Introduction

Indole alkaloids, one of the largest classes of naturally occurring alkaloids, have proven to be an important and rich source of pharmaceuticals1,2,3,4,5,6,7,8. Particularly, tetrahydrocarbolines are the common structural motif in most of the indole alkaloids and represent a key scaffold in a wide range of market drugs and clinical candidates9,10,11,12,13, such as hypotensive drug reserpine, antiphosphodiesterase-5 agent Tadalafil, and antiallergic therapeutics mebhydrolin and dimebolin (Fig. 1a)14,15,16,17,18. Therefore, the study of tetrahydrocarboline synthesis and drug discovery has inspired practitioners from a wide spectrum of the scientific community. Typically, the β-tetrahydrocarbolines biogenetically originate from tryptamine and secologanin via an enzymatic Pictet-Spengler reaction and sequential diversification19,20,21,22,23,24,25,26,27, while most of the chemical synthesis of these molecules uses the same Pictet-Spengler strategy, and these protocols dictate the functionality on C-1 position (Fig. 1b). Respecting the γ-tetrahydrocarbolines, which are less abundant in natural isolates, the most facile synthetic approach is the Fischer-indole reaction (Fig. 1b)28. Although the power of the aforementioned methods is undisrupted, these protocols still have limitations, including normally tedious synthetic procedures, and limited diversity in piperidine fragments. And more to be considered is that in the course of structure-activity relationship studies in medicinal chemistry, these alkaloids-relevant molecular optimization has to rely on long-step modifications wherein some designed molecules are synthetically unreachable. Given the prevalence of indole alkaloids and the limitations of present synthetic methods, the development of modular synthesis of tetrahydrocarboline type of indole alkaloids with broad diversity in a rapid and efficient manner would greatly enrich the toolbox of organic chemists, enlarge the compound library available for drug discovery and also accelerate the lead compound optimization process.

Fig. 1: Modular assembly of indole alkaloids through multicomponent reaction.
figure 1

a Representative approved drugs with the structure of tetrahydrocarboline type of indole alkaloids. b Conventional synthetic approaches to tetrahydrocarboline type of indole alkaloids. c Modular assembly of tetrahydrocarboline type of indole alkaloids delineated in this work.

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The rapid assembly of complex scaffolds in a single step from simple precursors identifies as an ideal reaction in terms of efficiency and sustainability. Typically, multicomponent reactions (MCRs) fall into this ideal reaction and have become powerful synthetic tools for the construction of complex and diverse chemical entities in a one-pot fashion from three or more starting materials29,30,31,32. Compared to conventional stepwise synthesis, the MCRs could rapidly assemble complex molecules with simple manipulation, high atom- and step-economy; therefore, they have found widespread applications in the facile synthesis of natural products, market drugs, biologically active compounds, and materials. The prominent MCRs, such as Ugi, Mannich, Strecker, Biginelli reaction, or Hantzsch dihydropyridine synthesis, have been robust preparative drug discovery methods33,34,35,36,37,38,39,40,41,42,43,44. However, the MCRs-promoted modular and straightforward synthesis of tetrahydrocarboline type of indole alkaloids still remains explored. On the other hand, most of the reported MCRs mechanistically relied on those activated carbon centers to render the multiple chemical bond formations and target molecules construction, whereas the invoking of unactivated carbon centers poses a substantial challenge.

Bearing these challenges in mind and inspired by the Mannich reaction for the biomimetic synthesis of tropinone alkaloids, as well as our previous studies on MCRs45,46,47, we speculated that if a functional group was incorporated into the N-atom of indole, the electron density distribution of indole moiety would be changed and this might have the opportunity to invoke the tethered unactivated carbon centers to involve in an MCR process toward the construction of indole alkaloids. Herein, we would like to report our successful implementation of this concept for the modular assembly of tetrahydrocarboline type of indole alkaloids through multicomponent reaction (Fig. 1c). This practical reaction could tolerate a broad range of functional groups so that a wide set of market drugs and drug-like alkaloids could be achieved in a single-step from simple precursors, and this could also greatly enlarge the alkaloid library and much simplified the optimization process in drug discovery.

