Total Synthesis of the Racemate of Laurolitsine

The total synthesis of laurolitsine was achieved for the first time. This reaction was accomplished in 14 steps with a 2.3% yield (this was calculated using 3-hydroxy-4-methoxybenzaldehyde as the starting material) starting from two simple materials, 3-hydroxy-4-methoxybenzaldehyde and 2-(3-hydroxy-4-methoxyphenyl)acetic acid, and the longest linear sequence consisted of 11 steps. The key steps included an electrophilic addition reaction in which a nitro group was reduced to an amino group using lithium tetrahydroaluminum and a Pd-catalyzed direct biaryl coupling reaction. In this paper, many of the experimental steps were optimized, and an innovative postprocessing method in which 2-(3-(benzyloxy)-4-methoxyphenyl)ethanamine is salted with oxalic acid was proposed.


Introduction
Laurolitsine is an alkaloid isolated from natural plants such as Litsea glutinosa that exhibits potent antidiabetic effects and hypoglycemic activity in vivo and has wide application prospects in the clinic [1,2].Laurolitsine was first discovered and named by Tatsuo Nakasato and Shozo Nomura from the leaves of Neolitsea sericea (Blume) Koidz. in 1959 [3].Sun et al. obtained laurolitsine by efficient isolation from the chloroform extract of Litsea cubeba [4].Through a series of experiments, Zhang et al. confirmed that laurolitsine, which is abundant in Litsea glutinosa bark, can exhibit potent antidiabetic effects with hypoglycemic activity in vivo.Laurolitsine improved insulin resistance, glucose tolerance and lipid metabolism; protected liver, renal and pancreatic functions; and promoted weight loss in db/db mice [1].
However, since laurolitsine must be isolated and extracted from plants, the cycle is long, and low yields are obtained; thus, the price of this material is relatively high, which greatly limits the development of related experiments on laurolitsine [4].At present, there are no reports on the total synthesis of laurolitsine.Therefore, exploring a new synthesis route is imperative, and reasonable and efficient chemical synthesis methods for obtaining laurolitsine quickly and in large quantities are very important for related research.
Laurolitsine is an aporphine alkaloid that has a special biphenyl tetracyclic structure with a chiral carbon atom at the 6a position (Figure 1) and a wide range of physiological activities due to its different oxidation states and substituents [5][6][7][8][9][10].Due to their remarkable pharmacological effects, aporphine alkaloids have attracted widespread attention in organic synthesis.However, directly synthesizing aporphine alkaloids is challenging due to their special benzene ring structure.The 1-benzyl-substituted tetrahydroisoquinoline can be used as a basic skeleton for the biomimetic synthesis of aporphine alkaloids.Therefore, establishing this parent nucleus structure is fundamental to the synthesis of aporphine alkaloids [11].Therefore, establishing this parent nucleus structure is fundamental to the synthesis of aporphine alkaloids [11].Lafrance et al. reported that a variety of aporphine alkaloids with 2-position substitutions can be directly synthesized using a Pd-catalyzed arylation reaction [12].This breakthrough not only highlights the utility of direct arylation in target-oriented synthesis reactions but also allows the synthesis of various compounds with different substituents.Researchers have used the same approach for the enantioselective synthesis of pronuciferine and nuciferin [11].Gao et al. described a highly efficient and practical multicomponent one-pot reaction.This reaction represents a streamlined pathway for synthesizing functionalized 1,2-dihydroisoquinolines, showcasing the versatility of multicomponent reactions in organic synthesis [13].Under different reaction conditions, a novel series of aporphine analogs were finally accomplished through intramolecular phenol ortho-arylation using Pd-mediated catalysis [14][15][16][17][18].However, the operation is complex, and most steps require silica gel column chromatography.The purpose of this study was to synthesize total amounts of laurolitsine for the first time and optimize the reaction steps.An innovative postprocessing method in which 2-(3-(benzyloxy)-4-methoxyphenyl)ethanamine is salted with oxalic acid is also proposed, which greatly reduces the time cost and increases the yield of intermediates.
Compounds 4 and 9 were synthesized by two different routes.Compound 3 was synthesized through a benzylation reaction and a nitration reaction, and 4 was subsequently produced by the reduction of 3. In relation to another reaction route, 9 was produced from 8 through substitution with Br2.Finally, 8 was obtained by esterification, benzylation, and hydrolysis.Lafrance et al. reported that a variety of aporphine alkaloids with 2-position substitutions can be directly synthesized using a Pd-catalyzed arylation reaction [12].This breakthrough not only highlights the utility of direct arylation in target-oriented synthesis reactions but also allows the synthesis of various compounds with different substituents.Researchers have used the same approach for the enantioselective synthesis of pronuciferine and nuciferin [11].Gao et al. described a highly efficient and practical multicomponent one-pot reaction.This reaction represents a streamlined pathway for synthesizing functionalized 1,2-dihydroisoquinolines, showcasing the versatility of multicomponent reactions in organic synthesis [13].Under different reaction conditions, a novel series of aporphine analogs were finally accomplished through intramolecular phenol ortho-arylation using Pd-mediated catalysis [14][15][16][17][18].However, the operation is complex, and most steps require silica gel column chromatography.The purpose of this study was to synthesize total amounts of laurolitsine for the first time and optimize the reaction steps.An innovative postprocessing method in which 2-(3-(benzyloxy)-4-methoxyphenyl)ethanamine is salted with oxalic acid is also proposed, which greatly reduces the time cost and increases the yield of intermediates.

