HETEROCYCLES

Paper | Special issue | Vol. 88, No. 1, 2014, pp. 363-375
Received, 14th June, 2013, Accepted, 24th June, 2013, Published online, 27th June, 2013.
DOI: 10.3987/COM-13-S(S)32

■ Isolation, Structure Characterization, and Synthesis of Stabilized 1,2,3,4-Tetrahydroisoquinoline Marine Natural Product from Potassium Cyanide Pretreated Thai Tunicate, Ecteinascidia thurstoni

Shinya Kimura, Waree Pangkruang, Masashi Yokoya, Amane Honda, Ploenthip Puthongking, Khanit Suwanborirux,* and Naoki Saito*

Department of Medicinal Chemistry, Pharmaceutical Chemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan

Abstract

A simple 1,2,3,4-tetrahydroisoquinoline marine natural product (2a) together with two known compounds (3 and 4) was isolated from a low polar fraction of the crude extract of KCN pretreated Thai tunicate Ecteinascidia thurstoni. The structure of 2a was determined by X-ray crystallographic analysis. We also reported an eight-step large-scale preparation of 2a from isovanillin (9).

INTRODUCTION
In our ongoing search for new anticancer metabolites from marine animals, we found that the Thai tunicate, Ecteinascidia thurstoni,1 produces ecteinascidin 743 (1a), a novel metabolite that has received considerable attention for its strong in vivo activity (Figure 1).2,3 However, there are few structure-activity relationship (SAR) studies of this fascinating target because it can be isolated in only trace amounts due to its instability, i.e., 1a easily decomposes during the extraction and isolation process.4,5 We solved this problem by converting 1a having a very unstable α-amino alcohol functionality at C-21 into a stable α-aminonitrile compound by pretreatment with KCN to generate 1b and its S-oxide 1c.1 The availability of 1b enabled us to prepare several ecteinascidin analogues having enhanced antitumor activity. We have already reported the preparation of 6’-O-ester derivatives6 and 2’-N-amide derivatives7 along with their biological activities.
In our continuing SAR studies of ecteinascidin marine natural products, we found simple 1,2,3,4-tetrahydroisoquinoline (2a) along with two known compounds (3 and 4). We present here the structure of 2a, which was elucidated by spectroscopic analyses and X-ray crystallographic analysis. We also report an eight-step synthesis of 2a from commercially available isovanillin (9) in 56% overall yield.

RESULTS AND DISCUSSION
The Thai tunicate E. thurstoni was collected by SCUBA diving around the southeastern coast of Phuket Island in October 2011. The collected animals (51.3 kg, wet weight) were homogenized with phosphate buffer solution to adjust pH to 7, and then aqueous KCN was added. The resulting mixture was macerated with methanol and a residue (32.28 g) was obtained by using our standard extraction and separation process. The residue was subjected to Sephadex LH-20 column chromatography to give ecteinascidins 770 (1b: 359.0 mg) and 786 (1c: 319.8 mg). The most lipophilic fraction (4.55 g) in the Sephadex LH-20 column chromatography was subjected to flash column chromatography on silica gel several times to give 2a (10.0 mg), 3 (8.7 mg), and 4 (3.6 mg) (Figure 2).
New compound 2a was confirmed to have the molecular formula C12H12N2O2 by HREIMS. All proton and carbon signals were assigned after extensive NMR measurements using COSY, HMQC, and HMBC techniques. The molecular formula of 2a indicated seven degrees of unsaturation and the detected resonance attributable to six olefinic carbons and one nitrile carbon in 2a accounted for five degrees of unsaturation. Thus, 2a was presumed to have two rings. It displayed simple 1H-NMR signals at δ 3.85 (3H, s, OCH3), δ 5.54 (1H, br s, D2O exchangeable OH proton), δ 3.70 and δ 3.71 (each 2H, s, methylene protons neighboring nitrogen), δ 6.51 and δ 6.81 (each 1H, s, aromatic protons), and δ 2.83 (4H, s like protons). Acetylation of 2a afforded monoacetate 2b, which confirmed the presence of one hydroxyl group. Both 1H- and 13C-NMR spectral data of 2a and 2b in Table 1 showed correspondence with the NMR data reported for the synthetic compound 2-(6,7-dimethoxy-3,4-dihydroisoquinoline- 2(1H)-yl)acetonitrile (5).8 The NMR data for 2a revealed the presence of only one methoxy group, suggesting that 2a is an O-demethyl analogue of 5. It was not possible to define the relative positions of the unassigned hydroxyl and methoxy groups at C-6 and C-7. Finally, the structure elucidation of 2a was completed by single crystal X-ray diffraction analysis (Figure 3).

