Multicomponent double Mannich alkylamination involving C(sp2)–H and benzylic C(sp3)–H bonds

Alkylamines are ubiquitous in pharmaceuticals, materials and agrochemicals. The Mannich reaction is a well-known three-component reaction for preparing alkylamines and has been widely used in academic research and industry. However, the nucleophilic components in this process rely on C(sp2)−H and activated C(sp3)−H bonds while the unactivated C(sp3)−H bonds involved Mannich alkylamination is a long-standing challenge. Here, we report an unprecedented multicomponent double Mannich alkylamination for both C(sp2)−H and unactivated benzylic C(sp3)−H bonds. In this process, various 3-alkylbenzofurans, formaldehyde and alkylamine hydrochlorides assemble efficiently to furnish benzofuran-fused piperidines. Mechanistic studies and density functional theory (DFT) calculations revealed a distinctive pathway that a multiple Mannich reaction and retro-Mannich reaction of benzofuran and dehydrogenation of benzylic C(sp3)−H bonds were key steps to constitute the alkylamination. This protocol furnishes a Mannich alkylamine synthesis from unusual C–H inputs to access benzofuran-fused piperidines with exceptional structural diversity, molecular complexity and drug-likeness. Therefore, this work opens a distinctive vision for the alkylamination of unactivated C(sp3)−H bonds, and provides a powerful tool in diversity-oriented synthesis (DOS) and drug discovery. The Mannich reaction is a three-component reaction for preparing alkylamines, but the nucleophilic components rely on C(sp2)−H and activated C(sp3)−H bonds. Here, the authors report an unprecedented multicomponent double Mannich alkylamination for both C(sp2)−H and unactivated benzylic C(sp3)−H bonds.

As noted, the wide prevalence of alkylamines in small molecule biological probes, pre-clinical candidates, and approved drugs is owing to their favorable physical and pharmacological properties 25,26 . In particular, cyclic alkylamines can enhance structural rigidity, thus improving target selectivity and druggability. For example, the cyclic six-membered piperidines represent the most commonly used nitrogen heterocycles among U.S. FDA-approved pharmaceuticals (Fig. 2). As a highly relevant synthetic strategy, the double Mannich reaction based on enolizable C−H bonds provides straightforward access to cyclic alkylamines. This was exemplified by Robinson for the facile synthesis of tropinone 27 . To engage unactivated C(sp 3 )−H bonds in this process would significantly expand the synthetic toolbox for the assembly of cyclic alkylamines and drug candidates. Based on serendipitous findings, herein, we report on the discovery of a multicomponent double Mannich reaction for both C(sp 2 )−H and unactivated benzylic C(sp 3 )−H bonds (Fig. 1c). In this process, various 3-alkylbenzofurans, formaldehyde, and primary alkylamine hydrochlorides assemble efficiently to furnish diverse benzofuran-fused piperidines. In addition, many bioactive molecules could be applicable in this protocol to prepare their analogs.

