Total Synthesis of 6-Hydroxymetatacarboline-d Discovered from Mycena metata via the Pictet-Spengler Reaction Followed by the Horner-Wadsworth-Emmons Reaction.

Total synthesis of a new β-carboline alkaloid, 6-hydroxymetatacarboline-d, which was isolated from fruiting bodies of Mycena metata was accomplished in 14 steps. The synthetic strategy features the Pictet-Spengler reaction to construct the tricyclic core followed by amide coupling and the Horner-Wadsworth-Emmons reaction.

Introduction

The β-carboline scaffold represents the core unit of several natural compounds and pharmaceutical agents. Compounds containing this subunit are ubiquitously present in plants, marine organisms, insects, and mammals including human tissues and body fluids in the form of alkaloids or hormones.[1]

β-Carbolines are frequently found in plant-derived beverages, foodstuffs (rice, wheat, corn, mushrooms, barley, soybeans, vinegar, and grapes), narcotic substances, such as tobacco (Nicotiana spp.), maracuja (Passiflora spp.), guarana (Pauliniacupana), and yopo (Anadenantheraperegrina).[2] They are also found in mammals, including in the liver, heart, kidney, and human brain tissues and blood plasma, and they are widely observed in marine organisms.[3−7] β-Carboline alkaloids were first isolated in 1841 from Peganum harmala (Zygophillaceae; Syrian rue), which is traditionally used in herbal remedies as an emmenagogueor abortifacient in the Middle East and Northern Africa.[8] Furthermore, for hundreds of years, they have been used for the treatment of alimentary tract cancers and malaria in North western China. Interestingly, eight plant families are known to produce more than 64 different types of β-carboline alkaloid.[9−11] By dry weight, the seeds of Peganumharmala contain 0.16–5.9% of β-carboline alkaloids,[12,13] while β-carboline frameworks also exhibit fluorescence properties, owing to the presence of significant conjugation. Indeed, it is owing to the presence of β-carbolines in the cuticle of scorpions that their skin is known to fluoresce when exposed to certain wavelengths of light.[14]

This privileged scaffold has displayed significant pharmacological properties, including anticancer, antibacterial, anxiolytic, antifungal,[15] antiviral, anti-HIV, anti-Alzheimer, antimalarial, and anticonvulsant activity.[16−21]

In 2013 Robert J. R. Jaeger et al[22] isolated an alkaloid, 6-hydroxymetatacarboline-d, from the fruiting bodies of Mycena metata and assigned its structure, on the basis of various range of spectroscopic studies. The total synthesis of 6-hydroxymetatacarboline-d has been reported previously via a different approach.[23] In the present research work, we wish to report the novel route for the synthesis of 6-hydroxymetatacarboline-d via the Pictet–Spengler and Horner–Wadsworth–Emmons (HWE) reactions.

Results and Discussion

6-Hydroxymetatacarboline-d was synthesized starting with 5-hydroxytryptophan. Further in the synthesis, the corresponding commercially available enantiomerically pure starting materials (methyl l-threoninate and tert-butyl l-prolinate) were used in the synthesis.

5-Hydroxytryptophan (1) was refluxed with SOCl2 in methanol to provide 5-hydroxytryptophan methyl esterhydrochloride (2) in 95% yield[24] (Scheme ). Having the compound (2) in hand, next, we envisioned to synthesize both cis and trans isomers of tetrahydro-β-carboline (THβC) derivatives with the Pictet–Spengler reaction of 5-hydroxytryptophan methyl ester with the respective aldehyde. The cis isomer is predominantly formed under kinetically controlled conditions. Whereas, the selectivity is transferred toward the trans isomer under thermodynamically controlled conditions, which exclusively depends upon the nature of reagents, solvents, reaction time, and temperature.[25,26] We observed that the Pictet–Spengler reaction[27] using 5-hydroxytryptophan methyl ester hydrochloride and 2,2-dimethoxyacetaldehyde in DCM with catalytic TFA at ambient temperature resulted in the formation of methyl 1-(dimethoxymethyl)-6-hydroxy-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (4) in 59% yields. The selectivity of cis/trans was not of much importance to us in the present scheme as we needed to do the C-ring oxidation involving both the chiral centers. Initially, the oxidation of the C-ring was not successful with KMnO4 in the presence of a free hydroxyl group as step (d) in Scheme .

Scheme 1

Synthesis of Methyl 6-((tert-Butyldimethylsilyl)oxy)-1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carboxylate (7)

The next approach was to protect the free hydroxyl group, which could be removed selectively. The protecting group, tert-butyldimethylsilyl, being selective to hydroxyl,[28] was taken. The hydroxyl protection was done with tert-butyldimethylsilyl chloride in the presence of imidazole in dichloromethane to obtain intermediate (6). On treating this protected moiety with KMnO4 in THF, we were successful in C-ring oxidation to obtain methyl 6-((tert-butyldimethylsilyl)oxy)-1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carboxylate (7) with 57% yield.

Further, the demasking of acetal was achieved with AcOH: H2O (1:1), which furnished methyl 6-((tert-butyldimethylsilyl)oxy)-1-formyl-9H-pyrido[3,4-b]indole-3-carboxylate (8) in excellent yield (89%)(Scheme ). The next step was chain extension on a formyl group, which was achieved with the Horner–Wadsworth–Emmons (HWE) reaction[29] using tert-butyl-2-(diethoxyphosphoryl)acetate (9) and NaH, to obtain methyl-1-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl)-6-((tert-butyldimethylsilyl)oxy)-9H-pyrido[3,4-b]indole-3-carboxylate (10) with 80% yield. Here, tert-butyl ester was taken for selective hydrolysis, which was done using TFA in DCM. Further, the amide coupling was done between obtained acid (11) and tert-butyl l-prolinate (12) using HATU to obtain the desired amide product (13) in 55% yield. The hydrolysis of the obtained amide product was challenging as it degraded under basic (LiOH, NaOH, KOH) and acidic conditions (aq HCl).