Compound 3 (mp 198-199.5 oC) was identified as the commercially available indole-3-carboxaldehyde based on its characteristic spectroscopic data.9 Loliolide 4 (C11H16O3) was also identified from its spectroscopic data and by comparison with published data,10,11 including its optical rotation.12 (-)-Loliolide (4) was previously isolated from a variety of higher plants and a few marine animals, including the sea hare Dolabella ecaudata,13 the marine sponge Spheciospongia sp.,14 and the soft coral Sinularia capillosa.15 However, as D. ecaudata is known to concentrate algal metabolites, 4 isolated from it may be of algal origin.16
We were very interested in the structure of 2a because its 1,2,3,4-tetrahydroisoquinoline ring has the same substituents as the northwestern part of the ecteinascidin framework. It also possesses a smallest cyanonitrile compound that enables the formation of potent electrophilic iminium ion species A, which has been implicated in the formation of covalent bonds with DNA and bio macromolecules (Chart 1).17 In this context, we directed our attention towards establishing a practical method for the synthesis of 2a.

6-Hydroxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline (6a) was obtained by selective demethylation of corresponding dimethoxy compound 7a18 or 7b via 6b,19 which was prepared by the Pomeranz-Fritsch cyclization. As we were able to prepare 6-hydroxy-7-methoxyisoquinolinemethanol (8)20 in ten steps from vanillin in 26% overall yield using an isopropyl group for phenol protection, we set our sights on developing another synthetic process for large-scale preparation (Figure 4).21

Alkylation of isovanillin (9) with isopropyl bromide in the presence of K2CO3 in DMF followed by the Henry condensation gave β-nitrostyrene (11) in 94% yield (Scheme 1). This material was then employed in a two-step reduction (1. Hydride reduction of alkene; 2. Nitro group reduction with NaBH4-NiCl422) to afford phenethylamine (12) in 95% overall yield. The construction of 1,2,3,4-tetrahydroisoquinoline (14) from 12 was accomplished by means of our modified Pictet-Spengler reaction.23 The reaction of 12 with a large excess of paraformaldehyde in the presence of K2CO3 in ethanol at 25 oC for 5 h gave O,N-acetal (13),24 which was subsequently treated with trifluoroacetic acid (TFA) at 25 oC for 1.5 h to provide 14 in 92% overall yield. The structure of 14 was determined from the 1H-NMR spectrum that displayed signals of H-5 as a singlet at δ 6.62 ppm and H-8 as a singlet at δ 6.51 ppm. Treatment of 14 with TiCl4 in CH2Cl2 at 25 oC for 5 h gave 6a (84%), which was identical with authentic spectral data.18 Finally, the reaction of 6a with 37% aqueous formaldehyde and KCN in 1N HCl solution at 25 oC for 5 h gave 2a in 81% yield. 2a was confirmed to be identical with the natural product by direct comparison of IR, EIMS, 1H-NMR, and 13C-NMR data, together with their melting points. Furthermore, its acetate (2b) prepared from synthetic 2a was also identical with that of authentic 2b prepared from natural 2a.

CONCLUSION
We found a stable analog of 1,2,3,4-tetrahydroisoquinoline marine natural product (2a) along with two known compounds, indole-3-carboxaldehyde (3) and loliolide (4), from KCN pretreated E. thurstoni. In addition, we developed a practical method for the synthesis of simple natural product 2a from isovanillin (9) in 56% overall yield. Unfortunately, 2a did not show any cytotoxicity when tested in vitro using three representative human solid tumor cell lines (HCT116 human colon carcinoma, QG56 human lung carcinoma, and DU145 prostate carcinoma). Nevertheless, we remain very interested in the biosynthetic precursors of ecteinascidin marine natural products and will continue to conduct studies related to them.

