HETEROCYCLES

Paper | Special issue | Vol. 84, No. 1, 2012, pp. 587-595
Received, 1st June, 2011, Accepted, 20th July, 2011, Published online, 29th July, 2011.
DOI: 10.3987/COM-11-S(P)27

■ Improved Synthesis of the New Furo[3,2-h]isoquinoline Alkaloids TMC-120B and TMC-120A, and Their Inhibitory Activities against IFN-γ and IL-4 Production

Tominari Choshi,* Teppei Kumemura, Haruto Fujioka, Yuhzo Hieda, and Satoshi Hibino*

Graduate School of Pharmacy and Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan

Abstract

In our synthetic route of TMC-120B (2), the synthetic intermediate, methyl [7-(methoxycarbonyl)-3-methyl-8-isoquinolyloxy]acetate (8) was newly synthesized in seven steps along with an improvement of the overall yield. The catalytic hydrogenation of TMC-120B (2) was also improved. Inhibition of interferon-γ and interleukin-4 production by TMC-120A (1), TMC-120B (2), and their derivative 4 was evaluated. The results indicated that these compounds are selective inhibitors of Th1 cell function.

INTRODUCTION
In our research, we have studied the synthesis of biologically active fused heteroaromatic compounds, including natural products, through the construction of functionalized frameworks based on the thermal electrocyclic reaction1,2 of a 6π-electron system or an aza 6π-electron system incorporating a principal aromatic or heteroaromatic moiety3-6 Recently, we selected three new furo[3,2-h]isoquinoline alkaloids, TMC-120A (1), TMC-120B (2), and TMC-120C (3), isolated from Aspergillus ustus TC1118,7,8 as target compounds (Scheme 1). We performed the first total synthesis of TMC-120B (2) by constructing the appropriate 3,7,8-trisubstituted isoquinoline framework 8 in 13 steps (7% overall yield), starting from 2,4-bis(methoxymethyloxy)benzaldehyde (5), based on the microwave-assisted thermal electrocyclic reaction5,6 of the aza 6π-electron system 6, followed by the formation of a furanone ring 9 and the introduction of an isopropylidene moiety in 16 steps (2.5% overall yield) (Scheme 2).9,10

In the present paper, we describe an improved synthesis of TMC-120B (2) through a new synthetic route of methyl [7-(methoxycarbonyl)-3-methyl-8-isoquinolyloxy]acetate 8, and TMC-120A (1) from 2. In addition, an aromatic benzylidene derivative 4 was synthesized by our reported procedure10 to examine inhibitory activities against interferon-γ (IFN-γ) and interleukin (IL)-4 production together with aliphatic derivatives 1 and 2. These results are also described here.

RESULTS AND DISCUSSION
We attempted a short-step synthesis of the 3,7,8-trisubstituted isoquinoline 8, starting from the known methyl 3-formyl-2,4-dihydroxybenzoate (10)11 as illustrated in Scheme 3.
Initially, a selective monobenzylation of the benzoate 10 with benzyl bromide and NaH in DMF at room temperature afforded the monobenzyl ether 11 on a less hindered hydroxy group at the 6-position, which was followed by further alkylation with methyl bromoacetate in the presence of K2CO3 at 50 oC to give the diester 12. Debenzylation of 12 with 10% Pd-C under H2 atmosphere smoothly reproduced the 4-hydroxybenzoate 13. After conversion of the phenol 13 into the triflate 14 with trifluoromethansulfonic anhydride (Tf2O) and pyridine at 0 oC, the Stille coupling reaction of the triflate 14 with tributyl propenylstannane and PdCl2(PPh3)2 in the presence of Et4NCl in DMF at 80 oC yielded the 2-propenylbenzaldehyde 15. Subsequent treatment of 15 with hydroxylamine methyl ether in EtOH at reflux temperature produced the oxime ether 16 as an aza 6π-electron system, which was then subjected to the microwave-assisted electrocyclic reaction9,10 in 1,2-dichlorobenzene at 180 oC to give the expected 3,7,8-trisubstituted isoquinoline 8 in 47% yield from 15. The microwave-assisted thermal electrocyclic reaction of 16 without purification was attempted, but it was assumed that the yield of 8 would be almost the same as that of a similar reaction of 7 from 6, as reported previously.9,10 The isoquinoline 8 was obtained in seven steps from 10 in 16% yield.