Scheme 2

Synthesis of (S)-1-(3-(2-(tert-Butoxycarbonyl)pyrrolidin-1-yl)-3-oxoprop-1-en-1-yl)-6-hydroxy-9H-pyrido[3,4-b]indole-3-carboxylic acid (14)

Different hydrolysis conditions were tried on intermediates (13) and (7) using different bases and acids at varying temperatures. Finally, successful hydrolysis of intermediate (7) was achieved under anhydrous conditions using trimethyltinhydroxide[30] at 110 °C in 1,2-dichloroethane to obtain the intermediate (15) with 65–70% yields. The duration of the reaction was decreased by increasing the mole concentration of trimethyltinhydroxide. As hydrolysis was not successful on intermediate (13), we had to change the route in Scheme .

Scheme 3

Synthesis of Methyl (6-((tert-Butyldimethylsilyl)oxy)-1-formyl-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threoninate (18)

Amide coupling between acid (15) and methyl l-threoninate (16) was done using HATU to obtain the desired amide (17) in 80–85% yield. Further demasking of acetal was achieved with AcOH:H2O (1:1), which furnished the desired formyl product (18) in 85–89% yields (Scheme ).

In Scheme , chain extension on the formyl group was achieved using the Horner–Wadsworth–Emmons (HWE) reaction with tert-butyl 2-(diethoxyphosphoryl)acetate (19) and NaH to obtain the desired product (20) with 85–90% yields. Further, the selective hydrolysis of tert-butyl ester was done using TFA in DCM to obtain the desired acid (21), which was coupled with tert-butyl l-prolinate (22) using HATU to get the desired amide product (23) in 66% yield.

Scheme 4

Synthesis of tert-Butyl ((E)-3-(6-((tert-Butyldimethylsilyl)oxy)-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido [3,4-b]indol-1-yl)acryloyl)-l-prolinate (23)

Then, the double bond was reduced using hydrogenation conditions with 10% Pd/C in 15–30 min to obtain the intermediate (24) with 93% yield. Now, our challenge was to selectively remove the protecting groups to obtain the desired final compound (Scheme ).

Scheme 5

Synthesis of 6-Hydroxymetatacarboline-d TFA (27)

Intermediate (24) was then treated with TBAF to knock off the TBDMS group to obtain the intermediate (25), which was further treated with tributyltinhydroxide in 1,2-dichloroethane to obtain the compound (26). For the target compound, the tertiary butyl group was removed using TFA to obtain 6-hydroxymetatacarboline-d (27) as TFA salt with 89% purity. The overall yield for the total synthesis was ∼3%, and it is summarized in Table .

Table 1

Stepwise Yield of the Synthesized Products and Characterization Techniques Useda

scheme	steps	yield (%)	characterization techniques of compounds
1	a	95	1H NMR, 13C NMR, LCMS
	b	59	1H NMR, 13C NMR, LCMS, IR
	c	65	1H NMR, 13C NMR, LCMS, IR
	e	57	1H NMR, 13C NMR, LCMS, IR
2	f	89	1H NMR, 13C NMR, LCMS, IR
	g	70	1H NMR, 13C NMR, LCMS, IR
	h	65	1H NMR, 13C NMR, LCMS, IR
	i	55	1H NMR, 13C NMR, LCMS, IR
3	k	70	1H NMR, 13C NMR, LCMS, IR
	l	85	1H NMR, 13C NMR, LCMS, IR
	m	89	1H NMR, 13C NMR, LCMS, IR
4	n	94	1H NMR, 13C NMR, LCMS, IR
	o	90	1H NMR, 13C NMR, LCMS, IR
	p	66	1H NMR, 13C NMR, LCMS, IR
5	q	93	1H NMR, 13C NMR, LCMS, IR
	r	88	1H NMR, 13C NMR, LCMS, IR
	s	81	1H NMR, 13C NMR, LCMS, IR
	t	89	1H NMR, 13C NMR, LCMS, ,HRMS, IR

Methods for steps d & j not successfully proceed as mentioned in Schemes and 2.

Conclusions

A concise and efficient strategy for the total synthesis of 6-hydroxymetatacarboline-d was developed from 5-hydroxytryptophan with the application of the Pictet–Spengler reaction and Horner–Wadsworth–Emmons (HWE) reaction as the key reactions. Hydrolysis of the methyl ester with trimethyltin hydroxide played an important role in our scheme as the normal basic and acidic conditions did not give us the desired results. This is the first report for the total synthesis of 6-hydroxymetatacarboline-d. The synthetic protocol developed can also be appropriately used for the preparation of other β-carboline analogues.