Results

Reaction optimization

We commenced our study by choosing the model substrates of indole derivatives 1, formaldehyde 2, and methyl glycine ester hydrochloride 3a. Each reaction parameter was examined including solvent, temperature, and the N-substitution of indole (Table 1, for details, see Supplementary information), and we identified that an intriguing multicomponent reaction of N-benzyl-2-methyl indole 1a, 2, and 3a in the solvent of N,N’-dimethylformamide (DMF) under air could well occur to furnish γ-tetrahydrocarboline 4a in excellent yield (92%). This meant it has overcome the intrinsic challenge of modular assembly of tetrahydrocarboline type of indole alkaloids in a single step, wherein multiple chemical bond formations were observed including an unactivated carbon center involved C(sp3)–C(sp3) connection. Besides, the N-substitution was important in this process wherein the alkyl groups were optimal while the hydrogen atom and electron-withdrawing groups (t-butoxycarbonyl, Boc; p-toluenesulfonyl, Ts) would lead to diminished reaction efficiency (Supplementary Table 2). This is probably because the N-substitution of indole would render drastic changes to the electron density distribution of indole moiety which is essential to this MCR process and the alkaloid formation.

Table 1 Reaction optimizationa
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Reaction scope study and tetrahydrocarboline synthesis

We next focused on the generality of this multicomponent one-pot synthesis of indole alkaloids using this operationally simple protocol. Firstly, a wide set of amine-building blocks were explored (Fig. 2). For instance, diverse amino acid derivatives could well engage in this process with 2-methyl indoles 1 and formaldehyde 2 to deliver the corresponding products 4a–4l, and the chirality and functionality from amino acid could be directly incorporated into the indole alkaloids which could not be accessed by conventional methods. Besides, numerous flexible aliphatic amines were compatible with this protocol to furnish the indole alkaloids in good to excellent yields (4m–4ak), with a diversity of substituents attached, including chloride, bromide, sulfone, trifluoromethyl, cyano, pathalimide, acetoxy, alkene, alkyne, three- to six-membered cyclic and heterocyclic rings, phenol. Notably, these functionalities were widely used in drug discovery and they could be intuitively mapped onto the indole alkaloid frameworks from simple materials in this protocol. The structures (4f, 4af) were unambiguously confirmed by single-crystal x-ray crystallography. We next examined the compatibility of this protocol with indole building blocks. A variety of substitutions in the indole scaffold were well tolerated in this multicomponent synthesis of tetrahydrocarboline alkaloids, including fluoro, chloro, bromo, ester, methyl, phenyl, and pinacolboranyl (4al-4av). Notably, the 7-azaindole, a bioisostere of indole, was also applicable in this protocol (4aw). Meanwhile, various substitutions in the N-position of indole were varied, such as methyl, para-methoxybenzyl (PMB), 2-phenylethyl, allyl, cinnamyl, and tert-butyl carbonate (Boc) (4ax-4aac). Besides, we were pleased to find that the multi-substituted pyrroles were also applicable in this protocol for leading to pyrrole-piperidine fused products (4aad and 4aae). These functionalities pervade the structure-activity relationship studies in medicinal chemistry and serve as a productive springboard for further skeletal modification strategies.

Fig. 2: Scope for the multicomponent synthesis of γ-tetrahydrocarboline type of indole alkaloids.
figure 2

Conditions: 1 (0.2 mmol), 2 (1.0 mmol), 3 (0.4 mmol), DMF (1.5 mL), at 60 °C for 3 h. Isolated yields are given. Phth pathalimide, PMB para-methoxybenzyl, Boc tert-butyl carbonate. aCH3CN (1.5 mL) was used as solvent, TsOH (0.1 mmol) was added as an additive and the reaction was conducted at 80 °C.

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Encouraged by the above results, we subsequently wondered whether β-tetrahydrocarboline could be assembled in the same manner using this MCR protocol (Fig. 3). Thus, the 3-methyl indole was employed as a building block for investigation, and the reaction parameters were examined including the substituents of indole, solvent, and reaction temperature. Gratifyingly, (Boc)2O, a widely used protecting group, was placed on the N-position of 3-methyl-5,6-dimethoxy indole (5) to render the occurrence of MCR with formaldehyde 2 and amine-building block 3. The generality was also examined, and a wide range of functionalities in the amines were well applicable in this process, including amino acid derivatives, alkene, alkyne, chloro, sulfone, cyano, phthalimide, and cyclobutane (6a–6n). Moreover, the prolonged aliphatic chain in the indole moiety with the functionality of ester and protected alcohol were also compatible to deliver the alkaloid products smoothly (6o–6p). Besides, a single group substitution in the indole skeleton such as methoxy, benzyloxy, or hydroxyl, would lead to diminished yields, probably due to the changed electron density distribution; however, when p-toluenesulfonic acid (p-TSA) was added as an additive, the corresponding β-tetrahydrocarbolines could be achieved in moderate to excellent yields (6q–6af; for details, see Supplementary information). In particular, We found that the reaction was dramatically affected by the N-substitutions, wherein the electron-withdrawing groups like benzyloxycarbonyl (Cbz) and N,N- dimethylformyl were optimal (6ag, 6ah, for details, see Supplementary information) and electron-donating groups were inferior. The structure (6v) was unambiguously confirmed by single-crystal x-ray crystallography. Considering the remarkable value of tetrahydrocarboline type of indole alkaloid in natural products and drug discovery, this protocol provides a rapid and distinct approach to new structured indole alkaloids available for drug discovery, and would definitely simplify the optimization process of lead molecule thus improving the accuracy and efficiency of candidate identification.

