Method for the on-line synthesis of cinnamic amide derivatives based on a continuous flow bioreactor

Abstract

The application provides a method for online synthesis of cinnamic amide derivatives based on a continuous flow bioreactor, in which cinnamyl methyl ester or methyl 4-chlorocinnamate and a biological amine are catalyzed by a lipase Lipozyme RM IM in a microfluidic channel reactor to synthesize cinnamic amide compounds online. The method not only greatly shortens the reaction time, but also has high conversion rate; meanwhile, the method first uses the economical lipase Lipozyme RM IM to catalyze the amidation reaction of cinnamyl methyl ester or methyl 4-chlorocinnamate and a biological amine, reduces the reaction cost, and has the advantages of economy and efficiency.

Description

Technical Field

[0001] This invention relates to a method for the online synthesis of cinnamamide derivatives based on a continuous flow bioreactor. Background Technology

[0002] Cinnamic acid is a natural organic acid commonly found in plants, possessing high safety and diverse pharmacological activities, including antioxidant, anti-inflammatory, antitumor, cell-protective, and tyrosinase-inhibiting effects. Among cinnamic acid derivatives, cinnamamides and their derivatives exhibit broad physiological activities, such as anticancer, antimicrobial, anti-inflammatory, and melanin-inhibiting effects. The combination of cinnamic acid with biogenic amines has led to the development of several novel drug molecule templates with potential pharmacological activities. Cinnamyl phenethylamine is a class of compounds with important biological activity within the cinnamamide class, capable of inducing apoptosis in U-937 cancer cells (lymphoma), making it a potential anticancer drug molecule. The ability of cinnamyl tryptamine to inhibit melanin production makes it a promising candidate for skin whitening agents.

[0003] However, in actual synthesis processes, condensation reagents such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), dicyclohexylcarbodiimide (DCC), or triazine reagents are typically used to synthesize cinnamamide derivatives in N,N-dimethylformamide (DMF) or tetrahydrofuran (THF). These methods suffer from environmental pollution, energy waste, complex post-processing, and may even lead to allergic reactions. Therefore, developing an efficient and green cinnamamide synthesis technology is of great significance.

[0004] Biocatalysis is an interdisciplinary field of biology and chemistry, and bioenzymes are non-toxic and environmentally friendly biocatalysts. Bioenzyme-catalyzed reactions have become a focus of green chemistry research due to their high efficiency, green nature, and high specificity, and are powerful tools for synthesizing active pharmaceutical ingredients. However, bioenzyme-catalyzed reactions are limited by factors such as solvent solubility of substrates and solvent polarity inhibiting enzyme activity, often resulting in long reaction times (24h–96h) and relatively low conversion rates for specific substrates. Therefore, developing a new synthetic technique for cinnamamide derivatives based on continuous flow bioreactor technology, building upon traditional enzymatic reactions, has become our research objective.

[0005] Compared to conventional chemical reactors, microfluidic reactors offer advantages such as high mixing efficiency, rapid mass and heat transfer, precise parameter control, high reaction selectivity, and good safety, leading to their widespread application in organic synthesis reactions. In continuous flow microreactors, many reactions can be rapidly screened under minute-scale conditions, allowing for safe reactions even under harsh experimental conditions. This significantly saves reaction materials, improves screening efficiency, and aligns more closely with the concept of green chemistry.

[0006] To date, research on the enzyme-catalyzed synthesis of cinnamamides is relatively limited. Novozym 435 can effectively catalyze the reaction, but this method requires a long reaction time (24 hours) and the use of methyl tert-butyl ether as a solvent poses certain risks to human health and the environment. To develop a new, efficient, and environmentally friendly method for the synthesis of cinnamamides, we investigated a novel online synthesis method for a series of cinnamamide derivatives based on a continuous flow bioreactor, aiming to find a new and highly efficient synthetic technique for cinnamamides. Summary of the Invention

[0007] The present invention aims to provide a novel method for the online synthesis of cinnamamide in a continuous flow bioreactor, which has the advantages of short reaction time and high yield.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] This invention provides a method for online synthesis of cinnamamide derivatives based on a continuous flow bioreactor. The method employs a microfluidic channel reactor, which includes a syringe, a reaction channel, and a product collector connected in sequence. The syringe is installed in an injection pump and is connected to the inlet of the reaction channel via a first connecting pipe. The product collector is connected to the outlet of the reaction channel via a second connecting pipe. The inner diameter of the reaction channel is 1.6-2.2 mm (preferably 2.0 mm), and the length of the reaction channel is 0.8-1.2 m (preferably 1.0 m).

[0010] The method includes:

[0011] Using tert-amyl alcohol as the reaction solvent, methyl cinnamate of formula (I) and biogenic amine of formula (II) or (IV) as raw materials, and lipase Lipozyme RM IM as the catalyst, a reaction system is constructed. The raw materials and the reaction solvent are placed in a syringe, and lipase Lipozyme RM IM is uniformly filled into the reaction channel of a microfluidic channel reactor. Under the synchronous push of the injection pump, the raw materials and the reaction solvent are continuously introduced into the reaction channel to carry out the amidation reaction. The reaction temperature is controlled at 35-55℃ (preferably 45℃), and the reaction time in which the reaction liquid flows continuously in the reaction channel is 30-50 min (preferably 40 min). The reaction liquid flowing out of the reaction channel is collected online by a product collector. The reaction liquid is post-treated to obtain the cinnamamide derivative of formula (III) or (V).

[0012]

[0013] In formulas (I) and (III), R1 is H or Cl (preferably H); in formula (II) or (III), R2 is H or OH (preferably H); in formula (IV) or (V), R3 is H or OCH3 (preferably H).

[0014] The molar ratio of methyl cinnamate of formula (I) to biogenic amine of formula (II) or (IV) is 1:0.5 to 4 (particularly preferred 1:2); the amount of catalyst added, based on the volume of the reaction solvent, is 0.030 g / mL to 0.060 g / mL (preferably 0.04 g / mL) within the maximum capacity of the reaction channel to accommodate the filled catalyst; and the concentration of methyl cinnamate of formula (I) in the reaction system is 0.1 mmol / mL to 0.4 mmol / mL (preferably 0.25 mmol / mL).

[0015] The lipase Lipozyme RM IM used is a commercial product manufactured by Novozymes. It is a food-grade lipase (EC 3.1.1.3) prepared by microorganisms, specifically for positions 1 and 3, on granular silica gel. It is produced by deep fermentation of a genetically modified Aspergillus oryzae microorganism obtained from Rhizomucor miehei. The lipase Lipozyme RM IM can be prepared by directly and uniformly immobilizing the granular catalyst in the reaction channel using physical methods.

[0016] Furthermore, in the microfluidic channel reactor used in this invention, the number of syringes can be one or more, depending on the specific reaction requirements. This invention uses two reaction materials, preferably two syringes. Specifically, the syringes are a first syringe and a second syringe. The first connecting pipe is a Y-shaped or T-shaped pipe. The first syringe and the second syringe are respectively connected to the two ports of the Y-shaped or T-shaped pipe and connected in series with the reaction channel through the Y-shaped or T-shaped pipe. The increased probability of contact and collision between reactant molecules in the microchannel allows the two reaction streams to mix and react in the common reaction channel. That is, the microfluidic channel reactor of this invention includes a first syringe, a second syringe, a reaction channel, and a product collector; the first syringe and the second syringe are connected to the inlet of the reaction channel via Y-shaped or T-shaped pipes, and the product collector is connected to the outlet of the reaction channel via a pipe.

[0017] Furthermore, methyl cinnamate of formula (I) and biogenic amine of formula (II) or (IV) are each dissolved in tert-amyl alcohol to obtain methyl cinnamate solution and biogenic amine solution, respectively, which are then introduced into the reaction channel via the first syringe and the second syringe. In the methyl cinnamate solution, the concentration of methyl cinnamate of formula (I) is 0.2 mmol / mL to 0.8 mmol / mL (preferably 0.5 mmol / mL), and the concentration of biogenic amine solution of formula (II) or (IV) is 0.25 to 2.0 mmol / mL (preferably 1 mmol / mL).

[0018] Furthermore, this invention recommends a method for online synthesis of cinnamamide derivatives based on a continuous flow bioreactor. The method employs a microfluidic channel reactor, which includes a syringe, a reaction channel, and a product collector connected in sequence. The syringe is installed in an injection pump and is connected to the inlet of the reaction channel via a first connecting pipe. The product collector is connected to the outlet of the reaction channel via a second connecting pipe. The inner diameter of the reaction channel is 1.6–2.2 mm (preferably 2.0 mm), and the length of the reaction channel is 0.8–1.2 m (preferably 1.0 m). The syringes are a first syringe and a second syringe. The first connecting pipe is a Y-type or T-type pipe. The first syringe and the second syringe are respectively connected to two ports of the Y-type or T-type pipe and connected in series with the reaction channel via the Y-type or T-type pipe.

[0019] The method is as follows: methyl cinnamate of formula (I) and biogenic amine of formula (II) or (IV) are dissolved in tert-amyl alcohol to obtain a methyl cinnamate solution of formula (I) with a concentration of 0.2 mmol / mL to 0.8 mmol / mL (preferably 0.5 mmol / mL) and a biogenic amine solution of formula (II) or (IV) with a concentration of 0.25 to 2.0 mmol / mL (preferably 1 mmol / mL); the methyl cinnamate solution of formula (I) and the biogenic amine solution of formula (II) or (IV) are respectively loaded into the first syringe and the second syringe, and the first syringe and the second syringe are loaded into the same injection pump; lipase Lipozyme After RMIM is uniformly filled into the reaction channel of the microfluidic channel reactor, the methyl cinnamate solution of formula (I) and the biogenic amine solution of formula (II) or (IV) are continuously and synchronously introduced into the reaction channel under the synchronous push of the injection pump to carry out the amidation reaction. The reaction temperature is controlled at 35-55°C (preferably 45°C), and the reaction time in which the reaction solution flows continuously in the reaction channel is 30-50 min (preferably 40 min). The reaction solution flowing out of the reaction channel is collected online by a product collector. The reaction solution is post-treated to obtain the cinnamamide derivative of formula (III) or (V). The concentration ratio of the methyl cinnamate solution of formula (I) to the biogenic amine solution of formula (II) or (IV) is 1:0.5-4 (particularly preferred 1:2). Within the maximum limit that the reaction channel can accommodate the filled catalyst, the amount of catalyst added is 0.03 g / mL to 0.06 g / mL (preferably 0.04 g / mL) based on the volume of the reaction medium.

[0020] Further, the post-treatment is as follows: after removing the solvent from the reaction solution under reduced pressure, silica gel column chromatography is performed using a mixed solution of ethyl acetate and petroleum ether at a volume ratio of 1:10 as the eluent, the eluent containing the target compound is collected, evaporated to dryness, and the cinnamamide derivative shown in formula (III) or (V) is obtained.

