common, while miltiorin D (4) was a 11,12-seco-abietane diterpene with a γ-lactone ring. Miltiorin D (4) is the first example of a 11,12-seco-abietane diterpene from natural sources. Anti-NA activities of the isolated diterpenes were evaluated. © 2015 Published by Elsevier B.V. 2. Experimental 43 2.1. General experimental procedures 44 Optical rotations were obtained on a JASCO DIP-370 digital polarimeter. NMR spectra were measured by a Bruker AVANCE500 instrument using tetramethylsilane as an internal standard. HRESIMS and ESIMS were recorded on a Waters LCT PREMIER 2695. Column chromatography was performed with silica gel 60N (63–210 μm, Kanto Kagaku, Japan), MIC gel CHP 20P (75– 150 μm, Mitsubishi Chemical, Japan), and YMC-gel ODS-A (S50 μm, YMC Co., Ltd., Japan). 45 46 2.2. Plant material 53 49 50 51 52 Radix Salviae miltiorrhizae (Lot. US302822) was provided by 54 UCHIDA WAKANYAKU Ltd., Japan. A voucher specimen was 55 deposited in the herbarium of the University of Tokushima. 56 2.3. Extraction and isolation ⁎ Corresponding author. Tel./fax: +81 88 633 7276. E-mail address: [email protected] (Y. Kashiwada). 47 48 57 The dried roots of Salvia miltiorrhiza (2.0 kg, dry) were 58 extracted with MeOH (3 × 10 L) at rt to give the extract (291 g), 59 which was partitioned with CHCl3 (6 × 1.5 L) and H2O (1.5 L). 60 http://dx.doi.org/10.1016/j.fitote.2015.01.013 0367-326X/© 2015 Published by Elsevier B.V. Please cite this article as: Hirata A, et al, Miltiorins A–D, diterpenes from Radix Salviae miltiorrhizae, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.01.013 80 81 82 83 84 85 86 87 88 89 90 91 92 93 t1:1 t1:2 2.5. Miltiorin B (2) 98 O F Pale yellow amorphous solid; [α]D −12.4 (c 0.23, CHCl3); 99 HRESIMS m/z 397.2000 [M + Na]+ (calcd for C22H30O5Na, 100 397.1991); 1H and 13C NMR data (Table 1). 101 2.6. Miltiorin C (3) 102 R O 78 79 Pale yellow amorphous solid; [α]D −17.6 (c 0.43, CHCl3); 95 HRESIMS m/z 367.2259 [M + Na]+ (calcd for C22H32O3Na, 96 367.2249); 1H and 13C NMR data (Table 1). 97 Pale yellow amorphous solid; [α]D −34.4 (c 1.48, CHCl3); 103 HRESIMS m/z 379.1873 [M + Na]+ (calcd for C22H28O4Na, 104 379.1885); 1H and 13C NMR data (Table 1). 105 2.7. Miltiorin D (4) 106 P 76 77 94 Pale yellow amorphous solid; [α]D ≈ 0 (c 0.28, CHCl3); 107 HRESIMS m/z 313.1447 [M − H]− (calcd for C19H21O4, 108 313.1440); 1H and 13C NMR data (Table 2). 109 D 74 75 2.4. Miltiorin A (1) 2.8. Methylations of miltiorins A–C T 72 73 C 70 71 E 68 69 R 66 67 110 A mixture of miltiorin A (1, 0.8 mg), CH3I (20 μL), and K2CO3 (13 mg) in dry acetone (400 μL) was stirred at rt for 3 h. After removal of the solvent, the residue was subjected to chromatography over silica gel (n-hexane/EtOAc, 95:5) to give 12-Omethyl miltiorin A (1a, 0.7 mg). Methylations of miltiorins B (2) and C (3) were carried out as for 1 to give 12-O-methyl miltiorins B (2a) and C (3a), respectively. Table 1 H and 13C NMR data for miltiorins A–C (1–3) in CDCl3. 1 t1:3 1 t1:4 Position t1:5 1 43.8 t1:6 t1:7 2 3 69.6 46.5 t1:8 t1:9 t1:10 4 5 6 34.7 49.8 18.9 t1:11 7 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 8 9 10 11 12 13 14 15 16 17 18 19 20 2-OAc R 65 δH (J in Hz) δC N U 29.5 126.4 147.0 39.0 110.6 151.2 132.1 126.7 26.7 22.5 22.7 33.2 22.3 25.4 171.3 21.5 2.55 (brd, 11.8) 1.43 (t, 11.8) 5.20 (tt, 11.8, 4.0) 1.84 (m) 1.31 (t, 11.8) – 1.37 (dd, 12.5, 2.0) 1.88 (m) 1.69 (qd, 12.5, 7.0) 2.89 (dd, 16.7, 6.0) 2.79 (dd, 16.7, 11.3) – – – 6.61 (s) – – 6.86 (s) 3.16 (sept, 6.7) 1.23 (3H, d, 6.7) 1.24 (3H, d, 6.7) 1.01 (3H, s) 1.02 (3H, s) 1.25 (3H, s) – 2.09 (3H, s) O 63 