EXPERIMENTAL
All melting points were determined with a Yanagimoto micro melting point apparatus and are uncorrected. Optical rotation was measured with a Horiba-SEPA polarimeter. IR spectra were obtained with a Shimadzu Prestige-21/IR Affinity-1 Fourier Transform Infrared (FT-IR) spectrometer. 1H- and 13C-NMR spectra were recorded on a JEOL JNM-LAMBDA 500 NMR spectrometer at 500 MHz for 1H and 125 MHz for 13C, and on a JEOL AL-400 spectrometer at 400 MHz for 1H and 100 MHz for 13C. NMR spectra were measured in CDCl3 or DMSO-d6 and the chemical shifts were recorded in δH values relative to (CH3)4Si (TMS) as the internal standard. Mass spectra were recorded on a 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.

Isolation of Three Minor Marine Natural Products from KCN Pretreated E. thurstoni. The tunicate Ecteinascidia thurstoni was collected by SCUBA around the southeastern coast of Phuket Island at depths range from 1 to 5 m in October 2011 and frozen until used. The collected animals (51.3 kg, wet weight) were homogenized with aqueous buffer solution and adjusted to pH between 6.8 and 7.2. 10% aqueous KCN was added and the resulting mixture was stirred at 30 oC for 5 h. Use of our standard extraction and separation process provided ecteinascidin 770 (1b: 359.0 mg, 7.0 x 10-5 % of wet weight) and ecteinascidin 786 (1c: 319.8 mg, 6.2 x 10-5 % of wet weight) from the crude extract (32.4 g). The most lipophilic fraction (4.55 g) was purified by silica gel flash column chromatography several times to give compounds 2 (10.0 mg), 3 (8.7 mg), and 4 (3.6 mg).
2-(6-Hydroxy-7-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)acetonitrile (2a). Recrystallization of colorless powder from benzene afforded 2a as pale yellow prisms, mp 165-166 oC. IR νmax (KBr) 3400～2600, 2247, 1611, 1535, 1427, 1402, 1382, 1348, 1290, 1252, 1215, 1204, 1130, 1118, 1094, 1024, 941, 862 cm-1. δH (400 MHz) 2.83 (4H, br s, 3-H2 and 4-H2), 3.70 (2H, s, NCH2), 3.71 (2H, s, 1-H2), 3.85 (3H, s, OCH3), 5.54 (1H, br s, 6-OH), 6.51 (1H, s, 8-H), 6.67 (1H, s, 5-H). δC (100 MHz) 28.3 (C-4), 46.0 (NCH2), 49.8 (C-3), 53.9 (C-1), 55.9 (OCH3), 108.5 (C-8), 114.2 (C-5), 114.7 (CN), 124.4 (C-10), 125.7 (C-9), 144.3 (C-6), 145.1 (C-7). EIMS m/z (%): 218 (M+, 74), 217 (58), 191 (8), 151 (11), 150 (100), 135 (11), 107 (10). HREIMS m/z 218.1058 (M+, calcd for C12H12N2O2, 218.1055).
Indole-3-carboxaldehyde (3). Recrystallization from EtOAc/hexane gave 3 as colorless needles, mp 198-199.5 oC (Lit.9 mp 193-198 oC). IR νmax (KBr) 3169, 2931, 1636, 1614, 1446, 1244, 1128, 1083 cm-1. δH (400 MHz, DMSO-d6) 7.20 (1H, dt, J = 6.8 and 1.5 Hz, C-5), 7.25 (1H, dt, J = 6.8 and 1.5 Hz, C-6), 7.50 (1H, dd, J = 6.8, 1.5 Hz, C-7), 8.07 (1H, dd, J = 6.8, 1.5 Hz, C-4), 8.29 (1H, s, C-2), 9.92 (1H, s, CHO), 12.13 (1H, br s, NH). EIMS m/z (%): 145 (M+, 100), 144 (87), 116 (18). Anal. Calcd for C9H7NO: C 74.47, H 4.86, N 9.65. Found: C 74.35, H 5.87, N 9.59.
Loliolide (4). Colorless amorphous powder. IR [α]D22 －64.4 (c, 0.06, MeOH); Lit.10 [α]D23 －67.9 (c, 0.88, MeOH).νmax (KBr) 3435, 2924, 1734, 1720, 1622, 1273, 1265, 1234, 1193, 1161, 1099, 1028, 962, 868 cm-1. δH (500 MHz) 1.27 and 1.47 (each 3H, s, 4-CH3 x 2), 1.54 (1H, dd, J = 14.7, 3.7 Hz, 5-H), 1.78 (3H, s, 7a-CH3), 1.78 (1H, dd, J = 13.3, 4.0 Hz, 7-H), 1.98 (1H, dt, J = 14.7, 2.9 Hz, 5-H), 2.46 (1H, dt, J = 13.3, 2.5 Hz, 7-H), 4.33 (1H, quint, J = 3.4 Hz, 6-H), 5.70 (1H, s, 3-H). δC (125 MHz) 26.5 (4-CH3), 27.0 (7a-CH3), 30.7 (4-CH3), 35.9 (C-4), 45.6 (C-7), 47.3 (C-5), 66.9 (C-6), 86.6 (C-7a), 113.0 (C-3), 171.8 (C-2), 182.3 (C-3a). EIMS m/z (%): 196 (M+, 40), 178 (95), 153 (24), 140 (56), 135 (35), 112 (23), 111 (100), 109 (27), 95 (24), 67 (21), 57 (20), 43 (48). HREIMS m/z 196.1105 (M+, calcd for C11H16O3, 196.1099).

