HETEROCYCLES

Paper | Special issue | Vol. 86, No. 1, 2012, pp. 317-330
Received, 25th April, 2012, Accepted, 25th May, 2012, Published online, 28th May, 2012.
DOI: 10.3987/COM-12-S(N)12

■ Selenium Oxide Oxidation of Hexahydro-1,5-imino-3-benzazocine-7,10-dione in Aliphatic Alcohol for Conversion of Renieramycin Marine Natural Products

Mayuko Mori, Naomi Daikuhara, Junya Yamada, and Naoki Saito*

Meiji College of Pharmacy, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan

Abstract

A selenium oxide oxidation of hexahydro-1,5-imino-3-benzazocine-7,10-dione derivatives in several aliphatic alcohols to generate C-6 ether derivatives stereoselectively is described. This procedure shows promise for the construction of several renieramycins, such as renieramycins B, D, and V. The results of cytotoxicity studies are also presented.

INTRODUCTION
Natural products belonging to the tetrahydroisoquinolinequinone family and their reduced forms, such as saframycin antibiotics and renieramycin marine natural products, together with jorumycin and jorunnamycin, have attracted considerable interest over the past 30 years for their unique structures and meager availability in nature, and for their potent antitumor activity.1 Monoquinone-type natural products, such as saframycins D-F (1d-f),2 saframycins Mx 1 (2a) and Mx 2 (2b),3 renieramycins N (3n),4 P (3p),5 and Q (3q),5 cribrostatin 4 (4),6 and jorunnamycin B (6b),7 have a p-quinone moiety at ring A, a hydroquinone moiety at ring E, and a variety of oxygenated functional groups at C-14 position (Figures 1 and 2).
In regard to the chemical transformation of these natural products, we have reported the introduction of a hydroxyl group at C-6 position8 of right-hand half model compound 79 stereoselectively.10 This method was used for the transformation of natural products, such as saframycins B (1b) to C (1c)10b and saframycins A (1g) to F (1f) via G (1g).11,12 It was also utilized as one of the crucial steps for the first total synthesis of cribrostatin 4 (4) by Danishefsky and co-workers,12 and adapted for the following synthesis (Figure 3: I → II).14-16

As part of our search for new metabolites via the isolation and characterization of biologically active compounds from Thai marine animals, we have isolated and elucidated the structure of renieramycin M (3m)4 in gram scale from the Thai blue sponge Xestospongia sp. along with many minor renieramycin-type derivatives after stabilization of the sponge homogenized in phosphate buffer solution by the addition of KCN.5,17 Recently, we succeeded in isolating renieramycin V (3v), the first example of renieramycin connected to a sterol moiety.18 Renieramycin V did not show any cytotoxicity; nevertheless, we were very interested in its structure because we had hypothesized that the sterol moiety at the C-14 position of 3m would play a role in protecting marine organism from predators. In order to devise new ways for the preparation of 3b, 3d, 3i, and 3v, we re-investigated the construction of an ether bond at C-6 position18 of right-hand half model compounds using a selenium oxide (SeO2) oxidation.

RESULTS AND DISCUSSION
We have reported that the reaction of 7 with SeO2 (1.1 equiv.) in dioxane under reflux for 4 h gave 8 in 80% yield (Figure 3).9 Our next challenge was to introduce an alkoxy group to the C-6 position of hexahydro-1,5-imino-3-benzazocine derivative 7.19 Treatment of 7 with SeO2 (1.1 equiv.) in ethanol/dioxane (1:1) under reflux for 30 h gave 9a and 8 in 25.6% and 41.8% yields, respectively. As this reaction proceeded slowly, water was added to accelerate the reaction. The reaction time of 7 with SeO2 (1.1 equiv.) in ethanol/H2O (1:1) under reflux was considerably reduced (10 h); however, the yields of 9a (16.0%) and 8 (63.4%) were still unsatisfactory. After several attempts, heating a solution of 7 in EtOH under reflux in the presence of 1.1 equiv. of SeO2 for 32 h was found to be best choice in terms of yield (9a: 86.0% and 8: 9.3%). All protons and carbons of 9a were assigned after extensive NMR measurements using COSY, NOESY, HMQC, and HMBC techniques. The stereochemistry of C-6 in 9a was readily identified on the basis of the chemical shifts of H-6 (δ 4.38) and H-5 (δ 3.58), along with the small coupling between H-6 and H-5 (J = 1.7 Hz), which revealed an H-6 proton orthogonal to the H-5 proton (Table 1). Heating 7 with SeO2 oxidation in the presence of primary alcohols gave corresponding ethers 9b-e in 69-97% yields (Table 1, entries 2-5). The usefulness of this procedure was further demonstrated by using several secondary alcohols; ethers 9f-h were obtained in 63-89% yields. These results indicate that the present method offers a facile entry to a general procedure for the stereoselective preparation of C-6 ethers.