Experimental Section

Melting points were measured on a Mettler Toledo-MP-80 automated melting point system and are uncorrected. Infrared spectra (ν max) were recorded on a Perkin–Elmer/Spectrum-2 FTIR spectrometer. Samples were analyzed as thin films on KBr plates. Proton (1H) and carbon (13C) NMR spectra were recorded at room temperature in CDCl3, CD3OD, or (CD3)2SO on a Varian spectrometer operating at 400 MHz for protonand 100 MHz for carbon nuclei. The signal due to residual CHCl3 appeared at δH 7.26, and the central resonance of the CDCl3 triplet appeared at δC 77.2 were used to reference 1H and 13C NMR spectra, respectively. For the spectra recorded in CD3OD, these were referenced to the signals at δH 3.31 and δC 49.0, respectively, while the equivalent resonances employed for the spectra recorded in CDCl3 were δH 7.26 and δC 77.16 ppm and for (CD3)2SO were δH 2.50 and δC 39.52 ppm. Low-resolution ESI mass spectra were recorded on a single quadruple liquid chromatograph–mass spectrometer, while high-resolution measurements were conducted on a time-off light instrument. Low and high-resolution EI mass spectra were recorded on a magnetic-sector machine. Analytical thin-layer chromatography (TLC) was performed on aluminum-backed 0.2 mm thick silica gel 60 F254 plates as supplied by Merck. Eluted plates were visualized using a 254 nm UV lamp and/or by treatment with a suitable dip followed by heating. The dip commonly used was potassium permanganate/potassium carbonate/5%sodium hydroxide aqueous solution/H2O (3 g:20 g:5 mL; 300 mL). Flash chromatographic separations were carried out on a CombiFlash with silica gel of 230–400 mesh as the stationary phase and using the LR-grade solvents as indicated. The melting points of solids purified by such means were recorded directly (i.e., after they had crystallized from the concentrated chromatographic fractions). The starting materials and reagents were generally available from Sigma–Aldrich, Merck, TCI, or Combi blocks and were used as supplied. Drying agents and other inorganic salts were purchased from JDH, Spectrochem, or Sigma Chemical Companies. Where necessary, reactions were performed under a nitrogen/argon atmosphere.

Synthesis of Methyl 2-Amino-3-(5-hydroxy-1H-indol-3-yl)propanoate Hydrochloride (2)

To a solution of 2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid (15 g, 0.068 mol) in methanol (150 mL) was added SOCl2 (5 mL) drop wise at 0–5 °C in 8–10 min. The reaction mass was slowly brought to room temperature and stirred to 65 °C for 6 h. After completion of the reaction as confirmed by TLC (100% Ethyl acetate, Rf: 0.2), it was cooled to room temperature and concentrated under vacuum. Ethyl acetate (50 mL) was added and stirred for 2–3 h to get solids. The solids were filtered and given diethyl ether (25 mL) washings to get the desired product methyl 2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoate hydrochloride (17.5 g, 95%) as a brown solid.

1H NMR (400 MHz, DMSO-d6) δ3.17 (d, 2H, J = 6.24 Hz), 3.68 (s, 3H), 4.19 (brd, 1H), 6.60–6.63 (dd, J = 1.96 Hz, J = 8.64 Hz, 1H), 6.76 (s, 1H), 7.11–7.16 (m, 2H), 8.40 (bs, 3H), 8.68 (s, 1H), 10.76 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 26.6, 52.5, 52.6, 101.9, 105.3, 111.6, 111.9, 125.4, 127.6, 130.7. ESI-MS (m/z): 235.12 [M + 1]+.

Synthesis of Methyl 1-(Dimethoxymethyl)-6-hydroxy-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (4)

To a stirred solution of l-tryptophan methyl ester HCl (5.0 g, 0.018 mol) in anhydrous dichloromethane (50 mL), 2,2-dimethoxy acetaldehyde (60% aqueous solution in water) (3.85 mL, 0.022 mol) was added at room temperature. Thereafter, a solution of TFA (5% TFA in DCM, 10 mL) was added dropwise and the reaction mixture was further stirred at room temperature for 2 h. After completion of the reaction as confirmed by TLC (5% MeOH in DCM, Rf: 0.2, 0.3), the reaction was quenched by slow addition of 10% aqueous NaHCO3 solution under stirring, till pH ≈ 7.0. The organic layer was separated, and the aqueous layer was further extracted with 10% methanol in dichloromethane (3 × 50 mL). The organic layers were combined and washed with saturated brine solution (50 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain methyl 1-(dimethoxymethyl)-6-hydroxy-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate as an off-white solid (3.5 g, 59%).

1H NMR (400 MHz, DMSO-d6) δ 2.37–2.39 (m, 1H), 2.52–2.57 (m, 1H), 2.71–2.75 (m, 1H), 2.81–2.87 (m, 1H), 3.39 (s, 1H), 3.40 (s, 1H), 3.43 (s, 2H), 3.44 (s, 2H), 3.64 (s, 1H), 3.73 (s, 2H), 4.11–4.18 (m, 2H), 4.39 (d, J = 6.0 Hz, 1H), 6.52–6.55 (m, 1H), 6.66–6.67 (s, 1H), 7.13–7.18 (d, J = 8.4 Hz, 1H), 8.53–8.55 (s, 1H), 9.84–9.93 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 24.5, 25.4, 51.7, 51.8, 51.9, 52.5, 54.1, 54.2, 54.2, 54.4, 55.1, 55.3, 55.7, 89.4, 101.5, 101.6, 101.7, 105.8, 105.9, 106.4, 106.5, 110.7, 110.8, 111.7, 111.9, 126.9, 130.6, 131.9, 132.4, 150.2, 150.3, 173.2, 173.9, 197.9. IR (KBr, cm–1): 3407, 2952, 2838, 1734, 1629, 1593, 1453, 1347, 1284, 1197, 1121, 1059. ESI-MS (m/z): 321.33 [M + 1]+.