Facile Construction of the Laurolitsine Skeleton
The protection of 1 with a benzyl (Bn) group furnished 2.Then, 3 was prepared via the Henry reaction and elimination reaction by adding 2, ammonium acetate, and nitromethane to an acetic acid solvent at 118 °C [19][20][21].First, we wanted to complete the reaction by increasing the amount of nitromethane and ammonium acetate, but despite our Scheme 1. Retrosynthetic analysis of laurolitsine.
Molecules 2024, 29, 745 3 of 12 Compounds 4 and 9 were synthesized by two different routes.Compound 3 was synthesized through a benzylation reaction and a nitration reaction, and 4 was subsequently produced by the reduction of 3. In relation to another reaction route, 9 was produced from 8 through substitution with Br 2 .Finally, 8 was obtained by esterification, benzylation, and hydrolysis.

Facile Construction of the Laurolitsine Skeleton
The protection of 1 with a benzyl (Bn) group furnished 2.Then, 3 was prepared via the Henry reaction and elimination reaction by adding 2, ammonium acetate, and nitromethane to an acetic acid solvent at 118 • C [19][20][21].First, we wanted to complete the reaction by increasing the amount of nitromethane and ammonium acetate, but despite our efforts to perform many experiments, the effect was not good, the product was sticky and difficult to separate and filter, and the yield and purity were poor.After many experimental alterations, we finally determined the reaction conditions (CH 3 NO 2 (4 equiv.),NH 4 OAc (1.3 equiv.),HOAc, 118 • C).The viscosity of the product may be related to the amount of ammonium acetate.If the amount of ammonium acetate is too high, side reactions increase and intermediate 3 becomes viscous and difficult to filter.A small amount of ammonium acetate causes the reaction to be incomplete.Regarding the next step of reduction, we attempted to reduce Pd/C, iron powder, sodium sulfide and sodium borohydride and did not achieve good results; subsequently, we succeeded by adding the strong reductant LiAlH 4 , which was converted to substituted phenyl ethanamine 4 via reduction.LiAlH 4 was selected to reduce the double bond and nitro group simultaneously.A much lower yield was obtained when LiAlH 4 was added at ambient temperature due to the generation of impurities including two compounds that only reduced the nitro group but not the double bond and only reduced the double bond but not the nitro group.Purifying the product was difficult because reduction of the double bond and nitro group was incomplete, and the polarity difference between the two impurities was very small [19][20][21].In terms of the feeding temperature, reaction temperature, and reductant ratio, we performed a large number of experiments and ultimately determined the most suitable reaction conditions (LiAlH 4 (4 equiv.),THF, 0 → 35 • C, THF = tetrahydrofuran).A method involving salt formation between oxalic acid and amino groups was used for purification.Compound 4 was combined with oxalic acid in methanol to form oxalate, after which the salt was hydrolyzed with sodium hydroxide solution, which achieved high purity and reduced the number of tedious purification steps.The specific method is described in detail later (Scheme 2).First, 5 was esterified with ethanol to furnish 6, and then 7 was formed by a benzylation reaction.Next, 8 was obtained by hydrolysis with sodium hydroxide solution, and 9 was obtained by reaction with Br2, acetic acid and sodium acetate [15].Notably, this reaction requires Br2 to be slowly added to the system at the end; otherwise, the reaction will not be complete (Scheme 3).First, 5 was esterified with ethanol to furnish 6, and then 7 was formed by a benzylation reaction.Next, 8 was obtained by hydrolysis with sodium hydroxide solution, and 9 was obtained by reaction with Br 2 , acetic acid and sodium acetate [15].Notably, this reaction requires Br 2 to be slowly added to the system at the end; otherwise, the reaction will not be complete (Scheme 3).
First, 5 was esterified with ethanol to furnish 6, and then 7 was formed by a benzylation reaction.Next, 8 was obtained by hydrolysis with sodium hydroxide solution, and 9 was obtained by reaction with Br2, acetic acid and sodium acetate [15].Notably, this reaction requires Br2 to be slowly added to the system at the end; otherwise, the reaction will not be complete (Scheme 3).9) under standard peptide-coupling conditions to afford amide 10 (1,1′-carbonyldiimidazole, THF, RT, 20 h).Unfortunately, we could not observe any reactions between 4 and 9.We varied the concentration of the reaction mixture and the number of equivalents of each reagent, and no good effects were observed.Considering that 4 contains a benzyl group and has a large steric hindrance, we subsequently changed the solvent to xylene and dehydrated and condensed the mixture with an oil-water separator at 135 °C; however, no satisfactory results were obtained.Finally, we tried to obtain 10 successfully by using DMF, HOBt and EDC.The use of the dehydrating condensation agent EDC results in mild reaction conditions and an easy process.