In order to examine the scope of this procedure, we explored the reaction of quinone 1012,20, an aminonitrile model, with SeO2. Subjecting 10 to SeO2 oxidation in EtOH, BuOH, and cyclohexanol under reflux led to the production of ethers [11a (11.1%), 11b (5.0%), and 11c (25.1%)] along with the recovery of a large amount of the starting material.
Finally, all synthetic compounds were tested in vitro for cytotoxicity using three representative human solid tumor cell lines (HCT116 human colon carcinoma, QG56 human lung carcinoma, and DU145 prostate carcinoma) following the standard MTT method (Table 2). Several compounds displayed micromolar inhibitory effects, but the data revealed that the introduction of the ether bond at C-6 almost did not change the in vitro activity of the model compounds.

In summary, the selenium oxide oxidation of 7 offers a facile entry to the preparation of C-6 ethers in a stereoselective manner. The application of this method to the transformation of renieramycins G to D along with the preparation of renieramycin V is under study in our laboratory.21

EXPERIMENTAL
All melting points were determined with a Yanagimoto micromelting point apparatus and uncorrected. IR spectra were obtained with a Shimadzu Prestige 21/RA Affinity-1 FT-IR spectrometer. 1H and 13C NMR spectra were recorded on a JEOL JNM-ECA 500 FT NMR spectrometer at 500 MHz for 1H and 125 MHz for 13C; and a JEOL JNM-AL 300 NMR spectrometer at 300 MHz for 1H and 75 MHz for 13C (ppm, J in Hz with TMS as internal standard). All proton and carbon signals were assigned by extensive NMR measurements using COSY, HMBC, and HMQC techniques. Mass spectra were recorded on a JEOL JMS 700 instrument with a direct inlet system operating at 70 eV. Elemental analyses were conducted on a YANACO MT-6 CHN CORDER elemental analyzer.

Oxidation of lactam 7 with selenium oxide and ethanol
Method A: A suspension of 7 (58.0 mg, 0.20 mmol) and SeO2 (24.4 mg, 0.22 mmol) in dioxane (2.0 mL) and EtOH (2.0 mL) was heated at reflux for 30 h. The reaction mixture was filtered and washed with CHCl3 (100 mL). The combined filtrates were concentrated in vacuo and the residue was diluted with water (30 mL) and extracted with CHCl3 (30 mL x 3). The combined extracts were washed with brine (30 mL), dried, and concentrated in vacuo to give a residue (138 mg), which was subjected to chromatography on silica gel with CH2Cl2-MeOH (80:1) to furnish 9a (17.1 mg, 25.6%) as a dark yellow solid. Further elution with CH2Cl2-MeOH (50:1) afforded 8 (25.6 mg, 41.8%) as pale yellow prisms.
Method B: Using the same procedure as that described above but with EtOH (2.0 mL) and water (2.0 mL) as solvent (11 h) gave a residue (67.1 mg), which was subjected to chromatography on silica gel with CH2Cl2-MeOH (80:1) to give 9a (10.7 mg, 16.0%) as a dark yellow solid. Further elution with CH2Cl2-MeOH (50:1) afforded 8 (38.8 mg, 63.4%) as pale yellow prisms.
Method C (General Procedure): A suspension of 7 (58.0 mg, 0.20 mmol) and SeO2 (24.4 mg, 0.22 mmol) in EtOH (10.0 mL) was heated at reflux for 32 h. The reaction mixture was filtered and washed with CHCl3 (100 mL). The combined filtrates were concentrated in vacuo and the residue was diluted with water (30 mL) and extracted with CHCl3 (30 mL x 3). The combined extracts were washed with brine (30 mL), dried, and concentrated in vacuo to give a residue (92.4 mg), which was subjected to chromatography on silica gel with CH2Cl2-MeOH (100:1) to furnish 9a (58.1 mg, 86.0%) as a dark yellow solid. Further elution with CH2Cl2-MeOH (80:1) afforded 8 (5.7 mg, 9.3%) as pale yellow prisms.

(1R*,5S*,6S*)-6-Ethoxy-2,3,5,6-tetrahydro-9-methoxyl-3,8,11-trimethyl-1,5-imino-3-benzazocine-4,7,10(1H)-trione (9a): Recrystallized from EtOAc-ether, mp 157-159 °C. IR νmax (KBr) 1655, 1610, 1312, 1236, 1094 cm-1. δH (500 MHz) 1.24 (3H, t, J = 7.1 Hz, OCH2CH3), 1.98 (3H, s, 8-CH3), 2.65 (3H, s, 11-CH3), 2.87 (3H, s, 3-CH3), 2.95 (1H, d, J = 13.0 Hz, 2-Hβ), 3.58 (1H, br s, 5-H), 3.85 (2H, q, J = 7.1 Hz, OCH2CH3), 3.97 (3H, s, 9-OCH3), 3.98 (1H, dd, J = 13.0, 5.1 Hz, 2-Hα), 4.08 (1H, d, J = 5.1 Hz, 1-H), 4.38 (1H, d, J = 1.7Hz, 6-H). δC (125 MHz) 8.8 (8-CH3), 15.4 (OCH2CH3), 34.1 (3-CH3), 41.0 (11-CH3), 47.7 (C2), 49.6 (C1), 60.9 (9-OCH3), 63.9 (C5), 67.2 (OCH2CH3), 71.1 (C6), 130.2 (C8), 138.2 (C6a), 139.2 (C10a), 155.3 (C9), 166.6 (C4), 182.3 (C10), 185.9 (C7). EIMS m/z (%): 334 (M+, 55), 305 (11), 290 (32), 289 (21), 234 (13), 219 (18), 218 (100), 205 (11). HREIMS m/z 334.1531 (M+, calcd for C17H22N2O5, 334.1529). Anal. Calcd for C17H22N2O5: C, 61.06; H, 6.63; N, 8.38. Found: C, 61.11; H, 6.58; N, 8.31.