Synthesis of Methyl 6-((tert-Butyldimethylsilyl)oxy)-1-(dimethoxymethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (6)

To a solution of methyl 1-(dimethoxymethyl)-6-hydroxy-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (3.5 g, 0.011 mol) in dichloromethane (100 mL) was added TBDMSCl (2.5 g, 0.016 mol) followed by imidazole (0.27 g, 0.055 mol) at 0–5 °C. The reaction mass was stirred at room temperature for 24 h. The reaction was monitored by TLC (50% ethyl acetate in n-hexane, R: 0.4). The reaction mass was quenched with DM water (100 mL) and extracted with 5% MeOH in dichloromethane (3 x 50 mL). Water (100 mL) and saturated brine solution (100 mL) were given washings to the combined organic layer. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to get crude mass. The crude compound was purified over a silica gel column in a CombiFlash, eluting with 0–35% ethyl acetate in n-heptane. The desired fractions were concentrated under vacuum to get methyl 6-((tert-butyldimethylsilyl)oxy)-1-(dimethoxymethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (3.1 g, 65%) as a yellow gum.

1H NMR (400 MHz, DMSO-d6) δ 0.15 (s, 6H), 0.96 (s, 9H), 2.38–2.41 (m, 1H), 2.52–2.58 (m, 1H), 2.73–2.76 (m, 1H), 2.87–2.90 (m, 1H), 3.39–3.41 (s, 2H), 3.44–3.45 (s, 4H)m, 3.64–3.72 (s, 3H), 4.12–4.19 (m, 1H), 4.39–4.40 (m, 1H), 6.56–6.59 (m, 1H), 6.78 (s, 1H), 7.20–7.27 (d, J = 8.04 Hz, 1H), 10.01–10.11 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 14.0, 17.9, 20.7, 24.5, 25.4, 25.7, 51.6, 51.7, 51.8, 52.4, 54.2, 54.4, 54.8, 55.1, 55.2, 55.7, 59.7, 105.9, 106.4, 106.7, 106.8, 106.9, 111.7, 112.0, 114.4, 114.5, 126.8, 131.7, 131.8, 132.4, 132.9, 147.7, 147.8, 170.2, 173.0, 173.8. IR (KBr, cm–1): 3463, 2955, 2932, 2898, 2858, 1739, 1627, 1590, 1569, 1472, 1454, 1390, 1362, 1328, 1296, 1257, 1208, 1126, 1072. ESI-MS (m/z): 435.40 [M + 1]+.

Synthesis of Methyl 6-((tert-Butyldimethylsilyl)oxy)-1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carboxylate (7)

To a solution of methyl 6-((tert-butyldimethylsilyl)oxy)-1-(dimethoxymethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (3.1 g, 0.007 mol) in tetrahydrofuran (100 mL) was added KMnO4 (9.2 g) lotwise in 30 min at room temperature. The reaction mass was stirred at room temperature for 20 h. The reaction was monitored by TLC (50% ethyl acetate in n-hexane, Rf: 0.5). The reaction mass was diluted with dichloromethane (100 mL) and stirred for 15 min. The solids were filtered through a celite bed and given dichloromethane washings (3 × 50 mL). The filtrate was concentrated under vacuum to get methyl 6-((tert-butyldimethylsilyl)oxy)-1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carboxylate (1.75 g, 57%) as a yellow gum.

1H NMR (400 MHz, DMSO-d6) δ 0.23 (s, 6H), 1.00 (s, 9H), 3.43 (s, 6H), 3.90 (s, 3H), 5.63 (s, 1H), 7.13–7.15 (dd, J = 2.4 Hz, J = 8.76 Hz, 1H), 7.63 (d, J = 8.76 Hz, 1H), 7.87 (d, J = 2.14 Hz, 1H), 8.96 (s, 1H), 11.37 (s, 1H). IR (KBr, cm–1): 3438, 3400, 2954, 2932, 2898, 2858, 2833, 1765, 1720, 1632, 1578, 1490, 1467, 1434, 1390, 1348, 1319, 1289, 1265, 1236, 1201, 1116, 1059, 1008. ESI-MS (m/z): 430.19 [M + 1]+.

Synthesis of 6-((tert-Butyldimethylsilyl)-oxy)-1-(dimethoxymethyl)-9H-pyrido-[3,4-b]indole-3-carboxylic Acid (15)

To a solution of methyl 6-((tert-butyldimethylsilyl)oxy)-1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carboxylate (2 g, 0.0046 mol) in 1,2-dichloroethane was added trimethyltinhydroxide (1.66 g, .0092 mol). The reaction mass was stirred at 110 °C for 24 h. The reaction was monitored by TLC (70% ethyl acetate in n-hexane, Rf:0.2). Upon completion, the reaction mass was cooled to room temperature. It was quenched with a 5% citric acid solution (50 mL) and extracted with dichloromethane (3 × 50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to get the desired product, 6-((tert-butyldimethylsilyl)-oxy)-1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (1.35 g, 70%) as a yellow solid.

1H NMR (400 MHz, DMSO-d6) δ 0.25 (s, 6H), 0.99 (s, 9H), 3.44 (s, 6H), 5.62 (s, 1H), 7.14 (dd, J = 2.16 Hz, J = 8.84 Hz, 1H), 7.63 (d, J = 8.76 Hz, 1H), 7.85 (s, 1H), 8.92 (s, 1H), 11.34 (s, 1H), 12.63 (bs, 1H).13C NMR (100 MHz, DMSO-d6) δ −4.5, 25.6, 54.2, 105.9, 111.1, 113.5, 118.1, 121.2, 122.5, 128.9, 134.4, 135.4, 136.5, 140.3, 148.9, 166.7. ESI-MS (m/z): 417.40 [M + 1]+.