64 The CHCl3-soluble portion (24.3 g) was subjected to silica gel column chromatography (n-hexane/EtOAc, 0:10 to 5:1 and then CHCl3/MeOH, 20:1 to 0:10) to give eleven fractions (frs. 1–11). Tanshinone IIA (443 mg) was obtained by crystallization of fr. 4 from EtOAc, while tanshinone I (34.6 mg) was crystallized from the EtOAc solution of fr. 5. The mother liquor of fr. 5 was applied to an ODS column (MeOH/H2O, 5:5 to 10:0) to give 18 fractions (frs. 5.1–18). Fr. 5.6 was separated by repeated silica gel column chromatography (nhexane/acetone, 99:1 to 90:10; toluene/EtOAc, 25:1) to afford norsalvioxide (8 mg). Fr. 5.9 was subjected to silica gel column chromatography (n-hexane/EtOAc 15:1 to 1:1), and then purified using ODS HPLC (COSMOSIL 5C18-AR-II, Nacalai tesque, 20 × 250 mm, MeOH/H2O, 85:15) to give miltiorin A (1, 15.3 mg). Fr. 6 was separated by silica gel column chromatography (CHCl3/MeOH, 50:1 to 0:100) to yield 15 fractions (frs. 6.1–15). Cryptotanshinone (277 mg) was crystallized from fr. 6.7 (acetone). Fr. 6.8 was loaded on a Toyopearl HW-40C column (toluene/MeOH, 5:1), an ODS column (MeOH/H2O, 30:70 to 100:0), and a silica gel column (toluene/EtOAc, 30:1) to give miltiorin B (2, 2.2 mg), 5,6dehydrosugiol (3.2 mg), and 2α-acetoxysugiol (7.7 mg). Separation of fr. 7 by Toyopearl HW-40C column chromatography (toluene/MeOH, 7:1) afforded 13 fractions (frs. 7.1–13). Fr. 7.2 was purified by RP HPLC (COSMOSIL πNAP, 20 × 250 mm, MeOH/H2O, 85:15) to furnish 15,16dihydrotanshinone (3.5 mg). Fr. 7.8 was subjected to silica gel column chromatography repeatedly (n-hexane/EtOAc, 5:1 to 1:1; CHCl3) to give (+)-danshexinkun A (5.5 mg). Fr. 7.4 was applied to a silica gel column (n-hexane/acetone, 5:1 to 1:1), and then purified by ODS HPLC (COSMOSIL 5C18-ARII, 20 × 250 mm, MeOH/H2O, 70:30) to yield miltiorins C (3, 14.8 mg) and D (4, 7.7 mg). C 61 62 A. Hirata et al. / Fitoterapia xxx (2015) xxx–xxx E 2 2 3 δC 43.7 δH (J in Hz) δC 38.2 170.5 124.7 2.68 (brd, 11.5) 1.64 (t, 11.5) 5.42 (tt, 12.0, 4.0) 2.01 (brd, 12.0) 1.50 (t, 12.0) – – 6.48 (s) 199.0 – 185.4 – 121.3 154.9 40.7 109.9 159.2 133.9 127.5 26.8 22.2 22.4 35.9 22.6 25.3 171.0 21.5 – – – 6.70 (s) – – 7.95 (s) 3.16 (sept, 7.0) 1.26 (3H, d, 7.0) 1.27 (3H, d, 7.0) 1.28 (3H, s) 1.32 (3H, s) 1.44 (3H, s) – 2.09 (3H, s) 122.7 152.8 41.9 110.8 158.7 134.5 125.2 26.9 22.3 22.5 32.6 29.9 33.3 171.0 21.4 – – – 6.94 (s) – – 8.01 (s) 3.26 (sept, 7.0) 1.25 (3H, d, 7.0) 1.27 (3H, d, 7.0) 1.29 (3H, s) 1.41 (3H, s) 1.56 (3H, s) – 2.05 (3H, s) 68.4 47.7 35.8 55.1 73.5 41.9 δH (J in Hz) 2.56 (m) 1.51 (t, 11.7) 5.17 (brt, 11.7) 1.84 (brd, 11.7) 1.39 (t, 11.7) – 1.85 (d, 12.5) 4.59 (d, 12.5) 67.7 44.7 Please cite this article as: Hirata A, et al, Miltiorins A–D, diterpenes from Radix Salviae miltiorrhizae, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.01.013 111 112 113 114 115 116 117 A. Hirata et al. / Fitoterapia xxx (2015) xxx–xxx Table 2 H and 13C NMR data for miltiorin D (4) in CDCl3. 1 4 t2:7 3 37.3 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 34.6 142.2 130.2 129.5 133.5 121.4 147.8 170.2 173.9 142.3 126.3 33.1 21.4 21.6 31.6 30.8 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 C E R 127 128 R 125 126 2.8.2. 12-O-methyl miltiorin B (2a) Colorless amorphous solid; HRESIMS m/z 411.2151 [M + Na]+ (calcd for C23H32O5Na, 411.2147); 1H NMR (CDCl3) δH 7.93 (1H, s, H-14), 6.70 (1H, s, H-11), 5.20 (1H, m, H-2), 4.60 (1H, dd, J = 12.6, 1.8 Hz, H-6), 3.91 (3H, s, 12-OMe), 3.25 (1H, sept, J = 7.0 Hz, H-15), 2.60 (1H, ddd, J = 12.0, 3.6, 2.4 Hz, H1a), 2.10 (3H, s, 2-OAc), 1.87 (1H, d, J = 12.6 Hz, H-5), 1.85 (1H, ddd, J = 12.7, 4.2, 2.6 Hz, H-3a), 1.47 (3H, s, H3-20), 1.33 (3H, s, H3-19), 1.28 (3H, s, H3-18), 1.22 (3H, d, J = 7.0 Hz, H3-17), and 1.20 (3H, d, J = 7.0 Hz, H3-16). O 123 124 C 122 2.8.1. 