Although, Kohno and coworkers7,8 reported that TMC-120A (1) was obtained from TMC-120B (2) in 36% yield with 10% Pd-C, we further attempted the conversion of TMC-120B (2) to TMC-120A (1). The catalytic reduction of TMC-120B (2) with 10% Pd-C under H2 atmosphere in EtOAc gave the racemic TMC-120A (1) in 99% yield. Asymmetric reduction of 2 with several chiral reagents failed. Furthermore, the benzylidene derivative 4 of TMC-120B (2) was synthesized by the reaction of the furanone 99,10 and 3,5-dimethoxybenzaldehyde with LDA, followed by treatment with methanesulfonyl chloride (MsCl) in the presence of DMAP in pyridine, to evaluate the biologic activities.

We tested the effect of TMC-120A (1), B (2), and 4 in several cellular assay systems and found that they have a potent inhibitory effect on IFN-γ production. IFN-γ production was induced by stimulating ovalbumin (OVA)–specific murine T cells with OVA peptide antigen. IFN-γ was detected in a peptide concentration-dependent manner, and was strongly inhibited by TMC-120A (1), B (2), and 4 at a concentration of 3 µM (Table 1). The peptide stimulation simultaneously induced IL-4 production. The effects of TMC-120A (1), B (2), and 4 on IL-4 production were relatively weak, in that inhibition was scarce at 3 µM (Table 2). All these compounds at a concentration of 3 µM showed no effect on the proliferative response as assessed by [3H] thymidine uptake, indicating that the suppression of IFN-γ production is not due to toxic effects of the compounds (Table 3).

CONCLUSION
An alternative synthesis of the synthetic intermediate 8 was improved in a seven-step sequence, and the overall yield of 8 increased to 16% from 10 by converting the starting material. In addition, the reduction of the C=C double bond of TMC-120B (2) according to the procedure reported by the Kohno group7,8 was achieved in excellent yield. As a result, the formal total synthesis of TMC-120B (2) together with TMC-120A (1) was completed.
The characteristic feature of these compounds is the selective inhibition of IFN-γ production compared with IL-4 production. Because IFN-γ and IL-4 represent Th1 and Th2 cytokines, respectively, it is possible that these compounds are selective inhibitors of Th1 cell function.

EXPERIMENTAL
All melting points were measured with a Yanagimoto micro-melting point apparatus MP-500D and are uncorrected. IR spectra were recorded with a Shimadzu FT-IR-8500 spectrophotometer. 1H-NMR (300 MHz) and 13C-NMR (75 MHz) spectra were taken with a JEOL AL-300 instrument using tetramethylsilane as an internal standard. Mass spectra (MS) were determined on a JEOL MStation 700 spectrometer. The microwave irradiation was carried out at 180 W and 2450 MHz with “Discover” of CEM corporation. All air sensitive reactions were run under an argon atmosphere. Solvents were distilled by normal methods (THF dried over sodium benzophenone ketyl, CH2Cl2 dried over CaH2, DMF dried over CaH2). Silica gel 60PF254 (60-100 mesh, Merck Art 7744) was used for column chromatography.

Methyl 4-benzyloxy-3-formyl-2-hydroxybenzoate (11)
A solution of the phenol 10 (475 mg, 2.42 mmol) in DMF (5 mL) was added to a suspension of 60% NaH (174 mg, 4.36 mmol) in DMF (12 mL) under cooling with ice-water. After being stirred at the room temperature for 30 min, benzyl bromide (0.35 mL, 2.91 mmol) was added under cooling with ice-water. The mixture was stirred at room temperature for 12 h, which was quenched with an aqueous NH4Cl solution (saturated). The mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 20 g) using EtOAc-hexane (3:7) as an eluent to give the ester 11 (430 mg, 62%), mp 90-91 oC (Et2O-hexane). IR (ATR) ν: 1720, 1619 cm-1.1H-NMR (CDCl3) δ: 3.92 (3H, s), 5.23 (3H, s), 6.54 (1H, d, J= 8.8 Hz), 7.36-7.43 (5H, m), 8.95 (1H, d, J=8.8 Hz), 10.46 (1H, s), 12.77 (1H, s). MS (EI) m/z: 286 (M+). HR-MS (EI) m/z: 286.0834 (M+) (Calcd for C16H14O5: 286.0841).

