Two-Step Tandem Synthesis of Coumarin Derivatives Containing Bioamide Skeleton Catalyzed by Lipozyme TL IM from Thermomyces lanuginosus in Sustainable Continuous-Flow Microreactors

Abstract

Due to their remarkable biological and pharmacological activities such as antibacterial, antifungal, anticoagulant, antioxidant, anticancer, and anti-inflammatory properties, synthesis of coumarins and their derivatives has attracted considerable attention in research and development among both organic and medicinal chemists. In this paper, we demonstrated for the first time a two-step tandem enzymatic synthesis of coumarin bioamide derivatives through sustainable continuous-flow technology. Salicylaldehyde and dimethyl malonate were firstly reacted to obtain coumarin carboxylate methyl derivatives, which were then reacted with various biogenic amines at 50 °C for about 40 min under the catalysis of lipase TL IM from Thermomyces lanuginosus to obtain coumarin bioamide derivatives in continuous-flow reactors. Reaction parameters such as reaction solvent, reaction catalyst type, reactant ratio, residence time, reaction temperature and comparative experiments with traditional batch process were studied. Ideal product yields (62.7–87.1%) were obtained. Environmentally friendly methanol was applied as the reaction medium. Substantially shorter reaction times as well as a significant increase in the product yield were obtained as compared to the batch process. This innovative approach provides a promising green, efficient and rapid synthesis strategy for pharmaceutical synthesis and further research on novel coumarin bioamide derivatives.

1. Introduction

Coumarin (2H-1-benzopyran-2-one), made up of fused benzene and α-pyrone rings, was first isolated in 1820, from Tonka bean (Dipteryxodorata) of the Fabaceae family. Coumarins are secondary metabolites found in a wide variety of plant species and most prevalent in the Apiaceae, Rutaceae, Asteraceae, and Fabaceae families [1,2]. They have attracted research attention, due to their remarkable biological and pharmacological activities, which include antibacterial, antifungal, anticoagulant, antioxidant, anticancer, and anti-inflammatory properties [3,4,5,6,7,8,9,10]. Synthesis of coumarins and their derivatives has attracted considerable attention in research and development among both organic and medicinal chemists.

Exploration of new heterocycles that can accommodate potency to multiple biological targets remains an intriguing scientific endeavor. Many hybrid molecules with coumarin-based ring systems have been synthesized utilizing novel synthetic methodologies. Some coumarin derivatives conjugated with nitrogen-containing heterocyclic moieties, such as triazole and pyridine, were synthesized and proved to possess antibacterial bioactivity [11,12]. It is noteworthy that coumarin scaffolds can bind to the peripheral anion site (PAS) of acetylcholine esterase as an acetylcholinesterase (AChE) inhibitor [13]. In recent years, acetylcholinesterase inhibitors based on the coumarin backbone have been frequently reported [14,15,16,17,18]. At the same time, an indole amine framework in the design of new AChE inhibitors or multi-target agents for Alzheimer’s disease (AD) therapy was developed. Coumarin bioamide complex drugs synthesized from coumarin and bioamines have been frequently reported, and these drugs have anti-cholinesterase, antioxidant and antitumor activities [19,20,21].

Coumarin bioamide derivatives are mainly based on coumarin main chains, which are connected by amide bonds. Coumarin bioamine derivatives have received much attention in the field of organic synthesis and drug research because of their amazing bioactivity. Biogenic amines (BAs) play a pivotal role in regulating fundamental physiological processes, functioning as pleiotropic regulatory molecules. Their indispensable contributions span the following: (1) cellular dynamics: modulating growth, proliferation, and homeostatic balance; (2) biomolecular architecture: coordinating the integration and stabilization of protein-lipid complexes; (3) neurodevelopmental trajectories: directing structural and functional maturation of the central nervous system; (4) genomic integrity: attenuating DNA damage through nucleotide cycle regulation and transcriptional fidelity; and (5) epigenetic governance: fine-tuning gene expression networks to maintain cellular identity [22,23,24]. Tyramine and tryptamine derivatives are a very active class of bioamines, which is related to the treatment of many diseases, including migraine, schizophrenia, and elevated blood pressure [25]. Ghanei-Nasab et al. designed and synthesized a series of coumarin tryptamine derivatives (1, Figure 1) and tested them for their pharmacological activity and found them to be strong inhibitors of acetylcholinesterase [19]. Coumarin tyramine derivatives (2a, Figure 1) have been widely researched for their strong antioxidant activities [20]. Coumarin phenylethylamine derivatives (2b, Figure 1) have been shown to have antitumor activity [21].

