CuBr₂-Catalyzed Mild Oxidation of 3,4-Dihydro-β-Carbolines and Application in Total Synthesis of 6-Hydroxymetatacarboline D

Abstract

A green chemical method for the conversion of 3,4-dihydro-β-carbolines to β-carbolines has been developed using air as the oxidant. With 15 mol % CuBr₂ as the catalyst, 3,4-dihydro-β-carbolines could be efficiently oxidized to β-carbolines in dimethyl sulfoxide at room temperature in the presence of 1,8-diazabicyclo[5,4,0]undec-7-ene (or Et₃N). By applying this method, the first total synthesis of 6-hydroxymetatacarboline D was performed through 12 steps in 22% overall yield starting from l-5-hydroxy-tryptophan.

1. Introduction

Aromatic β-carbolines are a kind of extremely important compounds not only due to their ubiquitousness in natural resources such as plants,¹ marine organisms,² bacteria,³ fungi,⁴ and foodstuffs⁵ but also due to their diverse biological activities.⁶ Therefore, development of mild and practical synthetic methods for aromatic β-carbolines is of considerable interest and has attracted much attention of chemists.⁷ Because 3,4-dihydro-β-carbolines can be readily obtained via Bischler–Napieralski cyclization,⁸ the aromatization of 3,4-dihydro-β-carbolines is an easy and good method to obtain β-carbolines. Normally, 3,4-dihydro-β-carbolines are converted to β-carbolines via metal-catalyzed (Pd, Ir) dehydrogenation⁹ or oxidation with stoichiometric oxidants such as dichlorodicianoquinone,¹⁰ KMnO₄,¹¹ MnO₂,¹² S,¹³ 2-iodoxybenzoic acid,¹⁴ and pyridinium dichromate.¹⁵ However, the dehydrogenation methods employed precious transition metals as the catalyst and the oxidation methods often suffered from use of strong oxidants, which sometimes are unfriendly to the environment. Therefore, development of novel mild, efficient, and environmentally benign methods for the conversion of 3,4-dihydro-β-carbolines to aromatic β-carbolines is highly desirable.

On the other hand, oxygen is an important component of air, which is one of the most abundant resources on the earth, so air is regarded as a clean and ecofriendly oxidant, having advantages like safeness, nontoxicity, cheapness, and ready availability. More and more researchers have carried out various oxidation reactions using air as the oxidant during the recent decades.¹⁶ Nevertheless, it is still a very challenging task to use air as the oxidant for the oxidative conversion of 3,4-dihydro-β-carbolines to β-carbolines due to its weak oxidizing ability and low solubility in most solvents. Fortunately, we found that when CuBr₂ was used as the catalyst, 3,4-dihydro-β-carbolines could be successfully converted to β-carbolines by air oxidation. Herein, we describe a very mild and efficient method for oxidative conversion of 3,4-dihydro-β-carbolines to β-carbolines.

2. Results and Discussion

Our investigation commenced with the optimization of reaction conditions for the Cu-catalyzed oxidative conversion of 3,4-dihydro-β-carbolines to β-carbolines. With oxidative conversion of 3,4-dihydro-1-phenyl-β-carboline 1a to 1-phenyl-β-carboline 2a as the model reaction, various conditions have been tested, and results are summarized in Table 1. As can be seen from Table 1, no reaction happened in the absence of copper catalyst (entry 1); cupric bromide, cupric chloride, cupric acetate, cupric sulfate, cupric carbonate, copper(II) oxide, and copper powder have been tested as catalysts for the model reaction (Table 1, entries 2–8). It was found that soluble cupric salts (entries 2–4) are much more effective than insoluble cupric compounds and copper powder (entries 5–8) and cupric bromide is the best catalyst for the reaction. When 15 mol % CuBr₂ was used as the catalyst, the aerobic oxidation of compound 1a produced desired product 2a in excellent yield at room temperature (entry 2). To obtain β-carboline 2a in high yield, an appropriate base was also needed here for the reaction (entry 2 vs entries 9–12); the reaction did not happen at all without a base (entry 13). 1,8-Diazabicyclo[5,4,0]undec-7-ene (DBU), 1,5-diazabi-cyclo[4,3,0]non-5-ene (DBN), 4-dimthylaminopyridine (DMAP), pyridine, and triethylamine have been tried for the model reaction, and it was found that DBU is the most suitable base and 2.0 equiv of DBU was necessary for the reaction. When 0.5 equiv of DBU was used, it took 4 days for the reaction to be complete. Several solvents such as dimethyl sulfoxide (DMSO), N,N-dimethylforamide (DMF), acetonitrile, ethanol, tetrahydrofuran (THF), dichloromethane, ethyl acetate, and acetone have also been tested for the model reaction (entries 2 and 14–20), and it was found that DMSO is the best solvent for the reaction.

Table 1. Optimization of Reaction Conditions for the Cu-Catalyzed Oxidation of 1-Phenyl-3,4-dihydro-β-carboline 1a to 1-Phenyl-β-carboline 2a^a.

entry	catalystb	basec	solvent	t (h)	yield (%)d
1	none	DBU	DMSO	18	0
2	CuBr2	DBUe	DMSOf	15	96
3	CuCl2	DBU	DMSO	15	91
4	Cu(OAc)2	DBU	DMSO	15	88
5	CuSO4	DBU	DMSO	15	12
6	CuCO3	DBU	DMSO	15	10
7	CuO	DBU	DMSO	18	6
8	Cu	DBU	DMSO	18	5
9	CuBr2	DBNg	DMSO	15	85
10	CuBr2	DMAPh	DMSO	20	10
11	CuBr2	Py	DMSO	20	9
12	CuBr2	Et3N	DMSO	20	8
13	CuBr2	none	DMSO	20	0
14	CuBr2	DBU	DMFi	16	87
15	CuBr2	DBU	CH3CN	16	62
16	CuBr2	DBU	EtOH	16	35
17	CuBr2	DBU	THFj	16	26
18	CuBr2	DBU	CH2Cl2	16	65
19	CuBr2	DBU	EtOAc	16	15
20	CuBr2	DBU	Me2CO	16	23

^aAll reactions were performed at 25 °C under air.

^b15 mol % catalyst was used.

^c2.0 equiv of the base was used.

^dIsolated yields.

^e1,8-Diazabicyclo[5,4,0]undec-7-ene.

^fDimethyl sulfoxide.

^g1,5-Diaza-bicyclo[4,3,0]non-5-ene.

^h4-Dimthylaminopyridine.

ⁱN,N-Dimethylforamide.

^jTetrahydrofurane.

Subsequently, we attempted the CuBr₂-catalyzed oxidative conversion of variously substituted 3,4-dihydro-β-carbolines to β-carbolines in DMSO, and the results are summarized in Table 2. The scope of the reaction is very wide; almost all of the tested 3,4-dihydro-β-carbolines could be smoothly converted to β-carbolines in good to excellent yields. As can be seen from Table 2, 18 β-carbolines 2a–r were obtained in 80–97% yields, and it was worth noting that a comparatively strong and expensive base, DBU, should be used for β-carbolines 2a–h, whereas a comparatively weak and cheap base, Et₃N, could be used for β-carbolines 2i–r probably due to the presence of electron-withdrawing ester groups (R² = COOR). We have observed that 3,4-dihydro-β-carbolines with electron-withdrawing carbonyl groups (COR or COOR) at the C-1 position could undergo oxidation in DMSO at room temperature under air (O₂) to afford β-carbolines in the presence of DBU without the copper catalyst.¹⁷ But when much weak and cheaper Et₃N instead of DBU was used here for oxidation of 3,4-dihydro-β-carbolines 1i–r, which also contain ester groups at the C-3 position, a cupric salt (CuBr₂) should be used as the catalyst for the reaction.

Table 2. CuBr₂-Catalyzed Oxidation of Variously Substituted 3,4-Dihydro-β-carbolines to β-Carbolines^a,b,c,d,e.

^aThe reactions were performed at 25 °C in DMSO with 15 mol % CuBr₂ as the catalyst in the presence of 2.0 equiv of the base.

^bDBU was used as the base for β-carbolines 2a–h.

^cReaction time.

^dIsolated yields.

^eEt₃N was used as the base for β-carbolines 2i–r.