12-O-methyl miltiorin A (1a) Colorless amorphous solid; HRESIMS m/z 381.2403 [M + Na]+ (calcd for C23H34O3Na, 381.2406); 1H NMR (CDCl3) δH 6.85 (1H, s, H-14), 6.67 (1H, s, H-11), 5.20 (1H, m, H-2), 3.80 (3H, s, 12-OMe), 3.22 (1H, sept, J = 7.0 Hz, H-15), 2.89 (1H, dd, J = 16.9, 6.1 Hz, H-7a), 2.79 (1H, ddd, J = 16.9, 11.1, 7.6 Hz, H7b), 2.57 (1H, brd, J = 12.4 Hz, H-1a), 2.08 (3H, s, 2-OAc), 1.89 (1H, m, H-6a), 1.84 (1H, brd, J = 12.7 Hz, H-3a), 1.70 (1H, m, H6b), 1.47 (1H, t, J = 12.4 Hz, H-1b), 1.38 (1H, dd, J = 12.6, 2.4 Hz, H-5), 1.27 (3H, s, H3-20), 1.19 (3H, d, J = 7.0 Hz, H3-17), 1.17 (3H, d, J = 7.0 Hz, H3-16), and 1.03 (6H, s, H3-18 and H319). N 120 121 5.17 (dd, 12.0, 5.0) 2.38 (m) 1.61 (qd, 12.0, 4.0) 1.92 (dt, 14.5, 4.0) 1.84 (td, 14.5, 4.0) – – 7.38 (d, 8.0) 7.44 (d, 8.0) – – – – – – 7.29 (s) 2.89 (sept, 6.5) 1.24 (3H, d, 6.5) 1.24 (3H, d, 6.5) 1.46 (3H, s) 1.17 (3H, s) U 118 119 δH (J in Hz) 2.8.3. 12-O-methyl miltiorin C (3a) Colorless amorphous solid; HRESIMS m/z 393.2047 [M + Na]+ (calcd for C23H30O4Na, 393.2042); 1H NMR (CDCl3) δH 7.99 (1H, s, H-14), 6.82 (1H, s, H-11), 6.47 (1H, s, H-6), 5.47 (1H, m, H-2), 3.91 (3H, s, 12-OMe), 3.29 (1H, sept, J = 7.0 Hz, H-15), 2.75 (1H, ddd, J = 12.5, 4.5, 2.6 Hz, H-1a), 2.10 (3H, s, 2OAc), 2.02 (1H, ddd, J = 12.6, 4.1, 2.4 Hz, H-3a), 1.66 (1H, t, J = 12.5 Hz, H-1b), 1.61 (3H, s, H3-20), 1.53 (1H, t, J = 12.6 Hz, H3b), 1.44 (3H, s, H3-19), 1.32 (3H, s, H3-18), 1.25 (3H, d, J = 7.0 Hz, H3-17), and 1.22 (3H, d, J = 7.0 Hz, H3-16). 2.9.1. 2-Deacetyl-12-O-methyl miltiorin A (1b) Colorless amorphous solid; HRESIMS m/z 339.2308 [M + Na]+ (calcd for C21H32O2Na, 339.2300); 1H NMR (CDCl3) δH 6.85 (1H, s, H-14), 6.73 (1H, s, H-11), 4.07 (1H, m, H-2), 3.79 (3H, s, 12-OMe), 3.22 (1H, sept, J = 7.0 Hz, H-15), 2.89 (1H, ddd, J = 17.5, 6.9, 1.3 Hz, H-7a), 2.79 (1H, ddd, J = 17.5, 11.0, 7.2 Hz, H-7b), 2.61 (1H, brd, J = 12.0 Hz, H-1a), 1.88 (1H, m, H-6a), 1.85 (1H, m, H-3a), 1.69 (1H, m, H-6b), 1.41 (1H, t, J = 12.0 Hz, H-1b), 1.35 (1H, dd, J = 12.6, 2.2 Hz, H5), 1.22 (3H, s, H3-20), 1.19 (3H, d, J = 7.0 Hz, H3-17), 1.17 (3H, d, J = 7.0 Hz, H3-16), 1.01 (3H, s, H3-18), and 0.97 (3H, s, H3-19). 159 160 2.9.2. 2-Deacetyl-12-O-methyl miltiorin B (2b) Colorless amorphous solid; HRESIMS m/z 369.2030 [M + Na]+ (calcd for C21H30O4Na, 369.2042); 1H NMR (CDCl3) δH 7.93 (1H, s, H-14), 6.77 (1H, s, H-11), 4.59 (1H, d, J = 13.2 Hz, H-6), 4.11 (1H, m, H-2), 3.90 (3H, s, 12-OMe), 3.25 (1H, sept, J = 7.1 Hz, H-15), 2.62 (1H, m, H-1a), 1.84 (1H, d, J = 13.2, H5), 1.41 (3H, s, H3-20), 1.30 (3H, s, H3-19), 1.28 (3H, s, H3-18), 1.22 (3H, d, J = 7.1 Hz, H3-17), and 1.21 (3H, d, J = 7.1 Hz, H316). 171 2.9.3. 2-Deacetyl-12-O-methyl miltiorin C (3b) Colorless amorphous solid; HRESIMS m/z 351.1938 [M + Na]+ (calcd for C21H28O3Na, 351.1936); 1H NMR (CDCl3) δH 7.99 (1H, s, H-14), 6.87 (1H, s, H-11), 6.47 (1H, s, H-6), 4.39 (1H, m, H-2), 3.91 (3H, s, 12-OMe), 3.29 (1H, sept, J = 6.9 Hz, H-15), 2.77 (1H, brd, J = 11.9 Hz, H-1a), 2.02 (1H, brd, J = 12.0 Hz, H-3a), 1.56 (3H, s, H3-20), 1.41 (3H, s, H3-19), 1.32 (3H, s, H3-18), 1.25 (3H, d, J = 6.9 Hz, H3-17), and 1.23 (3H, d, J = 6.9 Hz, H3-16). 180 181 R O O F 78.0 26.2 δC 151 152 P Position 1 2 A mixture of 12-O-methyl miltiorin A (1a, 0.6 mg) and K2CO3 (0.6 mg) in MeOH (300 μL) was stirred at rt for 10 h. The reaction mixture was diluted with H2O (2 mL), and extracted with CHCl3 (2 mL × 3). The CHCl3 layer was concentrated under reduced pressure to give 2-deacetyl12-O-methyl miltiorin A (1b, 0.5 mg). 