HETEROCYCLES

Paper | Special issue | Vol. 80, No. 1, 2010, pp. 455-462
Received, 30th June, 2009, Accepted, 24th July, 2009, Published online, 24th July, 2009.
DOI: 10.3987/COM-09-S(S)45

■ Chemical Confirmation of the Structure of a Mutagenic Aminophenylnorharman, 9-(4’-Aminophenyl)-9H-pyrido[3,4-b]indole: An Authentic Synthesis of 9-(4’-Nitrophenyl)-9H-pyrido[3,4-b]indole as Its Relay Compound

Yasuoki Murakami,* Nobuyuki Imai, Tsuyoshi Miura, Takashi Sugimura, Keiji Wakabayashi, Yukari Totsuka, Noriyasu Hada, Yuusaku Yokoyama, Hideharu Suzuki, and Katsuyoshi Mitsunaga

Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025, Japan

Abstract

9-(4’-Aminophenyl)-9H-pyrido[3,4-b]indole 2 is a mutagenic compound produced by non-mutagenic norharman 1 and aniline in the presence of S9 mix. 9-(4’-Nitrophenyl)-9H-pyrido[3,4-b]indole 4, the relay compound for synthesis of 2, was synthesized starting from ethyl indole-2-aldehyde 12 via initial N-(4-nitro)phenylation of the indole nucleus, elongation of the 2-aldehyde substituent, and then construction of the pyridine nucleus in order to ensure the nitrogen substitution in 2.

INTRODUCTION
Sugimura et al.1 reported that norharman 1 (9H-pyrido[3,4-b]indole, β-carboline) itself is not mutagenic to Salmonella strains, but becomes mutagenic to S. typhimurium TA98 and YG1024 with S9 mix in the presence of non-mutagenic aromatic amines such as aniline and o-toluidine. In a

subsequent report2 they isolated mutagenic compound 2 produced by the reaction between norharman and aniline with S9 mix (Scheme 1). In order to elucidate the structure, one of the potential structures, 9-(4’-aminophenyl)-9H-pyrido[3,4-b]indole 2, was synthesized2 via Ullmann reaction of norharman 1 with　4-bromonitrobenzene, followed by catalytic hydrogenation. The synthetic sample was identical to the natural one and the spectral data of the product supported the structure of 2 but not 2’. The synthetic strategy was based on the fact that Ullmann reaction of indoles with aryl halides proceeded at its NH position.3 However, if the reaction occurs on the pyridine nitrogen of 1 via its basicity or another resonance structure 3’, the product should be compound 4’ (Scheme 1 and 2), whose structure would be much more unstable than the structure 4, as it has neither benzene, indole, nor pyridine aromaticity any longer.　Thus, such a compound is thought to be difficult to produce. On the other hand, it was recently reported4 that NA-methylammonium harman 6 derived from harman 5 was basified to yield the compound 7, whose skeleton is the same as those of 2’ and 4’ (Scheme 2). In this paper we report the authentic synthesis of the relay compound 4 in order to ensure the nitrogen substitution of the substituted phenyl group in 2.

RESULTS AND DISCUSSION
The synthetic strategy for the synthesis of the relay compound ４ was designed as shown in Scheme 3. The key point is the initial (4-nitro)phenylation at the 1-nitrogen position in the indole nucleus, followed by elongation of the 2-substituent and cyclization to form the pyridine nucleus.
The usual construction of the pyridine ring in the indole nucleus for synthesis of the 9H-pyrido[3,4-b]indole nucleus is cyclization of the 3-substituent of the tryptamine derivative to the 2-position of the indole nucleus as seen in the Bischler-Napieralski reaction, Pictet-Spengler reaction and so on. On the other hand, there are few methods for cyclization of the 2-substituent to the 3-position of the indole nucleus. Several years ago we developed a method for 9H-pyrido[3,4-b]indole synthesis of the latter type in the course of the synthetic study of 4-oxo-β-carboline.5 We applied this method in the present strategy.