(1R*,5S*,6S*)-2,3,5,6-Tetrahydro-9-methoxyl-3,8,11-trimethyl-6-propoxy-1,5-imino-3-benzazocine-4,7,10(1H)-trione (9b): Yield 97%, pale yellow prisms (reflux, 26 h, recrystallization from EtOAc-ether), mp 97-100 °C. IR νmax (KBr) 1647, 1622, 1308, 1236, 1152, 1088 cm-1. δH (400 MHz) 0.92 (3H, t, J = 7.3 Hz, O(CH2)2CH3), 1.62 (2H, ddq, J = 7.3, 6.8, 6.3 Hz, OCH2CH2CH3), 1.98 (3H, s, 8-CH3), 2.64 (3H, s, 11-CH3), 2.87 (3H, s, 3-CH3), 2.95 (1H, d, J = 13.2 Hz, 2-Hβ), 3.55 (1H, br s, 5-H), 3.73 (1H, dt, J = 13.2, 6.8 Hz, OCH), 3.76 (1H, dt, J = 13.2, 6.3 Hz, OCH), 3.94 (1H, dd, J = 13.2, 5.4 Hz, 2-Hα), 3.97 (3H, s, 9-OCH3), 4.06 (1H, d, J = 5.4 Hz, 1-H), 4.35 (1H, d, J = 1.5 Hz, 6-H). δC (100 MHz) 8.8 (8-CH3), 10.5 (O(CH2)2CH3), 23.0 (OCH2CH2CH3), 34.1 (3-CH3), 41.0 (11-CH3), 47.8 (C2), 49.7 (C1), 60.9 (9-OCH3), 63.9 (C5), 71.4 (C6), 73.6 (OCH2), 130.2 (C8), 138.3 (C6a), 139.4 (C10a), 155.4 (C9), 166.8 (C4), 182.5 (C10), 185.9 (C7). EIMS m/z (%): 348 (M+, 45), 290 (49), 289 (24), 234 (12), 219 (20), 218 (100), 205 (11). HREIMS m/z 348.1683 (M+, calcd for C18H24N2O5, 348.1685). Anal. Calcd for C18H24N2O5: C, 62.05; H, 6.94; N, 8.04. Found: C, 62.33; H, 6.88; N, 7.91.

(1R*,5S*,6S*)-6-Butoxy-2,3,5,6-tetrahydro-9-methoxyl-3,8,11-trimethyl-1,5-imino-3-benzazocine-4,7,10(1H)-trione (9c): Yield 88%, dark brown amorphous powder (120 °C, 27 h). IR νmax (CHCl3) 1659, 1209, 1153 cm-1. δH (400 MHz) 0.88 (3H, t, J = 6.8 Hz, O(CH2)5CH3), 1.34 (2H, m, CH2CH3), 1.58 (2H, quintet, J = 7.1 Hz, OCH2CH2), 1.98 (3H, s, 8-CH3), 2.64 (3H, s, 11-CH3), 2.87 (3H, s, 3-CH3), 2.95 (1H, d, J = 12.7 Hz, 2-Hβ), 3.55 (1H, br s, 5-H), 3.76 (1H, dt, J = 13.7, 6.3 Hz, OCH), 3.81 (1H, dt, J = 13.7, 6.3 Hz, OCH), 3.94 (1H, dd, J = 12.7, 5.4 Hz, 2-Hα), 3.97 (3H, s, 9-OCH3), 4.06 (1H, d, J = 5.4 Hz, 1-H), 4.34 (1H, d, J = 1.5 Hz, 6-H). δC (100 MHz) 8.8 (8-CH3), 13.8 (O(CH2)3CH3), 19.3 (CH2CH3), 31.9 (OCH2CH2), 34.0 (3-CH3), 41.0 (11-CH3), 47.8 (C2), 49.7 (C1), 60.9 (9-OCH3), 63.9 (C5), 71.4 (C6), 71.7 (OCH2), 130.2 (C8), 138.3 (C6a), 139.4 (C10a), 155.4 (C9), 166.8 (C4), 182.5 (C10), 185.9 (C7). EIMS m/z (%): 362 (M+, 41), 291 (14), 290 (67), 289 (26), 234 (14), 219 (20), 218 (100), 206 (12). HREIMS m/z 362.1840 (M+, calcd for C19H26N2O5, 362.1842).