In this paper, we demonstrated for the first time a two-step tandem enzymatic synthesis of coumarin bioamide derivatives through sustainable continuous-flow technology. The key to coumarin bioamide derivatives’ synthesis is the construction of amide bonds. Traditional methods mainly activate acid components through toxic or harmful reagents, or require the use of stoichiometric coupling agents, resulting in poor atomic economics [26]. Reagents used to form amide bonds, such as EDC, DCC and BOP, must be added in stoichiometric amounts to form an acyl chloride to produce an activated acid that may be attacked by an amine nucleophile. The high costs and expenses incurred using these common amide bond formation processes have led to calls for more efficient methods [27,28]. In 2007, the American Chemical Society’s Green Chemistry Institute Drug Roundtable (ACS GCIPR) voted that avoiding the use of atomically and economically poor reagents to construct amide bonds was the biggest challenge facing organic chemistry [29]. In all cases, the enzyme is the key ingredient that enables the chemistry and helps to make it green, and nature has refined enzymes into remarkably efficient catalysts capable of enhancing reaction rates by factors as high as 10 in comparison to uncatalyzed reactions [30,31]. Biocatalysis, which uses enzymes to construct amide bonds, provides a powerful method for conducting coupling reactions between organic compounds. Compared with chemical catalysis, biocatalysis has more powerful advantages, such as mild reaction conditions, high stereoselectivity and high catalytic efficiency [32]. The following are some reports about the application of enzymatic methods in biocatalysis. The microbial Candida Antarctic lipase B (CAL-B), as a member of the serine hydrolase family, is the most commonly used lipase in amidation reactions [33]. Penicillin G acylase (PGA) can catalyze various activated acyl donors to conjugate with the corresponding nuclei to synthesize β-lactam antibiotics [34]. The enzyme reaction conditions are mild, and the reaction time required for optimal yield can be up to several days. Enzyme immobilization (EI) refers to the process of confining soluble enzymes within defined spatial matrices to generate insoluble, reusable forms that maintain high catalytic activity and enhanced stability under harsh environmental conditions. This technique simplifies downstream processing by facilitating efficient separation of enzymes from reaction mixtures. The immobilization of enzymes enables their utilization across multiple industries for producing diverse products in sectors including food manufacturing, fine chemical synthesis, cosmetics development, and pharmaceutical production. Notably, immobilized lipases have emerged as one of the most critical biocatalytic systems for industrial applications, particularly within the fuel, pharmaceutical, cosmetic, and food processing industries [35,36,37,38,39]. In order to explore the synthesis reaction of new coumarin derivatives containing bioamide skeleton catalyzed by enzyme and improve the efficiency of traditional enzyme-catalyzed reactions, it is important to combine biocatalysis technology with continuous-flow technology. In recent decades, continuous-flow technology has shown amazing applications in the production of fine chemicals and pharmaceuticals. In 2007, a roundtable co-organized by the American Chemical Society (ACS) listed several key areas for research to promote sustainable manufacturing and recognized the importance of continuous processing. The fundamental advantages of continuous-flow technology over conventional batch reactors are precise temperature and residence time control, enhanced heat and mass transfer, and the handling of hazardous reagents in a safe manner [40,41,42,43,44]. In this paper, a two-step tandem enzymatic synthesis of coumarin derivatives containing bioamide scaffolds was studied by means of a sustainable continuous-flow technique. The purpose was to develop a green and efficient method for continuous-flow synthesis of coumarin derivatives containing bioamide scaffolds using Lipozyme^® TL-IM produced by Novozymes (Beijing, China) and fixed on porous polymer beads. The parameters of the solvent, including substrate ratio, reaction temperature, enzyme reutilization rate, reaction flow rate, residence time and spatiotemporal yield, were investigated. The synthesis of coumarin bioamide derivatives 5a–5t were outlined in Scheme 1. The first step is the synthesis of coumarin-3-carboxylic acid methyl esters from salicylaldehyde derivatives 1a–1e with dimethyl malonate 2 in a continuous-flow microreactor. In the second step, coumarin-3-carboxylic acid methyl esters derivatives reacted directly with various bioamines 4a–4d under the catalysis of lipase TL IM, and twenty coumarin bioamide derivatives 5a–5t were obtained. This method provides a green, efficient way for the rapid synthesis of coumarin bioamide derivatives and the further development of highly active new drugs.

2. Results

2.1. Synthesis of Intermediate Coumarin Carboxylic Acid Methyl Ester Derivatives

We studied the reaction of coumarin carboxylic acid methyl ester derivatives co-catalyzed by lipase TL IM and potassium carbonate in a continuous-flow microreactor. By investigating the synthesis of coumarin carboxylic acid ester methyl derivative intermediates, we found that in a mixed catalyst (25 mg K₂CO₃/120 mg lipase TL IM), the highest yield was achieved after 10 min of reaction at 40 °C (

Table 1. Reaction results of intermediate coumarin-3-methyl carboxylate in a continuous-flow reactor ^a.

Entry	R1	R2	R3	Product	Yield b (%)
1	H	H	H	3a	86.5 ± 1.7
2	CH3	H	H	3b	95.5 ± 1.5
3	Cl	H	H	3c	82.6 ± 0.8
4	H	CH3	H	3d	93.1 ± 1.3
5	H	H	CH3	3e	89.3 ± 0.5

^a General experimental conditions: in the continuous-flow reactors, 5 mmol of salicylaldehyde derivatives (salicylaldehyde, 5-chlorosalicylaldehyde, 5-methylsalicylaldehyde, 3-methylsalicylaldehyde, 4-methylsalicylaldehyde) was dissolved in 10 mL DMSO solution (feed A), and 10 mmol of diester malonate derivatives in 10 mL DMSO solution (feed B). A mixture of catalysts (25 mg K₂CO₃ and 120 mg lipase TL IM) was filled in the PFA tube (ID = 2.0 mm, length = 100 cm). Materials A and B were mixed at a flow rate of 17.8 µL min⁻¹ in a y-type mixer at 40 °C, and the resulting liquid was connected to a sample bottle for collection of the final mixture. The reaction temperature was controlled by a water bath thermostat. ^b Isolated yield. Yield: 100 × (actually obtained amount/calculated amount). Data are presented as mean ± SD of three experiments.