[0021] Specifically, the silica gel column used in the silica gel column chromatography was prepared by wet packing with 200-300 mesh silica gel, with a column height of 35 cm and a column diameter of 4.5 cm. The specific operation was as follows: after evaporating the solvent, the resulting sample was dissolved in a small amount of eluent and then loaded onto the column using a wet packing method. The eluent was collected at a flow rate of 2 mL / min. -1 Meanwhile, TLC was used to track the elution process, and the resulting eluates containing a single target compound were combined, evaporated to dryness, and cinnamamide derivatives of formula (III) or (V) were obtained.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention enables the online synthesis of cinnamamide in a microfluidic channel reactor. This method not only significantly shortens the reaction time but also achieves high conversion rates. The post-processed product has a purity of over 99% and can be considered a pure product. Furthermore, it is the first time that the economical lipase Lipozyme RM IM has been used to catalyze the amidation reaction of methyl cinnamate with biogenic amines, reducing reaction costs and demonstrating economic and high efficiency. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the microfluidic channel reactor used in an embodiment of the present invention.

[0025] In the diagram, 1-first syringe, 2-second syringe, 3-reaction channel, 4-product collector, 5-water bath thermostat. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0027] Structural reference of the microfluidic channel reactor used in the embodiments of the present invention Figure 1 The system includes a syringe pump (not shown), two syringes 1 and 2, a reaction channel 3, a water bath thermostat (5, only its plan view is shown), and a product collector 4. The two syringes 1 and 2 are installed in the syringe pump and connected to the inlet of the reaction channel 3 through a Y-type interface. The reaction channel 3 is placed in the water bath thermostat 5, and the reaction temperature is controlled by the water bath thermostat 5. The inner diameter of the reaction channel 3 is 2.0 mm, and the tube length is 1.0 m. The outlet of the reaction channel 3 is connected to the product collector 4 through an interface.

[0028] Example 1: Synthesis of Cinnamyl Phenethylamine

[0029]

[0030] Device Reference Figure 1 Methyl cinnamate (5.0 mmol, 0.810 g) and phenylethylamine (10.0 mmol, 1.21 g) were dissolved in 10 mL of tert-amyl alcohol, and then each was placed into a 10 mL syringe for later use. 0.87 g of Lipozyme RM IM was uniformly filled into the reaction channel. Using a PHD 2000 syringe pump, the two reaction solutions were dispensed at a rate of 15.6 μL / min. -1 The flow rate is introduced into the reaction channel through the "Y" connector to carry out the reaction. The reactor temperature is controlled at 45℃ by a water bath constant temperature box. The reaction solution flows continuously in the reaction channel for 40 minutes. The reaction results are tracked and detected by thin-layer chromatography (TLC).

[0031] The reaction solution was collected online using a product collector. The solvent was removed by vacuum distillation. The solution was then packed into a column using a wet method with 200-300 mesh silica gel. The eluent was ethyl acetate:petroleum ether (v / v) = 1:10. The column height was 35 cm and the column diameter was 4.5 cm. The sample was dissolved in a small amount of the eluent and then loaded onto the column using a wet method. The eluent was collected at a flow rate of 2 mL / min. -1 Meanwhile, TLC was used to track the elution process, and the eluents containing a single product were combined and evaporated to dryness to obtain 1.142 g of white solid, with a separation yield of 91%.

[0032] The NMR characterization results are as follows:

[0033] 1 H NMR (400MHz, DMSO-d6) 1 H NMR(400MHz, DMSO-d6)δ8.21(t,J=5.6Hz,1H),7.57–7.50(m,2H),7.43–7.1 4(m,9H),6.59(d,J=15.9Hz,1H),3.40–3.27(m,2H),2.76(t,J=7.3Hz,2H).; 13 C NMR (101MHz, DMSO) δ165.16,139.62,138.82,135.04,129.66,129.14,128.84,128.56,127.71,126.33,122.31,40.56,35.30.

[0034] Examples 2-6

[0035] The solvent in the microfluidic microchannel reactor was changed, while other aspects remained the same as in Example 1. The results are shown in Table 1.

[0036] Table 1 Effect of solvent on reaction

[0037] Example solvent Conversion rate [%) Production [g] 1 tert-amyl alcohol (20 mL) 91 1.142 2 tert-Butanol (20 mL) 49 0.615 3 Acetonitrile (20 mL) 47 0.590 4 n-Hexane (20 mL) nd 0 5 DMF (20mL) nd 0 6 DMSO (20 mL) nd 0

[0038] The results in Table 1 show that when the molar ratio of methyl cinnamate to phenylethylamine is 1:2 and the flow rate is 15.6 μL·min, the desired effect is achieved. -1 The reaction time was 40 min and the reaction temperature was 45 °C. The conversion rate was optimal when tert-amyl alcohol was used as the organic solvent in the reactor. Therefore, tert-amyl alcohol is the optimal solvent in the microfluidic microchannel reactor of this invention.

[0039] Examples 7-10

[0040] The temperature of the microfluidic channel reactor was changed, and other aspects remained the same as in Example 1. The reaction results are shown in Table 2.

[0041] Table 2: Effect of temperature on the reaction

[0042] Example Temperature [°C] Conversion rate [%) Production [g] 7 35 65 0.816 8 40 78 0.979 1 45 91 1.142 9 50 80 1.004 10 55 67 0.841

[0043] The results in Table 2 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min for all reactions. Tert-amyl alcohol was used as the organic solvent in the reactor, and the molar ratio of methyl cinnamate to phenylethylamine was 1:2. The optimal conversion rate was achieved at a reaction temperature of 45°C; temperatures that were too high or too low would affect enzyme activity. Therefore, the optimal temperature in the microfluidic microchannel reactor of this invention was 45°C.

[0044] Examples 11-14

[0045] Using the amount of methyl cinnamate as a baseline, the molar ratio of methyl cinnamate to phenylethylamine in the microfluidic microchannel reactor was varied, while other aspects remained the same as in Example 1. The results are shown in Table 3.

[0046] Table 3 Effect of substrate molar ratio on the reaction

[0047]

[0048]

[0049] The results in Table 3 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min and the reaction temperature was 45 °C. Tert-amyl alcohol was used as the organic solvent in the reactor. As the reactant phenylethylamine increased, the conversion rate of the reaction also increased. The conversion rate was optimal when the substrate-to-methyl cinnamate-to-phenylethylamine ratio was 1:2. Therefore, the optimal substrate-to-methyl cinnamate ratio in the microfluidic microchannel reactor of this invention is 1:2.

[0050] Examples 15-20

[0051] The reaction time of the microfluidic channel reactor was changed, while other aspects remained the same as in Example 1. The reaction results are shown in Table 4.

[0052] Table 4. Effect of reaction time on the reaction

[0053] Example Time [min] Conversion rate [%) Production [g] 15 20 52 0.653 16 25 65 0.816 17 30 77 0.966 18 35 82 1.029 1 40 91 1.142 19 45 81 1.017 20 50 73 0.916

[0054] The results in Table 4 show that when the reactor uses tert-amyl alcohol as the organic solvent, the molar ratio of methyl cinnamate to phenylethylamine is 1:2, the reaction temperature is 45℃, and the reaction time is 40 min, the conversion rate is 91%. Therefore, the optimal reaction time in the microfluidic microchannel reactor of this invention is 40 min.

[0055] Comparative Examples 1-4

[0056] The type of amine in the microfluidic channel reactor was changed, replacing phenylethylamine with aniline, 4-chloroaniline, 4-methoxyaniline, and benzylamine, while other aspects remained the same as in Example 1. The reaction results are shown in Table 5.

[0057] Table 5. Effect of different amines on reaction conversion rate

[0058] Comparative Example amine Conversion rate [%) Production [g] 1 Aniline (10.0 mmol, 0.930 g) <5 0.038 2 4-Chloroaniline (10.0 mmol, 1.280 g) <5 0.044 3 4-Methoxyaniline (10.0 mmol, 1.230 g) <5 0.050 4 Benzylamine (10.0 mmol, 1.070 g) 20 0.251 Example 1 Phenylethylamine (10.0 mmol, 1.210 g) 91 1.142

[0059] The results showed that the structure of different amines had a significant impact on the conversion rate of methyl cinnamate. The conversion rate of methyl cinnamate was less than 5% in aniline, 4-chloroaniline or 4-methoxyaniline, while the conversion rate of methyl cinnamate reached 91% in phenethylamine.

[0060] Comparative Examples 5-8

[0061] The catalyst in the microfluidic microchannel reactor was changed to porcine pancreatic lipase PPL, lipase Novozym435, Bacillus subtilis alkaline protease, and lipase TM IM, respectively. Other aspects were the same as in Example 1. The results are shown in Table 6.

[0062] Table 6. Effects of different enzymes on reaction conversion rate and selectivity

[0063] Comparative Example enzyme source Conversion rate [%) Selectivity [%) Production [g] 5 PPL 15 100 0.188 6 Novozym 435 39 100 0.489 7 Bacillus subtilis alkaline protease <5 100 0.012 8 Lipase™ IM 44 100 0.552 Example 1 Lipase RM IM 91 100 1.142

[0064] The results showed that different enzymes had a significant impact on the amidation reaction of cinnamyl phenylethylamine in a microfluidic channel reactor. Using lipase™ IM to catalyze the reaction, the conversion rate of methyl cinnamate was 44%. However, using PPL to catalyze the reaction, the conversion rate of methyl cinnamate was only 15%. Therefore, the optimal enzyme source for the microfluidic microchannel reactor in this invention is lipase RM IM.

[0065] Example 21: Synthesis of Cinnamyl Tyramine

[0066]

[0067] Device Reference Figure 1 Methyl cinnamate (5.0 mmol, 0.810 mg) and tyramine (10.0 mmol, 1.370 g) were dissolved in 10 mL of tert-amyl alcohol, and then each was placed into a 10 mL syringe for later use. 0.87 g of Lipozyme RM IM was uniformly filled into the reaction channel. Using a PHD 2000 syringe pump, the two reaction solutions were dispensed at a rate of 15.6 μL / min. -1 The flow rate is introduced into the reaction channel through the "Y" connector to carry out the reaction. The reactor temperature is controlled at 45℃ by a water bath constant temperature box. The reaction solution flows continuously in the reaction channel for 40 minutes. The reaction results are tracked and detected by thin-layer chromatography (TLC).

[0068] The reaction solution was collected online using a product collector. The solvent was removed by vacuum distillation. The solution was then packed into a column using a wet method with 200-300 mesh silica gel. The eluent was ethyl acetate:petroleum ether (v / v) = 1:10. The column height was 35 cm and the column diameter was 4.5 cm. The sample was dissolved in a small amount of the eluent and then loaded onto the column using a wet method. The eluent was collected at a flow rate of 2 mL / min. -1 Meanwhile, TLC was used to track the elution process, and the eluents containing a single product were combined and evaporated to dryness to obtain 1.148 g of white solid, with a separation yield of 86%.