Acetylation of 2a. A solution of 2a (3.9 mg, 0.018 mmol) and acetic anhydride (0.2 mL) in dry pyridine (1.0 mL) was stirred at 25 oC for 1 h. The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined extracts were washed with brine (10 mL), dried, and concentrated in vacuo to give a residue (6.3 mg), which was subjected to chromatography on a silica gel (2 g) column with hexane-EtOAc (2:1) as the eluent to give 2b (3.5 mg, 75.0%) as a pale yellow solid.
νmax (KBr) 2941, 2841, 2234, 1755, 1622, 1516, 1464, 1429, 1369, 1333, 1294, 1267, 1227, 1200, 1132, 1096, 1022, 914, 868 cm-1. δH (400 MHz) 2.30 (3H, s, COCH3), 2.84 (4H, br s, 3-H2 and 4-H2), 3.70 (2H, s, NCH2), 3.76 (2H, s, 1-H2), 3.79 (3H, s, OCH3), 6.62 (1H, s, 8-H), 6.80 (1H, s, 5-H). δC (100 MHz) 20.6 (COCH3), 28.1 (C-4), 45.9 (NCH2), 49.6 (C-3), 54.0 (C-1), 56.0 (OCH3), 110.2 (C-8), 114.6 (CN), 122.7 (C-5), 125.4 (C-10), 131.6 (C-9), 138.3 (C-6), 149.3 (C-7), 169.2 (CO). EIMS m/z (%): 260 (M+, 22), 218 (49), 217 (53), 191 (11), 151 (11), 150 (100). HREIMS m/z 260.1163 (M+, calcd for C14H16N2O3, 260.1161).

X-Ray Structure Determination of Compound 2a. Crystals of 2a (C12H14N2O2) belong to orthorhombic space group P212121(#19) with a = 6.4469(2) Å, b = 8.8558(2) Å, c = 19.8617(5) Å, V = 1133.95(5) Å3, Z = 4, and Dcalcd = 1.278 g/cm3. X-Ray intensities were measured with a Rigaku R-AXIS RAPID diffractometer in the graphite-monochromatic CuKα radiation mode (λ = 1.54187 Å). The final cycle of the full-matrix least-squares refinement was based on 2080 unique reflections (2θ<136.4o) and 146 variable parameters and converged with unweighted and weighted agreement factors of R = 0.0507, Rw = 0.1164, and R1 = 0.0445 for I > 2.0σ (I) data. The drawing of the molecule was made by ORTEP as shown in Figure 3. CCDC-No. 898,447 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif.