); the STY was 4.06 for 210.4 g h⁻¹ L⁻¹ biocatalyst.

2.2. Effect of Reaction Solvent and Catalyst

Generally, the catalytic performance of the catalyst is affected by the reaction solvent. In this paper, all lipases utilized in catalytic reactions consist of immobilized enzymes manufactured by Novozymes. These biocatalysts are engineered through an immobilization process involving covalent binding to non-compressible silica-based matrices, which confers exceptional catalytic activity and operational stability under harsh environmental conditions while retaining enzymatic functionality in organic solvent systems. In this research, we used coumarin carboxylic acid methyl ester (3a) and phenethylamine (4a) as templates to synthesize coumarin phenylacetamide (5a) in a continuous-flow microreactor, and the results of a blank controlled experiment showed that the reaction did not proceed in the absence of the enzyme. We tested the reaction using different lipases (TL IM, Novozym^®435, Beijing, China) in different solvents (methanol, tert-amyl alcohol, DMSO, isopropyl alcohol, acetonitrile, acetone), and the results are shown in

Table 2. The effect of solvent on enzymatic synthesis of coumarin bioamide derivatives in a continuous-flow microreactor ^a.

Entry	Solvent	Catalysts	Log p	Yield b(%)
1	Methanol	None	−0.76	n.d.
2	Methanol	Lipozyme TL IM	−0.76	77.8 ± 1.1
3	Methanol	Novozym® 435	−0.76	55.2 ± 0.3
4	DMSO	Lipozyme TL IM	−1.3	n.d.
5	DMSO	Novozym® 435	−1.3	n.d.
6	Acetonitrile	Lipozyme TL IM	−0.33	42.5 ± 0.8
7	Acetonitrile	Novozym® 435	−0.33	n.d.
8	tert-amyl alcohol	Lipozyme TL IM	0.60	63.3 ± 1.5
9	tert-amyl alcohol	Novozym® 435	0.60	40.7 ± 0.6
10	Isopropanol	Lipozyme TL IM	0.39	32.3 ± 0.5
11	Isopropanol	Novozym® 435	0.39	n.d.
12	acetone	Lipozyme TL IM	−0.23	36.5 ± 0.7
13	acetone	Novozym® 435	−0.23	n.d.

^a General experimental conditions: in the continuous-flow reactors, feed 1, 5 mmol of coumarin-3-carboxylate methyl ester (3a) was dissolved in 10 mL solvent; feed 2, 10 mmol phenylethylamine (4a) was added to 10 mL of solvent at 50 °C, flow rate 17.8 µL min⁻¹, residence time 35 min, enzyme 870 mg. ^b Isolated yield. Yield: 100 × (actually obtained amount/ideal calculated amount). Using compound 5a as a representative case, the reaction was charged with 5 mmol of substrate 3a. The theoretical yield of 5a (molecular weight = 293.32 g mol⁻¹) was calculated as 1.467 g (5 mmol × 293.32 g mol⁻¹). However, due to competing side reactions and inherent mass transfer inefficiencies during purification, the isolated yield of 5a via silica gel column chromatography was reduced to 1.141 g. Yield (%) = 100 × (1.141/1.467) = 77.8%. The data are presented as average ± SD of triplicate experiments. n.d. means no reaction was found.

. As we can see from Table 2, methanol and TL IM lipases were the best reaction solvents and catalysts for the synthesis of coumarin bioamide derivatives. Therefore, methanol was selected as the best solvent, and TL IM enzyme was the best catalyst. Lipozyme^® TL IM demonstrates enhanced structural stability attributed to its compact lid architecture, which minimizes interference from external molecular interactions. During catalysis, substrate binding to the lipase’s active site facilitates optimal alignment for transition state stabilization, effectively lowering the activation energy barrier and thereby enhancing catalytic efficiency.

2.3. Effect of Reaction Parameters on Synthesis of Coumarin Bioamide Derivatives

For biocatalytic reactions, the substrate molar ratio has an important effect on the rate of catalysis and the final yield of the product. In this study, we conducted experiments on the molar ratio of six substrates (Figure 2). It was found that with the increase in phenylethylamine (4a), the yield of the product increased correspondingly. When the molar ratio of coumarin-3-methyl carboxylate (3a) and phenylethylamine (4a) was 1:2, the yield was the highest. Therefore, coumarin-3-carboxylate methyl ester (3a): phenethylamine (4a) = 1:2 was selected as the best substrate molar ratio.

The rate of chemical reaction increases with the increase in temperature, but it is different for biocatalysis. The essence of enzyme is a protein, so the activity is affected by temperature, and the reaction rate of enzyme-catalyzed reaction increases with the increase in temperature within a certain range. When the optimal temperature is reached, the reaction rate will gradually decrease with the increase in temperature. In methanol solvent, the reaction temperature was adjusted from 35 °C to 60 °C. It can be seen from Figure 3 that the optimal temperature was 50 °C. When the temperature exceeded 50 °C, the yield decreased slightly, so 50 °C was chosen as the optimal reaction temperature.