(1R*,5S*,6S*)-6-Hexyloxy-2,3,5,6-tetrahydro-9-methoxyl-3,8,11-trimethyl-1,5-imino-3-benzazocine-4,7,10(1H)-trione (9d): Yield 73%, dark brown amorphous powder (120 °C, 32 h). IR νmax (CHCl3) 1659, 1309, 1225, 1088 cm-1. δH (400 MHz) 0.91 (3H, t, J = 6.8 Hz, O(CH2)3CH3), 1.29 (6H, br, CH2 x 3), 1.34 (2H, m, CH2CH3), 1.59 (2H, quintet, J = 6.8 Hz, OCH2CH2), 1.98 (3H, s, 8-CH3), 2.64 (3H, s, 11-CH3), 2.87 (3H, s, 3-CH3), 2.95 (1H, d, J = 13.2 Hz, 2-Hβ), 3.55 (1H, br s, 5-H), 3.75 (1H, dt, J = 13.7, 6.3 Hz, OCH), 3.80 (1H, dt, J = 13.7, 6.3 Hz, OCH), 3.94 (1H, dd, J = 12.7, 5.4 Hz, 2-Hα), 3.97 (3H, s, 9-OCH3), 4.06 (1H, d, J = 5.4 Hz, 1-H), 4.34 (1H, d, J = 1.5 Hz, 6-H). δC (100 MHz) 8.8 (8-CH3), 14.0 (O(CH2)5CH3), 22.6 (C5’), 25.7 (C4’), 29.8 (C3’), 31.6 (C2’), 34.1 (3-CH3), 41.0 (11-CH3), 47.8 (C2), 49.7 (C1), 60.9 (9-OCH3), 64.0 (C5), 71.4 (C6), 72.0 (OCH2), 130.2 (C8), 138.3 (C6a), 139.4 (C10a), 155.4 (C9), 166.8 (C4), 182.5 (C10), 186.0 (C7). EIMS m/z (%): 390 (M+, 48), 375 (11), 305 (12), 291 (17), 290 (91), 289 (33), 234 (14), 219 (20), 218 (100), 206 (13). HREIMS m/z 390.2156 (M+, calcd for C21H30N2O5, 390.2155).

(1R*,5S*,6S*)-6-Cyclohexylmethoxy-2,3,5,6-tetrahydro-9-methoxyl-3,8,11-trimethyl-1,5-imino-3-benzazocine-4,7,10(1H)-trione (9e): Yield 69%, pale yellow prisms (120 °C, 26 h, recrystallized from EtOAc-ether), mp 112-114 °C. IR νmax (KBr) 1657, 1614, 1310, 1233, 1153, 1096 cm-1. δH (400 MHz) 0.89 (1H, dd, J = 12.2, 3.4 Hz, CH), 0.95 (1H, dd, J = 12.2, 3.4 Hz, CH), 1.10-1.26 (3H, m), 1.65-1.72 (6H, m), 1.98 (3H, s, 8-CH3), 2.53 (3H, s, 11-CH3), 2.87 (3H, s, 3-CH3), 2.95 (1H, d, J = 13.2 Hz, 2-Hβ), 3.54 (1H, br s, 5-H), 3.54 (1H, dd, J = 9.3, 6.3 Hz, OCH), 3.63 (1H, dd, J = 8.8, 6.3 Hz, OCH), 3.94 (1H, dd, J = 13.2, 5.4 Hz, 2-Hα), 3.96 (3H, s, 9-OCH3), 4.06 (1H, d, J = 5.4 Hz, 1-H), 4.34 (1H, d, J = 1.5 Hz, 6-H). δC (100 MHz) 8.9 (8-CH3), 25.8 (2 x CH2), 26.6 (CH2), 29.8 (CH2), 30.1 (CH2), 34.1 (3-CH3), 37.9 (C2’), 41.1 (11-CH3), 47.9 (C2), 49.7 (C1), 60.9 (9-OCH3), 63.8 (C5), 71.5 (C6), 77.6 (OCH2), 130.2 (C8), 138.3 (C6a), 139.4 (C10a), 155.4 (C9), 165.8 (C4), 182.5 (C10), 185.9 (C7). EIMS m/z (%): 402 (M+, 48), 375 (11), 305 (12), 291 (17), 290 (91), 289 (33), 234 (14), 219 (20), 218 (100), 206 (13). HREIMS m/z 402.2155 (M+, calcd for C22H30N2O5, 402.2155). Anal. Calcd for C22H30N2O5: C, 65.65; H, 7.51; N, 6.96. Found: C, 65.72; H, 7.66; N, 6.84.