A possible mechanism for the above CuBr₂-catalyzed oxidative conversion of 3,4-dihydro-β-carbolines 1 to β-carbolines 2 is proposed in Scheme 1. As can be seen from Scheme 1, two steps might be involved: 3,4-dihydro-β-carbolines 1 would first undergo reversible tautomerization to form enamine intermediates I-A via migration of double bonds in the presence of a base and then the unstable enamine intermediates I-A would undergo copper-catalyzed aerobic oxidation of the C–N single bond to the C=N double bond to afford β-carbolines 2 according to Adimurthy’s reports.¹⁸

Scheme 1. Possible Mechanism for the CuBr₂-Catalyzed Oxidation of 3,4-Dihydro-β-carbolines to β-Carbolines.

To showcase the synthetic utility of the above-described method for the CuBr₂-catalyzed oxidative conversion of 3,4-dihydro-β-carbolines to β-carbolines, we applied the methodology to the new total synthesis of β-carboline alkaloid 6-hydroxymetatacarboline D 3. It was recently discovered in 2013 by Spiteller and his colleagues¹⁹ from fruiting bodies of small mushrooms, called as Mycena metata, that are often found in coniferous and deciduous forests.

Nagarajan and his colleagues have recently reported total syntheses of metatacarbolines A, C, D, E, F via the Wittig reaction starting from methyl 1-formyl-β-carboline-3-carboxylate,²⁰ but 6-hydroxymetatacarboline D 3, as the most abundant β-carboline alkaloid¹⁹ in M. metata, has not been synthesized yet; thus, we herein developed the first total synthesis of 6-hydroxymetatacarboline D 3 staring from l-5-hydroxy-tryptophan, as depicted in Scheme 2.

Scheme 2. Total Synthesis of 6-Hydroxymetatacarboline D 3.

l-5-Hydroxy-tryptophan was first treated with 1.0 equiv of tert-butoxy-carboxylic anhydride and 2.0 equiv of NaHCO₃ at room temperature in DMF under an atmosphere of N₂ to protect the amino group and then the in situ treatment of intermediate I–B with 1.1 equiv of K₂CO₃ and 1.1 equiv of benzyl bromide furnished compound 4 in 90% yield over two steps. In the following step, compound 4 was treated with 2.0 equiv of K₂CO₃ and 1.2 equiv of benzyl bromide in dry acetone at reflux to afford compound 5 in 87% yield. Removal of the tert-butoxycarbonyl group of compound 5 via hydrolysis at 60 °C in a mixed solvent of ethyl acetate and 4 N HCl aqueous solution produced compound 6 in 93% yield. Condensation of compound 6 with 1.1 equiv of the freshly prepared succinic monochloride methyl monoester at 0 °C in dichloromethane in the presence of 2.0 equiv of triethylamine furnished amide 7 in 94% yield, which was then exposed to 3.0 equiv of POCl₃ in ethyl acetate at reflux to afford 3,4-dihydro-β-carboline 1r in 71% yield via Bischler–Napieralski cyclization. According to the above-described CuBr₂-catalyzed oxidation of 3,4-dihydro-β-carbolines to β-carbolines, compound 1r was treated with 0.15 equiv of cupric bromide and 2.0 equiv of triethylamine in DMSO at 25 °C under an atmosphere of air and β-carboline 2r was successfully obtained in 84% yield.

Next, when compound 2r was treated with 1.1 equiv of LiOH·H₂O in a mixed solvent of tetrahydrofuran and water (THF/H₂O = 5:1) at room temperature, highly selective hydrolysis of the methyl ester group occurred smoothly to furnish acid 8 in 91% yield. When compound 8 was treated with 1.5 equiv of methyl l-prolinate, 1.2 equiv of 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC·HCl), 1.2 equiv of N-hydroxybenzotriazole (HOBt), and 2.0 equiv of diisopropylethylamine (DIPEA) in CH₂Cl₂ at 0 °C to room temperature, amide 9 was obtained in 88% yield. Removal of both benzyl groups of compound 9 via Pd/C-catalyzed hydrogenation in methanol at reflux gave acid 10 in an almost quantitative yield. Acid 10 was then treated with 1.5 equiv of methyl l-threoninate, 1.2 equiv of EDC·HCl, 1.2 equiv of HOBt, and 2.0 equiv of DIPEA in anhydrous DMF at 0 °C to room temperature to furnish amide 11 in 85% yield.

Finally, compound 11 could be converted to the targeted β-carboline alkaloid 6-hydroxymetatacarboline D 3 via simultaneous hydrolysis of two ester groups. When compound 11 was treated with 5.0 equiv of LiOH·H₂O in methanol at 0 °C to room temperature under an atmosphere of N₂ and then was acidified with dilute aqueous solution of HCl to the isoelectric point (pH ≈ 4.2), compound 3 was thus obtained in 82% yield.

3. Conclusions

In conclusion, we have developed a novel CuBr₂-catalyzed aerobic oxidation of 3,4-dihydro-β-carbolines to β-carbolines. This method has following advantages: (a) it is mild and environmentally benign because the process was carried out at room temperature with air as the clean oxidant; (b) all products have good to excellent yields; (c) the scope of the reaction is very wide, and it could be applicable to all of the tested substrates; (d) experiments were easily performed on a gram scale. Moreover, by applying this method, we have conducted the first total synthesis of β-carboline alkaloid 6-hydroxymetatacarboline D 3 via 12 steps in 22% overall yield starting from l-5-hydroxy-tryptophan.

4. Experimental Section

4.1. General Method

¹H NMR and ¹³C NMR spectra were acquired on a Bruker AM-400 instrument, and chemical shifts are given on the δ scale as parts per million with tetramethylsilane as the internal standard. IR spectra were recorded with a Nicolet Magna IR-550 instrument. Mass spectra were recorded with a HP1100 LC-MS spectrometer. Melting points were determined on a Mel-TEMP II apparatus. Column chromatography was performed on silica gel (Qingdao Ocean Chemical Corp.). All chemicals were analytically pure.

4.2. General Procedure for CuBr₂-Catalyzed Oxidative Conversion of 3,4-Dihydro-β-carbolines (1) to β-Carbolines (2)

A round-bottom flask was charged with 3,4-dihydro-β-carboline 1 (3.0 mmol), DMSO (10 mL), DBU or Et₃N (6.0 mmol), and CuBr₂ (0.45 mmol). The mixture was stirred under air at 25 °C for a period of time as indicated in Table 2. After the reaction was complete (checked by thin-layer chromatography (TLC), eluent: EtOAc/hexane = 1:2–1:4), the mixture was poured into the mixed solution of ammonium hydroxide (0.5 N, 50 mL) and EtOAc (50 mL). The mixture was stirred for 15 min. Two phases were separated. The water layer was re-extracted twice with EtOAc (2 × 20 mL). The organic extracts were combined and washed twice with brine (2 × 10 mL) and dried over anhydrous MgSO₄. Evaporation of solvent gave a residue, which was purified by flash chromatography (eluent: EtOAc/CH₂Cl₂ = 1:4–1:20) to give pure compound 2 as crystals in 80–97% yields (see Table 2). Characterization data of β-carbolines 2a–r are as follows.

4.2.1. 1-Phenyl-9H-pyrido[3,4-b]indole (2a)

Pale yellow solid, mp 240–241 °C. ¹H NMR (DMSO-d₆, 400 MHz) δ 11.54 (br s, 1H, NH on the indole ring), 8.48 (d, J = 5.2 Hz, 1H, H-4), 8.27 (d, J = 7.9 Hz, 1H, H-8), 8.13 (d, J = 5.2 Hz, 1H, H-3), 8.05 (d, J = 7.8 Hz, 2H, ortho Ph-H), 7.47–7.72 (m, 5H, Ph-H, H-5, and H-6), 7.28 (dd, J₁ = 7.9 Hz, J₂ = 7.7 Hz, 1H, H-7). ¹³C NMR (DMSO-d₆, 100 MHz) δ 142.51, 141.44, 138.75, 138.69, 133.34, 129.49, 129.03, 128.80, 128.71, 128.46, 121.88, 121.16, 119.80, 114.17, 112.75. IR (KBr film) 3061, 2954, 2877, 1623, 1560, 1496, 1415, 1322, 1234, 737, 698 cm^–1. High-resolution mass spectrometry (HRMS) (electrospray ionization (ESI)) m/z calcd for C₁₇H₁₃N₂ [M + H]⁺: 245.1079; found: 245.1075.