Fig. 3: Scope for the multicomponent synthesis of β-tetrahydrocarboline type of indole alkaloids.
figure 3

Conditions: 1 (0.2 mmol), 2 (1.0 mmol), 3 (0.4 mmol), CH3CN (1.5 mL), at 80 °C for 8 h. Isolated yields are given. Phth pathalimide, Boc tert-butyl carbonate. aTsOH (0.1 mmol) was added as an additive and the reaction was conducted at 60 °C.

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To highlight the value of this protocol, a collective and concise synthesis of market drugs and clinical candidates was exemplified (Fig. 4). For instance, Mebhydrolin (8) and γ-tetrahydrocarboline block (9), with the key structure of γ-tetrahydrocarboline, could be rapidly achieved from the feedstock 2-methyl indole (7a) in short step-synthetic routes under this protocol; while AVN-101 (10) and Dimebolin (11) could also be accessed from feedstock 7 g in only two-step synthesis under this protocol; Setipiprant (13) could also be rapidly achieved under this strategy. A 5 mmol scale reaction was established to demonstrate the practicability. Meanwhile, various new structured β-tetrahydrocarboline types of drug-like compounds (14–17) could be rapidly assembled and further merged with peptide and click chemistry, which could occupy new chemical space and biological space for drug discovery. Moreover, once a structure optimization of indole alkaloids is needed, this protocol provides a distinctive shortcut rather than a long-step modification.

Fig. 4: Applications to the concise synthesis of market drugs and drug-like molecules.
figure 4

MCR multicomponent reaction, PCP para-cyanophenyl.

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Mechanistic investigations

Since the modular assembly of indole alkaloids and the unusual C(sp3)–C(sp3) connection occurrence in this process is impressive, the reaction mechanism was probed (Fig. 5). Control reaction, deuterium-labeling reaction, and cross-over reaction were set up to elucidate a multiple alkylamination cascade in this process48,49,50. For instance, the indole starting material 1a, the mono-alkylaminated intermediate 18, the di-alkylaminated intermediate 19, were individually subject to the reaction with deuterated formaldehyde (DCDO, [D2]-2) and glycine methyl ester hydrochloride 3a, and the equivalent amount of [D2]-2 and 3a was varied, and the results showed that the deuterated γ-tetrahydrocarboline 4a ([D4]-4a) could be formed with different deuteration ratio (Fig. 5a). Besides, when 18 was subjected to react with formaldehyde in the absence of 3a, there was not any product formation (Fig. 5b). Interestingly, either 18 or 19 was subjected to the reaction with formaldehyde and benzylamine hydrochloride (3af), 4af was formed as the major product rather than 4a. Meanwhile, a tertiary amine substituted indole 20 was prepared and subjected to reaction with 2 and 3a, the reaction did not deliver the γ-tetrahydrocarboline product. Additionally, kinetic isotope effect (KIE) studies were carried out and a KIE value of 3.25 was obtained, indicating that the C(sp3)−H bond cleavage might be involved in the rate-limiting step (Fig. 5c, for details, see Supplementary information). Taking together these results, a plausible reaction mechanism was proposed (Fig. 5d). At beginning, the indole ring triggered the iterative alkylaminations to deliver Int-2 and Int-3. Then, either 1a or Int-2 would undergo another alkylamination to deliver Int-4. At this stage, a further alkylamination via transition state TS-1 would induce the unactivated carbon center to undergo dehydrogenation and convert the unactivated C(sp3) to activated C(sp2) along with the formation of Int-5. The following iminium formation of Int-6, cyclization, and retro-Mannich reaction would achieve the construction of alkaloids and enable the cascade cycle51.

Fig. 5: Mechanistic studies for synthesis of γ-tetrahydrocarboline.
figure 5

a Deuterium-labeling and stepwise control reaction. b Cross-over reaction. c Kinetic isotope effect experiments. d Proposed mechanism.