3-Isopropoxy-4-methoxybenzaldehyde (10). A mixture of isovanillin (9) (10.0 g, 65.7 mmol), isopropyl bromide (18.7 mL, 0.197 mmol), and anhydrous K2CO3 (27.4 g, 0.197 mmol) in DMF (100 mL) was heated at 80 oC for 1 h. After the reaction mixture was cooled to 25 oC, it was diluted with water (200 mL) and extracted with Et2O (300 mL x 3). The combined extracts were washed with brine (70 mL), dried, and concentrated in vacuo to give a residue, the chromatography of which on a silica gel column with hexane-EtOAc (9:1-4:1) gave 10 (13.0 g, quantitative yield) as a colorless oil.
νmax (CHCl3) 3017, 2936, 2841, 1982, 1686, 1585, 1508, 1435, 1267, 1240, 1132, 1111, 1024, 1001, 808 cm-1. δH (400 MHz) 1.40 (6H, d, J = 6.0 Hz, CH(CH3)2), 3.94 (3H, s, OCH3), 4.65 (1H, sept, J = 6.0 Hz, CH(CH3)2), 6.98 (1H, d, J = 8.3 Hz, 5-H), 7.42 (1H, d, J = 2.0 Hz, 2-H), 7.45 (1H, dd, J = 8.3, 2.0 Hz, 6-H), 9.84 (1H, s, CHO). δC (100 MHz) 21.9 (CH(CH3)2), 56.1 (OCH3), 71.3 (OCH), 110.9 (CH), 112.7 (CH), 126.4 (CH), 130.0 (C), 147.8 (C), 155.6 (C), 190.9 (CHO). EIMS m/z (%): 194 (M+, 26), 153 (10), 152 (100), 151 (99). HREIMS m/z 194.0938 (M+, calcd for C11H14O3, 194.0943).

3-Isopropoxy-4-methoxy-β-nitrostyrene (11). A stirred solution of 10 (12.80 g, 65.9 mmol) and NH4OAc (5.24 g, 65.9 mmol) in nitromethane (192 mL) was heated under reflux for 3 h. After the reaction mixture was cooled to 0 oC, it was diluted with water (200 mL) and extracted with Et2O (500 mL x 3). The combined extracts were washed with brine (200 mL), dried, and concentrated in vacuo to give a residue, recrystallization of which from MeOH gave 11 (13.87 g, 88.7%) as pale yellow prisms. The mother liquor was purified by column chromatography with hexane-EtOAc (9:1) to give an additional amount of 11 (0.87 g, 5.6%) (14.74 g, 94.3%), mp 117-118 oC.
νmax (KBr) 3111, 2981, 2968, 1626, 1593, 1514, 1493, 1433, 1333, 1261, 1231, 1168, 1138, 1113, 1026, 1005, 959, 806 cm-1. δH (400 MHz) 1.40 (6H, d, J = 6.2 Hz, CH(CH3)2), 3.92 (3H, s, OCH3), 4.57 (1H, sept, J = 6.2 Hz, CH(CH3)2), 6.92 (1H, d, J = 8.3 Hz, 5-H), 7.05 (1H, d, J = 2.0 Hz, 2-H), 7.17 (1H, dd, J = 8.3, 2.0 Hz, 6-H), 7.51 (1H, d, J = 13.7 Hz, ArCH=CHNO2), 7.95 (1H, d, J = 13.7 Hz, ArCH=CHNO2). δC (100 MHz) 21.9 (CH(CH3)2), 56.0 (OCH3), 71.8 (OCH), 111.9 (CH), 115.0 (CH), 122.7 (C), 124.6 (CH), 135.0 (CH), 139.4 (CH), 147.8 (C), 154.2 (C). EIMS m/z (%): 237 (M+, 75), 195 (100), 149 (15), 148 (64), 133 (27), 89 (14). HREIMS m/z 237.0997 (M+, calcd for C12H15NO4, 237.1001). Anal. Calcd for C12H15NO4: C 60.75, H 6.37, N 5.90. Found: C 60.83, H 6.45, N 5.84.