4.2.2. 1-(3-Methoxyphenyl)-9H-pyrido[3,4-b]indole (2b)

Pale yellow solid, mp 120–121 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.81 (s, 1H, NH on the indole ring), 8.55 (d, J = 5.2 Hz, 1H, H-4), 8.16 (d, J = 7.9 Hz, 1H, H-5), 7.94 (d, J = 5.2 Hz, 1H, H-3), 7.57–7.47 (m, 4H, Ar-H), 7.44 (dd, J₁ = 8.0 Hz, J₂ = 7.2 Hz, 1H, H-7), 7.31 (dd, J₁ = 7.7 Hz, J₂ = 7.2, 1H, H-6), 7.00 (dd, J₁ = 8.2 Hz, J₂ = 2.5, 1H, H-7), 3.87 (s, 3H, OCH₃). ¹³C NMR (100 MHz, CDCl₃) δ 160.06, 142.84, 140.69, 139.68, 138.89, 133.65, 129.88, 129.86, 128.41, 121.69, 121.66, 120.45, 120.03, 114.64, 113.90, 113.51, 111.73, 55.22. IR (KBr film) 3123, 3058, 2956, 1624, 1601, 1563, 1496, 1453, 1413, 1322, 1223, 778, 742 cm^–1. HRMS (ESI) m/z calcd for C₁₈H₁₅N₂O [M + H]⁺: 275.1184; found: 275.1180.

4.2.3. 1-(3,5-Dimethoxyphenyl)-9H-pyrido[3,4-b]indole (2c)

Pale yellow solid, mp 172–173 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.84 (s, 1H, NH in indole ring), 8.54 (d, J = 5.2 Hz, 1H, H-4), 8.15 (d, J = 7.9 Hz, 1H, H-5), 7.93 (d, J = 5.2 Hz, 1H, H-3), 7.53 (dd, J₁ = 8.1 Hz, J₂ = 7.1, 1H, H-7), 7.48 (d, J = 8.1 Hz, 1H, H-8), 7.29 (dd, J₁ = 7.9 Hz, J₂ = 7.1, 1H, H-6), 7.09 (s, 2H, ortho Ph-H), 6.54 (s, 1H, para Ph-H), 3.84 (s, 6H, OCH₃). ¹³C NMR (100 MHz, CDCl₃) δ 161.26, 142.84, 140.57, 140.41, 139.08, 133.59, 129.89, 128.51, 121.80, 121.75, 120.16, 114.02, 111.75, 106.33, 100.75, 55.47. IR (KBr film) 3116, 3065, 2934, 2837, 1593, 1562, 1498, 1384, 1352, 1230, 1205, 1155, 1062, 826, 741 cm^–1. HRMS (ESI) m/z calcd for C₁₉H₁₇N₂O₂ [M + H]⁺: 305.1290; found: 305.1289.

4.2.4. 1-(Furan-2-yl)-9H-pyrido[3,4-b]indole (2d)

Pale yellow solid, mp 167–168 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.50 (s, 1H, NH on the indole ring), 8.37 (d, J = 5.1 Hz, 1H, H-4), 8.25 (d, J = 7.9 Hz, 1H, H-5), 8.07 (d, J = 5.1 Hz, 1H, H-3), 7.98 (d, J = 1.7 Hz, 1H, furanyl-H), 7.77 (d, J = 8.2 Hz, 1H, H-8), 7.57 (dd, J₁ = 8.2 Hz, J₂ = 7.2, 1H, H-7), 7.32–7.22 (m, 2H, H-6, and furanyl-H), 6.79 (dd, J₁ = 3.3 Hz, J₂ = 1.7, 1H, furanyl-H). ¹³C NMR (100 MHz, CDCl₃) δ 154.55, 142.80, 140.55, 138.93, 133.55, 131.43, 130.29, 128.63, 121.72, 121.33, 120.18, 113.72, 112.38, 111.71, 108.78. IR (KBr film) 3459, 3125, 3034, 2999, 1623, 1556, 1493, 1452, 1424, 1377, 1381, 1300, 1283, 1235, 1162, 996, 754 cm^–1. HRMS (ESI) m/z calcd for C₁₅H₁₁N₂O [M + H]⁺: 235.0871; found: 235.0865.

4.2.5. 1-(Benzo[d][1,3]dioxol-5-yl)-9H-pyrido[3,4-b]indole (2e)

White solid, mp 171–172 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.49 (s, 1H, NH on the indole ring), 8.42 (d, J = 5.1 Hz, 1H, H-4), 8.24 (d, J = 7.9 Hz, 1H, H-5), 8.06 (d, J = 5.1 Hz, 1H, H-3), 7.67 (d, J = 8.2 Hz, 1H, H-8), 7.61–7.52 (m, 3H, Ph-H, H-6, and H-7), 7.26 (d, J = 7.5 Hz, 1H, Ph-H), 7.15 (d, J = 7.5 Hz, 1H, Ph-H), 6.15 (s, 2H, OCH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 147.65, 147.62, 141.86, 141.05, 138.19, 132.74, 132.54, 129.10, 128.07, 122.38, 121.52, 120.87, 119.46, 113.52, 112.42, 108.60, 108.50, 101.29. IR (KBr film) 3426, 2910, 1626, 1503, 1471, 1451, 1418, 1244, 1227, 1039, 746 cm^–1. HRMS (ESI) m/z calcd for C₁₈H₁₃N₂O₂ [M + H]⁺: 289.0977; found: 289.0975.

4.2.6. 1-(4-Chlorophenyl)-9H-pyrido[3,4-b]indole (2f)

White solid, mp 195–196 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.56 (s, 1H, NH on the indole ring), 8.46 (d, J = 5.1 Hz, 1H, H-4), 8.27 (d, J = 7.8 Hz, 1H, H-5), 8.14 (d, J = 5.1 Hz, 1H, H-3), 8.06 (d, J = 8.4 Hz, 2H, Ph-H), 7.66 (d, J = 8.4 Hz, 2H, Ph-H), 7.65 (d, J = 7.2 Hz, 1H, H-8), 7.57 (dd, J₁ = 7.9 Hz, J₂ = 7.2, 1H, H-7), 7.27 (dd, J₁ = 7.8 Hz, J₂ = 7.2, 1H, H-6). ¹³C NMR (100 MHz, DMSO-d₆) δ 141.15, 140.84, 138.41, 137.17, 133.24, 133.00, 130.15, 129.39, 128.69, 128.27, 121.62, 120.81, 119.59, 114.17, 112.38. IR (KBr film) 3299, 3058, 1625, 1597, 1566, 1494, 1454, 1420, 1390, 1318, 1230, 1139, 1087, 1011, 830 cm^–1. HRMS (ESI) m/z calcd for C₁₇H₁₂ClN₂ [M + H]⁺: 279.0689; found: 279.0687.

4.2.7. 9H-Pyrido[3,4-b]indole (2g)

Pale yellow solid, mp 195–197 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.63 (s, 1H, NH on the indole ring), 8.92 (s, 1H, H-1), 8.34 (d, J = 5.2 Hz, 1H, H-4), 8.23 (d, J = 7.9 Hz, 1H, H-5), 8.10 (d, J = 5.2 Hz, 1H, H-3), 7.60 (d, J = 8.1 Hz, 1H, H-8), 7.54 (dd, J₁ = 8.1 Hz, J₂ = 7.1, 1H, H-7), 7.24 (dd, J₁ = 7.9 Hz, J₂ = 7.1, 1H, H-6). ¹³C NMR (100 MHz, DMSO-d₆) δ 140.58, 138.15, 136.04, 134.08, 128.13, 127.49, 121.80, 120.66, 119.27, 114.66, 111.98. IR (KBr film) 3129, 3044, 2938, 1627, 1561, 1505, 1449, 1329, 1284, 1243, 734 cm^–1. HRMS (ESI) m/z calcd for C₁₁H₉N₂ [M + H]⁺: 169.0766; found: 169.0760.