12-O-Methyl miltiorins B (2a) and C (3a) were hydrolyzed as for 1a to furnish the 2-deacetyl derivatives (2b and 3b, respectively). D t2:4 t2:5 t2:6 150 E t2:3 2.9. Alkaline hydrolyses of 12-O-methyl miltiorins A–C T t2:1 t2:2 3 153 154 155 156 157 158 161 162 163 164 165 166 167 168 169 170 172 173 174 175 176 177 178 179 182 183 184 185 186 187 188 2.10. Preparation of 2-(S)- and 2-(R)-MTPA esters of 12-O-methyl 189 miltiorins A–C 190 To CH2Cl2 solutions (300 μL) of each sample (1b–3b, 0.2 mg) were added 4-(dimethylamino)pyridine (0.3 mg), triethylamine (20 μL), and (R)-MTPACl (5 μL), and the mixture was stirred at rt for 1 h. After addition of MeOH (300 μL), the reaction mixture was concentrated by evaporation, and the residue was purified by silica gel column chromatography (nhexane/EtOAc, 95:5) to afford the 2-(S)-MTPA esters (1c–3c). Similarly, the 2-(R)-MTPA esters of 12-O-methyl miltiorins A–C (1d–3d) were prepared. 191 2.10.1. 2-(S)-MTPA ester of 12-O-methyl miltiorin A (1c) Colorless amorphous solid; HRESIMS m/z 555.2718 [M + Na]+ (calcd for C31H39O4F3Na, 555.2698); 1H NMR (CDCl3) δH 6.854 (1H, s, H-14), 6.637 (1H, s, H-11), 5.448 (1H, m, H-2), 200 Please cite this article as: Hirata A, et al, Miltiorins A–D, diterpenes from Radix Salviae miltiorrhizae, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.01.013 192 193 194 195 196 197 198 199 201 202 203 A. Hirata et al. / Fitoterapia xxx (2015) xxx–xxx F 4 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 2.10.3. 2-(S)-MTPA ester of 12-O-methyl miltiorin B (2c) Colorless amorphous solid; HRESIMS m/z 585.2468 [M + Na]+ (calcd for C31H37O6F3Na, 585.2440); 1H NMR (CDCl3) δH 7.935 (1H, s, H-14), 4.603 (1H, m, H-6), 6.661 (1H, s, H-11), 3.890 (3H, s, 12-OMe), 1.501 (3H, s, H3-20), 1.361 (3H, s, H319), and 1.280 (3H, s, H3-18). 253 254 D P R O A modified fluorometric assay using the fluorogenic substrate 2′-(4-methylumbellyferyl)-α-D-N-acetylneuraminic acid (MUNAN-A, Sigma) was used to determine the NA activity on the type A (H1N1) viruses [7]. All compounds were dissolved in DMSO and diluted to the corresponding concentrations in PBS. A 96-well plate containing a mixture of the diluted virus suspension (1 × 105 PFU, 50 L) and the different concentration of compound solution (50 μL) was incubated on ice for 1 h. MUNANA substrate solution {0.2 mM in 0.1 M acetate buffer (pH 4.6), 25 μL} was added and the mixture was incubated for 30 min at 37 °C. The enzymatic reaction was quenched by adding 100 μL of glycine-NaOH buffer solution (pH 10.7). The fluorescence intensity of the product (4methylumbellyferone) was measured on a spectrophotometer E T 218 219 C 216 217 E 214 215 252 R 212 213 2.10.2. 2-(R)-MTPA ester of 12-O-methyl miltiorin A (1d) Colorless amorphous solid; HRESIMS m/z 555.2711 [M + Na]+ (calcd for C31H39O4F3Na, 555.2698); 1H NMR (CDCl3) δH 6.843 (1H, s, H-14), 6.601 (1H, s, H-11), 5.447 (1H, m, H-2), 3.775 (3H, s, 12-OMe), 3.208 (1H, sept, J = 7.0 Hz, H-15), 2.882 (1H, dd, J = 15.5, 6.5 Hz, H-6a), 2.780 (1H, m, H-7b), 2.610 (1H, m, H-1a), 1.949 (1H, m, H-3a), 1.711 (1H, m, H-6a), 1.697 (1H, m, H-6b), 1.486 (1H, m, H-1b), 1.451 (1H, m, H-3b), 1.374 (1H, m, H-5), 1.302 (3H, s, H3-20), 1.182 (3H, d, J = 7.0 Hz, H3-17), 1.156 (3H, d, J = 7.0 Hz, H3-16), 1.047 (3H, s, H3-18), and 1.032 (3H, s, H3-19). 2.11. Influenza A neuraminidase inhibition assay R 211 248 2.10.4. 