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Similarly, control reaction and deuterium-labeling reaction were also conducted to investigate the mechanism for β-tetrahydrocarboline formation. For instance, the indole starting material 5a, the mono-alkylaminated intermediate 22, and the di-alkylaminated intermediate 23, were individually subject to the reaction with deuterated formaldehyde (DCDO, [D2]-2) and glycine methyl ester hydrochloride 3a, and the results showed that the deuterated γ-tetrahydrocarboline [D4]-6a could be formed with almost completed deuteration ratio (Fig. 6a). Besides, the one-pot reaction of 22, formaldehyde and benzylamine hydrochloride (3af) was set up, wherein 6f was formed as the major product rather than 6a (Fig. 6b). Additionally, kinetic isotope effect (KIE) studies were carried out and a KIE value of 2.64 was obtained, indicating that the C(sp3)−H bond cleavage might be involved in the rate-limiting step (Fig. 6c, for details, see Supplementary Information). These results indicated that the synthetic mechanism of β-tetrahydrocarboline is probably similar with γ-tetrahydrocarboline (Fig. 6a–c) and a plausible mechanism was then proposed (Fig. 6d). Initially, the indole ring triggered the iterative alkylaminations to deliver Int-7 and Int-8. Then, either 5a or Int-7 would undergo another alkylamination to deliver Int-9. At this stage, a further alkylamination via transition state TS-2 would induce the unactivated carbon center to undergo dehydrogenation and convert the unactivated C(sp3) to activated C(sp2) along with the formation of Int-10. The following iminium formation of Int-11, cyclization, and retro-Mannich reaction would achieve the construction of β-tetrahydrocarboline products 6 and enable the cascade cycle51.

Fig. 6: Mechanistic studies for synthesis of β-tetrahydrocarboline.
figure 6

a Deuterium-labeling and stepwise control reaction. b Cross-over reaction. c Kinetic isotope effect experiments. d Proposed mechanism.

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Discussion

In conclusion, we have achieved a modular assembly of tetrahydrocarbolines through multicomponent reaction of 2-substituted or 3-substituted indoles, formaldehyde, and amino hydrochlorides, and this protocol provides expedient access to these indole alkaloids. The chirality and a wide scope of functional groups were compatible in this process thus significantly enlarging the chemical space and biological space of tetrahydrocarbolines. Because tetrahydrocarbolines are widely encountered as pharmaceutically relevant substrates, we believe that this protocol would be a widely applicable strategy in indole-related drug discovery. Besides, this work also provides a new vision for the reaction of unactivated C(sp3) center and C(sp3)–C(sp3) bond formation.

Methods

General procedure for the synthesis of γ-tetrahydrocarboline 4a-4aac

A mixture of 1 (0.2 mmol), formaldehyde 2 (37% in water, 0.08 mL, 5 equiv.), and corresponding primary amine hydrochloride 3 (0.4 mmol, 2 equiv.) in DMF (1.5 mL) was stirred at 60 °C until the reaction was completed. The reaction was quenched by saturated aqueous NaHCO3. The aqueous layer was extracted with ethyl acetate (three times), and the combined organic layer was dried over Na2SO4 and concentrated. Purification by silica gel column chromatography to give corresponding γ-tetrahydrocarboline products 4a–4aac.

General procedure for the synthesis of 4aad and 4aae

A mixture of 1t (or 1u) (0.2 mmol), formaldehyde 2 (37% in water, 0.08 mL, 5 equiv.), TsOH (0.1 mmol, 0.5 equiv.) and methyl glycinate hydrochloride 3a (0.4 mmol, 2 equiv.) in MeCN (1.5 mL) were stirred at 80 °C until the reaction was completed. The reaction was quenched by saturated aqueous NaHCO3. The aqueous layer was extracted with ethyl acetate (three times), and the combined organic layer was dried over Na2SO4 and concentrated. Purification by silica gel column chromatography to give products 4aad and 4aae.

General procedure for the synthesis of β-tetrahydrocarboline 6a–6p

A mixture of 5 (0.2 mmol), formaldehyde 2 (37% in water, 0.08 mL, 5 equiv.), and corresponding primary amine hydrochloride 3 (0.4 mmol, 2 equiv.) in MeCN (1.5 mL) was stirred at 80 °C until the reaction was completed. The reaction was quenched by saturated aqueous NaHCO3. The aqueous layer was extracted with ethyl acetate (three times), and the combined organic layer was dried over Na2SO4 and concentrated. Purification by silica gel column chromatography to give corresponding β-tetrahydrocarboline products 6a–6p.

General procedure for the synthesis of 6q–6ah

A mixture of 5 (0.2 mmol), formaldehyde 2 (37% in water, 0.08 mL, 5 equiv.), TsOH (0.1 mmol, 0.5 equiv.) and corresponding primary amine hydrochloride 3 (0.4 mmol, 2 equiv.) in MeCN (1.5 mL) was stirred at 60 °C until the reaction was completed. The reaction was quenched by saturated aqueous NaHCO3. The aqueous layer was extracted with ethyl acetate (three times), and the combined organic layer was dried over Na2SO4 and concentrated. Purification by silica gel column chromatography to give corresponding products 6q–6ah.