(1R*,5S*,6S*)-6-Cyclohexyloxy-2,3,5,6-tetrahydro-9-methoxyl-3,8,11-trimethyl-1,5-imino-3-benzazocine-4,7,10(1H)-trione (9f): Yield 89%, pale yellow prisms (120 °C, 31 h, recrystallized from EtOAc-ether), mp 138-140 °C, dark brown amorphous powder (reflux, 30 h). IR νmax (KBr) 2936, 1661, 1622, 1504, 1445, 1371, 1335, 1312, 1234, 1148, 1078 cm-1. δH (500 MHz) 1.15-1.22 (1H, m), 1.26-1.41 (4H, m), 1.55 (1H, dt, J = 12.2, 3.5 Hz), 1.65 (1H, br s), 1.72-1.76 (1H, m), 1.97 (3H, s, 8-CH3), 2.05 (2H, br s), 2.63 (3H, s, 11-CH3), 2.87 (3H, s, 3-CH3), 2.95 (1H, d, J = 13.2 Hz, 2-Hβ), 3.53 (1H, dd, J = 1.5, 0.5 Hz, 5-H), 3.76 (1H, m, 1’-H), 3.94 (1H, dd, J = 13.2, 5.4 Hz, 2-Hα), 3.96 (3H, s, 9-OCH3), 4.06 (1H, dd, J = 5.4, 0.5 Hz, 1-H), 4.53 (1H, d, J = 1.5 Hz, 6-H). δC (125 MHz) 8.9 (8-CH3), 24.3 (CH2), 24.4 (CH2), 25.6 (CH2), 32.4 (CH2), 33.3 (CH2), 34.0 (3-CH3), 41.1 (11-CH3), 47.8 (C2), 49.7 (C1), 60.9 (9-OCH3), 65.4 (C5), 69.3 (C6), 79.6 (C1’), 130.2 (C8), 138.7 (C6a), 139.6 (C10a), 155.3 (C9), 167.0 (C4), 182.5 (C10), 186.0 (C7). EIMS m/z (%): 388 (M+, 96), 306 (58), 305 (54), 291 (27), 290 (86), 277 (14), 275 (16), 263 (14), 247 (14), 236 (20), 235 (18), 234 (34), 232 (12), 220 (14), 219 (29), 218 (100), 206 (33), 205 (14), 131 (13). HREIMS m/z 388.2000 (M+, calcd for C21H28N2O5, 388.1998). Anal. Calcd for C21H28N2O5: C, 64.93; H, 7.27; N, 7.21. Found: C, 64.83; H, 7.48; N, 7.05.

(1R*,5S*,6S*)-6-(Trans-4’-methylcyclohexyloxy-2,3,5,6-tetrahydro-9-methoxyl-3,8,11-trimethyl-1,5-imino-3-benzazocine-4,7,10(1H)-trione (9g): Yield 63%, pale yellow prisms (120 °C, 31 h, recrystallized from EtOAc-ether), mp 148-150 °C. IR νmax (KBr) 2926, 1651, 1617, 1449, 1312, 1238, 1152, 1092 cm-1. δH (400 MHz) 0.88 (3H, d, J = 6.3 Hz, 4’-CH3), 0.92-1.06 (1H, m, CH2), 1.19-1.37 (3H, m, CH2 + 4’CH), 1.69-1.75 (2H, m, CH2), 1.97 (3H, s, 8-CH3), 2.11-2.04 (2H, m, CH2), 2.63 (3H, s, 11-CH3), 2.86 (3H, s, 3-CH3), 2.93 (1H, dd, J = 13.2, 0.7 Hz, 2-Hβ), 3.51 (1H, dd, J = 1.5, 0.5 Hz, 5-H), 3.71 (1H, m, 1’-H), 3.93 (1H, dd, J = 13.2, 5.4 Hz, 2-Hα), 3.96 (3H, s, 9-OCH3), 4.05 (1H, dd, J = 5.4, 0.7 Hz, 1-H), 4.53 (1H, d, J = 2.0 Hz, 6-H). δC (100 MHz) 8.9 (8-CH3), 22.0 (4’-CH3), 32.0 (C4’), 32.4 (CH2), 33.3 (CH2), 33.4 (CH2), 33.5 (CH2), 34.0 (3-CH3), 41.1 (11-CH3), 47.7 (C2), 49.6 (C1), 60.9 (9-OCH3), 65.4 (C5), 69.4 (C6), 80.2 (C1’), 130.2 (C8), 138.7 (C6a), 139.6 (C10a), 155.3 (C9), 167.0 (C4), 182.5 (C10), 186.0 (C7). EIMS m/z (%): 402 (M+, 100), 307 (15), 306 (63), 305 (49), 291 (28), 290 (82), 289 (31), 277 (14), 275 (17), 263 (15), 247 (15), 236 (18), 235 (19), 234 (34), 232 (12), 220 (14), 219 (29), 218 (97), 206 (32), 205 (13), 204 (12), 131 (15). HREIMS m/z 402.2156 (M+, calcd for C22H30N2O5, 402.2155). Anal. Calcd for C22H30N2O5: C, 65.65; H, 7.51; N, 6.96. Found: C, 65.49; H, 7.67; N, 6.79.

