Design, Synthesis and Fungicidal Activity of Ester Derivatives of 4-(3,4-Dichloroisothiazole) 7-Hydroxy Coumarin

Abstract

The development of new fungicides is vital for safeguarding crops and ensuring sustainable agriculture. Building on our previous finding that 4-(3,4-dichloroisothiazole)-7-hydroxy coumarins can be used as fungicidal leads, 44 novel coumarin ester derivatives were designed and synthesized to evaluate whether esterification could enhance their fungicidal activity. In vitro fungicidal bioassays indicated that compound 2ai displayed good activity against Alternaria solani, Botrytis cinereal, Cercospora arachidicola, Physalospora piricola and Sclerotinia sclerotiorum, with an EC₅₀ value ranging from 2.90 to 5.56 μg/mL, comparable to the lead compound 1a, with its EC₅₀ value ranging from 1.92 to 9.37 μg/mL. In vivo bioassays demonstrated that compounds 1a, 2ar and 2bg showed comparable, excellent efficacy against Pseudoperonospora cubensis at a dose of 25 µg/mL. Our research shows that the esterification of 4-(3,4-dichloroisothiazole) 7-hydroxycoumarins results in a fungicidal activity equivalent to that of its lead compounds. Furthermore, our density functional theory (DFT) calculations and 3D-QSAR modeling provide a rational explanation of the structure–activity relationship and offer valuable insights to guide further molecular design.

1. Introduction

Agrochemicals are important strategical materials for crop protection and food safety production [1,2]. However, the unreasonable and unscientific use of some pesticides has also resulted in a series of risks [3,4], including increasing pest resistance [5], toxicity against non-target organisms and potential environmental pollution [6]. Therefore, the development of green and high-activity agricultural chemicals with good environmental behavior is always crucial for plant protection.

Natural products are usually metabolites or secondary metabolites of plants, microorganisms and animals, which have the advantages of structural diversity, biocompatibility, low resistance risk, a unique mode of action and a specific target [7,8,9,10,11]. They are widely used in the development of novel medicines and agrochemicals [12,13]. Coumarins are a highly significant class of natural products with bactericidal [14], anticoagulant [15], anti-inflammatory [16], antiviral [17], anti-HIV [18] and antitumor activity [19,20]; they are considered as phytoalexins for plant defense against pathogen attacks [1,21]. Several coumarin derivatives, such as osthole and coumoxystrobin, have been reported as fungicides [22,23] (Figure 1).

Lead optimization is an effective approach for the development of novel pesticides [24,25]. Among the different methods for novel pesticide development, intermediate derivatization is effective and widely adopted [26,27]. Previously, our group discovered 4-(3,4-dichloroisothiazole) 7-hydroxy coumarins as fungicidal leads by combining the naturally occurring coumarin and a substructure (i.e., 3,4-dichloroisothiazole) of the compounds with systemic acquired resistance. Although compounds 1a and 1b displayed favorable in vitro activity, their in vivo performance was suboptimal [1]. Subsequently, a series of 3,4-dichloroisothiazolocoumarin-containing strobilurins were developed and found to have poor solubility [2]. To continue the exploration of fungicidal leads and improve the physicochemical properties of a weak acidic moiety of 7-hydroxy group on the coumarins, the esterification of 7-hydroxy coumarins was carried out (Figure 2), the in vitro and in vivo fungicidal activity of the target compounds were simultaneously evaluated, and notably, compounds 2ai and 2ar were found to show good activity.

2. Results and Discussion

2.1. Chemical Synthesis

The synthetic route for the target compounds 2aa–2bv is outlined in Scheme 1. The target compounds 2aa–2bv were prepared via esterification using compound 1 with the corresponding acyl chloride. The specific structures of the target compounds are shown in

Table 1. Structures of target compounds 2aa–2bv.