Amide 10 was subjected to the Bischler-Napieralski reaction [22] to afford cyclized imine 11, which was subjected to NaBH4-mediated reduction without further purification to furnish secondary amine 12.The protection of secondary amine 12 with a tertbutoxycarbonyl (Boc) group furnished N-Boc-protected 13.Then, 14 was prepared by a Pd-catalyzed direct biaryl coupling methodology [23][24][25].If the reaction was carried out at a lower temperature for a long time, debrominated byproducts appeared.The selection of ligands was also particularly important, because different ligands had different stability, which will greatly affect the yield.In terms of the choice of solvent, we chose 1,4dioxane.Compared with DMA, DMF or DMSO, our advantage was that we can choose to directly steam the solvent after the reaction was complete, avoiding the product loss and tedious operation steps brought by extraction.We tested many reaction conditions and determined the most suitable reaction conditions (Pd(OAc)2 (0.2 equiv.),Cs2CO3 (3 equiv.),(t-Bu)2PMeHBF4 (0.4 equiv.),1,4-dioxane, 101 °C, (t-Bu)2PMeHBF4 = di-tert-butylmethylphosphine tetrafluor).It was worth noting that the reaction conditions were harsh and required very strict vacuum and nitrogen protection.The synthesis of 15 was achieved by deprotecting the benzyl (Bn) group of 14 by using Pd/C under a hydrogen atmosphere.Finally, the synthesis of laurolitsine ( 16) was achieved by deprotecting the Boc group of 15 by using anhydrous ZnBr2 [18].Spectral data, including 1 H NMR, 13 C NMR, were collected for both the natural and synthetic sample ( 16) and found to be in good agreement  9) under standard peptide-coupling conditions to afford amide 10 (1,1 ′carbonyldiimidazole, THF, RT, 20 h).Unfortunately, we could not observe any reactions between 4 and 9.We varied the concentration of the reaction mixture and the number of equivalents of each reagent, and no good effects were observed.Considering that 4 contains a benzyl group and has a large steric hindrance, we subsequently changed the solvent to xylene and dehydrated and condensed the mixture with an oil-water separator at 135 • C; however, no satisfactory results were obtained.Finally, we tried to obtain 10 successfully by using DMF, HOBt and EDC.The use of the dehydrating condensation agent EDC results in mild reaction conditions and an easy process.
Amide 10 was subjected to the Bischler-Napieralski reaction [22] to afford cyclized imine 11, which was subjected to NaBH 4 -mediated reduction without further purification to furnish secondary amine 12.The protection of secondary amine 12 with a tertbutoxycarbonyl (Boc) group furnished N-Boc-protected 13.Then, 14 was prepared by a Pd-catalyzed direct biaryl coupling methodology [23][24][25].If the reaction was carried out at a lower temperature for a long time, debrominated byproducts appeared.The selection of ligands was also particularly important, because different ligands had different stability, which will greatly affect the yield.In terms of the choice of solvent, we chose 1,4-dioxane.Compared with DMA, DMF or DMSO, our advantage was that we can choose to directly steam the solvent after the reaction was complete, avoiding the product loss and tedious operation steps brought by extraction.We tested many reaction conditions and determined the most suitable reaction conditions (Pd(OAc) 2 (0.2 equiv.),Cs 2 CO 3 (3 equiv.),(t-Bu) 2 PMeHBF 4 (0.4 equiv.),1,4-dioxane, 101 • C, (t-Bu) 2 PMeHBF 4 = di-tert-butylmethylphosphine tetrafluor).It was worth noting that the reaction conditions were harsh and required very strict vacuum and nitrogen protection.The synthesis of 15 was achieved by deprotecting the benzyl (Bn) group of 14 by using Pd/C under a hydrogen atmosphere.Finally, the synthesis of laurolitsine ( 16) was achieved by deprotecting the Boc group of 15 by using anhydrous ZnBr 2 [18].Spectral data, including 1 H NMR, 13 C NMR, were collected for both the natural and synthetic sample ( 16) and found to be in good agreement (Table 1) [4].All these compounds were well characterized by using 1 H NMR, 13 C NMR, and high-resolution (HR) ESI-MS, as showed in Supplementary Materials (Scheme 4).(Table 1) [4].All these compounds were well characterized by using 1 H NMR, 13 C NMR, and high-resolution (HR) ESI-MS, as showed in Supplementary Materials (Scheme 4).