Synthesis of Methyl (6-((tert-Butyldimethylsilyl)oxy)-1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threoninate (17)

To a solution of 6-((tert-butyldimethylsilyl)oxy)-1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (1.2 g, 0.0026 mol) in dimethylformamide (10 mL) was added l-threonine methyl esterhydrochloride (0.67 g, 0.0039 mol), followed by the addition of HATU (1.5 g, 0.0039 mol). The reaction mass was cooled to 0–5 °C and DIPEA was added (1.68 g, 0.013 mol). The reaction mass was stirred at room temperature for 20 h. The reaction was monitored by TLC (70% ethyl acetate in n-hexane, Rf: 0.7). The reaction mass was quenched with DM water (50 mL) and extracted with ethyl acetate (3 × 50 mL). Chilled water (2 × 50 mL) and saturated brine solution (50 mL) were given washings to the combined organic layer. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to get crude mass. The crude compound was purified over a silica gel column in a CombiFlash, eluting with 0–65% ethyl acetate in n-heptane. The desired fractions under vacuum were concentrated to get methyl (6-((tert-butyldimethylsilyl)oxy)-1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threoninate (1.3 g, 85%) as an off-white solid. Melting point: 76 °C, 1H NMR (400 MHz, DMSO-d6) δ 0.23 (s, 6H), 0.99 (s, 9H), 1.14 (d, J = 6.24 Hz, 3H), 3.47 (s, 3H), 3.48 (s, 3H), 3.69 (s, 3H), (4.30 (m, 1H), 4.57 (d, J = 9.04 Hz, 1H), 5.37 (d, J = 4.96 Hz, 1H), 5.70 (s, 1H), 7.15 (d, J = 9.0 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.87 (s, 1H), 8.56 (d, J = 8.8 Hz, 1H), 8.89 (s, 1H), 11.34 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ −4.6, 17.8, 20.5, 25.6, 38.21, 52.1, 54.0, 54.1, 57.7, 66.4, 105.3, 111.2, 113.5, 114.9, 121.2, 122.7, 129.5, 134.6, 136.7, 136.9, 139.3, 148.9, 164.8, 171.3. IR (KBr, cm–1): 3385, 2955, 2934, 2859, 1743, 1656, 1593, 1578, 1531, 1487, 1360, 1322, 1266, 1211, 1159, 1117, 1070, 1029. ESI-MS (m/z): 532.07 [M + 1]+.

Synthesis of Methyl (6-((tert-butyldimethylsilyl)oxy)-1-formyl-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threoninate (18)

To a solution of methyl (6-((tert-butyldimethylsilyl)oxy)-1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threoninate (1.3 g, 0.0024 mol) in DM water (10 mL) was added acetic acid (10 mL). The reaction mass was stirred at 100 °C for 30 min. (The reaction mass becomes clear, and then yellow solids fall out). The reaction was monitored by TLC (50% ethyl acetate in n-hexane, Rf: 0.6). The reaction mass was cooled to room temperature. It was quenched with chilled water (50 mL), and the solids were filtered. It was dried under vacuum to get methyl (6-((tert-butyldimethylsilyl)oxy)-1-formyl-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threoninate (1.05 g, 89%) as a yellow solid. Melting point: 205.5 °C, 1H NMR (400 MHz, DMSO-d6) δ 0.24 (s, 6H), 0.99 (s, 9H), 1.16 (d, J = 6.32 Hz, 3H), 3.70 (s, 3H), 4.33 (m 1H), 4.63 (d, J = 8.96 Hz, 1H), 5.41 (d, J = 5.2 Hz, 1H), 7.21 (d, J = 8.96 Hz, 1H), 7.70 (d, J = 8.76 Hz, 1H), 8.00 (s, 1H), 8.65 (d, J = 9.04 Hz, 1H), 9.23 (s, 1H), 10.29 (s, 1H), 12.29 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ −4.6, 17.9, 20.5, 25.6, 52.1, 57.8, 66.4, 111.9, 114.0, 119.1, 121.0, 123.4, 131.9, 133.9, 135.3, 137.7, 138.4, 149.8, 164.2, 171.2, 193.8. IR (KBr, cm–1): 3374, 2955, 2930, 2886, 2856, 1750, 1689, 1649, 1579, 1535, 1488, 1465, 1438, 1422, 1360, 1305, 1276, 1201, 1160, 1116, 1095, 1071, 1017. ESI-MS (m/z): 486.45 [M + 1]+.