Methyl [5-benzyloxy-6-formyl-2-(methoxycarbonyl)phenoxy]acetate (12)
A mixture of phenol 11 (535 mg, 1.87 mmol), K2CO3 (774 mg, 5.60 mmol), and methyl bromoacetate (0.37 mL, 3.74 mmol) in DMF (15 mL) were heated at 50 oC for 12 h. After being cooled to ambient temperature, which was quenched with water. The mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 30 g) using EtOAc-hexane (3:7) as an eluent to give the acetate 12 (519 mg, 78%), mp 108-109 oC (Et2O-hexane). IR (ATR) ν: 1754, 1720, 1681 cm-1.1H-NMR (CDCl3) δ: 3.83 (3H, s), 3.87 (3H, s), 4.75 (2H, s), 5.23 (2H, s), 6.89 (1H, d, J=9.2 Hz), 7.34-7.45 (5H, m), 8.05 (1H, d, J=9.2 Hz), 10.52 (1H, s). MS (EI) m/z: 358 (M+). HR-MS (EI) m/z: 358.1053 (M+) (Calcd for C19H18O7: 358.1053).

Methyl [6-formyl-5-hydroxy-2-(methoxycarbonyl)phenoxy]acetate (13)
A mixture of acetate 12 (100 mg, 0.28 mmol) and 10% Pd-C (10 mg) in EtOAc (10 mL) was stirred at room temperature for 2 h under H2 atmosphere. The reaction mixture was filtrated through a Celite pad, and the Celite pad was washed with EtOAc. The combined EtOAc was concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 15 g) using EtOAc-hexane (1:4) as an eluent to give the phenol 13 (73 mg, 98%), mp 96-97 oC (MeOH). IR (ATR) ν: 1751, 1716, 1650 cm-1. 1H-NMR (CDCl3) δ: 3.79 (3H, s), 3.90 (3H, s), 4.78 (2H, s), 6.78 (1H, d, J=9.2 Hz), 8.09 (1H, d, J= 9.2 Hz), 10.61 (1H, s), 12.38 (1H, s). MS (EI) m/z: 268 (M+). HR-MS (EI) m/z: 268.0596 (M+) (Calcd for C12H12O7: 268.0583).

Methyl [6-formyl-2-(methoxycarbonyl)-5-(trifluoromethanesulfonyloxy)phenoxy]acetate (14)
Tf2O (1.4 mL, 8.28 mmol) was added to a solution of the phenol 13 (1.9 g, 6.90 mmol) and pyridine (1.67 mL, 20.7 mmol) in CH2Cl2 (40 mL) under cooling with ice-water. After stirring at the same temperature for 1 h, the reaction mixture was quenched with saturated aqueous NaHCO3 solution. The mixture was extracted with CH2Cl2. The organic layer was washed with water, brine, and dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 40 g) using EtOAc-hexane (1:4) as an eluent to give the triflate 14 (2.2 g, 80%), mp 60-61 oC (Et2O-hexane). IR (ATR) ν: 1727, 1697, 1589, 1430, 1203 cm-1. 1H-NMR (CDCl3) δ: 3.79 (3H, s), 3.96 (3H, s), 4.79 (2H, s), 7.20 (1H, d, J=8.8 Hz), 8.17 (1H, d, J=8.8 Hz), 10.55 (1H, s). MS (EI) m/z: 400 (M+). HR-MS (EI) m/z: 400.009 (M+) (Calcd for C13H11F3O9S: 400.0076).