2-(3-Isopropoxy-4-methoxyphenyl)ethylamine (12). NaBH4 (8.03 g, 212 mmol) was added to a mixture of 11 (9.50 g, 65.9 mmol) and silica gel (96.20 g, 0.16 mol) in 2-propanol (180 mL) and CHCl3 (600 mL) over 15 min at 0 oC, and this mixture was vigorously stirred at 25 oC for 4 h. The reaction mixture was diluted with acetic acid (50 mL) at 0 oC. Then, a large amount of inorganic precipitate was removed by filtration and washing with CHCl3 and MeOH. The combined filtrates were concentrated in vacuo and the residue was diluted with 5% aqueous NaHCO3 solution (500 mL) and then extracted with CH2Cl2 (500 mL x 3). The combined extracts were washed with brine (500 mL), dried, and concentrated in vacuo to give a nitroalkane as a colorless oil, which was used without further purification. An analytical sample was obtained by column chromatography (elution with 9:1 hexane-EtOAc).
νmax (KBr) 2974, 1591, 1551, 1518, 1443, 1377, 1258, 1234, 1142, 1117, 1028, 964, 874 cm-1. δH (400 MHz) 1.36 (6H, d, J = 6.2 Hz, CH(CH3)2), 3.24 (2H, t, J = 7.3 Hz, ArCH2), 3.83 (3H, s, OCH3), 4.50 (1H, sept, J = 6.2 Hz, CH(CH3)2), 4.57 (2H, t, J = 7.3 Hz, CH2NO2), 6.74 (1H, d, J = 2.0 Hz, 2-H), 6.75 (1H, dd, J = 8.8, 2.0 Hz, 6-H), 7.82 (1H, d, J = 8.8 Hz, 5-H). δC (100 MHz) 22.0 (CH(CH3)2), 33.0 (ArCH2), 56.0 (OCH3), 71.6 (OCH), 76.6 (CH2NO2), 112.3 (CH), 116.5 (CH), 121.1 (CH), 128.0 (C), 147.5 (C), 149.8 (C). EIMS m/z (%): 239 (M+, 26), 197 (9), 151 (25), 150 (100), 135 (14), 91 (13). HREIMS m/z 239.1155 (M+, calcd for C12H17NO4, 239.1158).
A mixture of the above product and nickel chloride hexahydrate (38.80 g, 0.16 mol) in MeOH/THF (2:1, 690 mL) was stirred at -17 oC. NaBH4 (12.90 g, 368 mmol) was added to the above mixture over 30 min and stirring was continued at the same temperature for 30 min. The reaction mixture was diluted with water (450 mL) and filtered through a Celite pad. The filter cake was carefully washed with CHCl3 (200 mL) and the combined filtrates were concentrated in vacuo. The residue was diluted with benzene (300 mL) and extracted with 1N aqueous HCl (300 mL x 3). The combined aqueous layer was made alkaline with concentrated NH4OH and then extracted with MeOH/CHCl3 (1:19, 700 mL x 3). The combined extracts were washed with brine (500 mL), dried, and concentrated in vacuo. The residue was subjected to SiO2 chromatography with concentrated NH4OH-MeOH-CHCl3 (1 : 9 : 90) as eluent to give amine (12: 8.00 g, 95.0%) as a pale yellow oil.
νmax (CHCl3) 3011, 2980, 2936, 1587, 1512, 1443, 1423, 1373, 1260, 1138, 1109, 1030, 806 cm-1. δH (400 MHz) 1.36 (6H, d, J = 6.2 Hz, CH(CH3)2), 1.54 (2H, br s, NH2), 2.67 (2H, t, J = 6.8 Hz, ArCH2), 2.93 (2H, t, J = 6.8 Hz, CH2NO2), 3.83 (3H, s, OCH3), 4.52 (1H, sept, J = 6.2 Hz, CH(CH3)2), 6.74 (1H, dd, J = 7.8, 2.0 Hz, 6-H), 6.75 (1H, d, J = 2.0 Hz, 2-H), 6.82 (1H, d, J = 7.8 Hz, 5-H). δC (100 MHz) 22.1 (CH(CH3)2), 39.4 (ArCH2), 43.6 (CH2NH2), 56.0 (OCH3), 71.4 (OCH), 112.2 (CH), 116.9 (CH), 121.3 (CH), 132.3 (C), 147.2 (C), 148.9 (C). EIMS m/z (%): 209 (M+, 26), 180 (35), 179 (13), 138 (100), 137 (45), 123 (15). HREIMS m/z 209.1415 (M+, calcd for C12H19NO2, 209.1416).