2-(R)-MTPA ester of 12-O-methyl miltiorin B (2d) Colorless amorphous solid; HRESIMS m/z 585.2444 [M + Na]+ (calcd for C31H37O6F3Na, 585.2440); 1H NMR (CDCl3) δH 7.924 (1H, s, H-14), 6.623 (1H, s, H-11), 4.597 (1H, m, H-6), 3.882 (3H, s, 12-OMe), 1.489 (3H, s, H3-20), 1.368 (3H, s, H319), and 1.297 (3H, s, H3-18). O 209 210 2.758 (1H, brd, J = 14.5 Hz, H-1a), 2.1359 (1H, m, H-3a), 1.708 (1H, t, J = 14.5 Hz, H-1b), 1.642 (1H, m, H-3b), 1.629 (3H, s, H320), 1.476 (3H, s, H3-19), 1.337 (3H, s, H3-18), 1.243 (3H, d, J = 7.0 Hz, H3-17), and 1.211 (3H, d, J = 7.0 Hz, H3-16). C 207 208 3.780 (3H, s, 12-OMe), 3.216 (1H, sept, J = 7.0 Hz, H-15), 2.886 (1H, dd, J = 14.0, 7.0 Hz, H-7a), 2.789 (1H, m), 2.664 (1H, m, H1a), 1.872 (1H, m, H-3a), 1.727 (1H, m, H-6a), 1.699 (1H, m, H6b), 1.602 (1H, m, H-1b), 1.383 (1H, m, H-5), 1.344 (1H, m, H3b), 1.312 (3H, s, H3-20), 1.187 (3H, d, J = 7.0 Hz, H3-17), 1.164 (3H, d, J = 7.0 Hz, H3-16), 1.038 (3H, s, H3-18), and 1.015 (3H, s, H3-19). N 205 206 2.10.5. 2-(S)-MTPA ester of 12-O-methyl miltiorin C (3c) Colorless amorphous solid; HRESIMS m/z 567.2330 [M + Na]+ (calcd for C31H35O5F3Na, 567.2334); 1H NMR (CDCl3) δH 7.989 (1H, s, H-14), 6.794 (1H, s, H-11), 6.472 (1H, s, H-6), 5.699 (1H, m, H-2), 3.905 (3H, s, 12-OMe), 3.290 (1H, m, H-15), 2.834 (1H, brd, J = 14.5 Hz, H-1a), 2.065 (1H, m, H-3a), 1.799 (1H, t, J = 14.5 Hz, H-1b), 1.640 (3H, s, H3-20), 1.598 (1H, m, H3b), 1.464 (3H, s, H3-19), 1.313 (3H, s, H3-18), 1.247 (3H, d, J = 7.0 Hz, H3-16), and 1.217 (3H, d, J = 7.0 Hz, H3-16). U 204 O Chart 1. Structures of miltiorins A–D (1–4). 2.10.6. 2-(R)-MTPA ester of 12-O-methyl miltiorin C (3d) Colorless amorphous solid; HRESIMS m/z 567.2331 [M + Na]+ (calcd for C31H35O5F3Na, 567.2334); 1H NMR (CDCl3) δH 7.978 (1H, s, H-14), 6.764 (1H, s, H-11), 6.468 (1H, s, H-6), 5.699 (1H, m, H-2), 3.900 (1H, s, 12-OMe), 3.284 (1H, m, H-15), Fig. 1. (A) Selected 2D NMR correlations and (B) the relative stereochemistry for miltiorin A (1) (acetoxy group at C-2 and protons of methyl groups in B are not shown). Please cite this article as: Hirata A, et al, Miltiorins A–D, diterpenes from Radix Salviae miltiorrhizae, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.01.013 249 250 251 255 256 257 258 259 260 261 262 263 264 265 266 A. Hirata et al. / Fitoterapia xxx (2015) xxx–xxx 5 D P R O O F signals including six aromatic carbons (Table 1). These observations implied miltiorin A (1) to be a diterpene with an acetoxy group. Analysis of the 1H–1H COSY and HMBC spectra suggested that 1 has an abietane skeleton (Fig. 1A). The acetoxy group at C-2 was revealed by an HMBC correlation for H-2 to the acetoxy carbonyl carbon. The chemical shift of C-12 (δC 151.2) suggested the presence of a hydroxy group at C-12. NOESY correlations for H-2/H3-19, H-2/H3-20, and H3-19/H320 were indicative of the axial orientations for these protons as well as the trans junction between A- and B-rings. Therefore, the relative configuration of miltiorin A (1) was assigned as shown in Fig. 1B. The HRESIMS of miltiorin B (2) indicated the molecular formula of 2 to be C22H30O5 (m/z 397.2000 [M + Na]+). Analysis of the 1D NMR spectra implied 2 being an abietane diterpene structurally related to 1, and the signals of an sp3 oxymethine (CH-6) and a ketone carbonyl carbon (C-7) in 2 were discerned in place of the resonances of CH2-6 and CH2-7 in 1 (Table 1). A 1H–1H COSY cross-peak of H-5/H-6 and HMBC correlations for H-14 to C-7 and for H-6 to C-8 and C-10 disclosed the connectivities of C-5 to C-8 (Fig. 2A). A large coupling constant of 3JH-5/H-6 (12.5 Hz) indicated the pseudoaxial orientation of H-6. The