For this purpose, ethyl indole-2-carboxylate 8 was allowed to react with 4-fluoronitrobenzene to give ethyl N-(4’-nitrophenyl)indole-2-carboxylate 9. However, the reduction of ester carbonyl of 9 with LiAlH4 was not successful (Scheme 3). Thus, the reaction scheme to prepare the aldehyde 11 had to be changed. The synthetic route was changed as in Scheme 4.
The first N-(4-nitro)phenylation of indole-2-carboxaldehyde5 12 prepared from 8, which we feared to proceed with difficulty due to the sensitive reactivity of aldehyde functionality, went much better than expected (51% yield). The N-(4’-nitrophenyl)indole-2-carboxaldehyde 11 thus obtained was allowed to react with ethyl aminoacetate and then sodium cyanoborohydride to give the N-indolic aminoacetate 13. The cyclization of 13 with methanesulfonic acid gave the cyclized amino ketone 14. The aminoketone 14 was treated with tosyl chloride in the presence of pyridine to give the corresponding tosylamide 15 in good yield. The subsequent process of cyclic amino ketone resembling 14 to the target 9H-pyrido[3,4-b]indole nucleus has already been developed.6
The reduction of the ketone of 15 to the hydroxyl group with a large excess amount of sodium borohydride proceeded to give the alcohol 16 in good yield. The last and important dehydration and aromatization processes (two successive β-eliminations) were examined for the present reaction.

The reaction6 involved dehydration of the alcohol and β-elimination process around the sulfonyl group with HCl in MeOH, as shown in Scheme 5. In the present case the reaction did not proceed well with HCl in MeOH in several trials, probably due to its insolubility.　

After several acidic conditions were tried, the alcohol 16 was allowed to react with methanesulfonic acid. The target compound 4 was finally obtained from the basic layer in this reaction (20% yield). The product was identified with the already2 and freshly synthesized sample directly via the Ullmann reaction from 1 and 4-bromo-(or 4-fluoro)nitrobenzene in the presence of K2CO3. It was proved that the Ullmann reaction of 1 proceeded at the indolic NH position even on the 9H-pyrido[3,4-b]indole nucleus. It is worth noting that the Ullmann reaction of 1 with 4-fluoronitrobenzene without K2CO3 did not proceed at all. This means that the formation of nitrogen anion is necessary for Ullmann reaction of indole and pyridine nitrogen cannot take Ullmann reaction directly. Thus, the structure 2 was chemically determined. Using this scheme, it may be possible to develop a new strategy for 9H-pyrido[3,4-b]indole synthesis that involves cyclication of the 2-substituent toward the 3-position of the indole skeleton.

EXPERIMENTAL
All melting points were measured on a hot stage micro-melting points apparatus (Yanagimoto) and are uncorrected. Elemental analyses were conducted with a Yanaco CHN CORDER MT-6. The 1H-NMR spectra were measured with a Bruker UltrashieldTM 400 Plus (400MHz) spectrometer. Deuteriochloroform was used as the solvent with tetramethylsilane as an internal reference. MS spectra were measured on JEOL JMS-GC-mate II and JEOL JMS-600H spectrometers. IR spectra were recorded on a Shimadzu FTIR-8400S spectrometer. For column chromatography, Silica gel 60 (70-230 mesh ASTM; Merck) was used.