Synthesis of Methyl (1-((E)-3-(tert-Butoxy)-3-oxoprop-1-en-1-yl)-6-((tert-butyldimethylsilyl)oxy)-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threoninate (20)

To a solution of tert-butyl 2-(diethoxyphosphoryl)acetate (0.59 g, 0.0023 mol) in tetrahydrofuran (15 mL) was added NaH (0.093 g, 0.0023 mol) lotwise at 0–5 °C in 10 min. The reaction mass was stirred at 0–5 °C for 30 min. Methyl (6-((tert-butyldimethylsilyl)oxy)-1-formyl-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threoninate (0.75 g, 0.0016 mol) solution (in 10 mL THF) was added drop wise. The reaction mass turned to dark red color. The reaction mass was stirred at room temperature for 30 min. The reaction was monitored by TLC (50% ethyl acetate in n-hexane, Rf: 0.6). The reaction mass was quenched with chilled water (25 mL) and extracted with ethyl acetate (3 × 30 mL). The saturated brine solution (50 mL) was given washing to the combined organic layer. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to get crude mass. The crude compound was purified over a silica gel column in a CombiFlash, eluting with 0–35% ethyl acetate in n-heptane. The desired fractions were concentrated under vacuum to get methyl (1-((E)-3-(tert-butoxy)-3-oxoprop-1-en-1-yl)-6-((tert-butyldimethylsilyl)oxy)-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threoninate (0.85 g, 94%) as a yellow solid. Melting point: 76 °C, 1H NMR (400 MHz, DMSO-d6) δ 0.24 (s, 6H), 1.00 (s, 9H), 1.15 (d, J = 6.0 Hz), 1.56 (s, 9H), 3.70 (s, 3H), 4.31–4.32 (m 1H), 4.58–4.61 (m, 1H), 5.41 (d, J = 5.6 Hz, 1H), 7.09 (d, J = 15.2 Hz, 1H), 7.18 (dd, J = 2.0 Hz, J = 8.8 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.92 (d, J = 2.0 Hz, 1H), 8.28 (d, J = 15.6 Hz, 1H), 8.69 (d, J = 9.2 Hz, 1H), 8.94 (s, 1H), 12.34 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ −4.4, −4.3, 16.2, 16.3, 18.2, 20.3, 25.7, 27.8, 28.2, 52.8, 58.4, 62.5, 62.6, 67.8, 81.4, 111.1, 112.7, 114.5, 122.1, 123.2, 123.3, 130.2, 134.1, 136.1, 136.6, 137.4, 138.3, 150.1, 165.9, 167.1, 172.9. IR (KBr, cm–1): 3374, 2956, 2932, 2859, 1735, 1709, 1656, 1579, 1561, 1530, 1489, 1469, 1392, 1369, 1305, 1269, 1208, 1153, 1066, 1025. ESI-MS (m/z): 582.11 [M + 1]+.

Synthesis of (E)-3-(6-((tert-Butyldimethylsilyl)oxy)-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)acrylic Acid (21)

To a solution of methyl (1-((E)-3-(tert-butoxy)-3-oxoprop-1-en-1-yl)-6-((tert-butyldimethylsilyl)oxy)-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threoninate (0.4 g, 0.0007 mol) in dichloromethane (8 mL) was added trifluoroacetic acid (0.5 mL). It was stirred at 35 °C for 4 h. The reaction mass was concentrated under vacuum to get the crude product (E)-3-(6-((tert-butyldimethylsilyl)oxy)-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)acrylic acid (0.42 g, 90%) as a brown solid. Melting point: 268.4 °C, 1H NMR (400 MHz, DMSO-d6) δ 0.24 (s, 6H), 1.00 (s, 9H), 1.16 (d, J = 6.4 Hz, 3H), 1.21–1.27 (m, 1H), 3.69 (s, 3H), 4.01–4.05 (m, 1H), 4.31–4.32 (m, 1H), 4.57–4.59 (m, 1H), 5.40 (bs, 1H), 7.11–7.19 (m, 2H), 7.52 (d, J = 8.8 Hz, 1H), 7.92 (s, 1H), 8.33 (d, J = 15.2 Hz, 1H), 7.71–8. 75 (m, 1H), 8.93 (s, 1H), 12.36 (s, 1H), 12.75 (bs, 1H). 13C NMR (100 MHz, CDCl3) δ −4.6, 17.6, 20.6, 25.6, 52.1, 57.8, 66.5, 106.1, 111.7, 113.0, 114.9, 115.6, 119.3, 121.7, 121.8, 122.8, 122.9, 123.0, 129.9, 130.1, 134.2, 134.4, 135.4, 136.6, 136.8, 136.9, 137.7, 137.8, 138.1, 138.4, 149.3, 151.8, 164.7, 167.4, 171.2. ESI-MS (m/z): 528.46 [M + 1]+.

Synthesis of tert-Butyl ((E)-3-(6-((tert-Butyldimethylsilyl)oxy)-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)acryloyl)-L-prolinate (23)