4.2.8. 1-Propyl-9H-pyrido[3,4-b]indole (2h)

Pale yellow solid, mp 216–217 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.56 (s, 1H, NH on the indole ring), 8.25 (d, J = 5.3 Hz, 1H), 8.18 (d, J = 7.9 Hz, 1H, H-4), 7.91 (d, J = 5.3 Hz, 1H, H-3), 7.60 (d, J = 8.2 Hz, 1H, H-8), 7.53 (dd, J₁ = 8.2 Hz, J₂ = 7.1 Hz, 1H, H-7), 7.22 (dd, J₁ =7.9 Hz, J₂ = 7.1 Hz, 1H, H-6), 3.09 (t, J = 7.5 Hz, 2H, CCH₂), 1.91–1.78 (m, 2H, CH₂), 0.99 (t, J = 7.4 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 145.85, 140.35, 137.58, 134.17, 127.77, 127.08, 121.62, 121.09, 119.11, 112.56, 111.90, 35.48, 21.48, 13.99. IR (KBr film) 3444, 3063, 2954, 2870, 1626, 1566, 1507, 1428, 1325, 1248, 1056, 749 cm^–1. HRMS (ESI) m/z calcd for C₁₄H₁₅N₂ [M + H]⁺: 211.1235; found: 211.1232.

4.2.9. Methyl 1-(p-tolyl)-9H-pyrido[3,4-b]indole-3-carboxylate (2i)

White solid, mp 278–279 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.90 (s, 1H, NH on the indole ring), 8.90 (s, 1H, H-4), 8.41 (d, J = 7.8 Hz, 1H, H-5), 7.92 (d, J = 7.6 Hz, 2H, Ph-H), 7.70 (d, J = 8.2 Hz, 1H, H-8), 7.60 (dd, J₁ = 8.2 Hz, J₂ = 7.3 Hz, 1H, H-7), 7.44 (d, J = 7.6 Hz, 2H, Ph-H), 7.32 (dd, J₁ = 7.8 Hz, J₂ = 7.3 Hz, 1H, H-6), 3.93 (s, 3H, OCH₃), 2.44 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.08, 142.13, 141.41, 138.43, 136.58, 134.72, 134.49, 129.27, 129.01, 128.54, 128.47, 121.90, 121.12, 120.31, 116.44, 112.73, 51.99, 20.91. IR (KBr film) 3448, 3229, 2947, 1715 (C=O), 1624, 1430, 1387, 1351, 1255, 1218, 1100, 1047, 743 cm^–1. HRMS (ESI) m/z calcd for C₂₀H₁₇N₂O₂ [M + H]⁺: 317.1290; found: 317.1286.

4.2.10. Propyl 1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (2j)

White solid, mp 153–154 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.89 (s, 1H, NH on the indole ring), 8.85 (s, 1H, H-4), 8.41 (d, J = 7.8 Hz, 1H, H-5), 8.00 (d, J = 8.3 Hz, 2H, Ph-H), 7.70 (d, J = 8.1 Hz, 1H, H-8), 7.59 (dd, J₁ = 8.1 Hz, J₂ = 7.3, 1H, H-7), 7.32 (dd, J₁ = 7.8 Hz, J₂ = 7.3, 1H, H-6), 7.19 (d, J = 8.3 Hz, 2H, Ph-H), 4.31 (t, J = 6.6 Hz, 2H, OCH₂), 3.88 (s, 3H, OCH₃), 1.86–1.70 (m, 2H, CH₂), 1.02 (t, J = 7.3 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.67, 159.96, 141.99, 141.47, 136.88, 134.37, 130.08, 129.97, 128.99, 128.52, 121.93, 121.23, 120.32, 116.08, 114.18, 112.81, 66.06, 55.33, 21.87, 10.45. IR (KBr film) 3446, 3263, 2964, 1717 (C=O), 1611, 1513, 1386, 1350, 1250, 1218, 1100, 745 cm^–1. HRMS (ESI) m/z calcd for C₂₂H₂₁N₂O₃ [M + H]⁺: 361.1552; found: 361.1546.

4.2.11. Benzyl 1-(4-chlorophenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (2k)

White solid, mp 219–220 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.99 (s, 1H, NH on the indole ring), 8.96 (s, 1H, H-4), 8.43 (d, J = 7.9 Hz, 1H, H-5), 8.05 (d, J = 8.6 Hz, 2H, Ph-H), 7.70 (d, J = 8.2 Hz, 1H, H-8), 7.69 (d, J = 8.6 Hz, 2H, Ph-H), 7.61 (dd, J₁ = 8.2 Hz, J₂ = 7.2, 1H, H-7), 7.55 (d, J = 7.2 Hz, 2H, Ph-H), 7.42 (dd, J₁ = 7.6 Hz, J₂ = 7.2, 2H, Ph-H), 7.40–7.31 (m, 2H, H-6 and Ph-H), 5.46 (s, 2H, OCH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.30, 141.52, 140.87, 136.65, 136.54, 136.30, 134.62, 133.79, 130.43, 129.38, 128.81, 128.80, 128.54, 128.05, 128.04, 122.12, 121.11, 120.51, 117.13, 112.76, 66.09. IR (KBr film) 3447, 3241, 3059, 2924, 1719 (C=O), 1624, 1495, 1382, 1346, 1250, 1211, 1091, 1046, 745 cm^–1. HRMS (ESI) m/z calcd for C₂₅H₁₈ClN₂O₂ [M + H]⁺: 413.1057; found: 413.1055.

4.2.12. Methyl 1-(2-fluorophenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (2l)

White solid, mp 280–281 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.89 (s, 1H, NH on the indole ring), 9.00 (s, 1H, H-4), 8.45 (d, J = 7.8 Hz, 1H, H-5), 7.75 (dd, J₁ = 7.8 Hz, J₂ = 7.2 Hz, 1H, H-6), 7.70–7.56 (m, 3H, H-7, and Ph-H), 7.54–7.40 (m, 2H, H-8, and Ph-H), 7.35–7.30 (m, 1H, Ph-H), 3.93 (s, 3H, OCH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.99, 159.87 (¹J_F-C = 247.4 Hz), 141.31, 138.00, 136.59, 135.72, 132.02 (³J_F-C = 2.9 Hz), 131.25 (³J_F-C = 8.1 Hz), 128.90, 128.56, 125.28 (²J_F-C = 15.3 Hz), 124.87, 122.25, 120.96, 120.42, 117.31, 116.20 (²J_F-C = 21.4 Hz), 112.54, 52.09. IR (KBr film) 3450, 3276, 2949, 1719 (C=O), 1624, 1430, 1352, 1257, 1226, 1108, 1048, 739 cm^–1. HRMS (ESI) m/z calcd for C₁₉H₁₄FN₂O₂ [M + H]⁺: 321.1039; found: 321.1033.

4.2.13. Ethyl 1-methyl-9H-pyrido[3,4-b]indole-3-carboxylate (2m)

White solid, mp 217–218 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.04 (br s, 1H, NH on the indole ring), 8.77 (s, 1H, H-4), 8.36 (d, J = 7.9 Hz, 1H, H-5), 7.67 (d, J = 8.1 Hz, 1H, H-8), 7.60 (dd, J₁ = 8.0 Hz, J₂ = 7.9 Hz, 1H, H-6), 7.31 (dd, J₁ = 8.1 Hz, J₂ = 8.0 Hz, 1H, H-7), 4.38 (q, J = 7.1 Hz, 2H, OCH₂), 2.83 (s, 3H, CH₃), 1.38 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.13, 142.59, 141.21, 136.68, 136.61, 128.75, 127.22, 122.48, 121.82, 120.54, 116.30, 112.75, 60.89, 20.83, 14.84. IR (KBr film) 3317, 3041, 2978, 2932, 1709, 1624, 1596, 1568, 1500, 1345, 1256, 1239, 741 cm^–1. HRMS (ESI) m/z calcd for C₁₅H₁₅N₂O₂ [M + H]⁺: 255.1134, found: 255.1136.

4.2.14. Isopropyl 1-isopropyl-9H-pyrido[3,4-b]indole-3-carboxylate (2n)

White solid, mp 131–132 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.98 (s, 1H, NH on the indole ring), 8.72 (s, 1H, H-4), 8.34 (d, J = 7.8 Hz, 1H, H-5), 7.66 (d, J = 8.1 Hz, 1H, H-8), 7.58 (dd, J₁ = 8.1 Hz, J₂ = 7.2 Hz, 1H, H-7), 7.28 (dd, J₁ = 7.8 Hz, J₂ = 7.2 Hz, 1H, H-6), 5.19 (m, 1H, OCH), 3.68 (m, 1H, CCH), 1.40 (d, J = 6.8 Hz, 6H, two CH₃), 1.37 (d, J = 6.3 Hz, 6H, two CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.04, 150.26, 140.77, 136.57, 134.68, 128.27, 127.19, 121.90, 121.40, 120.04, 115.79, 112.33, 67.70, 31.25, 21.86, 21.26. IR (KBr film) 3510, 3458, 3224, 2982, 1703 (C=O), 1625, 1563, 1503, 1460, 1358, 1254, 1103, 741, 678 cm^–1. HRMS (ESI) m/z calcd for C₁₈H₂₁N₂O₂ [M + H]⁺: 297.1598; found: 297.1601.