6-Isopropoxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline (14). A mixture of amine 12 (7.49 g, 35.8 mmol), paraformaldehyde (3.39 g, 107 mmol), and K2CO3 (19.9 g, 143 mmol) in EtOH (300 mL) was stirred at 25 oC for 5 h. After the inorganic precipitate was removed by filtration, the filter cake was washed with EtOH (100 mL) and the combined filtrates were concentrated in vacuo to afford O,N-acetal (13),23 which was used in the next step without further purification.
A solution of the above product in trifluoroacetic acid (300 mL) was stirred at 25 oC for 1.5 h. The reaction mixture was poured into water (500 mL), pH was brought to 9-10 with concentrated NH4OH, and extraction was carried out with CHCl3 (700 mL x 4). The combined extracts were washed with brine (500 mL), dried, and concentrated in vacuo. The residue was subjected to silica gel chromatography with concentrated NH4OH-MeOH-CHCl3 (1 : 9 : 90) as eluent to give amine (14: 7.25 g, 92.0%) as a pale yellow oil.
νmax (CHCl3) 3017, 2978, 2933, 2837, 1609, 1514, 1466, 1383, 1327, 1300, 1254, 1206, 1103, 1026, 800 cm-1. δH (400 MHz) 1.35 (6H, d, J = 6.2 Hz, CH(CH3)2), 1.78 (1H, br s, NH), 2.69 (2H, t, J = 5.9 Hz, 4-H2), 3.11 (2H, t, J = 5.9 Hz, 3-H2), 3.81 (3H, s, OCH3), 3.93 (2H, s, 1-H2), 4.46 (1H, sept, J = 6.2 Hz, CH(CH3)2), 6.51 (1H, s, 8-H), 6.62 (1H, s, 5-H). δC (100 MHz) 22.1 (CH(CH3)2), 28.6 (C-4), 44.0 (C-3), 48.0 (C-1), 56.0 (OCH3), 71.6 (OCH), 109.8 (C-5), 117.0 (C-8), 126.5 (C), 128.5 (C), 145.6 (C), 148.8 (C). EIMS m/z (%): 221 (M+, 50), 179 (13), 178 (66), 151 (14), 150 (100), 135 (9). HREIMS m/z 221.1422 (M+, calcd for C13H19NO2, 221.1416).

6-Hydroxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline (6a). A stirred solution of 14 (1.10 g, 4.96 mmol) in dry CH2Cl2 (100 mL) was cooled in ice water and TiCl4 (1.6 mL, 14.87 mmol) was added dropwise for 10 min. This mixture was then stirred at 25 oC for 5 h. After the reaction mixture was poured into water (400 mL), pH was brought to 7 with saturated aqueous NaHCO3 solution. The mixture was extracted with MeOH/CHCl3 (1 : 9; 600 mL x 4). The combined extracts were washed with brine (600 mL), dried, and concentrated in vacuo to give a solid, recrystallization of which from EtOH afforded 6a (774.0 mg, 83.8%) as pale yellow needles, mp 211-212 oC (Lit.,25 mp 214-216 oC).
νmax (KBr) 3289, 2986, 2914, 2833, 1604, 1530, 1469, 1456, 1412, 1352, 1325, 1306, 1273, 1248, 1221, 1115, 1070, 1031, 962, 856, 839, 814 cm-1. δH (400 MHz, DMSO-d6) 2.69 (2H, m, 4-H2, the signals overlapped with solvent signal), 2.85 (2H, t, J = 5.9 Hz, 3-H2), 3.68 (3H, s, OCH3), 3.70 (2H, s, 1-H2), 6.43 (1H, s, 8-H), 6.51 (1H, s, 5-H). δC (100 MHz, DMSO-d6) 28.0 (C-4), 43.5 (C-3), 47.4 (C-1), 55.6 (OCH3), 109.9 (C-5), 115.6 (C-8), 126.6 (C), 126.8 (C), 144.4 (C), 145.7 (C). EIMS m/z (%): 179 (M+, 72), 178 (71), 151 (15), 150 (100), 135 (21), 107 (15). HREIMS m/z 179.0941 (M+, calcd for C10H13NO2, 179.0941). Anal. Calcd for C10H12NO2: C 67.02, H 7.31, N 7.82. Found: C 67.19, H 7.30, N 7.73.