To a solution of (E)-3-(6-((tert-butyldimethylsilyl)oxy)-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)acrylic acid (0.41 g, 0.0008 mol) in dimethylformamide (5 mL) was added tert-butyl-l-prolinate (0.16 g, 0.001 mol), followed by the addition of HATU (0.46 g, 0.0012 mol). The reaction mass was cooled to 0–5 °C, and DIPEA was added (0.52 g, 0.004 mol). The reaction was stirred at room temperature for 20 h. The reaction was monitored by TLC (70% ethyl acetate in n-hexane, Rf: 0.5). The reaction mass was quenched with chilled DM water (30 mL) and extracted with ethyl acetate (3 d7 30 mL). The chilled water (2 × 30 mL) and saturated brine solution (30 mL) were given washings to the combined organic layer. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to get crude mass. The crude compound was purified over a silica gel column in a CombiFlash, eluting with 0–60% ethyl acetate in n-heptane. The desired fractions were concentrated under vacuum to get tert-butyl ((E)-3-(6-((tert-butyldimethylsilyl)oxy)-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)acryloyl)-l-prolinate (0.34 g, 66%) as a yellow solid. Melting point: 183.6 °C, 1H NMR (400 MHz, DMSO-d6) δ 0.23 (s, 6H), 1.00 (s, 9H), 1.19 (d, J = 5.88 Hz, 3H), 1.43 (s, 9H), 1.86–1.91 (m, 1H), 1.98–2.02 (m, 1H), 2.21–2.26 (m, 1H), 3.69 (s, 3H), 3.81–3.82 (m, 2H), 4.28–4.35 (m, 1H), 4.37–4.38 (m, 1H), 4.50–4.52 (m, 1H), 5.39–5.40 (m, 1H), 7.17 (d, J = 8.96 Hz, 1H), 7.52–7.54 (d, J = 8.56 Hz, 1H), 7.68 (d, J = 15.04 Hz, 1H), 7.91 (s, 1H), 8.23 (d, J = 14.44 Hz, 1H), 8.87–8.89 (m, 2H), 12.37 (s, 1H). 13C NMR (100 MHz, CDCl3) δ −4.4, −4.3, 18.3, 20.6, 24.6, 25.7, 25.8, 27.8, 27.9, 29.3, 47.2, 50.7, 52.5, 52.8, 58.7, 60.1, 67.2, 81.7, 110.8, 112.9, 113.6, 121.7, 121.9, 123.1, 129.9, 134.0, 135.9, 136.2, 136.3, 136.9, 137.7, 149.8, 165.5, 166.0, 166.2, 171.6, 173.8. IR (KBr, cm–1): 3375, 2956, 2932, 2858, 1736, 1649, 1595, 1561, 1530, 1490, 1466, 1436, 1368, 1317, 1269, 1207, 1154, 1023. ESI-MS (m/z): 681.59 [M + 1]+.

Synthesis of tert-Butyl (3-(6-((tert-Butyldimethylsilyl)oxy)-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)propanoyl)-l-prolinate (24)

To a solution of tert-butyl ((E)-3-(6-((tert-butyldimethylsilyl)oxy)-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)acryloyl)-l-prolinate (0.15 g, 0.0002 mol) in MeOH (15 mL),charged 10% Pd/C (50% wet) (0.15 g) under a nitrogen atmosphere. The reaction mass was stirred under a hydrogen atmosphere (1 atm) for 20 min at room temperature. The reaction was monitored by TLC (70% ethyl acetate in n-hexane, Rf: 0.45). The reaction mass was filtered through a celite bed and given methanol (50 mL) washings. The filtrate was concentrated under vacuum to get the desired product tert-butyl (3-(6-((tert-butyldimethylsilyl)oxy)-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)propanoyl)-l-prolinate (0.14 g, 93%) as an off-white solid. Melting point: 125 °C, 1H NMR (400 MHz, DMSO-d6) δ 0.23 (s, 6H), 0.99 (s, 9H), 1.15 (d, J = 6.4 Hz, 3H), 1.32 (s, 9H), 1.75–1.82 (m, 1H), 1.87–1.98 (m, 2H), 2.10–2.22 (m, 1H), 2.88–2.98 (m, 1H), 3.02–3.14 (m, 1H), 3.36–3.44 (m, 2H), 3.61 (t, J = 6.8 Hz, 2H), 3.68 (s, 3H), 4.16 (dd, J = 3.6 Hz, J = 8.8 Hz, 1H), 4.24–4.34 (m, 1H), 4.53 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 5.36 (bs, 1H), 7.11 (dd, J = 2.0 Hz, J = 8.4 Hz, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.80 (s, 1H), 8.67 (m, 2H), 11.92 (s, 1H). 13C NMR (100 MHz, CDCl3) δ −4.37, 18.2, 20.1, 24.4, 25.8, 28.0, 29.6, 30.0, 47.4, 52.4, 57.7, 59.7, 69.6, 82.3, 110.2, 112.7, 121.3, 121.9, 127.2, 135.5, 136.2, 137.4, 141.0, 149.2, 166.2, 172.2, 172.4, 172.8. IR (KBr, cm–1): 3384, 3249, 2957, 2932, 2887, 2859, 1738, 1637, 1597, 1578, 1568, 1528, 1489, 1467, 1393, 1368, 1330, 1263, 1203, 1155, 1092, 1006. ESI-MS (m/z): 683.58[M + 1]+.

Synthesis of tert-Butyl (3-(6-Hydroxy-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)propanoyl)-l-prolinate (25)

To a cooled solution of tert-butyl (3-(6-((tert-butyldimethylsilyl)oxy)-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)propanoyl)-l-prolinate (0.11 g, 0.00016 mol) in THF (3 mL) was added a TBAF solution (1 M in THF, 0.2 mL). The reaction mass was stirred at 0 °C to room temperature for 30 min. The reaction was monitored by TLC (100% ethyl acetate, Rf: 0.3). Upon completion of the reaction, it was quenched with saturated ammonium chloride solution (10 mL) and extracted with ethyl acetate (15 mL × 2). Saturated brine solution (25 mL) was given washing to the combined organic layer. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to get desired product tert-butyl (3-(6-hydroxy-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)propanoyl)-l-prolinate (0.08 g, 88%) as an off-white solid.