In the continuous-flow microreactor, the residence time is crucial to the complete reaction. If the residence time is too short, the contact between substrate and catalyst will be affected, resulting in incomplete reaction. Prolonged residence time may lead to side reactions and unnecessary by-products. By increasing the residence time from 20 min to 50 min successively, the effect of residence time on the production of coumarin bioamide derivatives was studied. The results are shown in Figure 4. It can be seen from Figure 4 that when the residence time is 35 min, the yield reaches the maximum, and the yield decreases slightly as the residence time continues to increase. It may be that the generation of by-products affects the product yield, so 35 min is the best residence time.

Capitalizing on the cost-efficiency advantages inherent to immobilized enzyme systems, we systematically investigated the operational reusability of lipase TL IM under optimized reaction conditions. Through eight consecutive catalytic cycles, the immobilized enzyme retained robust activity, sustaining a bioamide coumarin derivative yield exceeding 52% even at the eighth iteration (Figure 5). This abbreviated reaction duration, coupled with the enzyme’s demonstrated recyclability, suggests substantial potential for enhancing the synthetic throughput of coumarin bioamide derivatives while maintaining catalytic performance. The preserved enzymatic efficiency across multiple cycles underscores the practical viability of this immobilized system for sustainable biocatalytic processes.

In order to compare the effects of a traditional shaker reactor and continuous-flow microreactor on enzymatic reaction, experiments were carried out on two kinds of reactors. As can be seen from

Table 3. Bioamide derivatives of coumarin were synthesized enzymically by continuous-flow microreactor and shaker reactor ^a.

Entry	Method	STY (g L−1 h−1)	Yield b (%)
1	A	195.8	77.8 ± 0.7
2	B	0.84	61.3 ± 1.1

^a General experimental conditions. Method A: in the continuous-flow reactor, feed 1, 5 mmol coumarin-3-carboxylate methyl ester (3a) was dissolved in 10 mL methanol; feed 2, 10 mmol phenylethylamine (4a) was dissolved in 10 mL methanol, flow rate 17.8 μL min⁻¹, residence time 35 min, enzyme 870 mg, 50 °C. Method B: in the shaker reactor, 5 mmol coumarin-3-carboxylate methyl ester (3a), 10 mmol phenylethylamine (4a) and 20 mL methanol were added to a 50 mL Erlenmeyer flask, enzyme 870 mg, 160 rpm, 50 °C, 24 h. ^b Isolated yield. Yield: 100 × (actually obtained amount/calculated amount). The data are presented as average ± SD of triplicate experiments.

, it takes only 35 min to achieve the optimal yield in continuous-flow microreactors (method A), while it takes more than 24 h reaction time to achieve the optimal yield in a shaking bed reactor (Method B). Spatiotemporal yield (STY) is a common constant used to evaluate the yield of different reaction systems normalized to 1 L volume, describing the amount of production formed at a given flow rate and reaction volume. The STY of continuous-flow microreactor is much higher than that of traditional shaker reactor, so the efficiency of synthesis of coumarin bioamide derivatives can be greatly improved by the continuous-flow microreactor (

Table 4. Effect of substrate structure on enzymatic synthesis of coumarin bioamide derivatives under continuous-flow conditions ^a.

Entry	R1	R2	R3	R4	Product	Yield b (%)
1	H	H	H		5a	77.8 ± 0.7
2	H	H	H		5b	72.1 ± 1.1
3	H	H	H		5c	77.9 ± 0.9
4	H	H	H		5d	68.1 ± 0.4
5	CH3	H	H		5e	84.5 ± 1.3
6	CH3	H	H		5f	87.1 ± 0.5
7	CH3	H	H		5g	82.8 ± 0.7
8	CH3	H	H		5h	79.3 ± 0.3
9	Cl	H	H		5i	65.4 ± 0.8
10	Cl	H	H		5j	62.7 ± 0.1
11	Cl	H	H		5k	<5
12	Cl	H	H		5l	63.6 ± 1.8
13	H	CH3	H		5m	83.2 ± 0.6
14	H	CH3	H		5n	85.7 ± 1.2
15	H	CH3	H		5o	79.9 ± 0.4
16	H	CH3	H		5p	80.2 ± 0.2
17	H	H	CH3		5q	80.3 ± 1.1
18	H	H	CH3		5r	74.1 ± 0.8
19	H	H	CH3		5s	70.8 ± 0.5
20	H	H	CH3		5t	75.2 ± 1.6

^a General experimental conditions: in the continuous-flow reactor, feed 1, 5 mmol coumarin-3-carboxylate methyl derivatives was dissolved in 10 mL methanol; feed 2, 10 mmol biogenic amine compounds was dissolved in 10 mL methanol, flow rate 17.8 μL min⁻¹, residence time 35 min, enzyme 870 mg, 50 °C. ^b Isolated yield. Yield: 100 × (actually obtained amount/calculated amount). The data are presented as average ± SD of triplicate experiments.