1-(4’-Nitrophenyl)indole-2-carboxaldehyde 11
A solution of indole-2-carboxaldehyde5 12 (1.80 g, 12.4 mmol)，4-fluoronitrobenzene (5.19 g, 36.8 mmol)，and powdered anhydrous K2CO3 (5.14 g, 37.2 mmol) in anhydrous DMF (27 mL) was heated with stirring at 100 ºC for 1.5 h. The reaction mixture was poured onto water (150 mL), and extracted with AcOEt. The organic layer was washed with water, dried over MgSO4, and evaporated in vacuo to dryness. The residue (6.38 g) was chromatographed over SiO2 with toluene as eluent to give the target compound 11 (1.70 g, 51%). Recrystallization of a part of the compound from a mixture of AcOEt and hexane gave pale yellow columns, mp 170-172 ºC. Anal. Calcd for C15H10N2O3: C, 67.67; H, 3.79; N, 10.52.　Found: C, 67.98; H, 3.95; N, 10.43. MS　(C15H10N2O3): m/z266 (M+). IR νmax(KBr)cm-1: 1683 (sh), 1672 (CO). 1H-NMR (CDCl3)δ: 7.23-7.58 (6H, m, C3,5,6,7,2’,6’-H), 7.83 (2H, m, C4-H), 8.41 (2H, d, J=8.0 Hz, C3’,C5’-H),　9.89 (1H, s, CHO).

Ethyl [1-(4’-Nitrophenyl)indole-2-ylmethyl]aminoacetate 13
To a muddy solution of 1-(4’-nitrophenyl)indole-2-carboxaldehyde 11 (724 mg, 2.77 mmol) and ethyl aminoacetate hydrochloride (1.12 g, 8.16 mmol) in ethanol (30 mL) was added triethylamine (1.17 mL, 8.16 mmol) and NaBH3CN (685 mg, 10.9 mmol) successively with stirring under ice-cooling. The reaction mixture (muddy state) was stirred under ice-cooling for 15 min and then at rt for an additional 3 h. Then, the reaction mixture was poured onto water and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4 and evaporated in vacuo to dryness to give a pale yellow residue. The crude products were chromatographed over SiO2. Elution with toluene, followed by toluene-AcOEt (10:1), gave the target compound 13 (713 mg, 74%) as a pale yellow oil. MS (C19H19N3O4): m/z 353 (M+). HRMS:　Calcd for C19H19N3O4, 353.1376; Found,　353.1378. IR νmax(CHCl3)cm-1: 3684, 3620 (NH), 1734 (C=O). 1H-NMR δ:1.23 (3H, t, J=8.0 Hz, -CH2CH3), 3.41 (2H, s, -CH2NH-), 3.94 (2H, s, -NCH2CO-), 4.12 (2H, J=8.0 Hz, -OCH2CH3), 6.75 (1H, s, C3-H), 7.18-7.26 (3H, m, C5,6,7-H), 7.63 (1H, m, C4-H), 7.73 (2H, J=8.0 Hz, C2’,6’-H), 8.41 (2H, C3’,5’-H).

9-(4’-Nitrophenyl)-1,2,3,9-tetrahydro-9H-pyrido[3,4-b]indole-4-one 14
A mixture of ethyl [1-(4’-nitrophenyl)indole-2-ylmethyl]aminoacetate 13 (513 mg, 1.45 mmol) and methanesulfonic acid (7 mL) was stirred at 45 ºC for 45 min, and then 70 ºC for 1 h. The reaction mixture was poured onto water (50 mL), basified with K2CO3, and extracted with AcOEt. The organic layer was washed with water and dried over MgSO4. Evaporation of the solvent in vacuo to dryness gave a solid (425 mg). This solid was chromatographed over SiO2 (12 g). Elution with CHCl3, followed by AcOEt, gave a small amount of the starting material and unknown compounds. Further elution with a mixture of AcOEt and EtOH (10:1) gave the target compound 14 (298 mg, 67%). A part of the sample was recrystallized from acetone to give pale yellow fine needles, mp 215-217 ºC (decomp). MS (C17H13N3O3): m/z307 (M+, 25% of base peak), 252(base peak). HRMS: Calcd for C17H13N3O3, 307.0957; Found,307.0964. IR νmax(KBr)cm-1:3326(NH), 1649(CO). 1H-NMR δ: 2.23 (1H, br.s, NH), 3.67 (2H, s, C3-H), 4.13 (2H, s, C1-H), 7.21-7.45 (3H, m, C6,7,8-H), 7.62 (2H, d, J=8.0 Hz, C2’,6’-H), 8.30 (1H, m, C5-H), 8.50 (2H, d, J=8.0 Hz, C3’,5’-H).