[0069] The NMR characterization results are as follows:

[0070] 1 H NMR(500MHz,DMSO-d6)δ9.19(s,1H),8.15(t,J=5.7Hz,1H),7.55(d,J=7.4Hz,2H),7.45–7.34(m,4H),7.0 2(d,J=8.2Hz,2H),6.72–6.65(m,2H),6.62(d,J=15.8Hz,1H),3.34–3.32(m,2H),2.66(t,J=7.5Hz,2H).; 13 C NMR (101MHz, DMSO) δ165.06,155.77,138.65,135.06,129.60,129.58,129.46,128.99,127.58,122.43,115.27,40.86,34.48.

[0071] Examples 22-26

[0072] The solvent in the microfluidic microchannel reactor was changed, while other aspects remained the same as in Example 21. The results are shown in Table 7.

[0073] Table 7 Effect of solvent on reaction

[0074] Example solvent Conversion rate [%) Production [g] 21 tert-amyl alcohol (20 mL) 86 1.148 22 tert-Butanol (20 mL) 44 0.587 23 Acetonitrile (20 mL) 41 0.547 24 n-Hexane (20 mL) nd 0 25 DMF (20mL) nd 0 26 DMSO (20 mL) nd 0

[0075] The results in Table 7 indicate that when the molar ratio of methyl cinnamate to tyramine is 1:2 and the flow rate is 15.6 μL·min, the desired effect is achieved. -1 The reaction time was 40 min and the reaction temperature was 45 °C. The conversion rate was optimal when tert-amyl alcohol was used as the organic solvent in the reactor. Therefore, tert-amyl alcohol is the optimal solvent in the microfluidic microchannel reactor of this invention.

[0076] Examples 27-30

[0077] The temperature of the microfluidic channel reactor was changed, and other aspects were the same as in Example 21. The reaction results are shown in Table 8.

[0078] Table 8 Effect of temperature on the reaction

[0079] Example Temperature [°C] Conversion rate [%) Production [g] 27 35 64 0.854 28 40 70 0.935 21 45 86 1.148 29 50 72 0.961 30 55 53 0.708

[0080] The results in Table 8 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min for all reactions. Tert-amyl alcohol was used as the organic solvent in the reactor, and the molar ratio of methyl cinnamate to tyramine was 1:2. The optimal conversion rate was achieved at a reaction temperature of 45°C; temperatures that were too high or too low would affect enzyme activity. Therefore, the optimal temperature in the microfluidic microchannel reactor of this invention was 45°C.

[0081] Examples 31-34

[0082] Using the amount of methyl cinnamate as a baseline, the molar ratio of methyl cinnamate to tyramine in the microfluidic microchannel reactor was varied, while other aspects remained the same as in Example 21. The results are shown in Table 9.

[0083] Table 9. Effect of substrate molar ratio on the reaction

[0084] Example Methyl cinnamate and tyramine Conversion rate [%) Production [g] 31 1:0.5(5.0mmol:2.5mmol,0.810g:0.343g) 38 0.507 32 1:1(5.0mmol:5.0mmol,0.810g:0.685g) 74 0.988 21 1:2(5.0mmol:10.0mmol,0.810g:1.370g) 86 1.148 33 1:3(5.0mmol:15.0mmol,0.810g:2.055g) 76 1.015 34 1:4(5.0mmol:20.0mmol,0.810g:2.740g) 71 0.948

[0085] The results in Table 9 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min and the reaction temperature was 45 °C. The reactor used tert-amyl alcohol as the organic solvent. As the reactant tyramine increased, the conversion rate of the reaction also increased. When the substrate-to-methyl cinnamate-to-tyramine ratio was 1:2, the conversion rate of the reaction was optimal. Therefore, the optimal substrate-to-material ratio in the microfluidic microchannel reactor of this invention is 1:2.

[0086] Examples 35-40

[0087] The reaction time of the microfluidic channel reactor was changed, and other aspects were the same as in Example 21. The reaction results are shown in Table 10.

[0088] Table 10 Effect of reaction time on the reaction

[0089] Example Time [min] Conversion rate [%) Production [g] 35 20 46 0.614 36 25 59 0.788 37 30 71 0.948 38 35 75 1.001 21 40 86 1.148 39 45 73 0.975 40 50 65 0.868

[0090] The results in Table 10 show that when the reactor uses tert-amyl alcohol as the organic solvent, the molar ratio of methyl cinnamate to tyramine is 1:2, the reaction temperature is 45℃, and the reaction time is 40 min, the reaction conversion rate is 86%. Therefore, the optimal reaction time in the microfluidic microchannel reactor of this invention is 40 min.

[0091] Comparative Examples 9-12

[0092] The type of amine in the microfluidic channel reactor was changed, replacing tyramine with aniline, 4-chloroaniline, 4-methoxyaniline, or benzylamine, while other aspects remained the same as in Example 21. The reaction results are shown in Table 11.

[0093] Table 11 Effect of different amines on reaction conversion rate

[0094] Comparative Example amine Conversion rate [%) Production [g] 1 Aniline (10.0 mmol, 0.930 g) <5 0.038 2 4-Chloroaniline (10.0 mmol, 1.280 g) <5 0.044 3 4-Methoxyaniline 10.0 mmol (1.230 g) <5 0.050 4 (Benzylamine 10.0 mmol, 1.070 g) 20 0.251 Example 21 Tyramine (10.0 mmol, 1.370 g) 86 1.148

[0095] The results showed that the structure of different amines had a significant impact on the conversion rate of methyl cinnamate. The conversion rate of methyl cinnamate was less than 5% in aniline, 4-chloroaniline or 4-methoxyaniline, while the conversion rate of methyl cinnamate reached 86% in tyramine.

[0096] Comparative Examples 13-16

[0097] The catalyst in the microfluidic microchannel reactor was changed to porcine pancreatic lipase PPL, lipase Novozym435, Bacillus subtilis alkaline protease, and lipase TM IM, respectively. Other aspects were the same as in Example 21. The results are shown in Table 12.

[0098] Table 12 Effects of different enzymes on reaction conversion rate and selectivity

[0099] Comparative Example enzyme source Conversion rate [%) Selectivity [%) Production [g] 9 PPL 10 100 0.133 10 Novozym 435 35 100 0.467 11 Bacillus subtilis alkaline protease <5 100 0.013 12 Lipase™ IM 40 100 0.534 Example 21 Lipase RM IM 86 100 1.148

[0100] The results in Table 12 show that different enzymes have a significant impact on the amidation reaction of cinnamoyltyramine in a microfluidic channel reactor. Using lipase™ IM to catalyze the reaction, the conversion rate of methyl cinnamate was 40%. However, using PPL to catalyze the reaction, the conversion rate of methyl cinnamate was only 10%. Therefore, the optimal enzyme source in the microfluidic microchannel reactor of this invention is lipase RM IM.

[0101] Example 41: Synthesis of Cinnamyl Tryptamine

[0102]

[0103] Device Reference Figure 1 Methyl cinnamate (5.0 mmol, 0.810 g) and tryptophan (10.0 mmol, 1.600 g) were dissolved in 10 mL of tert-amyl alcohol, and then each was placed in a 10 mL syringe for later use. 0.87 g of Lipozyme RM IM was uniformly filled into the reaction channel. Using a PHD 2000 syringe pump, the two reaction solutions were dispensed at a rate of 15.6 μL / min. -1The flow rate is introduced into the reaction channel through the "Y" connector to carry out the reaction. The reactor temperature is controlled at 45℃ by a water bath constant temperature box. The reaction solution flows continuously in the reaction channel for 40 minutes. The reaction results are tracked and detected by thin-layer chromatography (TLC).

[0104] The reaction solution was collected online using a product collector. The solvent was removed by vacuum distillation. The solution was then packed into a column using a wet method with 200-300 mesh silica gel. The eluent was ethyl acetate:petroleum ether (v / v) = 1:10. The column height was 35 cm and the column diameter was 4.5 cm. The sample was dissolved in a small amount of the eluent and then loaded onto the column using a wet method. The eluent was collected at a flow rate of 2 mL / min. -1 Meanwhile, TLC was used to track the elution process, and the eluents containing a single product were combined and evaporated to dryness to obtain 1.160 g of orange-yellow solid, with a separation yield of 80%.

[0105] The NMR characterization results are as follows:

[0106] 1 H NMR (400MHz, DMSO-d6) δ10.81–10.77(m,1H),8.20(t,J=5.7Hz,1H),7.55–7.49(m,3H),7.44–7.27(m,5H),7.13(d,J=2.3Hz,1H),7.03(dd d,J=8.1,6.9,1.2Hz,1H),6.94(ddd,J=8.0,6.9,1.1Hz,1H),6.60(d,J=15.8Hz,1H),3.44(td,J=7.4,5.7Hz,2H),2.85(t,J=7.4Hz,2H).; 13 C NMR(101MHz,DMSO)δ165.01,138.57,136.36,135.07,129.50,129.05,127.60,1 27.33,122.79,122.49,121.05,118.39,118.35,111.88,111.49,39.73,25.35.

[0107] Examples 42-46

[0108] The solvent in the microfluidic microchannel reactor was changed, and other aspects remained the same as in Example 41. The results are shown in Table 13.

[0109] Table 13 Effect of solvent on reaction

[0110] Example solvent Conversion rate [%) Production [g] 41 tert-amyl alcohol (20 mL) 80 1.160 42 tert-Butanol (20 mL) 30 0.435 43 Acetonitrile (20 mL) 31 0.450 44 n-Hexane (20 mL) nd 0 45 DMF (20mL) nd 0 46 DMSO (20 mL) nd 0

[0111] The results in Table 13 indicate that when the molar ratio of methyl cinnamate to tryptophan is 1:2 and the flow rate is 15.6 μL·min, the desired effect is achieved. -1The reaction time was 40 min and the reaction temperature was 45 °C. The conversion rate was optimal when tert-amyl alcohol was used as the organic solvent in the reactor. Therefore, tert-amyl alcohol is the optimal solvent in the microfluidic microchannel reactor of this invention.

[0112] Examples 47-50

[0113] The temperature of the microfluidic channel reactor was changed, and other aspects were the same as in Example 41. The reaction results are shown in Table 14.

[0114] Table 14 Effect of Temperature on the Reaction

[0115] Example Temperature [°C] Conversion rate [%) Production [g] 47 35 40 0.580 48 40 64 0.928 41 45 80 1.160 49 50 67 0.972 50 55 45 0.653

[0116] The results in Table 14 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min for all reactions. Tert-amyl alcohol was used as the organic solvent in the reactor, and the molar ratio of methyl cinnamate to tryptophan was 1:2. The conversion rate was optimal at a reaction temperature of 45°C; temperatures that were too high or too low would affect enzyme activity. Therefore, the optimal temperature in the microfluidic microchannel reactor of this invention was 45°C.