Results
Reaction design and optimization for double Mannich reaction. As mentioned above, the challenge for unactivated C(sp 3 )−H bonds to involve in Mannich reactions is attributed to their difficult deprotonation and therefore non-nucleophilic properties. In continuation of our interest in multi-component reactions and drug discovery [28][29][30][31] , we hypothesized that the benzylic C(sp 3 )−H bond might possess sufficient reactivity through the electronic effect of (hetero)arenes, thus enabling Mannich type alkylamination. After considerable exploration and optimization of conditions, we realized this hypothesis using electron-rich benzofurans as heteroaryl substrates. As shown in Table 1, the model reaction was explored with benzofuran 1a, formaldehyde 2, and glycine methyl ester hydrochloride 3a. In the solvent of acetic acid (AcOH), these three components were mixed in a one-pot and kept at 60°C for 3 h (entries 1-6). The amounts of 2 and 3a were varied while a major product was formed and identified as benzofuran-fused piperidine 4a in 74% yield (entry 6). This indicated an efficient double Mannich alkylamination involving both the benzylic C(sp 3 )−H bond and the aryl C(sp 2 )−H bond, furnishing the cyclic amine. Increasing the reaction temperature would decrease the yield (entries 7-8) and only 25% yield could be obtained when conducted at room temperature (entry 9). The survey of solvents showed that acetonitrile (CH 3 CN) worked equally well (entry 10), while the utilization of isopropanol, hexafluoroisopropanol (HFIP), and HFIP/AcOH would give inferior results (entries [11][12][13]. Variation of 3a to glycine methyl ester would lead to a decreased yield (entry 14). Conducting this reaction on 5 mmol scales could afford 4a with slightly improved yield (entry 15), thus underscoring the scalability of this process.
Substrate scope in the synthesis of benzofuran-fused piperidines. Under the optimized conditions, we examined the substrate scope of this three-component double Mannich alkylamination. Various amino acid derivatives, including glycine, L-alanine, Lphenylalanine, L-valine, L-leucine, L-serine, L-cysteine, L-methionine, L-aspartic acid, L-glutamic acid, L-tyrosine, and L-isoleucine, all reacted smoothly with benzofuran 1a and formaldehyde 2 to furnish the corresponding fused piperidines in moderate to good yields (4a-4o) (Fig. 3). The structure of 4b and 4n was confirmed by X-ray analysis. Notably, the chirality and functionalities of the amino acids were retained in this process thus bestowing versatile physical and pharmacological properties on the products (see Supplementary Information). Besides, numerous primary amines hydrochlorides could engage in this process at 90°C, and benzyl, alkene, alkyne, trifluoromethyl, ester, alcohol, sulfone, amino and bromoalkyl functionalities were well tolerated. This offers ample opportunity for further functionalization towards drug discovery and small molecule biological probe development. The structure of 4r was confirmed by X-ray analysis. Moreover, three-, four-, five-, and six-membered ring-substituted amines were used to furnish the products in moderate to good yields (4z-4ac). Furthermore, a primary amine substituted with a quaternary carbon center was used, and also in this case the cyclization took place, giving 4ad in moderate yield. Additionally, the late-stage modification of drug derivatives could be achieved using this method. For instance, the important neurotransmitter GABA (γaminobutyric acid) methyl ester hydrochloride 3ae, the antiepileptic drug Pregabalin methyl ester hydrochloride 3af, the monoamine oxidase inhibitor Tranylcypromine hydrochloride 3ag, and the antiarrhythmic drug Mexiletine hydrochloride 3ah, could all be efficiently modified under this protocol to produce the corresponding piperidine-hybrids in moderate yields (4ae-4ah). In addition, the utilization of CH 3 CN as solvent also works well with extended reaction time.
With respect to heteroarenes, a variety of electron-rich 3alkylbenzofurans underwent the three-component double Mannich alkylaminaion efficiently, occurring at the 2-position of the benzofuran and the benzylic moiety (Fig. 4). Diverse functionalized substituents were tolerated in this process, including alkyloxy, allyloxy, propargyloxy, hydroxy, alkylthio, alkyl, chloro, bromo, and boronate (5a-5m). It should be noted that these functionalities not only bestow the products with remarkable structural diversity and favorable physical properties, but also provides ample potential for further derivatization. For example, the boronate compound 5m might be transformed to a variety of molecules through Suzuki coupling reaction and Chan-Lam coupling reaction. When  benzofurans with a substituted benzyl group like 3-ethyl, 3-(2phenyl)ethyl, 3-(4-hydroxy)butyl, and 3-(4-amino)butyl were used, interestingly, the double Mannich reaction proceeded well to give the poly-substituted and functionalized piperidines in excellent yields (5n-5r). Besides, the hydroxy and amino group in the substituted chain would offer numerous late-stage functionalization opportunities. Moreover, compound 5r was further subjected to a Huisgen click reaction to yield a triazole derivative 5s, whose structure was confirmed by X-ray analysis. Considering that the piperidines are prevalent motifs in natural products as well as pharmaceuticals and are important synthetic targets for drug discovery and development 32 , this process provides an efficient and direct approach to establish libraries with high drug-likeness, broad chemical space, and modifiable physicochemical properties.
On the basis of these observations, a plausible mechanism was proposed with starting materials 1a, formaldehyde 2, and methyl glycinate hydrochloride 3a as model substrates (Fig. 6a). Initially, the electron-rich 3-methylbenzofuran 1a, formaldehyde 2, and methyl glycinate hydrochloride 3a undergo the first Mannich alkylamination to deliver intermediate A (compd. 6). A undergoes the secondary Mannich reaction to furnish the cationic intermediate B.  Based on this mechanistic proposal, the electron-rich property of the benzofuran ring makes the multiple Mannich reaction and retro-Mannich reaction energetically feasible and eventually enables cyclization/alkylamination involving the benzylic C−H moiety. Consistent with this, when benzofurans with 3-(CH 2 R) substitution are used as inputs, the alkene intermediates C with poly-substituted alkene moiety are likely stabilized, thus explaining the high yields despite steric congestion (5n-5r).
Density functional theory calculations. To further explore the plausibility of our proposal, we performed a series of density functional theory (DFT) calculations. We focus on the reaction triggered by the protonation at 2-position of the benzofuran due to its prominent nucleophilicity 45  int-D is the on-cycle resting state, and the rate-determining step is the cyclization process via TS-D. These calculations validated the revised reaction mechanism, which discarded the original proposal involving the protonation of iminium intermediate. These DFT calculations revealed that the proposed reaction mechanism is rational.

Discussion
In summary, we have presented an unprecedented multicomponent double Mannich alkylamination for both C(sp 2 )−H and unactivated benzylic C(sp 3 )−H bonds. A series of 3-alkylbenzofurans, formaldehyde, and primary alkylamine hydrochlorides could rapidly assemble to produce the piperidines. Mechanistic studies and DFT calculations revealed a distinctive pathway that a multiple Mannich reactions and retro-Mannich reaction of benzofuran and ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-28088-z dehydrogenation of benzylic C(sp 3 )−H bonds were key steps to constitute the alkylamination, which opens a distinctive vision for alkylamination and functionalization of unactivated C(sp 3 )−H bonds. In addition, this protocol also provides a powerful tool in diversity-oriented synthesis and drug discovery.

Data availability
Materials and methods, experimental procedures, useful information, mechanistic studies, optimization studies, 1 H NMR spectra, 13 C NMR spectra, and mass spectrometry data are available in the Supplementary Information. Additional data are available from the corresponding author upon request. Crystallographic data for compounds 4b, 4n, 4r, 5s, are available from the Cambridge Crystallographic Data Center under reference number CCDC 2020613, 2020614, 2020615, and 2027076. These data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/.