(1R*,5S*,6S*)-2,3,5,6-Tetrahydro-9-methoxyl-6-(2’-butoxy)-3,8,11-trimethyl-1,5-imino-3-benzazocine-4,7,10(1H)-trione (9h: a mixture of 2’-epimers): Yield 70%, dark brown amorphous powder (120 °C, 37 h). IR νmax (CHCl3) 1660, 1310, 1236, 1075 cm-1. δH (500 MHz) 0.85 and 0.94 (each 3H, t, J = 7.4 Hz, C4’H3), 1.24 (2 x 3H, d, J = 6.2 Hz, C1’H3), 1.42-1.54 and 1.57-1.72 (each 2H, m, C3’H2), 1.97 (2 x 3H, s, 8-CH3), 2.63 and 2.64 (each 3H, s, 11-CH3), 2.86 and 2.87 (each 3H, s, 3-CH3), 2.94 and 2.95 (each 1H, dd, J = 13.2, 0.8 Hz, 2-Hβ), 3.49 and 3.51 (each 1H, br s, 5-H), 3.79 and 3.90 (each 1H, sextet, J = 6.2 Hz, C2’H), 3.94, 3.98 (each 1H, dd, J = 13.2, 5.2 Hz, 2-Hα), 3.95 and 3.97 (each 3H, s, 9-OCH3), 4.06 and 4.07 (each 1H, d, J = 5.2, Hz, C1H), 4.47 and 4.48 (each 1H, s, 6-H). δC (125 MHz) 8.9 (2 x 8-CH3), 9.8 and 10.1 (each C4’), 19.5 and 20.1 (each C1’), 29.1 and 29.7 (each C3’), 34.0 (2 x 3-CH3), 40.9 and 41.1 (each 11-CH3), 47.4 and 47.7 (each C2), 49.6 and 49.8 (each C1), 60.9 (2 x 9-OCH3), 64.7 and 65.5 (each C5), 69.5 and 69.9 (each C6), 78.5 and 78.9 (each C2’), 130.2 and 130.3 (each C8), 138.4 and 138.7 (each C6a), 139.4 and 140.1 (each C10a), 155.3 (2 x C9), 166.9 (2 x C4), 182.4 and 182.5 (each C10), 185.9 and 186.0 (each C7). EIMS m/z (%): 362 (M+, 86), 306 (32), 305 (63), 291 (17), 290 (52), 289 (32), 277 (11), 275 (12), 247 (13), 236 (17), 235 (14), 234 (32), 232 (11), 220 (13), 219 (27), 218 (100), 206 (30), 205 (15), 131 (13). HREIMS m/z 348.1838 (M+, calcd for C19H26N2O5, 362.1842).

(1R*,4S*,5S*,6S*)-6-Ethoxy-1,2,3,4,5,6,7,10-octahydro-9-methoxyl-3,8,11-trimethyl-7,10-dioxo-1,5-imino-3-benzazocine-4-carbonitrile (11a). A suspension of 10 (60.2 mg, 0.20 mmol) and SeO2 (24.4 mg, 0.22 mmol) in EtOH (10.0 mL) was heated at 120 °C for 86 h. The reaction mixture was filtered and washed with CHCl3 (100 mL). The combined filtrates were concentrated in vacuo and the residue was diluted with water (30 mL) and extracted with CHCl3 (30 mL x 3). The combined extracts were washed with brine (30 mL), dried, and concentrated in vacuo to give a residue (71.1 mg), which was subjected to chromatography on silica gel with hexane-EtOAc (8:1) to furnish 11a (7.7 mg, 11.1%) as a dark yellow amorphous powder. Further elution with hexane-EtOAc (3:2) afforded 10 (24.8 mg, 41.2% recovered) as a pale yellow amorphous powder.
IR νmax (CHCl3) 2361, 1655, 1639, 1610, 1452, 1309, 1229, 1157, 1078 cm-1. δH (400 MHz) 1.20 (3H, t, J = 7.0 Hz, OCH2CH3), 1.99 (3H, s, 8-CH3), 2.25 (3H, s, 3-CH3), 2.34 (1H, d, J = 11.7 Hz, 2-Hβ), 2.55 (3H, s, 11-CH3), 2.75 (1H, dd, J = 11.7, 3.5 Hz, 2-Hα), 3.39 (1H, d, J = 1.4 Hz, 5-H), 3.69 (1H, d, J = 2.0 Hz, 4-H), 3.72 and 3.78 (each 1H, q, J = 7.0 Hz, OCH), 3.90 (1H, br s, 6-H), 3.91 (1H, d, J = 3.5 Hz, 1-H), 3.99 (3H, s, 9-OCH3). δC (100 MHz) 8.9 (8-CH3), 15.6 (OCH2CH3), 42.8 (11-CH3), 43.2 (3-CH3), 51.5 (C1), 51.6 (C2), 57.9 (C4), 60.3 (C5), 60.8 (9-OCH3), 67.4 (C6), 68.5 (OCH2), 115.2 (CN), 129.8 (C8), 137.4 (C10a), 139.9 (C6a), 155.1 (C9), 182.7 (C10), 186.2 (C7). EIMS m/z (%): 345 (M+, 21), 219 (14), 218 (100). HREIMS m/z 345.1692 (M+, calcd for C18H23N3O4, 345.1689).