[0117] Examples 51-54

[0118] Using the amount of methyl cinnamate as a baseline, the molar ratio of methyl cinnamate to tryptophan in the microfluidic microchannel reactor was varied, while other aspects remained the same as in Example 41. The results are shown in Table 15.

[0119] Table 15 Effect of substrate molar ratio on the reaction

[0120] Example Methyl cinnamate and tryptophan Conversion rate [%) Production [g] 51 1:0.5(5.0mmol:2.5mmol,0.810g:0.400g) 19 0.276 52 1:1(5.0mmol:5.0mmol,0.810g:0.800g) 61 0.885 41 1:2(5.0mmol:10.0mmol,0.810g:1.600g) 80 1.160 53 1:3(5.0mmol:15.0mmol,0.810g:2.400g) 72 1.044 54 1:4(5.0mmol:20.0mmol,0.810g:3.200g) 66 0.957

[0121] The results in Table 15 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min and the reaction temperature was 45 °C. The reactor used tert-amyl alcohol as the organic solvent. As the reactant tryptamine increased, the conversion rate of the reaction also increased. When the substrate-to-methyl cinnamate-to-tryptamine ratio was 1:2, the conversion rate of the reaction was optimal. Therefore, the optimal substrate-to-substrate ratio in the microfluidic microchannel reactor of this invention is 1:2.

[0122] Examples 55-60

[0123] The reaction time of the microfluidic channel reactor was changed, and other aspects were the same as in Example 41. The reaction results are shown in Table 16.

[0124] Table 16 Effect of reaction time on the reaction

[0125] Example Time [min] Conversion rate [%) Production [g] 55 20 25 0.363 56 25 43 0.624 57 30 50 0.725 58 35 67 0.972 41 40 80 1.160 59 45 75 1.088 60 50 66 0.957

[0126] The results in Table 16 show that when the reactor uses tert-amyl alcohol as the organic solvent, the molar ratio of methyl cinnamate to tryptophan is 1:2, the reaction temperature is 45℃, and the reaction time is 40 min, the reaction conversion rate is 80%. Therefore, the optimal reaction time in the microfluidic microchannel reactor of this invention is 40 min.

[0127] Comparative Examples 17-20

[0128] The type of amine in the microfluidic channel reactor was changed, replacing tryptamine with aniline, 4-chloroaniline, 4-methoxyaniline, and benzylamine, while other aspects remained the same as in Example 41. The reaction results are shown in Table 17.

[0129] Table 17 Effect of different amines on reaction conversion rate

[0130] Comparative Example amine Conversion rate [%) Production [g] 1 Aniline (10.0 mmol, 0.930 g) <5 0.038 2 4-Chloroaniline (10.0 mmol, 1.280 g) <5 0.044 3 4-Methoxyaniline (10.0 mmol, 1.230 g) <5 0.050 4 Benzylamine (10.0 mmol, 1.070 g) 20 0.251 Example 41 Tryptophan (10.0 mmol, 1.600 g) 80 1.160

[0131] The results showed that the structure of different amines had a significant impact on the conversion rate of methyl cinnamate. The conversion rate of methyl cinnamate was less than 5% in aniline, 4-chloroaniline or 4-methoxyaniline, while the conversion rate of methyl cinnamate reached 80% in tryptophan.

[0132] Comparative Examples 21-24

[0133] The catalyst in the microfluidic microchannel reactor was changed to porcine pancreatic lipase PPL, lipase Novozym435, Bacillus subtilis alkaline protease, and lipase TM IM, respectively. Other aspects were the same as in Example 41. The results are shown in Table 18.

[0134] Table 18 Effects of different enzymes on reaction conversion rate and selectivity

[0135] Comparative Example enzyme source Conversion rate [%) Selectivity [%) Production [g] 13 PPL 8 100 0.116 14 Novozym 435 30 100 0.435 15 Bacillus subtilis alkaline protease <5 100 0.013 16 Lipase™ IM 32 100 0.464 Example 41 Lipase RM IM 80 100 1.160

[0136] The results in Table 18 show that different enzymes have a significant impact on the amidation reaction of cinnamic acid methyl ester in the microfluidic channel reactor. Using lipase™ IM to catalyze the reaction, the conversion rate of methyl cinnamate was 32%. However, using PPL to catalyze the reaction, the conversion rate of methyl cinnamate was only 8%. Therefore, the optimal enzyme source in the microfluidic microchannel reactor of this invention is lipase RM IM.

[0137] Example 61: Synthesis of Cinnamyl 5-Methoxytryptamine

[0138]

[0139] Device Reference Figure 1Methyl cinnamate (5.0 mmol, 0.810 g) and 5-methoxytryptamine (10.0 mmol, 1.930 g) were dissolved in 10 mL of tert-amyl alcohol, and then each was placed into a 10 mL syringe for later use. 0.87 g of Lipozyme RM IM was uniformly filled into the reaction channel. Using a PHD 2000 syringe pump, the two reaction solutions were dispensed at a rate of 15.6 μL / min. -1 The flow rate is introduced into the reaction channel through the "Y" connector to carry out the reaction. The reactor temperature is controlled at 45℃ by a water bath constant temperature box. The reaction solution flows continuously in the reaction channel for 40 minutes. The reaction results are tracked and detected by thin-layer chromatography (TLC).

[0140] The reaction solution was collected online using a product collector. The solvent was removed by vacuum distillation. The solution was then packed into a column using a wet method with 200-300 mesh silica gel. The eluent was ethyl acetate:petroleum ether (v / v) = 1:10. The column height was 35 cm and the column diameter was 4.5 cm. The sample was dissolved in a small amount of the eluent and then loaded onto the column using a wet method. The eluent was collected at a flow rate of 2 mL / min. -1 Meanwhile, the elution process was tracked by TLC, and the eluents containing a single product were combined and evaporated to dryness to obtain 1.168 g of yellow solid, yielding cinnamyl 5-methoxytryptamine with a separation yield of 73%.

[0141] The NMR characterization results are as follows:

[0142] 1 H NMR(400MHz,DMSO-d6)δ10.66(s,1H),8.23(q,J=4.8,4.2Hz,1H),7.55(dd d,J=8.1,3.4,1.7Hz,2H),7.48–7.34(m,4H),7.25–7.19(m,1H),7.13(d,J= 2.5Hz,1H),7.04(d,J=2.5Hz,1H),6.74–6.68(m,1H),6.68–6.60(m,1H),3. 76–3.72(m,3H),3.46(td,J=7.5,4.2Hz,2H),2.85(td,J=7.3,2.5Hz,2H).; 13 C NMR (101MHz, DMSO) δ165.01,153.08,138.56,135.07,131.49,129.50,129.05,127. 67,127.59,123.47,122.52,112.13,111.75,111.21,100.20,55.39,40.51,25.35.

[0143] Examples 62-66

[0144] The solvent in the microfluidic microchannel reactor was changed, while other aspects remained the same as in Example 61. The results are shown in Table 19.

[0145] Table 19 Effect of solvent on reaction

[0146] Example solvent Conversion rate [%) Production [g] 61 tert-amyl alcohol (20 mL) 73 1.168 62 tert-Butanol (20 mL) 30 0.480 63 Acetonitrile (20 mL) 34 0.544 64 n-Hexane (20 mL) nd 0 65 DMF (20mL) nd 0 66 DMSO (20 mL) nd 0

[0147] The results in Table 19 indicate that when the molar ratio of methyl cinnamate to 5-methoxytryptamine is 1:2 and the flow rate is 15.6 μL·min, the desired effect is achieved. -1 The reaction time was 40 min and the reaction temperature was 45 °C. The conversion rate was optimal when tert-amyl alcohol was used as the organic solvent in the reactor. Therefore, tert-amyl alcohol is the optimal solvent in the microfluidic microchannel reactor of this invention.

[0148] Examples 67-70

[0149] The temperature of the microfluidic channel reactor was changed, and other aspects were the same as in Example 61. The reaction results are shown in Table 20.

[0150] Table 20 Effect of Temperature on the Reaction

[0151] Example Temperature [°C] Conversion rate [%) Production [g] 67 35 32 0.512 68 40 52 0.832 61 45 73 1.168 69 50 61 0.976 70 55 40 0.640

[0152] The results in Table 20 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min for all reactions. Tert-amyl alcohol was used as the organic solvent in the reactor, and the molar ratio of methyl cinnamate to 5-methoxytryptamine was 1:2. The conversion rate was optimal at a reaction temperature of 45°C; temperatures that were too high or too low would affect enzyme activity. Therefore, the optimal temperature in the microfluidic microchannel reactor of this invention was 45°C.

[0153] Examples 71-74

[0154] Using the amount of methyl cinnamate as a baseline, the molar ratio of methyl cinnamate to 5-methoxytryptamine in the microfluidic microchannel reactor was varied, while other aspects remained the same as in Example 61. The results are shown in Table 21.

[0155] Table 21 Effect of substrate molar ratio on the reaction

[0156] Example Methyl cinnamate and 5-methoxytryptamine Conversion rate [%) Production [g] 71 1:0.5(5.0mmol:2.5mmol,0.810g:0.483g) 13 0.208 72 1:1(5.0mmol:5.0mmol,0.810g:0.965g) 54 0.864 61 1:2(5.0mmol:10.0mmol,0.810g:1.930g) 73 1.168 73 1:3(5.0mmol:15.0mmol,0.810g:2.895g) 62 0.992 74 1:4(5.0mmol:20.0mmol,0.810g:3.860g) 46 0.736

[0157] The results in Table 21 show that when the flow rate is 15.6 μL·min -1The reaction time was 40 min and the reaction temperature was 45 °C. The reactor used tert-amyl alcohol as the organic solvent. As the reactant 5-methoxytryptamine increased, the conversion rate of the reaction also increased. When the substrate-to-methyl cinnamate-to-5-methoxytryptamine ratio was 1:2, the conversion rate of the reaction was optimal. Therefore, the optimal substrate-to-material ratio in the microfluidic microchannel reactor of this invention is 1:2.

[0158] Examples 75-80

[0159] The reaction time of the microfluidic channel reactor was changed, and other aspects were the same as in Example 61. The reaction results are shown in Table 22.

[0160] Table 22 Effect of reaction time on the reaction

[0161]

[0162]

[0163] The results in Table 22 show that when the reactor uses tert-amyl alcohol as the organic solvent, the molar ratio of methyl cinnamate to 5-methoxytryptamine is 1:2, the reaction temperature is 45℃, and the reaction time is 40 min, the reaction conversion rate is 73%. Therefore, the optimal reaction time in the microfluidic microchannel reactor of this invention is 40 min.