Methyl [6-formyl-2-(methoxycarbonyl)-5-(prop-1-en-1-yl)phenoxy]acetate (15)
A mixture of the triflate 14 (1.5 g, 3.71 mmol), tributyl(1-propenyl)tin (1.5 g, 4.45 mmol), Et4NCl (738 mg, 4.45 mmol) and PdCl2(PPh3)2 (26 mg, 0.037 mmol) in DMF (20 mL) was heated at 80 oC for 50 min. After being cooled to ambient temperature, an aqueous 30% KF solution (20 mL) was added to the reaction mixture. The mixture was stirred at room temperature for 30 min, which was filtered through a Celite pad, and then the Celite pad was washed with EtOAc. The combined filtrate was extracted with EtOAc, which was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 30 g) using EtOAc-hexane (1:9) as an eluent to give the alkene 15 (1.0 g, 94%), mp 87-88 oC (EtOAc-hexane). IR (ATR) ν: 1758, 1724, 1697 cm-1. 1H-NMR (CDCl3) δ: 1.74 (3/2H, dd, J=1.8, 7.3 Hz), 1.96 (3/2H, dd, J= 1.6, 6.6 Hz), 3.81 (3/2H, s), 3.82 (3/2H, s), 3.91 (3/2H, s), 3.93 (3/2H, s), 4.71 (2/2H, s), 4.75 (2/2H, s), 5.99 (1/2H, dq, J=7.3, 11.7 Hz), 6.31 (1/2H, dq, J=6.6, 15.8 Hz), 6.81 (1/2H, dd, 1.8, 11.7 Hz), 7.17 (1/2H, d, J=8.1 Hz), 7.20 (1/2H, dd, J=1.6, 15.8 Hz), 7.36 (1/2H, d, J=8.4 Hz), 7.99 (1/2H, d, J=8.4 Hz), 8.02 (1/2H, d, J= 8.1 Hz), 10.55 (1/2H, s), 10.65 (1/2H, s). MS (EI) m/z: 292 (M+). HR-MS (EI) m/z: 292.0938 (M+) (Calcd for C15H16O6: 292.0947).

Methyl [7-(methoxycarbonyl)-3-methyl-8-isoquinolyloxy]acetate (8)
A mixture of alkene 15 (1.7 g, 5.86 mmol), MeONH2・HCl (734 mg, 8.79 mmol), and AcONa (720 mg, 8.79 mmol) in EtOH (30 mL) was heated at 80 oC for 1 h. After being cooled to ambient temperature, the mixture was quenched with water. After removal of solvent under reduced pressure, the residue was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. A solution of the crude oxime ether in 1,2-dichlorobenzene (55 mL) was heated at 180 oC for 15 min under microwave irradiation (180 W). After removal of solvent under reduced pressure, the residue was purified by column chromatography (silica gel, 40 g) using EtOAc-hexane (4:1) as an eluent to give the isoquinoline 8 (801 mg, 47%), mp 92-93 oC (MeOH). IR (ATR) ν: 1712, 1623 cm-1. 1H-NMR (CDCl3) δ: 2.73 (3H, s), 3.86 (3H, s), 3.97 (3H, s), 4.84 (2H, s), 7.48 (1H, s), 7.52 (1H, d, J=8.6 Hz), 8.05 (1H, d, J=8.6 Hz), 9.73 (1H, s). 13C-NMR (CDCl3) δ: 169.0, 165.5, 157.8, 154.8, 149.0, 140.0, 131.5, 122.3, 121.8, 119.0, 118.1, 72.5, 52.5, 52.3, 24.4. MS (EI) m/z: 289 (M+). HR-MS (EI) m/z: 289.0919 (M+) (Calcd for C15H15NO5: 289.0950).

TMC-120A (1)
A mixture of TMC-120B (2) (8.5 mg, 0.036 mmol) and 10% Pd-C (2 mg) in EtOAc (3 mL) were stirred at room temperature for 1 h under H2 atmosphere. The reaction mixture was filtrated through a Celite pad, and the Celite pad was washed with EtOAc. The combined EtOAc was concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 10 g) using EtOAc-hexane (2:3) as an eluent to give TMC-120A (1) (8.5 mg, 99%), mp 110-112 oC (MeOH) (Lit.,7,8 mp 115-116 oC). IR (ATR) ν: 1708 cm-1. 1H-NMR (CDCl3) δ: 0.93 (3H, d, J=7.0 Hz), 1.26 (3H, d, J=7.0 Hz), 2.47-2.54 (1H, m), 2.76 (3H, s), 4.69 (1H, d, J=3.7 Hz), 7.32 (1H, d, J=8.4 Hz), 7.55 (1H, s), 7.72 (1H, d, J=8.4 Hz), 9.56 (1H, s). 13C-NMR (CDCl3) δ: 199.6, 173.9, 157.5, 146.6, 142.3, 123.9, 120.1, 119.5, 117.5, 115.2, 91.5, 31.1, 24.7, 18.8, 15.7. MS (EI) m/z: 241 (M+). HR-MS (EI) m/z: 241.1104 (M+) (Calcd for C15H15O2: 241.1103).