9-(4’-Nitrophenyl)-2-tosyl-1,2,3,9-tetrahydro-9H-pyrido[3,4-b]indole-4-one 15
To a suspension of 9-(4’-nitrophenyl)-1,2,3,9-tetrahydro-9H-pyrido[3,4-b]indole 14 (265 mg, 0.862 mmol) in pyridine (7 mL) was added TsCl (493 mg, 2.59 mmol) under ice-cooling. The mixture was stirred under ice-cooling for 15 min and at rt for an additional 1 h. The reaction mixture was poured onto water, extracted with CHCl3, washed with dil. HCl aq. and water, and dried over MgSO4. Evaporation of the solvent in vacuo to dryness gave the target compound 15 (349 mg, 88%). A part of the compound was recrystallized from a mixture of DMF and EtOH to give almost colorless very fine needles, mp 253-258 ºC (decomp). Anal. Calcd for C24H19N3O5S: C, 62.46; H, 4.15; N, 9.11: Found: C; 62.42, H; 4.18, N; 8.58. MS: m/z 461 (M+,15% of base peak), 306 (base peak). HRMS:Calcd for C24H19N3O5S, 461.1045; Found,461.1043. IR νmax(KBr)cm-1:1664(CO). 1H-NMR δ: 2.50 (3H, s, arom-CH3), 4.11 (2H, s, C3-H), 4.76 (2H, s, C1-H), 7.20-7.45 (7H, m, arom-H), 7.87 (1H, dd, J=8.0 and 2.0 Hz, C5-H), 7.92 (2H, d, J=8.0 Hz, Ts-ortho-H), 8.56 (2H, d, J=8.0 Hz, C3’,5’-H).

9-(4’-Nitrophenyl)-2-tosyl-2,3,4,9-tetrahydro-9H-pyrido[3,4-b]indole-4-ol 16
The tosyl ketone 15 (33 mg, 0.0715 mmol) was added to a mixture of CHCl3 (1.5 mL) and MeOH (4 mL). To the resulting suspension was added NaBH4 (270 mg, 7.15 mmol) under ice-cooling to prevent generation of heat at the beginning and then the whole was stirred for 4.5 h at rt. The reaction mixture was poured onto water and extracted with CHCl3. The organic layer was washed with brine and dried over MgSO4. Evaporation of the solvent in vacuo to dryness gave the target alcohol 16 as yellowish powder (31 mg, 94%). This sample showed one spot on TLC (SiO2, toluene-AcOEt = 2:1), and was used for the next reaction. A part of the powder was recrystallized from acetone-MeOH to give pale yellow powder, mp 175-177 ºC (decomp). Anal. Calcd for C24H21N3O5S: C,　62.19; H、4.57; N,　9.07. Found:　C, 61.83; H,　4.68; N,　8.85. MS: 463 (M+, 5.9% of the base peak), 252 (base peak). HRMS; Calcd, 463.1202; Found, 463.1208. IR νmax(KBr)cm-1: 3482(OH). 1H-NMR δ: 2.44 (3H, s, arom-CH3), 3.16,3.96,4.55 (4H, aliph-H), 5.12 (1H, br. d, J=12.0 Hz, C4-H), 7.24-7.78 (10H, m, arom-H), 8.46 (2H, d, J=8.0 Hz, C3’, C5’-H).