[0164] Comparative Examples 25-28

[0165] The type of amine in the microfluidic channel reactor was changed, replacing 5-methoxytryptamine with aniline, 4-chloroaniline, 4-methoxyaniline, and benzylamine, while other aspects remained the same as in Example 61. The reaction results are shown in Table 23.

[0166] Table 23 Effect of different amines on reaction conversion rate

[0167] Comparative Example amine Conversion rate [%) Production [g] 1 Aniline (10.0 mmol, 0.930 g) <5 0.038 2 4-Chloroaniline (10.0 mmol, 1.280 g) <5 0.044 3 4-Methoxyaniline (10.0 mmol, 1.230 g) <5 0.050 4 Benzylamine (10.0 mmol, 1.070 g) 20 0.251 Example 61 5-Methoxytryptamine (10.0 mmol, 1.930 g) 73 1.168

[0168] The results showed that the structure of different amines had a significant impact on the conversion rate of methyl cinnamate. The conversion rate of methyl cinnamate was less than 5% in aniline, 4-chloroaniline or 4-methoxyaniline, while the conversion rate of methyl cinnamate reached 73% in 5-methoxytryptamine.

[0169] Comparative Examples 29-32

[0170] The catalyst in the microfluidic microchannel reactor was changed to porcine pancreatic lipase PPL, lipase Novozym435, Bacillus subtilis alkaline protease, and lipase TM IM, respectively. Other aspects were the same as in Example 61. The results are shown in Table 24.

[0171] Table 24 Effects of different enzymes on reaction conversion rate and selectivity

[0172] Comparative Example enzyme source Conversion rate [%) Selectivity [%) Production [g] 17 PPL 5 100 0.080 18 Novozym 435 25 100 0.401 19 Bacillus subtilis alkaline protease <5 100 0.016 20 Lipase™ IM 30 100 0.480 Example 61 Lipase RM IM 73 100 1.168

[0173] The results in Table 24 show that different enzymes have a significant impact on the amidation reaction of cinnamoyl 5-methoxytryptamine in a microfluidic channel reactor. Using lipase™ IM to catalyze the reaction, the conversion rate of methyl cinnamate was 30%. However, using PPL to catalyze the reaction, the conversion rate of methyl cinnamate was only 5%. Therefore, the optimal enzyme source in the microfluidic microchannel reactor of this invention is lipase RM IM.

[0174] Example 81: Synthesis of 4-chlorocinnamoyl phenethylamine

[0175]

[0176] Device Reference Figure 1 4-Chlorocinnamate (5.0 mmol, 0.985 g) and phenethylamine (10.0 mmol, 1.21 g) were dissolved in 10 mL of tert-amyl alcohol, and then each was placed into a 10 mL syringe for later use. 0.87 g of Lipozyme RM IM was uniformly filled into the reaction channel. Using a PHD 2000 syringe pump, the two reaction solutions were dispensed at a rate of 15.6 μL / min. -1 The flow rate is introduced into the reaction channel through the "Y" connector to carry out the reaction. The reactor temperature is controlled at 45℃ by a water bath constant temperature box. The reaction solution flows continuously in the reaction channel for 40 minutes. The reaction results are tracked and detected by thin-layer chromatography (TLC).

[0177] The reaction solution was collected online using a product collector. The solvent was removed by vacuum distillation. The solution was then packed into a column using a wet method with 200-300 mesh silica gel. The eluent was ethyl acetate:petroleum ether (v / v) = 1:10. The column height was 35 cm and the column diameter was 4.5 cm. The sample was dissolved in a small amount of the eluent and then loaded onto the column using a wet method. The eluent was collected at a flow rate of 2 mL / min. -1 Meanwhile, the elution process was tracked by TLC, and the eluents containing a single product were combined and evaporated to dryness to obtain 1.359 g of white solid, yielding 4-chlorocinnamoyl phenylethylamine with a separation yield of 95%.

[0178] The NMR characterization results are as follows:

[0179] 1H NMR(400MHz, DMSO-d6)δ8.18(t,J=5.7Hz,1H),7.58–7.51(m,2H),7.46–7.41(m,2H),7.36(d,J=1 5.8Hz,1H),7.30–7.13(m,5H),6.58(d,J=15.8Hz,1H),3.42–3.34(m,2H),2.74(t,J=7.3Hz,2H).

[0180] ; 13 C NMR (101MHz, DMSO) δ164.82,139.53,137.33,133.99,133.93,129.33,129.08,128.75,128.47,126.24,123.12,40.47,35.21.

[0181] Examples 82-86

[0182] The solvent in the microfluidic microchannel reactor was changed, while other aspects remained the same as in Example 81. The results are shown in Table 25.

[0183] Table 25 Effect of solvent on reaction

[0184] Example solvent Conversion rate [%) Production [g] 81 tert-amyl alcohol (20 mL) 95 1.359 82 tert-Butanol (20 mL) 53 0.758 83 Acetonitrile (20 mL) 50 0.715 84 n-Hexane (20 mL) nd 0 85 DMF (20mL) nd 0 86 DMSO (20 mL) nd 0

[0185] The results in Table 25 indicate that when the molar ratio of methyl 4-chlorocinnamate to phenylethylamine is 1:2 and the flow rate is 15.6 μL·min, the desired effect is achieved. -1 The reaction time was 40 min and the reaction temperature was 45 °C. The conversion rate was optimal when tert-amyl alcohol was used as the organic solvent in the reactor. Therefore, tert-amyl alcohol is the optimal solvent in the microfluidic microchannel reactor of this invention.

[0186] Examples 87-90

[0187] The temperature of the microfluidic channel reactor was changed, and other aspects were the same as in Example 81. The reaction results are shown in Table 26.

[0188] Table 26 Effect of Temperature on the Reaction

[0189] Example Temperature [°C] Conversion rate [%) Production [g] 87 35 70 1.001 88 40 83 1.187 81 45 95 1.359 89 50 86 1.230 90 55 71 1.015

[0190] The results in Table 26 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min for all reactions. Tert-amyl alcohol was used as the organic solvent in the reactor, and the molar ratio of methyl 4-chlorocinnamate to phenylethylamine was 1:2. The optimal conversion rate was achieved at a reaction temperature of 45°C; temperatures that were too high or too low would affect enzyme activity. Therefore, the optimal temperature in the microfluidic microchannel reactor of this invention was 45°C.

[0191] Examples 91-94

[0192] Using the amount of methyl 4-chlorocinnamate as a baseline, the molar ratio of methyl 4-chlorocinnamate to phenylethylamine in the microfluidic microchannel reactor was varied, while other aspects remained the same as in Example 81. The results are shown in Table 27.

[0193] Table 27 Effect of substrate molar ratio on the reaction

[0194]

[0195]

[0196] The results in Table 27 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min and the reaction temperature was 45 °C. The reactor used tert-amyl alcohol as the organic solvent. As the reactant phenylethylamine increased, the conversion rate of the reaction also increased. When the substrate ratio of methyl 4-chlorocinnamate to phenylethylamine was 1:2, the conversion rate of the reaction was optimal. Therefore, the optimal substrate molar ratio in the microfluidic microchannel reactor of this invention is 1:2.

[0197] Examples 95-100

[0198] The reaction time of the microfluidic channel reactor was changed, and other aspects were the same as in Example 81. The reaction results are shown in Table 28.

[0199] Table 28 Effect of reaction time on the reaction

[0200] Example Time [min] Conversion rate [%) Production [g] 95 20 57 0.815 96 25 69 0.987 97 30 81 1.158 98 35 86 1.231 81 40 95 1.359 99 45 83 1.187 100 50 76 1.087

[0201] The results in Table 28 show that when the reactor uses tert-amyl alcohol as the organic solvent, the molar ratio of methyl 4-chlorocinnamate to phenylethylamine is 1:2, the reaction temperature is 45℃, and the reaction time is 40 min, the conversion rate is 95%. Therefore, the optimal reaction time in the microfluidic microchannel reactor of this invention is 40 min.

[0202] Comparative Examples 33-36

[0203] The type of amine in the microfluidic channel reactor was changed, replacing phenethylamine with aniline, 4-chloroaniline, 4-methoxyaniline, and benzylamine, while other aspects remained the same as in Example 81. The reaction results are shown in Table 29.

[0204] Table 29 Effect of different amines on reaction conversion rate

[0205] Comparative Example amine Conversion rate [%) Production [g] 21 Aniline (10.0 mmol, 0.930 g) <5 0.046 22 4-Chloroaniline (10.0 mmol, 1.280 g) <5 0.054 23 4-Methoxyaniline (10.0 mmol, 1.230 g) <5 0.063 24 Benzylamine (10.0 mmol, 1.070 g) 35 0.500 Example 81 Phenethylamine 95 1.359

[0206] The results showed that the structure of different amines had a significant impact on the conversion rate of methyl 4-chlorocinnamate. The conversion rate of methyl 4-chlorocinnamate was less than 5% in aniline, 4-chloroaniline or 4-methoxyaniline, while the conversion rate of methyl 4-chlorocinnamate reached 95% in phenethylamine.

[0207] Comparative Examples 37-40

[0208] The catalyst in the microfluidic microchannel reactor was changed to porcine pancreatic lipase PPL, lipase Novozym435, Bacillus subtilis alkaline protease, and lipase TM IM, respectively. Other aspects were the same as in Example 81. The results are shown in Table 30.

[0209] Table 30 Effects of different enzymes on reaction conversion rate and selectivity

[0210] Comparative Example enzyme source Conversion rate [%) Selectivity [%) Production [g] 25 PPL 20 100 0.286 26 Novozym 435 40 100 0.572 27 Bacillus subtilis alkaline protease <5 100 0.143 28 Lipase™ IM 45 100 0.644 Example 81 Lipase RM IM 95 100 1.359

[0211] The results in Table 30 show that different enzymes have a significant impact on the amidation reaction of 4-cinnamoylphenylethylamine in a microfluidic channel reactor. Using lipase™ IM to catalyze the reaction, the conversion rate of methyl 4-chlorocinnamate was 45%. However, using PPL to catalyze the reaction, the conversion rate of methyl 4-chlorocinnamate was only 20%. Therefore, the optimal enzyme source for the microfluidic microchannel reactor in this invention is lipase RM IM.