). In the following equation, m_p is the mass of the generated product (g), T is the residence time (h), and V_R is the reactor volume (L).

$$\mathrm{STY}={\displaystyle \frac{{\mathrm{m}}_{\mathrm{p}}}{\mathrm{T}\times {\mathrm{V}}_{\mathrm{R}}}}$$

3. Materials and Methods

3.1. Materials

Unless otherwise specified, all compounds are purchased from commercial sources. Lipozyme^® TL IM and Novozym^® 435 was purchased from Novo Nordisk (Copenhagen, Denmark). Salicylaldehyde, 3-methylsalicylaldehyde, 5-chloro-salicylaldehyde, 5-methylsalicylaldehyde, phenylethylamine, tyramine, tryptamine are all purchased from Maclean (Shanghai, China). 4-methylsalicylaldehyde was purchased from Sinopharm (Shanghai, China). 5-methoxytryptamine from ShanghaiShaoyuan Co., Ltd. (Shanghai, China). Dimethyl-malonate was purchased from Meryer (Shanghai, China). Harvard Instrument PHD 2000 syringe pump was purchased from Harvard University (Holliston, MA, USA).

3.2. Experimental Setup and Experiment Conditions

The equipment diagram for synthesizing coumarin bioamide derivatives in a continuous-flow microreactor is shown in Figure 6. The experimental setup consists of two substrate syringes, an injection pump (Harvard Apparatus Dr 2000, Shanghai, China), a Y-mixer, a flow reactor with a 100 cm × 2 mm PFA tube, and a product collector. The silicone tube was filled with 870 mg Lipozyme^® TL IM (the lower limit of reactivity was 9000 PLU g⁻¹) and placed in a constant temperature water bath at 50 °C. An amount of 5 mmol coumarin-3-carboxylate methyl ester derivatives was dissolved in 10 mL methanol (feed 1) and 10 mmol amine in 10 mL methanol (feed 2). Feed 1 and 2 were delivered to the Y-mixer at a flow rate of 17.8 μL min⁻¹ with a residence time of 35 min. The resulting liquid was connected to the product collector to collect the final mixture. The main products were separated by silica gel chromatography and confirmed by ¹H NMR and ¹³C NMR.

3.3. Analytical Methods

The product obtained by column chromatography separation and purification was subjected to ¹H NMR and ¹³C NMR.

2-oxo-N-phenethyl-2H-chromene-3-carboxamide (5a). Yellow crystal. ¹H NMR (400 MHz, Chloroform-d) δ 8.25 (s, 1H), 7.38 (t, J = 7.4 Hz, 2H), 7.33 (s, 1H), 7.28–7.22 (m, 2H), 7.20 (d, J = 8.1 Hz 2H), 7.10 (s, 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.88 (t, J = 7.4 Hz, 1H), 3.88 (q, J = 7.1 Hz, 2H), 3.04 (t, J = 7.1 Hz, 2H). ¹³C NMR (101 MHz, Chloroform-d) δ 165.10, 161.18, 139.28, 137.04, 133.77, 132.16, 131.22, 128.94, 128.52, 126.40, 118.76, 118.51, 117.65, 117.01, 61.10, 37.44.

N-(4-hydroxyphenethyl)-2-oxo-2H-chromene-3-carboxamide (5b). Yellow oily liquid. ¹H NMR (400 MHz, Chloroform-d) δ 8.17 (s, 1H), 7.27 (d, J = 7.3 Hz, 1H), 7.16 (d, J = 7.7 Hz, 1H), 7.04 (d, J = 7.8 Hz, 2H), 6.94 (d, J = 8.4 Hz, 1H), 6.88 (s, 1H), 6.82 (t, J = 7.5 Hz, 2H), 6.78 (s, 1H), 6.76 (s, 1H), 3.80–3.75 (m, 2H), 2.91 (t, J = 7.0 Hz, 2H). ¹³C NMR (101 MHz, Chloroform-d) δ 165.03, 161.77, 154.94,134.59, 132.33, 131.28, 130.51, 129.94, 118.59, 118.36, 117.20, 116.85, 115.52, 60.98, 40.66, 36.49.

N-(2-(1H-indol-3-yl)ethyl)-2-oxo-2H-chromene-3-carboxamide (5c). Yellow oily liquid. ¹H NMR (400 MHz, Chloroform-d) δ 8.13 (s, 1H), 8.00 (d, J = 2.8 Hz, 1H), 7.62 (s, 1H), 7.60 (s, 1H), 7.35–7.3 (m, 1H), 7.28 (s, 1H), 7.20 (s, 1H), 7.12 (dd, J = 7.8, 2.3 Hz, 1H), 7.10 (s, 1H), 7.02 (q, J = 7.6 Hz, 1H), 6.93 (s, 1H), 6.81 (s, 1H), 3.87–3.82 (m, 2H), 3.13 (t, J = 7.2 Hz, 2H). ¹³C NMR (101 MHz, Chloroform-d) δ 164.99, 161.62, 136.34, 132.23, 131.26, 127.27, 122.45, 122.04, 119.36, 118.75, 118.66, 118.41, 117.14, 113.18, 111.33, 59.51, 26.90.

N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-2-oxo-2H-chromene-3-carboxamide (5d). Yellow oily liquid. ¹H NMR (400 MHz, Chloroform-d) δ 8.20 (t, J = 1.3 Hz, 1H), 7.93 (s, 1H), 7.36–7.28 (m, 2H), 7.22 (s, 1H), 7.17 (dd, J = 7.7, 1.7 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H), 7.04–6.95 (m, 2H), 6.92–6.81 (m, 2H), 3.94 (q, J = 8.7 Hz, 2H) 3.88 (s, 3H), 3.16 (t, J = 6.8 Hz, 2H). ¹³C NMR (101 MHz, Chloroform-d) δ 164.98, 161.47, 154.01, 132.15, 131.45, 131.25, 127.69, 123.25, 118.77, 117.15, 113.18, 112.25, 111.92, 100.52, 59.88, 55.95, 26.91.