1H NMR (400 MHz, DMSO-d6) δ 1.16 (d, J = 6.4 Hz, 3H), 1.33 (s, 9H), 1.77–1.85 (m, 1H), 1.91–1.95 (m, 2H), 2.09–2.16 (m, 1H), 2.88–2.96 (m, 1H), 3.04–3.14 (m, 1H), 3.23–3.55 (m, 4H), 3.60–3.64 (t, J = 6.8 Hz, 2H), 3.69 (s, 3H), 4.15–4.18 (m, 1H), 4.28–4.32 (m, 1H), 4.51–5.54 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 5.33 (d, J = 5.2 Hz, 1H), 7.09–7.11 (dd, J = 2.0 Hz, J = 8.8 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.57 (s, 1H), 8.54 (s, 1H), 8.66 (d, J = 8.8 Hz, 1H), 9.23 (s, 1H), 11.77 (s, 1H). 13C NMR (100 MHz, MeOD) δ 19.3, 24.2, 27.1, 28.8, 30.5, 30.6, 30.8, 51.5, 57.7, 59.8, 67.4, 81.2, 105.1, 112.4, 112.5, 118.1, 122.5, 127.7, 135.6, 136.6, 136.7, 136.8, 142.9, 151.4, 167.0, 171.4, 172.0, 172.2. ESI-MS (m/z): 569.31[M + 1]+. HRMS m/z found 569.2599, C29H36N4O8 [M + H]+ requires 569.2533.

Synthesis of (1-(3-((S)-2-(tert-Butoxycarbonyl)pyrrolidin-1-yl)-3-oxopropyl)-6-hydroxy-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threonine (26)

To a solution of tert-butyl(3-(6-hydroxy-3-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-9H-pyrido[3,4-b]indol-1-yl)propanoyl)-l-prolinate (0.05 g, 0.088 mmol) in 1,2-dichloroethane was added trimethyltinhydroxide (0.05 g, 0.264 mmol). The reaction was stirred at 110 °C for 24 h. The reaction was monitored by TLC (10% MeOH in DCM, Rf: 0.2). The reaction was cooled to room temperature. It was quenched with 1 N HCl solution till pH ≈ 5 and extracted with dichloromethane (3 × 15 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to get crude product. The crude compound was purified over silica a gel column in a CombiFlash, eluting with 8% methanol in dichloromethane. The desired fractions were concentrated under vacuum to get the desired product, (1-(3-((S)-2-(tert-butoxycarbonyl)pyrrolidin-1-yl)-3-oxopropyl)-6-hydroxy-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threonine (0.040 g, 81%) as a yellow solid.

1H NMR (400 MHz, DMSO-d) δ 1.11 (d, J = 6.0 Hz, 3H), 1.33 (s, 9H), 1.72–1.95 (m, 4H), 2.09–2.18 (m, 1H), 2.88–2.96 (m, 1H), 3.04–3.14 (m, 1H), 3.23–3.55 (m, 4H), 3.60–3.68 (t, J = 6.8 Hz, 2H), 4.15–4.18 (m, 1H), 4.28–4.32 (m, 1H), 4.51–5.54 (m, 1H), 7.09–7.11 (d, J = 7.6 Hz, 1H), 7.35–7.37 (d, J = 8.0 Hz, 1H), 7.45 (s, 1H), 8.53 (s, 1H), 8.67–8.70 (d, J = 8.4 Hz, 1H), 9.25 (s, 1H), 11.75 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 20.4, 22.1, 24.2, 27.5, 28.1, 28.8, 30.8, 30.9, 46.5, 57.5, 59.1, 59.3, 66.5, 80.1, 81.3, 105.8, 112.1, 112.8, 118.4, 122.1, 127.1, 134.9, 136, 136.1, 137.7, 143.1, 143.3, 151.4, 164.8, 169.9, 170.1, 171.4, 171.7, 172.8. ESI-MS (m/z): 555.10 [M + 1]+.

Synthesis of (3-(3-(((1S,2R)-1-Carboxy-2-hydroxypropyl)carbamoyl)-6-hydroxy-9H-pyrido[3,4-b]indol-1-yl)propanoyl)-l-proline TFA (27)

To a solution of (1-(3-((S)-2-(tert-butoxycarbonyl)pyrrolidin-1-yl)-3-oxopropyl)-6-hydroxy-9H-pyrido[3,4-b]indole-3-carbonyl)-l-threonine (0.025 g, 0.045 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (0.2 mL). It was stirred at 35 °C for 4 h. The reaction mass was concentrated under vacuum to get the desired product (3-(3-(((1S,2R)-1-carboxy-2-hydroxypropyl)carbamoyl)-6-hydroxy-9H-pyrido[3,4-b]indol-1-yl)propanoyl)-l-proline TFA (0.025 g, 89%) as a yellow solid.

1H NMR (400 MHz, DMSO-d6) δ 1.14 (d, J = 6.0 Hz, 3H), 2.88–2.19 (m, 3H), 2.12–2.17 (m, 1H), 2.89–2.98 (m, 1H), 3.05–3.14 (m, 2H), 3.34–3.47 (m, 3H), 4.22–4.31 (m, 2H), 4.45 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.58 (s, 1H), 8.56 (s, 1H), 8.64 (d, J = 8.8 Hz, 1H), 9.25 (bs, 1H), 11.80 (s, 1H), 12.5 (bs, 1H). 13C NMR (100 MHz, MeOD) δ 20.8, 23.5, 25.6, 27.9, 30.3, 32.2, 32.5, 59.6, 60.3, 60.9, 68.9, 106.5, 114.1, 114.6, 117.4, 120.5, 123.6, 129.8, 136.9, 137.6, 144.1, 153.1, 158.7, 159.2, 167.3, 173.4, 173.8, 176.0. ESI-MS (m/z): 499.32[M + 1]+.HRMS m/z found 499.1816,C24H26N4O8 [M + H]+ requires 499.1751.