[0212] Example 101: Synthesis of 4-chlorocinnamoyltyramine

[0213]

[0214] Device Reference Figure 1 4-Chlorocinnamate (5.0 mmol, 0.985 g) and tyramine (10.0 mmol, 1.370 g) were dissolved in 10 mL of tert-amyl alcohol, and then each was placed into a 10 mL syringe for later use. 0.87 g of Lipozyme RM IM was uniformly filled into the reaction channel. Using a PHD 2000 syringe pump, the two reaction solutions were dispensed at a rate of 15.6 μL / min. -1 The flow rate is introduced into the reaction channel through the "Y" connector to carry out the reaction. The reactor temperature is controlled at 45℃ by a water bath constant temperature box. The reaction solution flows continuously in the reaction channel for 40 minutes. The reaction results are tracked and detected by thin-layer chromatography (TLC).

[0215] The reaction solution was collected online using a product collector. The solvent was removed by vacuum distillation. The solution was then packed into a column using a wet method with 200-300 mesh silica gel. The eluent was ethyl acetate:petroleum ether (v / v) = 1:10. The column height was 35 cm and the column diameter was 4.5 cm. The sample was dissolved in a small amount of the eluent and then loaded onto the column using a wet method. The eluent was collected at a flow rate of 2 mL / min.-1 Meanwhile, the elution process was tracked by TLC, and the eluents containing a single product were combined and evaporated to dryness to obtain 1.359 g of white solid, yielding 4-chlorocinnamoyltyramine with a separation yield of 90%.

[0216] The NMR characterization results are as follows:

[0217] 1 H NMR(400MHz, DMSO-d6)δ9.18(s,1H),8.16(t,J=5.7Hz,1H),7.60–7.53(m,2H),7.50–7.42(m,2H),7.38(d,J=1 5.8Hz,1H),7.04–6.97(m,2H),6.71–6.63(m,2H),6.61(d,J=15.8Hz,1H),3.36(s,2H),2.64(t,J=7.4Hz,2H).; 13 C NMR (101MHz, DMSO) δ164.75,155.76,137.25,134.01,133.91,129.60,129.54,129.32,129.07,123.19,115.23,40.83,34.43.

[0218] Examples 102-106

[0219] The solvent in the microfluidic microchannel reactor was changed, while other aspects remained the same as in Example 101. The results are shown in Table 31.

[0220] Table 31 Effect of solvent on reaction

[0221] Example solvent Conversion rate [%) Production [g] 101 tert-amyl alcohol (20 mL) 90 1.359 102 tert-Butanol (20 mL) 48 0.725 103 Acetonitrile (20 mL) 42 0.634 104 n-Hexane (20 mL) nd 0 105 DMF (20mL) nd 0 106 DMSO (20 mL) nd 0

[0222] The results in Table 31 indicate that when the molar ratio of methyl 4-chlorocinnamate to tyramine is 1:2 and the flow rate is 15.6 μL·min, the desired effect is achieved. -1 The reaction time was 40 min and the reaction temperature was 45 °C. The conversion rate was optimal when tert-amyl alcohol was used as the organic solvent in the reactor. Therefore, tert-amyl alcohol is the optimal solvent in the microfluidic microchannel reactor of this invention.

[0223] Examples 107-110

[0224] The temperature of the microfluidic channel reactor was changed, and other aspects were the same as in Example 101. The reaction results are shown in Table 32.

[0225] Table 32 Effect of Temperature on the Reaction

[0226] Example Temperature [°C] Conversion rate [%) Production [g] 107 35 68 1.027 108 40 79 1.193 101 45 90 1.359 109 50 78 1.178 110 55 62 0.936

[0227] The results in Table 32 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min for all reactions. Tert-amyl alcohol was used as the organic solvent in the reactor, and the molar ratio of methyl 4-chlorocinnamate to tyramine was 1:2. The optimal conversion rate was achieved at a reaction temperature of 45°C; temperatures that were too high or too low would affect enzyme activity. Therefore, the optimal temperature in the microfluidic microchannel reactor of this invention was 45°C.

[0228] Examples 111-114

[0229] Using the amount of methyl 4-chlorocinnamate as a baseline, the molar ratio of methyl 4-chlorocinnamate to tyramine in the microfluidic microchannel reactor was varied, while other aspects remained the same as in Example 101. The results are shown in Table 33.

[0230] Table 33 Effect of substrate molar ratio on the reaction

[0231] Example 4-Chlorocinnamate and Tyramine Conversion rate [%) Production [g] 111 1:0.5(5.0mmol:2.5mmol,0.985g:0.343g) 42 0.634 112 1:1(5.0mmol:5.0mmol,0.985g:0.685g) 79 1.193 111 1:2(5.0mmol:10.0mmol,0.985g:1.370g) 90 1.359 113 1:3(5.0mmol:15.0mmol,0.985g:2.055g) 79 1.193 114 1:4(5.0mmol:20.0mmol,0.985g:2.740g) 70 1.057

[0232] The results in Table 33 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min and the reaction temperature was 45 °C. The reactor used tert-amyl alcohol as the organic solvent. As the reactant tyramine increased, the conversion rate of the reaction also increased. When the substrate-to-methyl 4-chlorocinnamate ratio to tyramine was 1:2, the conversion rate of the reaction was optimal. Therefore, the optimal substrate-to-material ratio in the microfluidic microchannel reactor of this invention is 1:2.

[0233] Examples 115-120

[0234] The reaction time of the microfluidic channel reactor was changed, while other aspects remained the same as in Example 101. The reaction results are shown in Table 34.

[0235] Table 34 Effect of reaction time on the reaction

[0236] Example Time [min] Conversion rate [%) Production [g] 115 20 50 0.755 116 25 63 0.951 117 30 72 1.087 118 35 79 1.193 101 40 90 1.359 119 45 81 1.223 120 50 61 0.921

[0237] The results in Table 34 show that when the reactor uses tert-amyl alcohol as the organic solvent, the molar ratio of methyl 4-chlorocinnamate to tyramine is 1:2, the reaction temperature is 45℃, and the reaction time is 40 min, the reaction conversion rate is 90%. Therefore, the optimal reaction time in the microfluidic microchannel reactor of this invention is 40 min.

[0238] Comparative Examples 41-44

[0239] The type of amine in the microfluidic channel reactor was changed, replacing tyramine with aniline, 4-chloroaniline, 4-methoxyaniline, or benzylamine, while other aspects remained the same as in Example 101. The reaction results are shown in Table 35.

[0240] Table 35 Effect of different amines on reaction conversion rate

[0241] Comparative Example amine Conversion rate [%) Production [g] 21 Aniline (10.0 mmol, 0.930 g) <5 0.046 22 4-Chloroaniline (10.0 mmol, 1.280 g) <5 0.054 23 4-Methoxyaniline (10.0 mmol, 1.230 g) <5 0.063 24 Benzylamine (10.0 mmol, 1.070 g) 35 0.500 Example 101 Tyramine (10.0 mmol, 1.370 g) 90 1.359

[0242] The results showed that the structure of different amines had a significant impact on the conversion rate of methyl 4-chlorocinnamate. The conversion rate of methyl 4-chlorocinnamate was less than 5% in aniline, 4-chloroaniline or 4-methoxyaniline, while the conversion rate of methyl 4-chlorocinnamate reached 90% in phenethylamine.

[0243] Comparative Examples 45-48

[0244] The catalyst in the microfluidic microchannel reactor was changed to porcine pancreatic lipase PPL, lipase Novozym435, Bacillus subtilis alkaline protease, and lipase TM IM, respectively, while other aspects remained the same as in Example 101. The results are shown in Table 36.

[0245] Table 36 Effects of different enzymes on reaction conversion rate and selectivity

[0246] Comparative Example enzyme source Conversion rate [%) Selectivity [%) Production [g] 29 PPL 16 100 0.242 30 Novozym 435 27 100 0.408 31 Bacillus subtilis alkaline protease 10 100 0.151 32 Lipase™ IM 41 100 0.619 Example 101 Lipase RM IM 90 100 1.359

[0247] The results in Table 36 show that different enzymes have a significant impact on the amidation reaction of 4-chlorocinnamoyltyramine in a microfluidic channel reactor. Using lipase™ IM to catalyze the reaction, the conversion rate of methyl 4-chlorocinnamate was 73%. However, using PPL to catalyze the reaction, the conversion rate of methyl 4-chlorocinnamate was only 15%. Therefore, the optimal enzyme source for the microfluidic microchannel reactor in this invention is lipase RM IM.

[0248] Example 121: Synthesis of 4-chlorocinnamoyltryptamine

[0249]

[0250] Device Reference Figure 1 4-Chlorocinnamate (5.0 mmol, 0.985 g) and tryptophan (10.0 mmol, 1.600 g) were dissolved in 10 mL of tert-amyl alcohol, and then each was placed into a 10 mL syringe for later use. 0.87 g of Lipozyme RM IM was uniformly filled into the reaction channel. Using a PHD 2000 syringe pump, the two reaction solutions were dispensed at a rate of 15.6 μL / min. -1 The flow rate is introduced into the reaction channel through the "Y" connector to carry out the reaction. The reactor temperature is controlled at 45℃ by a water bath constant temperature box. The reaction solution flows continuously in the reaction channel for 40 minutes. The reaction results are tracked and detected by thin-layer chromatography (TLC).

[0251] The reaction solution was collected online using a product collector. The solvent was removed by vacuum distillation. The solution was then packed into a column using a wet method with 200-300 mesh silica gel. The eluent was ethyl acetate:petroleum ether (v / v) = 1:10. The column height was 35 cm and the column diameter was 4.5 cm. The sample was dissolved in a small amount of the eluent and then loaded onto the column using a wet method. The eluent was collected at a flow rate of 2 mL / min. -1 Meanwhile, the elution process was tracked by TLC, and the eluents containing a single product were combined and evaporated to dryness to obtain 1.268 g of yellow solid, which yielded 4-chlorocinnamoyltryptamine with a separation yield of 78%.

[0252] The NMR characterization results are as follows:

[0253] 1 H NMR (400MHz, DMSO-d6) δ10.83–10.77(m,1H),8.22(t,J=5.8Hz,1H),7.55(dd,J= 11.4,8.2Hz,3H),7.49–7.44(m,2H),7.40(d,J=15.8Hz,1H),7.32(d,J=8.1Hz,1H ),7.15(d,J=2.3Hz,1H),7.05(ddd,J=8.1,6.9,1.2Hz,1H),6.96(ddd,J=7.9,7. 0,1.1Hz,1H),6.62(d,J=15.8Hz,1H),3.50–3.41(m,2H),2.87(t,J=7.3Hz,2H).; 13 C NMR(101MHz,DMSO)δ164.77,137.20,136.34,134.04,133.87,129.28,129.05,1 27.30,123.30,122.77,121.02,118.36,118.33,111.83,111.46,39.71,25.29.

[0254] Examples 122-126

[0255] The solvent in the microfluidic microchannel reactor was changed, while other aspects remained the same as in Example 121. The results are shown in Table 37.