4.2.15. Ethyl 1-(3,4-dimethoxyphenyl)-6-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indole-3-carboxylate (2o)

Pale yellow solid, mp 207–208 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.72 (s, 1H, NH on the indole ring), 8.88 (s, 1H, H-4), 8.00 (s, 1H, H-5), 7.69–7.42 (m, 3H, H-7, H-8, and Ph-H), 7.23 (d, J = 8.9 Hz, 1H, Ph-H), 7.20 (d, J = 8.9 Hz, 1H, Ph-H), 4.40 (q, J = 7.1 Hz, 2H, CH₂), 3.90 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 1.39 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.66, 154.14, 149.63, 148.84, 142.32, 136.22, 136.21, 134.90, 130.32, 128.75, 121.73, 121.20, 118.80, 116.48, 113.67, 112.02, 111.82, 103.50, 60.60, 55.71, 55.66, 55.51, 14.44. IR (KBr film) 3449, 3262, 2937, 1704 (C=O), 1514, 1495, 1463, 1303, 1266, 1240, 1219, 1104, 1033, 778, 759 cm^–1. HRMS (ESI) m/z calcd for C₂₃H₂₃N₂O₅ [M + H]⁺: 407.1607; found: 407.1609.

4.2.16. Ethyl 6-ethoxy-1-phenyl-9H-pyrido[3,4-b]indole-3-carboxylate (2p)

Pale yellow solid, mp 228–229 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.75 (s, 1H, NH on the indole ring), 8.92 (s, 1H, H-4), 8.06–7.96 (m, 3H, H-5, H-7, and para Ph-H), 7.64 (d, J = 7.4 Hz, 2H, ortho Ph-H), 7.61–7.53 (m, 2H, meta Ph-H), 7.22 (d, J = 8.9 Hz, 1H, H-8), 4.41 (q, J = 7.1 Hz, 2H, CH₂), 4.15 (q, J = 6.9 Hz, 2H, CH₂), 1.40 (t, J = 6.9 Hz, 3H, CH₃), 1.39 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.62, 153.35, 142.09, 137.69, 136.34, 136.21, 134.96, 129.00, 128.96, 128.81, 128.59, 121.65, 119.29, 116.94, 113.63, 104.29, 63.61, 60.61, 14.83, 14.45. IR (KBr film) 3448, 3227, 2980, 1712 (C=O), 1525, 1462, 1306, 1206, 1151, 1103, 1027, 725, 696 cm^–1. HRMS (ESI) m/z calcd for C₂₂H₂₁N₂O₃ [M + H]⁺: 361.1552; found: 361.1549.

4.2.17. Ethyl 6-ethoxy-1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (2q)

Pale yellow solid, mp 180–181 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.74 (s, 1H, NH on the indole ring), 8.92 (s, 1H, H-4), 7.99 (s, 1H, H-5), 7.63–7.49 (m, 4H, H-7, and Ph-H), 7.22 (d, J = 8.9 Hz, 1H, H-8), 7.15–7.11 (m, 1H, Ph-H), 4.40 (q, J = 7.1 Hz, 2H, CH₂), 4.15 (q, J = 6.9 Hz, 2H, CH₂), 3.89 (s, 3H, OCH₃), 1.40 (t, J = 6.9 Hz, 3H, CH₃), 1.39 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.58 (CO), 159.45, 153.33, 141.93, 139.04, 136.22, 136.18, 134.95, 129.91, 128.99, 121.64, 120.88, 119.25, 116.97, 114.49, 114.00, 113.63, 104.25, 63.59, 60.59, 55.15, 14.80, 14.42. IR (KBr film) 3450, 3259, 2974, 1707 (C=O), 1577, 1493, 1463, 1384. 1299, 1239, 1199, 1107, 1038, 795, 770 cm^–1. HRMS (ESI) m/z calcd for C₂₃H₂₃N₂O₄ [M + H]⁺: 391.1658; found: 391.1658.

4.2.18. Benzyl 6-(benzyloxy)-1-(3-methoxy-3-oxo-propyl)-9H-pyrido[3,4-b]indole-3-carboxylate (2r)

Pale yellow solid, mp 160–161 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.76 (s, 1H, NH on the indole ring), 8.66 (s, 1H, H-4), 7.62 (s, 1H, H-5), 7.56–7.44 (m, 5H, Ar-H), 7.44–7.31 (m, 6H, Ar-H), 7.27 (d, J = 9.2 Hz, 1H, H-8), 5.49 (s, 2H, CH₂Ph), 5.17 (s, 2H, CH₂Ph), 3.63 (s, 3H, OCH₃), 3.48 (t, J = 6.3 Hz, 2H, CH₂), 2.97 (t, J = 6.3 Hz, 2H, CH₂COOMe). ¹³C NMR (100 MHz, CDCl₃) δ 175.08, 175.03, 166.13, 153.73, 144.24, 137.09, 136.74, 136.32, 136.26, 135.78, 128.65, 128.57, 128.49, 128.23, 128.04, 127.60, 122.38, 119.52, 116.81, 113.16, 104.75, 70.91, 67.03, 52.00, 32.30, 28.59. IR (KBr film) 3356, 3032, 2948, 1738, 1716, 1670, 1495, 1452, 1370, 1302, 1229, 1189, 1117, 990, 840, 731, 648 cm^–1. HRMS (ESI) m/z calcd for C₃₀H₂₇N₂O₅ [M + H]⁺: 495.1920; found: 495.1917.

4.3. Total Synthesis of 6-Hydroxymetatacarboline D (3)

4.3.1. (S)-Benzyl 2-((tert-butoxycarbonyl)amino)-3-(5-hydroxy-1H-indol-3-yl)propanoate (4)

l-5-Hydroxy-tryptophan (10.01 g, 45.45 mmol) was dissolved in DMF (60 mL), and the solution was cooled to 0 °C using an ice bath. After NaHCO₃ (7.636 g, 90.90 mmol) was added, tert-butoxycarboxylic anhydride (9.919 g, 45.45 mmol) was added. The ice bath was removed, and the mixture was further stirred at room temperature for 6 h under a nitrogen atmosphere. Then, K₂CO₃ (6.910 g, 50.00 mmol) and benzyl bromide (8.552 g, 50.00 mmol) were added successively. The mixture was further continuously stirred at room temperature for 5 h under a nitrogen atmosphere. After TLC showed that the reaction was complete, the reaction was quenched by adding a dilute aqueous solution of citric acid until pH 5–6, and ethyl acetate (200 mL) and water (200 mL) were also added and stirred for 10 min, after which the mixture was transferred into a separatory funnel. Two phases were separated, and the aqueous phase was extracted again with ethyl acetate (100 mL). The organic extracts were combined and washed with brine (100 mL). The organic solution was dried over anhydrous MgSO₄. The solvent was evaporated under vacuum to give a solid crude product, which was purified by flash chromatography (eluent: EtOAc/hexane = 1:2) to produce compound 4 (16.79 g, 40.91 mmol) in 90% yield over two steps. White solid, mp 94–95 °C; [α]_D²⁰ = −4.3 (c 4.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.93 (s, 1H, NH on the indole ring), 7.36–7.28 (m, 3H, Ph-H), 7.26–7.19 (m, 2H, Ph-H), 7.16 (d, J = 8.7 Hz, 1H, H-7), 6.92 (s, 1H, H-4), 6.77 (s, 1H, H-2), 6.76 (d, J = 8.7 Hz, 1H, H-6), 5.13 (d, J = 12.3 Hz, 1H, CHH), 5.09 (s, 1H, CONH), 5.03 (d, J = 12.3 Hz, 1H, CHH), 4.67 (dd, J₁ = 7.8 Hz, J₂ = 4.8 Hz, 1H, CHCOO), 3.21–3.18 (m, 2H, CH₂), 1.42 (s, 9H, C(CH₃)₃). ¹³C NMR (100 MHz, CDCl₃) δ 172.64, 155.63, 149.99, 135.29, 131.33, 128.54, 128.41, 128.34, 128.28, 124.00, 112.21, 111.96, 109.03, 103.14, 80.29, 67.21, 54.33, 28.37, 28.14. IR (KBr film) 3408, 2977, 2931, 1690, 1498, 1457, 1367, 1200, 1162, 1057, 936, 797, 752, 698 cm^–1. HRMS (ESI) m/z calcd for C₂₃H₂₆N₂O₅Na [M + Na]⁺: 433.1739; found: 433.1736.