Compd.	R1	R2	Compd.	R1	R2	Compd.	R1	R2	Compd.	R1	R2
2aa	Cl		2al	Cl		2ba	H		2bl	H
2ab	Cl		2am	Cl		2bb	H		2bm	H
2ac	Cl		2an	Cl		2bc	H		2bn	H
2ad	Cl		2ao	Cl		2bd	H		2bo	H
2ae	Cl		2ap	Cl		2be	H		2bp	H
2af	Cl		2aq	Cl		2bf	H		2bq	H
2ag	Cl		2ar	Cl		2bg	H		2br	H
2ah	Cl		2as	Cl		2bh	H		2bs	H
2ai	Cl		2at	Cl		2bi	H		2bt	H
2aj	Cl		2au	Cl		2bj	H		2bu	H
2ak	Cl		2av	Cl		2bk	H		2bv	H

.

2.2. X-ray Diffraction of Compound 2be

The crystal structure cell stacking diagram of 2be, shown in Figure 3, was identified via X-ray diffraction analysis for structure validation. The crystal belongs to the monoclinic system with the space group P2_1/c, and each unit cell contains four molecules. The unit cell parameters are a = 16.9633(6) Å, b = 7.2717(3) Å, c = 14.8314(6) Å; α = 90, β = 90.164(4), γ = 90; and Z = 4, V = 1829.48(12) ų. In addition, the torsional angles (all near 180° or 0°) and the measured dihedral angles between the benzene and pyranone rings indicate that the entire coumarin ring was coplanar. Each molecule has a coumarin ring and an isothiazole moiety with a measured dihedral angle of 55.1° between these two moieties. There are no hydrogen bonds to be found in the crystal structure. The additional data for 2be were alternatively obtained from the Cambridge Crystallographic Data Center (www.ccdc.cam.ac.uk, accessed on 1 June 2023), denoted as CCDC 2256560.

2.3. In Vitro Fungicidal Activity

The in vitro fungicidal activity of target compounds 2aa–2bv against seven representative plant pathogens at 50 μg/mL are shown in

Table 2. In vitro fungicidal activity of target compounds 2 against the test phytopathogens.