relative stereochemistry of 2 was confirmed by NOESY analysis (Fig. 2B). Therefore, the structure of miltiorin B was elucidated to be 2. The molecular formula of miltiorin C (3), C22H28O4, was elucidated by the HRESIMS (m/z 379.1873 [M + Na]+). The 1H and 13C NMR spectra of 3 were similar to those of 1 except for the signals from B-ring (C-5–C-10). In place of the resonances of one sp3 methine (CH-5) and two sp3 methylenes (CH2-6 and CH2-7) in 1, the signals due to a β-substituted α,β-unsturated ketone {δH 6.48 (s); δC 185.4, 152.7, and 122.7} were observed 3. Results and discussion 275 276 The dried roots of S. miltiorrhiza (2.0 kg, dry) were extracted with MeOH to give the extract (291 g), which was partitioned with CHCl3 and water. The CHCl3-soluble material was found to show a NA inhibitory activity (IC50 94.1 μg/mL). Repeated chromatographic separations of the CHCl3-soluble material afforded four new diterpenes, miltiorins A (1, 15.3 mg), B (2, 2.2 mg), C (3, 14.8 mg), and D (4, 7.7 mg) (Chart 1), together with eight known diterpenes, 2α-acetoxysugiol [9], 5,6-dehydrosugiol [10], norsalvioxide [11], tanshinone IIA [12], cryptotanshinone [12], tanshinone I [12], 15,16-dihydrotanshinone [12], and (+)-danshexinkun A [13,14]. The structures of known diterpenes were identified by comparison of their spectral data with the reported data. Miltiorin A (1) was obtained as an optically active pale yellow amorphous solid {[α]D −17.6 (c 0.43, CHCl3)}. The molecular formula of 1, C22H32O3, was established by the HRESIMS (m/z 367.2259 [M + Na]+). The 1H NMR spectrum showed the resonances of two aromatic protons, one oxygenated methine, one acetyl group, one isopropyl group, and three tertiary methyls, while the 13C NMR spectrum displayed 22 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 E R R O C 279 280 N 277 278 U 271 272 T 274 269 270 C 273 with excitation and emission wavelengths of 360 and 440 nm, respectively. The drug concentrations required to inhibit 50% of the NA activity (IC50) were determined by plotting the percent inhibition of NA activity as a function of the drug concentrations [8]. Oseltamivir carboxylate was used as a positive control, which inhibited influenza A neuraminidase with an IC50 value of 0.005 ± 0.001 μg/mL. 267 268 E Fig. 2. (A) Selected 2D NMR correlations and (B) the relative stereochemistry for miltiorin B (2) (acetoxy group at C-2 and protons of methyl groups in B are not shown). Fig. 3. (A) Selected 2D NMR correlations and (B) the relative stereochemistry for miltiorin C (3) (acetoxy group at C-2 and protons of methyl groups in B are not shown). Please cite this article as: Hirata A, et al, Miltiorins A–D, diterpenes from Radix Salviae miltiorrhizae, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.01.013 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 A. Hirata et al. / Fitoterapia xxx (2015) xxx–xxx R O O F 6 Fig. 4. Structures of 12-O-methyl miltiorins A–C (1a–3a), 2-deacetyl-12-O-methyl miltiorins A–C (1b–3b), and the 3-O-(S)- and 3-O-(R)-MTPA esters (1c–3c and 1d– 3d, respectively) derived from 1b–3b. Δδ values [Δδ (in ppm) = δS − δR] obtained for 1c–3c and 1d–3d are shown. 341 342 343 344 345 346 347 348 349 350 351 352 353 Fig. 5. Selected 2D NMR correlations for miltiorin D (4). 354 355 Acknowledgment 388 We thank Mr. Hitoshi Katagiri, UCHIDA WAKANYAKU Ltd. for providing Radix Salviae miltiorrhizae. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 389 390 D P carboxy carbons. The presence of a tetrahydronaphthalene ring (C-1–C-10) with a geminal dimethyl group at C-4 was implied by interpretation of the 1H–1H COSY and HMBC spectra (Fig. 5). HMBC correlations for H-15 with C-12, C-13, and C-14 were indicative of the connectivities among C-12, C-13, and the isopropyl group (C-15) via C-14, while the connectivity of C-8 to C-13 was disclosed by HMBC cross-peaks of H-13 to C-7 and C-9. A carboxy group at C-9 was deduced by an HMBC crosspeak of H-7 with C-11. The down-field shifted chemical shifts for H-1 (δH 5.17) implied that C-1 was connected to C-11, forming a γ-lactone ring. The geometry of the olefin (C-13–C14) was deduced to be Z by a NOESY correlation for H-13/H-15. Thus, the gross structure of miltiorin D (4) was elucidated as shown in Chart 1. Though miltiorin D (4) was not optically active, it was not clear whether 4 was racemic or homochiral. Investigation of the CHCl3-soluble materials, which possessed an anti-NA activity, from Radix Salviae miltiorrhizae resulted in the isolation of four new diterpenes, miltiorins A–D (1–4), together with eight known diterpenes. The structures of 1–4 were elucidated by spectroscopic analysis including application of the modified Mosher's method. Miltiorins A–C (1–3) are abietane diterpenes possessing a 2α-acetoxy group and a 12-hydroxy group in common, while 4 was a 11,12-secoabietane diterpene with a γ-lactone ring. Miltiorin D (4) might be biogenetically derived from miltirone [16,17], a 20-norabietane diterpene with an ortho-quinone C-ring. Hydroxylation at C-1 of miltirone [18] was followed by C-11/C-12 oxidative cleavage and lactonization between C-1 and C-11 to give 4. Miltiorin D (4) is the first example of the isolation of a 11,12-seco-abietane diterpene from natural sources. In an influenza A neuraminidase inhibitory assay for the isolated diterpenes, a nor-abietane diterpene, (+)-danshexinkun A (5), showed a NA inhibitory activity (IC50 39.5 μg/mL), while the other diterpenes did not exhibit activity. E T C E 339 340 R 337 338 R 336 O 334 335 C 332 333 N 330 331 in 3. The presence of the β-substituted α,β-unsaturated ketone in ring B was supposed by HMBC cross-peaks of H-14 to C-7, H3-20 to C-5, and H-6 to C-8 and C-10 (Fig. 3A). A NOESY correlation for H-2 with H3-20 implied that these protons were located on the same side of the molecule (Fig. 3B). Therefore, the relative stereochemistry of miltiorin C (3) was assigned as shown in Fig. 3B. To assign the absolute configurations of C-2 for miltiorins A–C (1–3), the modified Mosher's method [15] was applied as follows. Miltiorin A (1) was converted into the 12-O-methyl derivative (1a), which was subsequently treated with K2CO3 in MeOH to give 2-deacetyl-12-O-methyl miltiorin A (1b). The Δδ values obtained for the 2-(S)- and 2-(R)-MTPA esters (1c and 1d, respectively) of 1b revealed that the absolute configuration of C-2 was S (Fig. 4). Therefore, the absolute configurations at three chiral centers in miltiorin A (1) were elucidated to be 2S, 5S, and 10S. Similarly, the absolute stereochemistry for miltiorins B (2) and C (3) was determined as shown in Chart 1. Miltiorin D (4) was isolated as colorless amorphous solid, and the molecular formula of 4 was assigned as C19H22O4 by the HRESIMS (m/z 313.1447 [M − H]−). The 1H NMR spectrum displayed the resonances due to a pair of ortho-coupled aromatic protons, one trisubstituted olefine, and one isopropyl group as well as the signals of one oxygenated sp3 methine, two sp3 methylenes, and two tertiary methyls. The 13C NMR spectrum revealed the existence of 19 carbons including two U 328 329 Please cite this article as: Hirata A, et al, Miltiorins A–D, diterpenes