General Experimental Details
All chemicals were purchased from commercial sources.Silica gel (200-300 mesh, Qingdao Marine Chemistry Co. Ltd., Qingdao, Tsingtao, China) was used for column

General Experimental Details
All chemicals were purchased from commercial sources.Silica gel (200-300 mesh, Qingdao Marine Chemistry Co. Ltd., Qingdao, Tsingtao, China) was used for column chromatography.Reactions were monitored by thin-layer chromatography (TLC).Silica gel plates (Qingdao Marine Chemistry Co. Ltd., GF254, 0.20-0.25 mm) were used for the TLC analyses, which were visualized under model ZF-20D ultraviolet analyzing equipment (Shanghai Baoshan Gucun Photoelectricity Instrument, Shanghai, China) at 254 nm. 1 H and 13 C NMR experiments were performed on a JNM-ECZ400S NMR Spectrometer (Nippon Electric Company Limited, Tokyo, Japan) at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR.Chemical shifts (δ) are given in parts per million (ppm), and coupling constants are given in Hz.The multiplicity of 1 H NMR signals is reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, dd = doublet of doublets.The HRMS (ESI) spectroscopic data were obtained from an Agilent 1290II Mass Spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA).

Scheme 3 .
Scheme 3. Conditions: (d) EtOH, H2SO4, 78 °C, 90%; (e) K2CO3, BnCl, CH3CN, 82 °C, 82%; (f) NaOH, EtOH, 78 °C, 93%; (g) Br2, NaOAc, HOAc, 25 °C, 61%.We thus attempted to repeat the work reported by Sharma et al.[18] by coupling 2-(3-(benzyloxy)-4-methoxyphenyl)ethanamine (4) with 2-(5-(benzyloxy)-2-bromo-4-methoxyphenyl)acetic acid (9) under standard peptide-coupling conditions to afford amide 10 (1,1′-carbonyldiimidazole, THF, RT, 20 h).Unfortunately, we could not observe any reactions between 4 and 9.We varied the concentration of the reaction mixture and the number of equivalents of each reagent, and no good effects were observed.Considering that 4 contains a benzyl group and has a large steric hindrance, we subsequently changed the solvent to xylene and dehydrated and condensed the mixture with an oil-water separator at 135 °C; however, no satisfactory results were obtained.Finally, we tried to obtain 10 successfully by using DMF, HOBt and EDC.The use of the dehydrating condensation agent EDC results in mild reaction conditions and an easy process.Amide 10 was subjected to the Bischler-Napieralski reaction [22] to afford cyclized imine 11, which was subjected to NaBH4-mediated reduction without further purification to furnish secondary amine 12.The protection of secondary amine 12 with a tertbutoxycarbonyl (Boc) group furnished N-Boc-protected 13.Then, 14 was prepared by a Pd-catalyzed direct biaryl coupling methodology[23][24][25].If the reaction was carried out at a lower temperature for a long time, debrominated byproducts appeared.The selection of ligands was also particularly important, because different ligands had different stability, which will greatly affect the yield.In terms of the choice of solvent, we chose 1,4dioxane.Compared with DMA, DMF or DMSO, our advantage was that we can choose to directly steam the solvent after the reaction was complete, avoiding the product loss and tedious operation steps brought by extraction.We tested many reaction conditions and determined the most suitable reaction conditions (Pd(OAc)2 (0.2 equiv.),Cs2CO3 (3 equiv.),(t-Bu)2PMeHBF4 (0.4 equiv.),1,4-dioxane, 101 °C, (t-Bu)2PMeHBF4 = di-tert-butylmethylphosphine tetrafluor).It was worth noting that the reaction conditions were harsh and required very strict vacuum and nitrogen protection.The synthesis of 15 was achieved by deprotecting the benzyl (Bn) group of 14 by using Pd/C under a hydrogen atmosphere.Finally, the synthesis of laurolitsine (16) was achieved by deprotecting the Boc group of 15 by using anhydrous ZnBr2[18].Spectral data, including 1 H NMR,13 C NMR, were collected for both the natural and synthetic sample (16) and found to be in good agreement