6-methyl-2-oxo-N-phenethyl-2H-chromene-3-carboxamide (5e) [45]. Yellow crystal. ¹H NMR (400 MHz, Chloroform-d) δ 8.18 (s, 1H), 7.38–7.31 (m, 1H), 7.32–7.27 (m, 2H), 7.25–7.22 (m, 2H), 7.20–7.15 (m, 1H), 7.11 (dd, J = 8.4, 2.3 Hz, 1H), 6.97 (d, J = 2.2 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 3.88–3.80 (m, 2H), 3.01 (t, J = 7.1 Hz, 2H), 2.28 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 165.07, 158.87, 139.35, 138.08, 133.44, 132.95, 131.27, 128.94, 128.51, 127.54, 126.37, 118.42, 117.42, 116.73, 61.19, 37.47, 20.36.

6-methyl-2-oxo-N-(4-hydroxyphenethyl)-2H-chromene-3-carboxamide (5f). Yellow solid. ¹H NMR (400 MHz, Chloroform-d) δ 8.10 (s, 1H), 7.17 (m, 1H) 7.10 (dd, J = 8.4, 2.3 Hz, 1H), 7.04 (d, J = 8.0 Hz, 2H), 6.97–6.92 (m, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.76 (d, J = 7.9 Hz, 2H), 6.71 (d, J = 8.1 Hz, 1H), 3.77 (t, J = 7.0 Hz, 2H), 2.90 (t, J = 6.9 Hz, 2H), 2.25 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 165.05, 160.37, 154.60, 133.71, 133.51 131.32, 130.75, 130.01, 127.30, 117.93, 117.55 117.44, 117.32, 115.52, 60.42, 36.41, 20.34.

6-methyl-2-oxo-N-(2-(1H-indol-3-yl)ethyl)-2H-chromene-3-carboxamide (5g). Yellow oily liquid. ¹H NMR (400 MHz, Chloroform-d) δ 8.13 (d, J = 1.4 Hz, 1H), 8.08 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.22–7.18 (s, 1H), 7.16–7.12 (m, 1H), 7.08 (s, 1H), 7.03–7.01 (d, J = 7.84 Hz 1H), 6.99 (d, J = 2.2 Hz, 1H), 6.94 (d, J = 2.2 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 3.90 (t, J = 6.9 Hz, 2H), 3.16 (t, J = 6.9 Hz, 2H), 2.26 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 164.93, 159.09, 136.32, 132.92, 131.23, 129.20, 127.71, 127.46, 127.28, 122.39, 121.99, 119.31, 118.64, 118.43, 116.76, 113.27, 111.26, 59.69, 40.96, 26.91, 20.34.

N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-6-methyl-2-oxo-2H-chromene-3-carboxamide (5h). Yellow oily liquid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.68–10.63 (m, 1H), 8.40 (s, 1H), 7.22 (d, J = 8.7 Hz, 1H), 7.15 (d, J = 2.2 Hz, 1H), 7.13 (s, 1H), 7.13–7.10 (m, 1H), 7.09 (s, 1H), 7.04 (d, J = 2.4 Hz, 1H), 6.77 (d, J = 8.3 Hz, 1H), 6.70 (dd, J = 8.7, 2.4 Hz, 1H), 3.91–3.83 (m, 2H), 3.74 (s, 3H), 3.03 (t, J = 6.9 Hz, 2H), 2.22 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.04, 158.99, 153.45, 133.88, 133.28, 131.83, 131.78, 129.32, 127.93, 127.33, 124.07, 118.75, 116.71, 115.80, 112.45, 112.03, 111.63, 100.58, 59.65, 55.71, 27.07, 20.37.

6-chloro-2-oxo-N-phenethyl-2H-chromene-3-carboxamide (5i). Yellow crystal. ¹H NMR (400 MHz, Chloroform-d) δ 13.46 (s, 1H), 8.15 (d, J = 5.8 Hz, 1H), 7.33 (t, J = 7.3 Hz, 2H), 7.28 (d, J = 4.8 Hz, 1H), 7.25 (d, J = 4.2 Hz, 2H), 7.22 (s, 1H), 7.17 (d, J = 2.5 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 3.88 (t, J = 7.0 Hz, 2H), 3.04 (t, J = 7.0 Hz, 2H). ¹³C NMR (101 MHz, Chloroform-d) δ 163.96, 159.81, 139.00, 132.01, 130.33, 128.91, 128.77, 128.57, 126.51, 123.04, 119.45, 118.60, 61.02, 37.27.

6-chloro-N-(4-hydroxyphenethyl)-2-oxo-2H-chromene-3-carboxamide (5j). Yellow oily liquid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.20 (s, 1H), 8.43 (d, J = 5.6 Hz, 1H), 7.47 (d, J = 2.7 Hz, 1H), 7.32 (dd, J = 8.9, 2.7 Hz, 1H), 7.05–7.00 (m, 2H), 6.98 (s, 1H), 6.88 (d, J = 8.8 Hz, 1H), 6.71–6.66 (m, 2H), 3.82–3.74 (m, 2H), 2.83 (t, J = 7.0 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.18, 160.48, 156.13, 132.38, 130.91, 130.61, 130.13, 129.65, 128.81, 122.00, 119.93, 119.14, 118.33, 115.58, 60.26, 36.14.