4.3.2. (S)-Benzyl 3-(5-(benzyloxy)-1H-indol-3-yl)-2-((tert-butoxycarbonyl)amino)propanoate (5)

Compound 4 (15.00 g, 36.54 mmol) was dissolved in 150 mL of acetone. After K₂CO₃ (10.10 g, 73.08 mmol) and benzyl bromide (7.500 g, 43.85 mmol) were added, the mixture was then heated and stirred at reflux for 36 h. After TLC showed that the reaction was complete (eluent: EtOAc/hexane = 1:3), the mixture was cooled to room temperature and filtered. The cake was washed three times with EtOAc (3 × 20 mL). The filtrate was concentrated under vacuum to give a residue, which was dissolved in EtOAc (100 mL). An aqueous solution of citric acid (50 mL, 1 N) was added, and the mixture was vigorously stirred for 5 min. Two phases were separated, and the aqueous phase was extracted again with EtOAc (30 mL). The organic extracts were combined and dried over anhydrous MgSO₄. The solvent was removed under vacuum to give the crude product as a colorless oil, which gradually solidified on standing. The solid was washed with a mixed solvent of ethyl acetate and hexane (EtOAc/hexane = 1:4) to produce compound 5 (15.91 g, 31.78 mmol) in 87% yield as white crystals, mp 115–116 °C. [α]_D²⁰ = −6.1 (c 4.5, CH₃OH). ¹H NMR (400 MHz, CDCl₃) δ 7.98 (s, 1H, NH on the indole ring), 7.46 (d, J = 7.2 Hz, 2H, Ph-H), 7.38 (t, J = 7.2 Hz, 2H, Ph-H), 7.34–7.26 (m, 4H, Ph-H), 7.25–7.19 (m, 3H, Ph-H and H-7), 7.12 (s, 1H, H-4), 6.92 (d, J = 8.8 Hz, 1H, H-6), 6.78 (s, 1H, H-2), 5.19–4.97 (m, 5H, two CH₂Ph and CONH), 4.72–4.66 (m, 1H, CH), 3.26–3.22 (m, 2H, CH₂), 1.41 (s, 9H, C(CH₃)₃). ¹³C NMR (100 MHz, CDCl₃) δ 172.31, 155.39, 153.43, 137.62, 135.37, 131.57, 128.59, 128.56, 128.46, 128.37, 128.11, 127.86, 127.73, 123.84, 113.04, 112.00, 109.72, 102.32, 80.00, 71.02, 67.10, 54.41, 28.40, 28.20. IR (KBr film) 3379, 2978, 2929, 1738, 1707, 1583, 1516, 1486, 1292, 1206, 1168, 1071, 1013, 947, 758, 698 cm^–1. HRMS (ESI) m/z calcd for C₃₀H₃₂N₂O₅Na [M + Na]⁺: 523.2209; found: 523.2203.

4.3.3. (S)-Benzyl 2-amino-3-(5-(benzyloxy)-1H-indol-3-yl)-propanoate (6)

Compound 5 (15.00 g, 29.96 mmol) was dissolved in EtOAc (250 mL). An aqueous solution of HCl (4 N, 30 mL) was added, and the mixture was heated to 60 °C and stirred for 8 h. After the reaction was complete (checked by TLC, eluent:EtOAc/hexane = 1:2), the mixture was then cooled to room temperature. The solvent was removed under vacuum to give a solid residue, which was washed with a mixed solvent of ethyl acetate and hexane (EtOAc/hexane = 1:2). The solid was transferred into a round-bottom flask, and EtOAc (120 mL) and an aqueous solution of K₂CO₃ (10% w/w, 80 mL) were added. After the mixture was vigorously stirred for 10 min, two phases were separated and the aqueous phase was extracted again with EtOAc (50 mL). The organic extracts were combined and dried over anhydrous MgSO₄. The solvent was removed under vacuum to produce compound 6 (11.16 g, 27.87 mmol) in 93% yield as a white solid, mp 104–105 °C. [α]_D²⁰ = −10.2 (c 3.0, CH₃OH). ¹H NMR (400 MHz, CDCl₃) δ 8.03 (s, 1H, NH on the indole ring), 7.45 (d, J = 7.2 Hz, 2H, Ph-H), 7.40–7.29 (m, 6H, Ph-H), 7.29–7.24 (m, 2H, Ph-H), 7.22 (d, J = 8.8 Hz, 1H, H-7), 7.14 (s, 1H, H-4), 6.93 (d, J = 8.8 Hz, 1H, H-6), 6.90 (s, 1H, H-2), 5.11 (s, 2H, CH₂), 5.06 (s, 2H, CH₂), 3.84 (dd, J₁ = 7.3 Hz, J₂ = 5.0 Hz, 1H, CHCOO), 3.22 (dd, J₁ = 14.4 Hz, J₂ = 5.0 Hz, 1H, CHH), 3.04 (dd, J₁ = 14.4 Hz, J₂ = 7.3 Hz, 1H, CHH), 1.59 (br s, 2H, NH₂). ¹³C NMR (100 MHz, CDCl₃) δ 180.35, 157.32, 143.00, 141.27, 136.73, 133.56, 133.53, 133.11, 133.01, 132.97, 132.86, 132.81, 129.66, 117.24, 116.83, 114.94, 107.15, 75.09, 70.70, 60.52, 36.14. IR (KBr film) 3348, 3147, 3034, 2927, 1728, 1583, 1487, 1455, 1298, 1217, 1201, 1118, 1081, 1009, 733, 695 cm^–1. HRMS (ESI) m/z calcd for C₂₅H₂₅N₂O₃ [M + H]⁺: 401.1865; found: 401.1861.

4.3.4. (S)-Methyl 4-((1-(benzyloxy)-3-(5-(benzyloxy)-1H-indol-3-yl)-1-oxopropan-2-yl)amino)-4-oxobutanoate (7)

4-Methoxy-4-oxobutanoic acid (2.904 g, 21.98 mmol) was dissolved in CH₂Cl₂ (30 mL), and SOCl₂ (3.922 g, 32.97 mmol) was added. The resulting solution was then heated and stirred at reflux for 4 h. The reaction solution was concentrated under vacuum to dryness, an oily residue was then dissolved in dry CH₂Cl₂ (15 mL), and the solution was immediately used below. Compound 6 (8.002 g, 19.98 mmol) and Et₃N (4.044 g, 39.96 mmol) were dissolved in CH₂Cl₂ (80 mL), and the solution was cooled down to 0 °C using an ice bath. The above freshly prepared solution of succinic monochloride methyl monoester was added over 5 min. After the addition was finished, stirring was continued at 0 °C for 30 min. When the reaction was complete (checked by TLC, eluent: EtOAc/hexane = 1:1), the reaction mixture was transferred into a separatory funnel. The organic solution was then washed successively with an aqueous solution of HCl (2 N, 50 mL) and an aqueous solution of K₂CO₃ (10% w/w, 30 mL). The organic solution was dried over anhydrous MgSO₄ and then concentrated under vacuum to give a crude product as colorless oil, which gradually solidified on standing. The solid was washed with a mixed solvent of ethyl acetate and hexane (EtOAc/hexane = 1:4) to produce compound 7 (9.664 g, 18.78 mmol) in 94% yield as white crystals, mp 101–102 °C. [α]_D²⁰ = +4.3 (c 5.5, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.97 (s, 1H, NH on the indole ring), 7.45 (d, J = 7.3 Hz, 2H, Ph-H), 7.37 (dd, J₁ = 7.6 Hz, J₂ = 7.3 Hz, 2H, Ph-H), 7.34–7.28 (m, 4H, Ph-H), 7.27–7.20 (m, 3H, Ph-H, and H-7), 7.09 (s, 1H, H-4), 6.93 (d, J = 8.8 Hz, 1H, H-6), 6.78 (s, 1H, H-2), 6.16 (d, J = 7.8 Hz, 1H, CONH), 5.07 (s, 2H, CH₂), 5.05 (s, 2H, CH₂), 4.99–4.95 (m, 1H, CH), 3.62 (s, 3H, OCH₃), 3.29–3.25 (m, 2H, CH₂), 2.68 (t, J = 6.8 Hz, 2H, CH₂), 2.43 (t, J = 6.8 Hz, 2H, CH₂CON). ¹³C NMR (100 MHz, CDCl₃) δ 173.31, 171.86, 171.18, 153.44, 137.61, 135.23, 131.50, 128.62, 128.53, 128.44, 128.38, 128.14, 127.83, 127.65, 123.98, 113.02, 112.10, 109.40, 102.06, 70.93, 67.23, 53.12, 51.86, 30.75, 29.07, 27.68. IR (KBr film) 3399, 3379, 3033, 2939, 1736, 1641, 1537, 1482, 1448, 1354, 1225, 1066, 1028, 799, 734, 699 cm^–1. HRMS (ESI) m/z calcd for C₃₀H₃₀N₂O₆Na [M + Na]⁺: 537.2002; found: 537.1999.