Scheme 3 .
Scheme 3. Conditions: (d) EtOH, H 2 SO 4 , 78 • C, 90%; (e) K 2 CO 3 , BnCl, CH 3 CN, 82 • C, 82%; (f) NaOH, EtOH, 78 • C, 93%; (g) Br 2 , NaOAc, HOAc, 25 • C, 61%.We thus attempted to repeat the work reported by Sharma et al.[18] by coupling 2-(3-(benzyloxy)-4-methoxyphenyl)ethanamine (4) with 2-(5-(benzyloxy)-2-bromo-4-methoxyph enyl)acetic acid (9) under standard peptide-coupling conditions to afford amide 10 (1,1 ′carbonyldiimidazole, THF, RT, 20 h).Unfortunately, we could not observe any reactions between 4 and 9.We varied the concentration of the reaction mixture and the number of equivalents of each reagent, and no good effects were observed.Considering that 4 contains a benzyl group and has a large steric hindrance, we subsequently changed the solvent to xylene and dehydrated and condensed the mixture with an oil-water separator at 135 • C; however, no satisfactory results were obtained.Finally, we tried to obtain 10 successfully by using DMF, HOBt and EDC.The use of the dehydrating condensation agent EDC results in mild reaction conditions and an easy process.Amide 10 was subjected to the Bischler-Napieralski reaction [22] to afford cyclized imine 11, which was subjected to NaBH 4 -mediated reduction without further purification to furnish secondary amine 12.The protection of secondary amine 12 with a tertbutoxycarbonyl (Boc) group furnished N-Boc-protected 13.Then, 14 was prepared by a Pd-catalyzed direct biaryl coupling methodology[23][24][25].If the reaction was carried out at a lower temperature for a long time, debrominated byproducts appeared.The selection of ligands was also particularly important, because different ligands had different stability, which will greatly affect the yield.In terms of the choice of solvent, we chose 1,4-dioxane.Compared with DMA, DMF or DMSO, our advantage was that we can choose to directly steam the solvent after the reaction was complete, avoiding the product loss and tedious operation steps brought by extraction.We tested many reaction conditions and determined the most suitable reaction conditions (Pd(OAc) 2 (0.2 equiv.),Cs 2 CO 3 (3 equiv.),(t-Bu) 2 PMeHBF 4 (0.4 equiv.),1,4-dioxane, 101 • C, (t-Bu) 2 PMeHBF 4 = di-tert-butylmethylphosphine tetrafluor).It was worth noting that the reaction conditions were harsh and required very strict vacuum and nitrogen protection.The synthesis of 15 was achieved by deprotecting the benzyl (Bn) group of 14 by using Pd/C under a hydrogen atmosphere.Finally, the synthesis of laurolitsine (16) was achieved by deprotecting the Boc group of 15 by using anhydrous ZnBr 2[18].Spectral data, including 1 H NMR,13 C NMR, were collected for both the natural and synthetic sample (16) and found to be in good agreement (Table1)[4].All these compounds were well characterized by using 1 H NMR,13 C NMR, and high-resolution (HR) ESI-MS, as showed in Supplementary Materials (Scheme 4).

Table 1 .
Comparison of the13C NMR data of 16 with literature data[4].

Table 1 .
Comparison of the13C NMR data of 16 with literature data[4].