2-Cyanomethyl-6-hydroxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline (2a). A stirred solution of 6a (400.0 mg, 2.232 mmol) and 37% aqueous formaldehyde (0.5 mL, 6.696 mml) in 1N aqueous HCl (3.2 mL) solution was cooled on ice-water. KCN (741.5 mg, 11.16 mmol) was added in one portion and the resulting mixture was stirred at 25 oC for 5 h. The reaction mixture was diluted with water (7.0 mL) and extracted with CHCl3 (20 mL x 3). The combined extracts were washed with brine (20 mL), dried, and concentrated in vacuo to give a residue (409.8 mg). The aqueous layer was neutralized with 1N aqueous HCl solution and extracted with MeOH/CHCl3 (1:9; 20 mL x 4). The combined extracts were washed with brine, dried, and concentrated in vacuo to give an additional residue (94.1 mg). The combined crude products were recrystallized from benzene to give 2a (396.0 mg, 81.0%) as dark yellow prisms, mp 165.5-167 oC.
νmax (KBr) 3400～2600, 2247, 1611, 1535, 1427, 1402, 1383, 1348, 1325, 1290, 1252, 1215, 1203, 1130, 1119, 1094, 1024, 941, 862 cm-1. δH (400 MHz) 2.84 (4H, br s, 3-H2 and 4-H2), 3.70 (2H, s, NCH2), 3.70 (2H, s, 1-H2), 3.85 (3H, s, OCH3), 5.52 (1H, br s, OH), 6.51 (1H, s, 8-H), 6.67 (1H, s, 5-H). δC (100 MHz) 28.3 (C-4), 45.9 (NCH2), 49.9 (C-3), 54.1 (C-1), 56.0 (OCH3), 108.6 (C-8), 114.2 (C-5), 114.7 (CN), 124.4 and 125.7 (C-9 and C-10), 144.3 (C-6), 145.1 (C-7). EIMS m/z (%): 218 (M+, 57), 217 (54), 151 (12), 150 (100), 135 (13), 107 (9). HREIMS m/z 218.1058 (M+, calcd for C12H14N2O2, 218.1055). Anal. Calcd for C12H14N2O2: C 66.03, H 6.47, N 12.84. Found: C 66.40, H 6.45, N 12.76.

6-Acetoxy-2-cyanomethyl-7-methoxy-1,2,3,4-tetrahydroisoquinoline (2b). A solution of 2a (50.0 mg, 0.23 mmol) and acetic anhydride (0.3 mL) in dry pyridine (1.2 mL) was stirred at 0 oC for 7 h. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (25 mL x 3). The combined extracts were washed with brine (20 mL), dried, and concentrated in vacuo to give a solid, recrystallization of which from Et2O afforded 2b (55.8 mg, 94.0%) as colorless prisms, mp 124-125 oC.
νmax (KBr) 2943, 2843, 2814, 2229, 1761, 1622, 1520, 1468, 1433, 1368, 1333, 1292, 1267, 1226, 1206, 1196, 1128, 1096, 1022, 914, 868 cm-1. δH (500 MHz) 2.30 (3H, s, COCH3), 2.84 (4H, br s, 3-H2 and 4-H2), 3.70 (2H, s, NCH2), 3.76 (2H, s, 1-H2), 3.79 (3H, s, OCH3), 6.62 (1H, s, 8-H), 6.80 (1H, s, 5-H). δC (125 MHz) 20.6 (COCH3), 28.1 (C-4), 45.9 (NCH2), 49.6 (C-3), 54.0 (C-1), 56.0 (OCH3), 112.7 (C-8), 114.6 (CN), 122.7 (C-5), 125.4 (C-10), 131.6 (C-9), 138.4 (C-6), 149.3 (C-7), 169.2 (CO). EIMS m/z (%): 260 (M+, 22), 218 (49), 217 (53), 191 (11), 151 (11), 150 (100). HREIMS m/z 260.1160 (M+, calcd for C14H16N2O3, 260.1161). Anal. Calcd for C14H16N2O3: C 64.60, H 6.20, N 10.76. Found: C 64.76, H 6.28, N 10.71.

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 the Promotion of Science (JSPS) Asia-Africa Science Platform Program (2010-2012). We would like to thank Dr. Kazuhiko Takatori of Meiji Pharmaceutical University for the X-ray crystallographic analysis of 2a.

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