4.3.5. (S)-Benzyl 6-(benzyloxy)-1-(3-methoxy-3-oxopropyl)-4,9-dihydro-3H-pyrido[3,4-b]indole-3-carboxylate (1r)

Compound 7 (2.001 g, 3.889 mmol) was dissolved in EtOAc (30 mL), and then phosphorus oxychloride (1.789 g, 11.67 mmol) was added into the mixture slowly. The resulting solution was then heated and stirred at reflux for 4 h. After the reaction was complete (checked by TLC, eluent: CH₂Cl₂/acetone = 30:1), the mixture was cooled to 0 °C by an ice bath. A saturated NaHCO₃ aqueous solution (50 mL) was then added. After the mixture was vigorously stirred for 5 min, two phases were separated, and the aqueous phase was extracted again with EtOAc (20 mL). The organic extracts were combined, dried over anhydrous MgSO₄, and then concentrated under vacuum to give the crude product, which was purified by flash chromatography (eluent: CH₂Cl₂/acetone = 60:1) to afford compound 1r (1.372 g, 2.763 mmol) in 71% yield as pale yellow crystals, mp 156–157 °C. [α]_D²⁰ = +6.2 (c 5.0, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 9.06 (s, 1H, NH on the indole ring), 7.39 (d, J = 7.3 Hz, 2H, Ph-H), 7.32 (dd, J₁ = 7.6 Hz, J₂ = 7.3 Hz, 2H, Ph-H), 7.28–7.20 (m, 7H, Ph-H, and H-8), 6.96 (s, 1H, H-5), 6.93 (d, J = 8.8 Hz, 1H, H-7), 5.16 (s, 2H, CH₂), 5.01 (s, 2H, CH₂), 4.48–4.44 (m, 1H, H-3), 3.59 (s, 3H, CH₃), 3.15–2.99 (m, 2H, H-4), 2.98–2.84 (m, 2H, CH₂), 2.82–2.65 (m, 2H, CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 174.60, 173.18, 160.89, 153.73, 137.34, 135.89, 132.59, 129.24, 128.60, 128.57, 128.23, 128.12, 127.94, 127.60, 125.48, 116.73, 115.06, 113.34, 102.11, 70.72, 66.86, 61.11, 52.01, 30.75, 29.50, 22.52. IR (KBr film) 3398, 3031, 1737, 1607, 1552, 1494, 1451, 1334, 1298, 1220, 1177, 733 cm^–1. HRMS (ESI) m/z calcd for C₃₀H₂₈N₂O₅Na [M + Na]⁺: 519.1896; found: 519.1893.

4.3.6. 3-(6-(Benzyloxy)-3-((benzyloxy)carbonyl)-9H-pyrido[3,4-b]indol-1-yl)propanoic Acid (8)

Compound 2r (2.502 g, 5.059 mmol) was dissolved in a mixed solvent of tetrahydrofurane and water (25 mL, THF/H₂O = 5:1), and LiOH·H₂O (0.233 g, 5.552 mmol) was added in batches. The resulting solution was then stirred at room temperature for 2 h. After the reaction was complete (checked by TLC, eluent: CH₂Cl₂/acetone = 30:1), the mixture was cooled to 0 °C using an ice bath, and an aqueous solution of HCl (2 N, 5 mL) was added dropwise into the reaction mixture and stirred for 10 min. The solution was concentrated under vacuum to give the crude product, which was washed with cooled water (10 mL) to afford compound 8 (2.212 g, 4.603 mmol) in 91% yield as pale yellow crystals, mp 139–140 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.02 (s, 1H, NH on the indole ring), 9.08 (s, 1H, H-4), 8.25 (s, 1H, H-5), 7.70 (d, J = 8.9 Hz, 1H, H-8), 7.58 (d, J = 7.3 Hz, 2H, Ph-H), 7.53 (d, J = 7.3 Hz, 2H, Ph-H), 7.49–7.32 (m, 7H, Ph-H, and H-7), 5.52 (s, 2H, CH₂Ph), 5.22 (s, 2H, CH₂Ph), 3.60 (t, J = 7.6 Hz, 2H, CH₂), 2.90 (t, J = 7.6 Hz, 2H, CH₂COO). ¹³C NMR (100 MHz, DMSO-d₆) δ 173.84, 164.65, 153.11, 144.19, 137.17, 136.36, 136.15, 135.99, 128.47, 128.36, 128.00, 127.97, 127.78, 127.24, 121.56, 119.70, 116.70, 113.33, 104.91, 69.85, 66.11, 31.50, 27.65. IR (KBr film) 3396, 2914, 2865, 1725, 1623, 1578, 1500, 1452, 1305, 1263, 1192, 1021, 855, 753, 697 cm^–1. HRMS (ESI) m/z calcd for C₂₉H₂₅N₂O₅ [M + H]⁺: 481.1763; found: 481.1762.

4.3.7. (S)-Benzyl 6-(benzyloxy)-1-(3-(2-(methoxycarbonyl)-pyrrolidin-1-yl)-3-oxopropyl)-9H-pyrido[3,4-b]indole-3-carboxylate (9)

Compound 8 (1.862 g, 3.875 mmol) was dissolved in CH₂Cl₂ (35 mL), and the solution was cooled to 0 °C using an ice bath. HOBt (0.629 g, 4.655 mmol), EDC·HCl (0.892 g, 4.653 mmol), methyl l-prolinate (0.751 g, 5.814 mmol), and DIPEA (1.002 g, 7.752 mmol) were added successively. The resulting solution was stirred at 0 °C to room temperature for 24 h. After the reaction was complete (checked by TLC, eluent: EtOAc), an aqueous solution of K₂CO₃ (5% w/w, 25 mL) was added. Two phases were separated, and the aqueous phase was extracted again with CH₂Cl₂ (35 mL). The organic extracts were combined and dried over anhydrous MgSO₄. The solvent was evaporated under vacuum to give the crude product, which was purified by flash chromatography (eluent: EtOAc/CH₂Cl₂ = 1:2) to afford compound 9 (2.018 g, 3.411 mmol) in 88% yield as white crystals, mp 194–195 °C. [α]_D²⁰ = −45.9 (c 1.7, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 10.61 (s, 1H, NH on the indole ring), 8.67 (s, 1H, H-4), 7.61 (s, 1H, H-5), 7.54 (d, J = 7.2 Hz, 2H, Ph-H), 7.51–7.45 (m, 3H, two Ph-H, and H-8), 7.44–7.32 (m, 6H, Ph-H), 7.27 (d, J = 8.1 Hz, 1H, H-7), 5.50 (s, 2H, CH₂Ph), 5.16 (s, 2H, CH₂Ph), 4.47 (dd, J₁ = 8.4 Hz, J₂ = 3.6 Hz, 1H, CHCO₂Me), 3.75–3.62 (m, 2H, CH₂CON), 3.61–3.53 (m, 1H, CHHN), 3.59 (s, 3H, COOCH₃), 3.51–3.42 (m, 1H, CHHN), 2.96 (t, J = 5.8 Hz, 2H, CH₂), 2.17–1.86 (m, 4H, CH₂CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 172.56, 166.08, 153.47, 145.30, 137.19, 137.10, 136.45, 136.06, 135.77, 128.61, 128.58, 128.52, 128.21, 127.97, 127.91, 127.59, 127.58, 122.20, 119.28, 116.80, 113.33, 104.56, 70.89, 66.90, 59.01, 52.17, 47.13, 33.09, 29.21, 28.61, 24.64. IR (KBr film) 3253, 2949, 2878, 1748, 1712, 1632, 1566, 1495, 1447, 1337, 1300, 1227, 1189, 1108, 1005, 724 cm^–1. HRMS (ESI) m/z calcd for C₃₅H₃₄N₃O₆ [M + H]⁺: 592.2448; found: 592.2451.