(1R*,4S*,5S*,6S*)-6-Butoxy-1,2,3,4,5,6,7,10-octahydro-9-methoxyl-3,8,11-trimethyl-7,10-dioxo-1,5-imino-3-benzazocine-4-carbonitrile (11b). A suspension of 10 (60.2 mg, 0.20 mmol) and SeO2 (24.4 mg, 0.22 mmol) in BuOH (10.0 mL) was heated at 120 oC for 41 h. The reaction mixture was filtered and washed with CHCl3 (100 mL). The combined filtrates were concentrated in vacuo and the residue was diluted with water (30 mL) and extracted with CHCl3 (30 mL x 3). The combined extracts were washed with brine (30 mL), dried, and concentrated in vacuo to give a residue (140.9 mg), which was subjected to chromatography on silica gel with hexane-EtOAc (5:1) to furnish 11b (3.7 mg, 5.0%) as a dark yellow amorphous powder. Further elution with hexane-EtOAc (3:2) afforded 10 (39.6 mg, 65.8% recovered) as a pale yellow amorphous powder.
IR νmax (CHCl3) 2359, 1655, 1617, 1449, 1310, 1229, 1150, 1094 cm-1. δH (500 MHz) 0.91 and 0.92 (3H, t, J = 7.3 Hz, O(CH2)3CH3), 1.28-1.38 (2H, m, C3’H2), 1.48-1.56 (2H, m, C2’H2), 1.99 (3H, s, 8-CH3), 2.24 (3H, s, 3-CH3), 2.33 (1H, dd, J = 11.6, 2.0 Hz, 2-Hβ), 2.53 (3H, s, 11-CH3), 2.73 (1H, dd, J = 11.6, 3.4 Hz, 2-Hα), 3.36 (1H, d, J = 1.4 Hz, 5-H), 3.60 (1H, dt, J = 9.6, 6.8 Hz, OCH), 3.67 (1H, d, J = 2.2 Hz, 4-H), 3.75 (1H, dt, J = 9.6, 6.2 Hz, OCH), 3.86 (1H, br s, 6-H), 3.89 (1H, m, 1-H), 3.99 (3H, s, 9-OCH3). δC (125 MHz) 8.9 (8-CH3), 13.8 (4’-H3), 19.4 (C3’), 32.1 (C2’), 42.8 (11-CH3), 43.2 (3-CH3), 51.5 (C1), 51.7 (C2), 58.1 (C4), 60.1 (C5), 60.9 (9-OCH3), 68.6 (C6), 71.8 (OCH2), 115.4 (CN), 129.8 (C8), 137.4 (C10a), 139.9 (C6a), 155.2 (C9), 182.8 (C10), 186.2 (C7). EIMS m/z (%): 373 (M+, 20), 219 (15), 218 (100). HREIMS m/z 373.2000 (M+, calcd for C20H27N3O4, 373.2002).

(1R*,4S*,5S*,6S*)-6-Cyclohexyloxy-1,2,3,4,5,6,7,10-octahydro-9-methoxyl-3,8,11-trimethyl-7,10-dioxo-1,5-imino-3-benzazocine-4-carbonitrile (11c). A suspension of 10 (60.2 mg, 0.20 mmol) and SeO2 (24.4 mg, 0.22 mmol) in cyclohexanol (10.0 mL) was heated at 120 °C for 83 h. The reaction mixture was filtered and washed with CHCl3 (100 mL). The combined filtrates were concentrated in vacuo and the residue was diluted with water (30 mL) and extracted with CHCl3 (30 mL x 3). The combined extracts were washed with brine (30 mL), dried, and concentrated in vacuo to give a residue (103.7 mg), which was subjected to chromatography on silica gel with hexane-EtOAc (5:1) to furnish 11c (20.0 mg, 25.1%) as a dark yellow amorphous powder. Further elution with hexane-EtOAc (3:2) afforded 10 (24.8 mg, 41.2% recovered) as a pale yellow amorphous powder.
IR νmax (CHCl3) 2359 (weak), 1655, 1616, 1449, 1308, 1229, 1150, 1067 cm-1. δH (400 MHz) 1.21-1.38 (5H, m), 1.55 (1H, m), 1.74 (2H, br s), 1.81-1.84 (1H, m), 1.97 (3H, s, 8-CH3), 1.96-2.00 (1H, m), 2.05 (2H, br s), 2.25 (3H, s, 3-CH3), 2.33 (1H, dd, J = 11.7, 1.0 Hz, 2-Hβ), 2.56 (3H, s, 11-CH3), 2.75 (1H, dd, J = 11.7, 3.4 Hz, 2-Hα), 3.36 (1H, s, 5-H), 3.62 (1H, m, 1’-H), 3.72 (1H, d, J = 2.0 Hz, 4-H), 3.91 (1H, br s, 1-H), 3.99 (3H, s, 9-OCH3), 4.06 (1H, br s, 6-H). δC (100 MHz) 8.9 (8-CH3), 24.1 (CH2), 24.3 (CH2), 25.6 (CH2), 32.2 (CH2), 42.9 (11-CH3), 43.2 (3-CH3), 51.5 (C1), 51.7 (C2), 57.7 (C4), 60.8 (9-OCH3), 61.6 (C5), 66.5 (C6), 79.6 (OCH), 115.3 (CN), 129.9 (C8), 137.4 (C10a), 140.3 (C6a), 155.1 (C9), 182.8 (C10), 186.2 (C7). EIMS m/z (%): 399 (M+, 15), 219 (17), 218 (100). HREIMS m/z 399.2153 (M+, calcd for C22H29N3O4, 399.2158).