from Radix Salviae miltiorrhizae, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.01.013 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 391 Q3 392 393 A. Hirata et al. / Fitoterapia xxx (2015) xxx–xxx C O R R E C T E D P R O O F [1] Wu YB, Ni ZY, Shi QW, Dong M, Kiyota H, Gu YC, et al. Constituents from Salvia species and their biological activities. Chem Rev 2012;112: 5976–6026. [2] Kashiwada Y, Ahmed FA, Kurimoto S, Kim SY, Shibata H, Fukioka T, et al. New α-glucosides of caffeoyl quinic acid from the leaves of Moringa oleifera Lam. J Nat Med 2012;66:217–21. [3] Sasaki H, Kashiwada Y, Shibata H, Takaishi Y. Prenylated flavonoids from Desmodium caudatum and evaluation of their anti-MRSA activity. Phytochemistry 2012;82:136–42. [4] Kashiwada Y, Omichi Y, Kurimoto S, Shibata H, Miyake Y, Kirimoto T, et al. Conjugates of a secoiridoid glucoside with a phenolic glucoside from the flower buds of Lonicera japonica Thunb. Phytochemistry 2013;96:423–9. [5] Kurimoto S, Takaishi Y, Ahmed FA, Kashiwada Y. Triterpenoids from the fruits of Azadirachata indica (Meliaceae). Fitoterapia 2014;92:200–5. [6] Sasaki H, Shibata H, Imabayashi K, Takaishi Y, Kashiwada Y. Prenylated flavonoids from the stems and leaves of Desmodium caudatum and evaluation of their inhibitory activity against the film-forming growth of Zygosaccharomyces rouxii F51. J Agric Food Chem 2014;62:6345–53. [7] Ullah N, Ahmed S, Muhanmmad P, Ahmed Z, Nawaz HR, Malik A. Coumarinolignoid glycoside from Daphne oleoides. Phytochemistry 1999; 51:103–5. [8] Shi SY, Zhou Q, Peng H, Zhou CX, Hu MH, Tao QF, et al. Four new constituents from Taraxacum mongolicum. Chin Chem Lett 2007;18: 1367–70. [9] Don MJ, Shen CC, Syu WJ, Ding YH, Sun CM. Cytotoxic and aromatic constituents from Salvia miltiorrhiza. Phytochemistry 2006;67:497–503. N 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 446 [10] Kusumoto N, Ashitani T, Hayasaka Y, Murayama T, Ogiyama K, Takahashi K. Antitermitic activities of abietane-type diterpenes from Taxodium distichum Cones. J Chem Ecol 2009;35:635–42. [11] Ginda H, Kusumi T, Ishitsuka MO, Kakisawa H, Weijie Z, Jun C, et al. Salviolon, a cytotoxic bisnorditerpene with a benzotropolone chromophore from a Chinese drug dan-shen (Salvia miltiorrhiza). Tetrahedron Lett 1988;29:4603–6. [12] Lee SY, Choi DY, Woo ER. Inhibition of osteoclast differentiation by tanshinones from the root of Salvia miltiorrhiza Bunge. Arch Pharm Res 2005;28:909–13. [13] Ikeshiro Y, Hashimoto I, Iwamoto Y, Mase I, Tomita Y. Diterpenoids from Salvia miltiorrhiza. Phytochemistry 1991;30:2791–2. [14] Danheiser RL, Casebier DS, Loebach JL. Total synthesis of dan shen diterpenoid quinones. Tetrahedron Lett 1992;33:1149–52. [15] Ohtani I, Kusumi T, Kashman Y, Kakisawa H. High-field FT MR application of Mosher's method. The absolute configurations of marine terpenoids. J Am Chem Soc 1991;113:4092–6. [16] Kakisawa H, Hayashi T. The structure of miltiorone, a new diterpenoid quinone. J Chem Soc Chem Commun 1970:299. [17] Nasipuri D, Mitra AK. Synthetic studies in the diterpene series. Part VIII. Synthesis of miltiorone, a diterpenoid quinone. J Chem Soc Perkin Trans 1973;1:285–7. [18] Michavila A, De La Torre MC, Rodriguez B. 20-Nor-abietane and rearranged abietane diterpenoids from the root of Salvia argentea. Phytochemistry 1986;25:1935–7. References U 394 7 Please cite this article as: Hirata A, et al, Miltiorins A–D, diterpenes from Radix Salviae miltiorrhizae, Fitoterapia (2015), http:// dx.doi.org/10.1016/j.fitote.2015.01.013 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445