4.3.8. (S)-6-Hydroxy-1-(3-(2-(methoxycarbonyl)-pyrrolidin-1-yl)-3-oxopropyl)-9H-pyrido[3,4-b]indole-3-carboxylic Acid (10)

Compound 9 (2.003 g, 3.385 mmol) was dissolved in MeOH (30 mL), and Pd/C (0.201 g, 10% w/w) was added. The mixture was then heated and stirred at reflux for 8 h under a H₂ atmosphere. After the reaction was complete (checked by TLC, eluent: EtOAc/hexane = 1:1), the hot reaction solution was immediately filtered through a thin layer of celite (2 cm) to remove the black catalyst and the filter cake was washed twice with warm methanol (2 × 10 mL). The filtrate was concentrated under vacuum to give compound 10 (1.337 g, 3.250 mmol) in 96% yield as a pale yellow solid, mp 215–216 °C. [α]_D²⁰ = −71.3 (c 1.0, CH₃OH). ¹H NMR (400 MHz, DMSO) δ 11.83 (s, 1H, NH on the indole ring), 9.29 (s, 1H, OH), 8.63 (s, 1H, H-4), 7.59 (s, 1H, H-5), 7.47 (d, J = 8.7 Hz, 1H, H-8), 7.11 (d, J = 8.7 Hz, 1H, H-7), 4.29 (dd, J₁ = 8.7 Hz, J₂ = 4.2 Hz, 1H, CHCOOMe), 3.67 (t, J = 6.8 Hz, 2H, CH₂), 3.58 (s, 3H, OCH₃), 3.50–3.24 (m, 2H, CH₂N), 3.11–2.87 (m, 2H, CH₂), 2.21–1.80 (m, 4H, CH₂CH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.61, 170.46, 166.73, 151.51, 143.99, 136.30, 135.28, 134.91, 126.89, 122.01, 118.58, 115.48, 112.90, 105.79, 58.23, 51.64, 46.50, 31.16, 28.81, 27.84, 24.37. IR (KBr film) 3169, 2953, 2757, 1741, 1642, 1607, 1577, 1447, 1354, 1241, 1204, 1179, 839, 798, 724 cm^–1. HRMS (ESI) m/z calcd for C₂₁H₂₁N₃O₆Na [M + Na]⁺: 434.1328; found: 434.1329.

4.3.9. (S)-Methyl 1-(3-(6-hydroxy-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)propanoyl)pyrrolidine-2-carboxylate (11)

Compound 10 (1.200 g, 2.917 mmol) was dissolved in anhydrous DMF (12 mL), and the solution was cooled to 0 °C with an ice bath. HOBt (0.473 g, 3.501 mmol), EDC·HCl (0.671 g, 3.500 mmol), methyl l-threoninate (0.583 g, 4.378 mmol), and DIPEA (0.754 g, 5.834 mmol) were added successively. The mixture was stirred at 0 °C for 2 h and then stirred at room temperature for 10 h. After the reaction was complete (checked by TLC, eluent: EtOAc), EtOAc (100 mL) and an aqueous solution of NaHCO₃ (5% w/w, 60 mL) were added and the mixture was then vigorously stirred for 5 min. Two phases were separated with a separatory funnel, and the aqueous solution was extracted again with EtOAc (60 mL). Organic extracts were combined and dried over anhydrous MgSO₄. The solvent was evaporated under vacuum to give the crude product, which was purified by flash chromatography (eluent: CH₂Cl₂/CH₃OH = 1:10) to furnish compound 11 (1.306 g, 2.480 mmol) in 85% yield as a white amorphous solid, mp 131–132 °C. [α]_D²⁰ = +13.1 (c 1.8, CHCl₃). ¹H NMR (400 MHz, DMSO-d₆) δ 11.78 (s, 1H, NH on the indole ring), 9.25 (s, 1H, ArOH), 8.66 (d, J = 9.0 Hz, 1H, CONH), 8.55 (s, 1H, H-4), 7.58 (s, 1H, H-5), 7.47 (d, J = 8.7 Hz, 1H, H-8), 7.11 (d, J = 8.7 Hz, 1H, H-7), 5.35 (d, J = 5.1 Hz, 1H, OH), 4.54 (dd, J₁ = 9.0 Hz, J₂ = 3.6 Hz, 1H, NCHCOOMe), 4.35–4.23 (m, 2H, two CH), 3.69 (s, 3H, OCH₃), 3.68–3.59 (m, 2H, CH₂), 3.55 (s, 3H, OCH₃), 3.48–3.32 (m, 2H, CH₂), 3.12–2.90 (m, 2H, CH₂), 2.22–1.80 (m, 4H, CH₂CH₂), 1.15 (d, J = 6.3 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.62, 171.33, 170.08, 165.04, 151.42, 143.21, 137.07, 136.16, 134.96, 127.10, 122.05, 118.56, 112.85, 112.33, 105.76, 66.43, 58.25, 57.63, 52.00, 51.60, 46.43, 30.76, 28.71, 27.39, 24.35, 20.50. IR (KBr film) 3374, 2976, 2954, 1740, 1629, 1529, 1450, 1460, 1365, 1276, 1198, 1088, 812, 667, 619 cm^–1. HRMS (ESI) m/z calcd for C₂₆H₃₁N₄O₈ [M + H]⁺: 527.2142; found: 527.2141.

4.3.10. 6-Hydroxymetatacarboline D (3)

Compound 11 (1.001 g, 1.901 mmol) was dissolved in MeOH (15 mL) and cooled to 0 °C using an ice bath. LiOH·H₂O (0.399 g, 9.508 mmol) was added in batches. The ice bath was removed, and the mixture was stirred at 0 °C to room temperature for 5 h under a N₂ atmosphere. After the reaction was complete (checked by TLC, eluent: EtOAc), the solution was concentrated under vacuum. The residue was dissolved in H₂O (6 mL) and cooled to 0 °C by an ice bath. An aqueous solution of HCl (2 N) was slowly added until pH ≈ 4.2 (isoelectric point). A pale yellow solid precipitated, and the mixture was further stirred at 0 °C for 1 h. The cooled suspension was then filtered, and the filter cake was washed twice with pure cool water (2 × 3 mL). After drying under vacuum for 8 h, final product 6-hydroxymetatacarboline D 3 (0.776 g, 1.557 mmol) was obtained in 82% yield as a white amorphous solid, mp 269–270 °C. [α]_D²⁰ = +23.5 (c 1.0, CH₃OH). ¹H NMR (400 MHz, DMSO-d₆) δ 11.77 (s, 1H, NH on the indole ring), 9.25 (s, 1H, ArOH), 8.63 (d, J = 9.0 Hz, 1H, CONH), 8.55 (s, 1H, H-4), 7.58 (s, 1H, H-5), 7.47 (d, J = 8.7 Hz, 1H, H-8), 7.11 (d, J = 8.7 Hz, 1H, H-7), 4.45 (dd, J₁ = 9.0 Hz, J₂ = 3.0 Hz, 1H, CH), 4.35–4.30 (m, 1H, CH), 4.28–4.23 (m, 1H, CH), 3.68–2.62 (m, 2H, CH₂), 3.52–3.44 (m, 2H, CH₂), 3.20–3.11 (m, 1H, CHHN), 2.96–2.86 (m, 1H,CHHN), 2.16–1.85 (m, 4H, CH₂CH₂). 1.14 (d, J = 6.3 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 173.86, 172.53, 170.16, 165.23, 151.54, 143.37, 137.51, 136.31, 135.08, 127.21, 122.25, 118.65, 113.04, 112.39, 105.93, 66.72, 58.50, 57.57, 46.60, 30.87, 28.96, 27.61, 24.45, 20.98. IR (KBr film) 3382, 2971, 1735, 1587, 1540, 1500, 1473, 1399, 1237, 1203, 1081, 799, 626 cm^–1. HRMS (ESI) m/z calcd for C₂₄H₂₆N₄O₈Na [M + Na]⁺: 521.1648; found: 521.1652.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b01908.

• ¹H and ¹³C NMR spectra of all compounds (PDF)

The authors declare no competing financial interest.