9-(4’-Nitrophenyl)-9H-pyrido[3,4-b]indole 4 from 9-(4’-nitrophenyl)-2-(toluene-4’’-sulfonyl)-2,3,4,9-tetrahydro-9H-pyrido[3,4-b]indole-4-ol 16
The above-mentioned alcohol 16 (40 mg, 0.086 mmol) was dissolved in methanesulfonic acid (3 mL) and stirred for 4 h at rt. The reaction mixture was poured onto water and extracted out with AcOEt. The aqueous layer was basified with K2CO3 and extracted with AcOEt. The organic layer was washed with water and dried over MgSO4. Evaporation of the solvent in vacuo to dryness gave the crude product. The crude product was purified with column-chromatography [SiO2 (8 g), CHCl3] to give yellow powder (5 mg, 20%), mp 188-190 ºC. This sample was identified with the relay compound2 derived from Ullmann reaction of norharman 1 and 4-bromonitrobenzene (or 4-fluoronitrobenzene) as described below, based on their NMR spectra and TLC behavior. 1H-NMR δ: 7.44 (1H, m, C6-H), 7.56-7.62 (2H, m, C7,8-H), 7.85 (2H, d, J=8 Hz, C2’,6’-H), 8.05 (1H, d, J=4.0 Hz, C4-H), 8.23 (1H,d, J=8.0 Hz, C5-H), 8.53 (2H, d, J=8.0 Hz, C3’,5’-H), 8.59 (1H, br. d, J=4.0 Hz, C3=H), 8.95(1H, s, C1-H).

9-(4’-Nitrophenyl)-9H-pyrido[3,4-b]indole 4 via Ullmann reaction2 from norharman 1
A mixture of norharman 1 (40 mg, 0.238 mmol), 4-fluoronitrobenzene (66 mg, 0.476 mmol) and powdered anhydrous K2CO3 (99 mg, 714 mmol) was added to DMF (3 mL) and the whole was heated at 100 ºC with stirring for 3 h. The reaction mixture was poured onto water (60 mL) and extracted with AcOEt. The organic layer was washed with water, dried over MgSO4, and evaporated in vacuo to dryness. The resulting mass was purified over column chromatography (SiO2, CHCl3) to give the target compound (79 mg, quantitative). This sample was recrystallized from CHCl3-MeOH and then treated with CHCl3 to give pale yellow needles, mp 192-192.5 ºC. The sample obtained from recrystallization from CHCl3-MeOH contained MeOH in its crystals. The crystals were dried at 100 ºC in vacuo over night for elemental analysis. Anal. Calcd for C17H11N3O2: C; 70.58, H; 3.83, N; 14.53. Found: C; 70.60, H; 3.97, N; 14.53.

Ethyl 1-(4’-nitrophenyl)indole-2-carboxylate 9
In anhydrous DMF (3 mL) was added ethyl indole-2-carboxylate (299 mg, 1.58 mmol), 4-fluoronitrobenzene (417 mg, 3 mmol), and powdered anhydrous K2CO3 (304 mg, 2.2 mmol). The whole was heated at 100 ºC under stirring for 14.5 h. The reaction mixture was poured onto water, and extracted with AcOEt. The organic layer was washed with water, dried over MgSO4, and evaporated to dryness in vacuo. The residue (702 mg) was chromatographed over SiO2 and eluted with toluene to give the target compound 9 (266 mg, 54%). A part of this compound was recrystallized from AcOEt-hexane to give pale yellow plates, mp 133-135 ºC. Anal. Calcd for C17H14N2O4: C, 65.80; H, 4.55; N, 9.03. Found: C, 65.99; H, 4.64; N, 8.76. MS (C17H14N2O4): 310 (M+). IR νmax(KBr)cm-1: no NH, 1704 (CO). 1H-NMR δ: 1.29 (3H, t, J=8.0 Hz, -CH2CH3), 4.25 (2H, t, J=8.0 Hz, -OCH2CH3), 7.13 (1H, d, J=1.5 Hz, C3-H), 7.23-7.36 (3H, m, indolic Hs), 7.52-7.56 (3H, m, C2’,C6’,an indolic H), 7.76 (1H, d, J=9.0 Hz, C4-H), 8.40 (2H, d, J=9.0 Hz, C3’,C5’-H).

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