Compd.	A. s	B. c	C. a	F. g	P. p	R. s	S. s	Compd.	A. s	B. c	C. a	F. g	P. p	R. s	S. s
2aa	4 ± 2	8 ± 1	0	0	39 ± 3	0	7 ± 2	2bc	7 ± 1	0	14 ± 3	0	9 ± 2	0	27 ± 2
2ab	12 ± 2	16 ± 1	4 ± 2	6 ± 2	18 ± 3	0	16 ± 2	2bd	7 ± 1	7 ± 2	13 ± 2	0	23 ± 2	0	11 ± 0
2ac	6 ± 1	4 ± 1	0	0	10 ± 3	0	6 ± 2	2be	4 ± 1	7 ± 3	0	0	10 ± 0	0	12 ± 2
2ad	7 ± 1	5 ± 3	7 ± 0	0	62 ± 3	0	6 ± 2	2bf	7 ± 1	8 ± 2	5 ± 2	0	31 ± 2	0	4 ± 2
2ae	6 ± 1	0	7 ± 3	0	3 ± 0	0	4 ± 2	2bg	63 ± 3	69 ± 2	50 ± 2	23 ± 1	56 ± 0	29 ± 1	63 ± 2
2af	3 ± 1	0	4 ± 2	0	10 ± 0	0	12 ± 1	2bh	42 ± 3	44 ± 2	35 ± 3	19 ± 3	41 ± 3	25 ± 3	45 ± 3
2ag	4 ± 1	0	9 ± 2	0	0	0	11 ± 1	2bi	4 ± 1	0	0	0	14 ± 2	0	5 ± 0
2ah	5 ± 2	4 ± 2	0	0	4 ± 0	0	8 ± 1	2bj	0	16 ± 2	10 ± 3	9 ± 3	23 ± 2	0	10 ± 2
2ai	77 ± 2	76 ± 0	86 ± 2	20 ± 2	69 ± 2	46 ± 3	76 ± 0	2bk	0	15 ± 2	13 ± 2	9 ± 1	24 ± 0	0	14 ± 3
2aj	71 ± 1	68 ± 0	76 ± 2	26 ± 1	78 ± 2	35 ± 3	63 ± 2	2bl	0	0	11 ± 2	0	19 ± 2	0	0
2ak	38 ± 2	30 ± 0	22 ± 2	0	29 ± 2	0	24 ± 2	2bm	26 ± 3	49 ± 2	26 ± 2	18 ± 2	63 ± 2	10 ± 2	52 ± 3
2al	23 ± 2	26 ± 1	17 ± 0	15 ± 2	77 ± 0	24 ± 3	27 ± 3	2bn	0	10 ± 2	9 ± 2	6 ± 2	28 ± 3	0	10 ± 2
2am	0	8 ± 1	11 ± 2	8 ± 2	54 ± 3	0	14 ± 0	2bo	0	5 ± 1	0	0	10 ± 0	0	4 ± 2
2an	0	7 ± 3	13 ± 2	11 ± 3	10 ± 3	0	11 ± 2	2bp	6 ± 2	13 ± 1	14 ± 0	14 ± 0	35 ± 2	0	8 ± 3
2ao	44 ± 2	43 ± 1	48 ± 2	23 ± 2	68 ± 0	0	35 ± 3	2bq	0	0	5 ± 2	0	8 ± 2	0	4 ± 2
2ap	0	0	0	0	24 ± 3	0	0	2br	0	0	0	0	8 ± 2	0	4 ± 2
2aq	0	0	0	0	16 ± 2	0	9 ± 2	2bs	0	4 ± 1	0	0	18 ± 3	0	6 ± 2
2ar	66 ± 1	70 ± 0	77 ± 2	28 ± 2	77 ± 2	76 ± 1	62 ± 1	2bt	8 ± 3	8 ± 1	5 ± 2	0	12 ± 2	0	8 ± 3
2as	25 ± 3	23 ± 1	19 ± 2	0	23 ± 2	0	28 ± 2	2bu	0	10 ± 2	6 ± 0	0	5 ± 0	0	11 ± 0
2at	17 ± 2	17 ± 1	4 ± 2	12 ± 3	23 ± 3	0	29 ± 3	2bv	30 ± 2	17 ± 0	32 ± 3	16 ± 1	53 ± 0	18 ± 3	17 ± 0
2au	17 ± 0	12 ± 1	15 ± 2	10 ± 3	24 ± 0	0	18 ± 2	1a *	70 ± 2	85 ± 2	84 ± 3	77 ± 1	93 ± 2	98 ± 1	78 ± 2
2av	0	0	5 ± 2	0	17 ± 3	0	14 ± 3	1b *	72 ± 3	83 ± 1	79 ± 3	80 ± 1	91 ± 2	83 ± 2	65 ± 1
2ba	5 ± 2	8 ± 3	4 ± 2	8 ± 2	17 ± 2	0	6 ± 2	osthole *	14 ± 1	58 ± 1	10 ± 1	17 ± 1	15 ± 1	70 ± 2	33 ± 1
2bb	7 ± 1	16 ± 2	15 ± 2	6 ± 0	18 ± 0	0	12 ± 2	isotianil *	25 ± 2	9 ± 2	20 ± 1	22 ± 1	24 ± 1	45 ± 1	18 ± 2

A. s, Alternaria solani; B. c, Botrytis cinerea; C. a, Cercospora arachidicola; F.g, Fusarium graminearum; P. p, Physalospora piricola; R. s, Rhizoctonia solani; S. s, Sclerotinia sclerotiorum. * Data from [1]. The drugs used for testing are pure drugs.