7-Methyl-2-[(3,5-dimethoxyphenyl)methylidene]furo[3,2-h]isoquinoline-3-one (4)
A solution of furanone 9 (20 mg, 0.10 mmol) in THF (1.5 mL) was added to a solution of LDA [prepared from i-Pr2NH (0.070 mL, 0.40 mmol) and n-BuLi (2.6 M in Hexane, 0.15 mL, 0.40 mmol) in THF (1.5 mL)] at -78 oC. After stirring at the same temperature for 30 min, 3,5-dimethoxybenzaldehyde (100 mg, 0.60 mmol) in THF (1 mL) was added to the reaction mixture, the mixture was allowed to stand slowly to rt, and further stirred for 4 h. The reaction mixture was quenched with saturated NH4Cl aqueous solution, and then extracted with EtOAc. The EtOAc was washed with water and brine, dried over Na2SO4, and concentrated under reduced pressure. To a mixture of the residue and DMAP (3 mg, 0.015 mmol) in pyridine (2 mL) was added methanesulfonyl chloride (0.077 mL, 1.0 mmol) under cooling with ice-water. After stirring at room temperature for 1 h, the reaction mixture was quenched with water, and then extracted with EtOAc. The EtOAc was washed with water and brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel 10 g) using EtOAc-hexane (4:1 v/v) as an eluent to give the furoisoquinoline 4 (21 mg, 61%), mp 207-209 oC (MeOH). IR (ATR) ν: 1697 cm-1. 1H-NMR (CDCl3) δ: 2.78 (9/4H, s), 3.42 (3/4H, s), 3.90 (6/4H, s), 3.91 (18/4H, s), 6.58 (3/4H, t, J=2.2 Hz), 6.78 (1/4H, t, J=2.2 Hz), 6.96 (4/4H, s), 7.18 (6/4H, d, J=2.2 Hz), 7.34 (2/4H, d, J=2.2 Hz), 7.49 (3/4H, d, J=8.4 Hz), 7.61 (3/4H, s), 7.65 (1/4H, s), 7.68 (1/4H, d, J=8.8 Hz), 7.89 (3/4H, d, J=8.4 Hz), 8.01 (1/4H, d, J=8.8 Hz), 9.63 (3/4H, s), 9.69 (1/4H, s). 13C-NMR (CDCl3) δ: 183.5, 182.9, 165.9, 165.6, 161.0, 160.9, 157.3, 155.4, 154.9, 147.3, 147.0, 146.0, 138.2, 138.1, 133.5, 133.5, 124.2, 124.1, 122.9, 119.7, 119.6, 119.3, 119.1, 117.6, 117.5, 114.6, 114.4, 109.8, 109.4, 107.5, 106.5, 103.5, 55.7, 55.56., 24.8, 24.6. MS (EI) m/z: 347 (M+). HR-MS (EI) m/z: 347.1175 (M+) (Calcd for C21H17NO4: 347.1158).

Effect on T cell activation
To evaluate antigen-dependent activation of murine T cells, a mixture of lymph node and spleen cells were prepared from DO11.10 transgenic mice expressing transgenic T cell receptors specific for OVA 323-339 epitope.12 The cells were incubated with different concentrations of epitope peptide in RPMI1640 medium supplemented with 10% fetal bovine serum and 50 µM β-mercaptoethanol in a humidified atmosphere containing 5% CO2 at 37 oC. The test compounds were dissolved in DMSO and added simultaneously with the peptide (final DMSO concentration: 0.2%). Culture supernatants were collected after 48 h of culture and subjected to measurement of IL-4 and IFN-γ concentrations by enzyme-linked immunosorbent assay kit (BD Pharmingen). Cell proliferation was examined by [3H] thymidine uptake during the last 16 h of the 64-h culture.

ACKNOWLEDGEMENT
This work was supported in part by Grant-in Aid for Scientific Research (C) (No.155900333) from Japan Society for the Promotion of Science (JSPS). We thanks to Dr. Mamoru Kiniwa and Dr. Koichi Ikizawa for the assistance of biological evaluation.

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