Preparation of mesylate 12: Methanesulfonyl chloride (MsCl, 39.1 μL, 0.5 mmol) was added dropwise to a stirred solution of 8 (30.6 mg, 0.1 mmol), TEA (70 μL, 0.5 mmol), and DMAP (6.0 mg, 0.05 mmol) in CH2Cl2 (2 mL) at 0 °C for 5 min, and the reaction mixture was stirred for 1 h at room temperature. The reaction mixture was diluted with water (15 mL) and extracted with CH2Cl2 ( 3 x 10 mL). The combined extracts were washed with brine (20 mL), dried, and concentrated in vacuo to give a residue (38.1 m), which was subjected to chromatography on silica gel with hexane-EtOAc (1:1) to furnish 13 (11.0 mg, 33.9%) as a dark yellow amorphous powder. Further elution with hexane-EtOAc (1:2) afforded 12 (3.0 mg, 7.8%) as a pale yellow amorphous powder.
(1R*,5S*,6S*)-2,3,5,6-Tetrahydro-6-methanesulfoxy-9-methoxyl-3,8,11-trimethyl-1,5-imino-3-benzazocine-4,7,10(1H)-trione (12):
IR νmax (CHCl3) 1661, 1616, 1449, 1364, 1261, 1177, 1096, 1022 cm-1. δH (400 MHz) 1.92 (3H, s, 8-CH3), 2.60 (3H, s, 11-CH3), 2.81 (3H, s, 3-CH3), 2.90 (1H, d, J = 12.7 Hz, 2-Hβ), 3.18 (3H, s, SO2CH3), 3.93 (1H, dd, J = 12.7, 4.6 Hz, 2-Hα), 3.95 (1H, br s, 5-H), 3.97 (3H, s, 9-OCH3), 4.03 (1H, d, J = 4.6 Hz, 1-H), 5.41 (1H, s, 6-H). δC (100 MHz) 9.0 (8-CH3), 34.3 (3-CH3), 38.5 (SO2CH3), 40.8 (11-CH3), 48.1 (C2), 49.8 (C1), 61.2 (9-OCH3), 64.8 (C5), 71.4 (C6), 129.6 (C8), 134.1 (C6a), 142.5 (C10a), 155.5 (C9), 164.4 (C4), 181.5 (C10), 184.3 (C7). EIMS m/z (%): 384 (M+, 18), 305 (18), 290 (29), 289 (36), 259 (14), 219 (18), 218 (100), 204 (13). HREIMS m/z 384.0992 (M+, calcd for C16H20N2O7S, 384.0991).
(1R*,5S*,6S*)-6-Chloro-2,3,5,6-tetrahydro-9-methoxyl-3,8,11-trimethyl-1,5-imino-3-benzazocine-4,7,10(1H)-trione (13): IR νmax (CHCl3) 1659, 1310, 1234, 1152, 1074 cm-1. δH (400 MHz) 1.92 (3H, s, 8-CH3), 2.63 (3H, s, 11-CH3), 2.82 (3H, s, 3-CH3), 2.90 (1H, d, J = 13.3 Hz, 2-Hβ), 3.58 (1H, br s, 5-H), 3.93 (3H, s, 9-OCH3), 3.94 (1H, dd, J = 13.3, 5.1 Hz, 2-Hα), 4.06 (1H, d, J = 5.1 Hz, 1-H), 5.04 (1H, s, 6-H). δC (100 MHz) 9.1 (8-CH3), 34.3 (3-CH3), 40.8 (11-CH3), 47.6 (C2), 49.7 (C1), 50.5 (C6), 61.1 (9-OCH3), 66.4 (C5), 130.0 (C8), 138.2 (C6a), 139.1 (C10a), 155.3 (C9), 165.4 (C4), 181.5 (C10), 184.1 (C7). EIMS m/z (%): 326 (M+ + 2, 12), 324 (M+, 35), 290 (12), 289 (34), 219 (14), 218 (100). HREIMS m/z 324.0876 (M+, calcd for C15H17N2O435Cl, 324.0877).

Cell growth inhibition assay: A single-cell suspension (2 x 103 cells/well) was added to serially diluted test compounds in a microplate. The cells were then cultured for 4 days. Cells were enumerated with a cell counting kit (DOJINDO, Osaka, Japan). IC50 was expressed as the concentration at which cell growth was inhibited by 50% compared with the untreated control.

ACKNOWLEDGEMENTS
This work was supported by a Grant-in-Aid (No. 23590019) for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. This work was also partially supported by the Japan Society for Promotion of Science (JSPS) Asia and Africa Science Platform Program (2010-2012), and a grant from the High-Tech Research Center Project, MEXT, Japan (No. S0801043). We are grateful to Dr. Takuo Tsukuda (Chugai Pharmaceutical Company, Kamakura Research Center) for conducting the cytotoxicity assay.

References

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