[0256] Table 37 Effect of solvent on reaction

[0257] Example solvent Conversion rate [%) Production [g] 121 tert-amyl alcohol (20 mL) 78 1.268 122 tert-Butanol (20 mL) 33 0.536 123 Acetonitrile (20 mL) 30 0.488 124 n-Hexane (20 mL) nd 0 125 DMF (20mL) nd 0 126 DMSO (20 mL) nd 0

[0258] The results in Table 37 indicate that when the molar ratio of methyl 4-chlorocinnamate to tryptophan is 1:2 and the flow rate is 15.6 μL·min, the desired effect is achieved. -1The reaction time was 40 min and the reaction temperature was 45 °C. The conversion rate was optimal when tert-amyl alcohol was used as the organic solvent in the reactor. Therefore, tert-amyl alcohol is the optimal solvent in the microfluidic microchannel reactor of this invention.

[0259] Examples 127-130

[0260] The temperature of the microfluidic channel reactor was changed, and other aspects were the same as in Example 121. The reaction results are shown in Table 38.

[0261] Table 38 Effect of Temperature on the Reaction

[0262] Example Temperature [°C] Conversion rate [%) Production [g] 127 35 41 0.666 128 40 60 0.975 121 45 78 1.268 129 50 63 1.024 130 55 45 0.731

[0263] The results in Table 38 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min for all reactions. Tert-amyl alcohol was used as the organic solvent in the reactor, and the molar ratio of methyl 4-chlorocinnamate to tryptophan was 1:2. The conversion rate was optimal at a reaction temperature of 45°C; temperatures that were too high or too low would affect enzyme activity. Therefore, the optimal temperature in the microfluidic microchannel reactor of this invention was 45°C.

[0264] Examples 131-134

[0265] Using the amount of methyl 4-chlorocinnamate as a baseline, the molar ratio of methyl 4-chlorocinnamate to tryptophan in the microfluidic microchannel reactor was varied, while other aspects remained the same as in Example 121. The results are shown in Table 39.

[0266] Table 39 Effect of substrate molar ratio on the reaction

[0267] Example 4-Chlorocinnamate and tryptophan Conversion rate [%) Production [g] 131 1:0.5(5.0mmol:2.5mmol,0.985g:0.400g) 17 0.276 132 1:1(5.0mmol:5.0mmol,0.985g:0.800g) 60 0.975 121 1:2(5.0mmol:10.0mmol,0.985g:1.600g) 78 1.268 133 1:3(5.0mmol:15.0mmol,0.985g:2.400g) 68 1.105 134 1:4(5.0mmol:20.0mmol,0.985g:3.200g) 61 0.991

[0268] The results in Table 39 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min and the reaction temperature was 45 °C. The reactor used tert-amyl alcohol as the organic solvent. As the reactant tryptamine increased, the conversion rate of the reaction also increased. When the substrate-to-tryptamine ratio of methyl 4-chlorocinnamate was 1:2, the conversion rate of the reaction was optimal. Therefore, the optimal substrate-to-tryptamine ratio in the microfluidic microchannel reactor of this invention is 1:2.

[0269] Examples 135-140

[0270] The reaction time of the microfluidic channel reactor was changed, while other aspects remained the same as in Example 121. The reaction results are shown in Table 40.

[0271] Table 40 Effect of reaction time on the reaction

[0272] Example Time [min] Conversion rate [%) Production [g] 135 20 24 0.390 136 25 40 0.650 137 30 46 0.748 138 35 64 1.040 121 40 78 1.268 139 45 70 1.138 140 50 63 1.023

[0273] The results in Table 40 show that when the reactor uses tert-amyl alcohol as the organic solvent, the molar ratio of methyl 4-chlorocinnamate to tryptophan is 1:2, the reaction temperature is 45℃, and the reaction time is 40 min, the reaction conversion rate is 78%. Therefore, the optimal reaction time in the microfluidic microchannel reactor of this invention is 40 min.

[0274] Comparative Examples 49-52

[0275] The type of amine in the microfluidic channel reactor was changed, replacing tryptamine with aniline, 4-chloroaniline, 4-methoxyaniline, and benzylamine, while other aspects remained the same as in Example 121. The reaction results are shown in Table 41.

[0276] Table 41 Effect of different amines on reaction conversion rate

[0277] Comparative Example amine Conversion rate [%) Production [g] 21 Aniline (10.0 mmol, 0.930 g) <5 0.046 22 4-Chloroaniline (10.0 mmol, 1.280 g) <5 0.054 23 4-Methoxyaniline (10.0 mmol, 1.230 g) <5 0.063 24 Benzylamine (10.0 mmol, 1.070 g) 35 0.500 Example 121 Tryptophan (10.0 mmol, 1.600 g) 78 1.268

[0278] The results showed that the structure of different amines had a significant impact on the conversion rate of methyl 4-chlorocinnamate. The conversion rate of methyl 4-chlorocinnamate was less than 5% in aniline, 4-chloroaniline or 4-methoxyaniline, while the conversion rate of methyl 4-chlorocinnamate reached 78% in tryptophan.

[0279] Comparative Examples 53-56

[0280] The catalyst in the microfluidic microchannel reactor was changed to porcine pancreatic lipase PPL, lipase Novozym435, Bacillus subtilis alkaline protease, and lipase TM IM, respectively, while other aspects remained the same as in Example 121. The results are shown in Table 42.

[0281] Table 42 Effects of different enzymes on reaction conversion rate and selectivity

[0282] Comparative Example enzyme source Conversion rate [%) Selectivity [%) Production [g] 33 PPL 13 100 0.211 34 Novozym 435 30 100 0.488 35 Bacillus subtilis alkaline protease <5 100 0.011 36 Lipase™ IM 34 100 0.553 Example 121 Lipase RM IM 78 100 1.268

[0283] The results in Table 42 show that different enzymes have a significant impact on the amidation reaction of 4-chlorocinnamoyltryptamine in a microfluidic channel reactor. Using lipase™ IM to catalyze the reaction, the conversion rate of methyl 4-chlorocinnamate was 34%. However, using PPL to catalyze the reaction, the conversion rate of methyl 4-chlorocinnamate was only 13%. Therefore, the optimal enzyme source in the microfluidic microchannel reactor of this invention is lipase RM IM.

[0284] Example 141: Synthesis of 4-chlorocinnamoyl 5-methoxytryptamine

[0285]

[0286] Device Reference Figure 14-Chlorocinnamate (5.0 mmol, 0.985 g) and 5-methoxytryptamine (10.0 mmol, 1.930 g) were dissolved in 10 mL of tert-amyl alcohol, and then each was placed into a 10 mL syringe for later use. 0.87 g of Lipozyme RM IM was uniformly filled into the reaction channel. Using a PHD 2000 syringe pump, the two reaction solutions were dispensed at a rate of 15.6 μL / min. -1 The flow rate is introduced into the reaction channel through the "Y" connector to carry out the reaction. The reactor temperature is controlled at 45℃ by a water bath constant temperature box. The reaction solution flows continuously in the reaction channel for 40 minutes. The reaction results are tracked and detected by thin-layer chromatography (TLC).

[0287] The reaction solution was collected online using a product collector. The solvent was removed by vacuum distillation. The solution was then packed into a column using a wet method with 200-300 mesh silica gel. The eluent was ethyl acetate:petroleum ether (v / v) = 1:10. The column height was 35 cm and the column diameter was 4.5 cm. The sample was dissolved in a small amount of the eluent and then loaded onto the column using a wet method. The eluent was collected at a flow rate of 2 mL / min. -1 Meanwhile, the elution process was tracked by TLC, and the eluents containing the single product were combined and evaporated to dryness to obtain 1.243 g of yellow solid, which yielded 4-chlorocinnamoyl 5-methoxytryptamine with a separation yield of 70%.

[0288] The NMR characterization results are as follows:

[0289] 1 H NMR(400MHz,DMSO-d6)δ10.62(d,J=2.3Hz,1H),8.21(t,J=5.8Hz,1H),7.58–7 .51(m,2H),7.48–7.42(m,2H),7.39(d,J=15.8Hz,1H),7.19(d,J=8.7Hz,1H),7 .09(d,J=2.3Hz,1H),7.00(d,J=2.4Hz,1H),6.67(dd,J=8.7,2.4Hz,1H),6.61 (d,J=15.9Hz,1H),3.71(s,3H),3.43(q,J=6.8Hz,2H),2.82(t,J=7.3Hz,2H).; 13 C NMR (101MHz, DMSO) δ164.81,153.08,137.21,134.04,133.89,131.49,129.29,129. 07,127.66,123.46,123.32,112.12,111.71,111.20,100.22,55.40,40.02,25.31.

[0290] Examples 142-146

[0291] The solvent in the microfluidic microchannel reactor was changed, while other aspects remained the same as in Example 141. The results are shown in Table 43.

[0292] Table 43 Effect of solvent on reaction

[0293] Example solvent Conversion rate [%) Production [g] 61 tert-amyl alcohol (20 mL) 70 1.243 62 tert-Butanol (20 mL) 31 0.550 63 Acetonitrile (20 mL) 30 0.533 64 n-Hexane (20 mL) nd 0 65 DMF (20mL) nd 0 66 DMSO (20 mL) nd 0

[0294] The results in Table 43 indicate that when the molar ratio of methyl 4-chlorocinnamate to 5-methoxytryptamine is 1:2 and the flow rate is 15.6 μL·min, the desired effect is achieved. -1 The reaction time was 40 min and the reaction temperature was 45 °C. The conversion rate was optimal when tert-amyl alcohol was used as the organic solvent in the reactor. Therefore, tert-amyl alcohol is the optimal solvent in the microfluidic microchannel reactor of this invention.

[0295] Examples 147-150

[0296] The temperature of the microfluidic channel reactor was changed, and other aspects were the same as in Example 141. The reaction results are shown in Table 44.

[0297] Table 44 Effect of Temperature on the Reaction

[0298] Example Temperature [°C] Conversion rate [%) Production [g] 147 35 30 0.534 148 40 45 0.799 141 45 70 1.243 149 50 59 1.047 150 55 40 0.711

[0299] The results in Table 44 show that when the flow rate is 15.6 μL·min -1 The reaction time was 40 min for all reactions. Tert-amyl alcohol was used as the organic solvent in the reactor, and the molar ratio of methyl 4-chlorocinnamate to 5-methoxytryptamine was 1:2. The optimal conversion rate was achieved at a reaction temperature of 45°C; temperatures that were too high or too low would affect enzyme activity. Therefore, the optimal temperature in the microfluidic microchannel reactor of this invention was 45°C.

[0300] Examples 151-154

[0301] Using the amount of methyl 4-chlorocinnamate as a baseline, the molar ratio of methyl 4-chlorocinnamate to 5-methoxytryptamine in the microfluidic microchannel reactor was varied, while other aspects remained the same as in Example 141. The results are shown in Table 45.