6-chloro-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-2-oxo-2H-chromene-3-carboxamide (5l). Yellow oily liquid. ¹H NMR (400 MHz, Chloroform-d) δ 8.47 (s, 1H), 7.95 (s, 1H), 7.20 (d, J = 3.2 Hz, 1H), 7.18 (d, J = 3.2 Hz, 1H), 7.02 (dd, J = 13.6, 2.6 Hz, 2H), 6.91 (s, 1H), 6.86 (d, J = 8.6 Hz, 1H), 6.85 (s, 1H), 6.83 (d, J = 2.4 Hz, 1H), 3.85 (s, 3H), 3.11–3.08 (m, 2H), 2.55 (t, J = 6.8 Hz, 2H). ¹³C NMR (101 MHz, Chloroform-d) δ 163.92, 160.44, 153.89, 153.39, 132.07, 131.57, 130.33, 128.54, 127.61, 123.42, 122.74, 119.36, 118.72, 112.49, 112.19, 111.98, 100.49, 59.40, 55.92, 40.80, 26.76.

7-methyl-2-oxo-N-phenethyl-2H-chromene-3-carboxamide (5m). Yellow crystal. ¹H NMR (400 MHz, DMSO-d₆) δ 8.41 (s, 1H), 7.30 (d, J = 7.4 Hz, 1H), 7.28 (s, 1H), 7.27 (s, 1H), 7.26 (t, J = 2.4 Hz, 2H), 7.23 (d, J = 4.5 Hz, 1H), 7.22–7.17 (m, 1H), 6.71–6.65 (m, 2H), 3.81 (td, J = 7.1, 1.1 Hz, 2H), 2.94 (t, J = 7.1 Hz, 2H), 2.27 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.08, 161.34, 143.09, 139.90, 131.86, 130.65, 129.28, 128.76, 126.57, 125.22, 121.18, 119.90, 117.27, 116.65, 60.03, 37.18, 21.78.

N-(4-hydroxyphenethyl)-7-methyl-2-oxo-2H-chromene-3-carboxamide (5n). Yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.19 (s, 1H), 8.38 (s, 1H), 7.23 (d, J = 8.2 Hz, 1H), 7.05–6.98 (m, 1H), 6.94 (s, 1H), 6.89 (d, J = 5.3 Hz, 2H), 6.67 (d, J = 6.4 Hz, 2H), 6.65 (t, J = 7.6 Hz, 1H), 3.74 (t, J = 7.0 Hz, 2H), 2.83–2.76 (m, 2H), 2.26 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.48, 161.04, 155.63, 142.61, 131.41, 130.25, 129.69, 129.40, 120.15, 119.38, 116.85, 116.78, 116.18, 115.09, 59.92, 35.95, 21.34.

N-(2-(1H-indol-3-yl)ethyl)-7-methyl-2-oxo-2H-chromene-3-carboxamide (5o). Yellow solid. ¹H NMR (400 MHz, Chloroform-d) δ 8.15 (s, 1H), 8.06 (s, 1H), 7.67 (d, J = 7.7 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.31–7.26 (m, 1H), 7.17 (t, J = 5.4 Hz, 1H), 7.15–7.11 (m, 1H), 7.06 (d, J = 7.8 Hz, 1H), 7.01 (d, J = 2.3 Hz, 1H), 6.82 (t, J = 7.5 Hz, 1H), 6.68 (dd, J = 7.8, 1.6 Hz, 1H), 3.96–3.84 (m, 2H), 3.18 (t, J = 6.9 Hz, 2H), 2.36 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 164.63, 161.81, 143.15, 136.33, 131.06, 127.29, 123.99, 122.42, 122.01, 121.20, 119.51, 119.33, 118.66, 117.77, 117.55, 116.38, 113.27, 111.28, 59.34, 26.94, 21.83.

N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-7-methyl-2-oxo-2H-chromene-3-carboxamide (5p). Yellow oily liquid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.68 (d, J = 2.6 Hz, 1H), 8.38 (s, 1H), 7.23 (d, J = 8.8 Hz, 1H), 7.21 (d, J = 7.7 Hz, 1H), 7.10 (d, J = 2.4 Hz, 1H), 7.05 (d, J = 2.5 Hz, 1H), 6.75–6.70 (m, 1H), 6.70 (s, 1H), 6.67 (d, J = 8.1 Hz, 1H), 6.64 (d, J = 1.6 Hz, 1H), 3.84 (t, J = 7.0 Hz, 2H), 3.74 (s, 3H), 3.03 (t, J = 6.9 Hz, 2H), 2.26 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.37, 161.35, 153.04, 142.63, 131.42, 131.37, 130.03, 127.51,126.49, 123.63, 119.30, 116.94,116.81, 116.22, 112.04, 111.61, 111.22, 100.15, 58.84, 55.27, 26.66, 21.35.