[0302] Table 45 Effect of substrate molar ratio on the reaction

[0303] Example 4-Chlorocinnamate and 5-methoxytryptamine Conversion rate [%) Production [g] 151 1:0.5(5.0mmol:2.5mmol,0.985g:0.483g) 12 0.213 152 1:1(5.0mmol:5.0mmol,0.985g:0.965g) 50 0.888 141 1:2(5.0mmol:10.0mmol,0.985g:1.930g) 70 1.243 153 1:3(5.0mmol:15.0mmol,0.985g:2.895g) 58 1.029 154 1:4(5.0mmol:20.0mmol,0.985g:3.860g) 42 0.746

[0304] The results in Table 45 show that when the flow rate is 15.6 μL·min -1The reaction time was 40 min and the reaction temperature was 45 °C. The reactor used tert-amyl alcohol as the organic solvent. As the reactant 5-methoxytryptamine increased, the conversion rate of the reaction also increased. When the substrate-to-methyl 4-chlorocinnamate ratio to 5-methoxytryptamine was 1:2, the conversion rate of the reaction was optimal. Therefore, the optimal substrate-to-material ratio in the microfluidic microchannel reactor of this invention is 1:2.

[0305] Examples 155-160

[0306] The reaction time of the microfluidic channel reactor was changed, while other aspects remained the same as in Example 141. The reaction results are shown in Table 46.

[0307] Table 46 Effect of reaction time on the reaction

[0308]

[0309]

[0310] The results in Table 46 show that when the reactor uses tert-amyl alcohol as the organic solvent, the molar ratio of methyl 4-chlorocinnamate to 5-methoxytryptamine is 1:2, the reaction temperature is 45℃, and the reaction time is 40 min, the reaction conversion rate is 70%. Therefore, the optimal reaction time in the microfluidic microchannel reactor of this invention is 40 min.

[0311] Comparative Examples 57-60

[0312] The type of amine in the microfluidic channel reactor was changed, replacing 5-methoxytryptamine with aniline, 4-chloroaniline, 4-methoxyaniline, or benzylamine, while other aspects remained the same as in Example 141. The reaction results are shown in Figure 47.

[0313] Table 47 Effect of different amines on reaction conversion rate

[0314] Comparative Example amine Conversion rate [%) Production [g] 21 Aniline (10.0 mmol, 0.930 g) <5 0.046 22 4-Chloroaniline (10.0 mmol, 1.280 g) <5 0.054 23 4-Methoxyaniline (10.0 mmol, 1.230 g) <5 0.063 24 Benzylamine (10.0 mmol, 1.070 g) 35 0.500 Example 141 5-Methoxytryptamine (10.0 mmol, 1.930 g) 70 1.243

[0315] The results showed that the structure of different amines had a significant impact on the conversion rate of methyl 4-chlorocinnamate. The conversion rate of methyl 4-chlorocinnamate was less than 5% in aniline, 4-chloroaniline or 4-methoxyaniline, while the conversion rate of methyl 4-chlorocinnamate reached 70% in 5-methoxytryptamine.

[0316] Comparative Examples 61-64

[0317] The catalyst in the microfluidic microchannel reactor was changed to porcine pancreatic lipase PPL, lipase Novozym435, Bacillus subtilis alkaline protease, and lipase TM IM, respectively, while other aspects remained the same as in Example 141. The results are shown in Table 48.

[0318] Table 48 Effects of different enzymes on reaction conversion rate and selectivity

[0319] Comparative Example enzyme source Conversion rate [%) Selectivity [%) Production [g] 37 PPL 8 100 0.142 38 Novozym 435 30 100 0.533 39 Bacillus subtilis alkaline protease <5 100 0.101 40 Lipase™ IM 32 100 0.568 Example 141 Lipase RM IM 70 100 1.243

[0320] The results in Table 48 show that different enzymes have a significant impact on the amidation reaction of 4-chlorocinnamoyl 5-methoxytryptamine in a microfluidic channel reactor. Using lipase™ IM to catalyze the reaction, the conversion rate of methyl 4-chlorocinnamate was 32%. However, using PPL to catalyze the reaction, the conversion rate of methyl 4-chlorocinnamate was only 8%. Therefore, the optimal enzyme source for the microfluidic microchannel reactor in this invention is lipase RM IM.

Claims

1. A method for online synthesis of cinnamamide derivatives based on a continuous flow bioreactor, characterized in that: The method employs a microfluidic channel reactor, which includes a syringe, a reaction channel, and a product collector connected in sequence. The syringe is installed in an injection pump and is connected to the inlet of the reaction channel via a first connecting pipe. The product collector is connected to the outlet of the reaction channel via a second connecting pipe. The inner diameter of the reaction channel is 1.6-2.2 mm, and the length of the reaction channel is 0.8-1.2 m. The method includes: Using tert-amyl alcohol as the reaction solvent, methyl cinnamate of formula (I) and biogenic amine of formula (II) or (IV) as raw materials, and lipase Lipozyme RM IM as the catalyst, a reaction system was constructed. The raw materials and the reaction solvent were placed in a syringe, and lipase Lipozyme RM IM was uniformly filled into the reaction channel of a microfluidic channel reactor. Under the synchronous push of the injection pump, the raw materials and the reaction solvent were continuously introduced into the reaction channel to carry out the amidation reaction. The reaction temperature was controlled at 35-55°C, and the reaction time of the reaction liquid flowing continuously in the reaction channel was 30-50 min. The reaction liquid flowing out of the reaction channel was collected online by a product collector. The reaction liquid was post-treated to obtain the cinnamonamide derivative of formula (III) or (V). In formulas (Ⅰ) and (III), R1 is H or Cl; in formulas (II) or (III), R2 is H or OH; and in formulas (IV) or (V), R3 is H or OCH3. The molar ratio of methyl cinnamate of formula (I) to biogenic amine of formula (II) or (IV) is 1:0.5 to 4; the amount of catalyst added, based on the volume of the reaction solvent, is 0.030 g / mL to 0.060 g / mL within the maximum capacity of the reaction channel to accommodate the filled catalyst; and the concentration of methyl cinnamate of formula (I) in the reaction system is 0.1 mmol / mL to 0.4 mmol / mL.

2. The method for online synthesis of cinnamamide derivatives based on a continuous flow bioreactor as described in claim 1, characterized in that: R1 is H, R2 is H, and R3 is H.

3. The method for online synthesis of cinnamamide derivatives based on a continuous flow bioreactor as described in claim 1, characterized in that: The molar ratio of methyl cinnamate as shown in formula (I) to the biogenic amine as shown in formula (II) or (IV) is 1:

2.

4. The method for online synthesis of cinnamamide derivatives based on a continuous flow bioreactor as described in claim 1, characterized in that: The amount of catalyst added was 0.04 g / mL based on the volume of the reaction solvent.

5. The method for online synthesis of cinnamamide derivatives based on a continuous flow bioreactor as described in claim 1, characterized in that: In the reaction system, the concentration of methyl cinnamate as shown in formula (I) is 0.25 mmol / mL.

6. The method for online synthesis of cinnamamide derivatives based on a continuous flow bioreactor as described in claim 1, characterized in that: The number of syringes is two.

7. The method for online synthesis of cinnamamide derivatives based on a continuous flow bioreactor as described in claim 6, characterized in that: The syringes are a first syringe and a second syringe. The first connecting tube is a Y-shaped or T-shaped tube. The first syringe and the second syringe are respectively connected to two ports of the Y-shaped or T-shaped tube and connected in series with the reaction channel through the Y-shaped or T-shaped tube.

8. The method for online synthesis of cinnamamide derivatives based on a continuous flow bioreactor as described in claim 7, characterized in that: Methyl cinnamate of formula (I) and biogenic amine of formula (II) or (IV) are each dissolved in tert-amyl alcohol to obtain methyl cinnamate solution and biogenic amine solution, respectively, which are then introduced into the reaction channel through the first syringe and the second syringe; in the methyl cinnamate solution, the concentration of methyl cinnamate of formula (I) is 0.2 mmol / mL to 0.8 mmol / mL, and the concentration of biogenic amine solution of formula (II) or (IV) is 0.25 to 2.0 mmol / mL.

9. The method for online synthesis of cinnamamide derivatives based on a continuous flow bioreactor as described in claim 6, characterized in that: The method employs a microfluidic channel reactor, which includes a syringe, a reaction channel, and a product collector connected in sequence. The syringe is installed in an injection pump and is connected to the inlet of the reaction channel via a first connecting pipe. The product collector is connected to the outlet of the reaction channel via a second connecting pipe. The inner diameter of the reaction channel is 1.6–2.2 mm, and the length of the reaction channel is 0.8–1.2 m. The syringes are a first syringe and a second syringe. The first connecting pipe is a Y-type or T-type pipe. The first syringe and the second syringe are respectively connected to two ports of the Y-type or T-type pipe and connected in series with the reaction channel via the Y-type or T-type pipe. The method is as follows: methyl cinnamate of formula (I) and biogenic amine of formula (II) or (IV) are dissolved in tert-amyl alcohol to obtain a methyl cinnamate solution of formula (I) with a concentration of 0.2 mmol / mL to 0.8 mmol / mL and a biogenic amine solution of formula (II) or (IV) with a concentration of 0.25 to 2.0 mmol / mL; the methyl cinnamate solution of formula (I) and the biogenic amine solution of formula (II) or (IV) are respectively loaded into the first syringe and the second syringe, and the first syringe and the second syringe are loaded into the same injection pump; lipase Lipozyme RM is then added. After IM is uniformly filled into the reaction channel of the microfluidic channel reactor, the methyl cinnamate solution of formula (I) and the biogenic amine solution of formula (II) or (IV) are continuously and synchronously introduced into the reaction channel under the synchronous push of the injection pump to carry out the amidation reaction. The reaction temperature is controlled at 35-55°C, and the reaction time in which the reaction solution flows continuously in the reaction channel is 30-50 min. The reaction solution flowing out of the reaction channel is collected online by the product collector. The reaction solution is post-treated to obtain the cinnamamide derivative of formula (III) or (V). The concentration ratio of the methyl cinnamate solution of formula (I) to the biogenic amine solution of formula (II) or (IV) is 1:0.5-4. Within the maximum limit that the reaction channel can accommodate the filled catalyst, the amount of catalyst added is 0.03 g / mL to 0.06 g / mL based on the volume of the reaction medium.

10. The method for online synthesis of cinnamamide derivatives based on a continuous flow bioreactor as described in any one of claims 1 to 9, characterized in that: The post-processing is as follows: after removing the solvent from the reaction solution under reduced pressure, silica gel column chromatography is performed using a mixed solution of ethyl acetate and petroleum ether at a volume ratio of 1:10 as the eluent. The eluent containing the target compound is collected, evaporated to dryness, and the cinnamamide derivative shown in formula (III) or (V) is obtained.

Images