8-methyl-2-oxo-N-phenethyl-2H-chromene-3-carboxamide (5q). Yellow oily liquid. ¹H NMR (600 MHz, DMSO-d₆) δ 8.45 (d, J = 1.5 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.29 (s, 2H), 7.28–7.24 (m, 2H), 7.21 (t, J = 1.4 Hz, 1H), 7.20 (s, 1H), 7.19 (s, 1H), 6.80–6.75 (m, 1H), 3.87–3.83 (m, 2H), 2.96 (t, J = 7.1 Hz, 2H), 2.16 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 167.46, 166.70, 159.53, 139.86, 133.49, 132.58, 129.74, 129.25, 128.78, 128.02, 126.60, 125.35, 118.37, 118.14, 59.97, 37.12, 15.69.

N-(4-hydroxyphenethyl)-8-methyl-2-oxo-2H-chromene-3-carboxamide (5r). Yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.19 (s, 1H), 8.44 (s, 1H), 7.23–7.17 (m, 2H), 7.05 (d, J = 2.0 Hz, 1H), 7.03 (d, J = 2.1 Hz, 1H), 6.80–6.73 (m, 1H), 6.73–6.66 (m, 2H), 6.58 (s, 1H), 3.82–3.74 (m, 2H), 2.84 (t, J = 7.1 Hz, 2H), 2.16 (s, 3H). ¹3C NMR (101 MHz, DMSO-d₆) δ 166.55, 159.63, 156.08, 133.45, 133.05, 130.11, 129.82, 129.72, 125.36, 124.11, 118.31, 118.13, 117.35, 115.56, 60.35, 36.35, 15.72.

N-(2-(1H-indol-3-yl)ethyl)-8-methyl-2-oxo-2H-chromene-3-carboxamide (5s). Yellow oily liquid. ¹H NMR (400 MHz, Chloroform-d) δ 8.18 (d, J = 1.5 Hz, 1H), 8.08 (s, 1H), 7.69 (dd, J = 11.8, 8.6 Hz, 1H), 7.58 (s, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.28 (dd, J = 7.1, 1.4 Hz, 1H), 7.26–7.24 (m, 1H), 7.24–7.18 (m, 1H), 7.05 (dd, J = 7.8, 1.7 Hz, 1H), 6.98 (d, J = 2.2 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 3.96–3.92 (m, 2H), 3.21 (t, J = 6.9 Hz, 2H), 2.39 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 165.26, 160.09, 136.37, 133.27, 129.03, 127.30, 126.39, 126.10, 125.69, 124.11, 122.51, 122.03, 119.35, 118.68, 117.97, 116.22, 113.13, 111.39, 59.41, 26.97, 15.71.

N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-8-methyl-2-oxo-2H-chromene-3-carboxamide (5t). Yellow oily liquid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.69 (d, J = 2.6 Hz, 1H), 8.41 (s, 1H), 7.36 (s, 1H), 7.24 (d, J = 8.7 Hz, 1H), 7.18 (d, J = 7.8 Hz, 1H), 7.15 (d, J = 1.8 Hz, 1H), 7.12 (d, J = 2.4 Hz, 1H), 7.06 (d, J = 2.4 Hz, 1H), 6.76 (d, J = 7.5 Hz, 1H), 6.73–6.68 (m, 1H), 3.88 (t, J = 6.9 Hz, 2H), 3.74 (s, 3H), 3.05 (t, J = 6.9 Hz, 2H), 2.17 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 165.92, 159.44, 153.04, 132.96, 131.38,131.01, 129.25, 127.54, 127.52, 124.97, 123.63, 117.76, 117.72, 112.05, 111.64, 111.24, 100.13, 58.92, 55.23, 26.64, 15.29.

4. Conclusions

Synthesis of coumarins and their derivatives has attracted considerable attention in research and development among both organic and medicinal chemists. In summary, we demonstrated for the first time a two-step tandem enzymatic synthesis of coumarin bioamide derivatives through sustainable continuous-flow technology. The first step is to synthesize coumarin-3-carboxylate methyl ester from salicylaldehyde derivatives (salicylaldehyde, 3-methylsalicylaldehyde, 5-chloro-salicylaldehyde, 4-methylsalicylaldehyde, 5-methylsalicylaldehyde) and dimethyl malonate. In the second step, coumarin-3-methyl carboxylate derivatives reacted directly with a variety of bioamines (phenethylamine, tyramine, tryptamine, 5-methoxytryptamine) under the catalysis of lipase TL IM to obtain 20 coumarin bioamide derivatives through continuous-flow technology. This methodology exhibits notable advantages through its operation under ambient pressure conditions utilizing methanol as the sole solvent in a moderate thermal regime (50 °C), coupled with rapid reaction kinetics (35 min). The protocol was further enhanced by employing a commercially accessible heterogeneous catalyst that demonstrated retained catalytic efficiency over multiple operational cycles. Lipase TL IM was used for the synthesis of coumarin bioamine for the first time, and the use of enzyme as catalyst can greatly reduce the pollution to the environment, avoiding the cumbersome reaction process and follow-up treatment. Furthermore, compared with a batch reactor, the new coumarin derivatives containing bioamide skeleton were synthesized by combining continuous-flow technology and biocatalysis technology, which had good atomic economy and greatly improved reaction efficiency. This method provides a green, efficient, rapid and valuable synthesis method for further screening the drug activity of coumarin bioamide derivatives.