. The osthole, isotianil and compounds 1a and 1b were used as positive controls. Compound 2ai showed more than 75% inhibition against Alternaria solani, Botrytis cinerea, Cercospora arachidicola and Sclerotinia sclerotiorum, being similar to the positive control compound 1a. Meanwhile, compound 2aj showed more than 75% inhibition against C. arachidicola (76%) and Physalospora piricola (78%), and compound 2ar showed more than 75% inhibition against C. arachidicola (77%), P. piricola (77%) and Rhizoctonia solani (76%).

In order to further explore the fungicidal activity of the compounds, their median effective concentration (EC₅₀) values were examined. As shown in

Table 3. The in vitro fungicidal EC₅₀ values of the selected compounds.

Compd.	Fungi	Regression Equation	R2	EC50 (μg/mL)	95% Confidence Interval
2ai	A. s.	y = 4.6354 + 0.7213x	0.9671	3.20	2.03~5.06
	B. c.	y = 4.7569 + 0.5255x	0.9738	2.90	1.90~4.43
	C. a.	y = 4.4115 + 1.0116x	0.9528	3.82	2.49~5.85
	P. p.	y = 4.6117 + 0.6906x	0.9799	3.65	2.77~4.81
	S. s.	y = 4.2996 + 0.9405x	0.9704	5.56	4.15~7.44
2aj	A. s.	y = 3.7955 + 0.9777x	0.9643	17.06	13.42~21.68
	B. c.	y = 3.4733 + 1.1522x	0.9555	21.14	14.13~31.63
	C. a.	y = 4.2600 + 1.0479x	0.9752	5.08	4.00~6.47
	P. p.	y = 4.1607 + 0.7510x	0.9502	13.11	8.86~19.39
	S. s.	y = 4.5100 + 0.7458x	0.9773	4.54	3.75~5.49
2ar	A. s.	y = 3.6741 + 1.2509x	0.9886	11.48	9.54~13.82
	B. c.	y = 3.3040 + 1.5643x	0.9530	12.14	9.29~15.86
	C. a.	y = 3.9396 + 1.3025x	0.9580	6.52	5.07~8.38
	P. p.	y = 3.6776 + 1.2105x	0.9635	12.37	9.76~15.68
	R. s.	y = 4.3684 + 1.1211x	0.9641	3.66	2.78~4.82
	S. s.	y = 3.8039 + 1.1299x	0.9740	11.44	9.42~13.90
2bg	A. s.	y = 4.2795 + 0.6054x	0.9624	15.49	12.15~19.76
	B. c.	y = 4.2014 + 0.7378x	0.9866	12.09	10.50~13.92
	S. s.	y = 4.6013 + 0.5269x	0.9827	5.71	4.75~6.87
1a *	A. s.	y = 4.2794 + 0.9061x	0.9696	6.24	4.79~8.12
	B. c.	y = 4.7981 + 0.7129x	0.9716	1.92	1.39~2.65
	C. a.	y = 4.2191 + 1.0934x	0.9676	5.18	3.68~6.95
	P. p.	y = 4.2839 + 0.7369x	0.9691	9.37	5.85~15.03
	R. s.	y = 4.9015 + 0.8031x	0.9718	1.33	0.93~1.90
	S. s.	y = 4.4350 + 1.0010x	0.9586	3.67	2.72~4.95
1b *	A. s.	y = 3.3158 + 1.7602x	0.9841	9.05	7.78~10.53
	B. c.	y = 4.2055 + 1.0298x	0.9734	5.90	4.82~7.24
	C. a.	y = 3.5992 + 1.5235x	0.9555	8.31	6.46~10.68
	P. p.	y = 3.9657 + 1.3588x	0.9018	5.77	3.15~10.58
	R. s.	y = 3.9626 + 1.3553x	0.9932	5.83	5.31~6.39
	S. s.	y = 3.8143 + 1.1971x	0.9499	9.78	7.00~13.67
Osthole *	B. c.	y = 3.5371 + 1.6848x	0.9899	7.38	6.50~8.39
	P. p.	y = 0.5260 + 2.3891x	0.9727	74.59	68.27~85.63
	R. s.	y = 3.7903 + 1.3147x	0.9948	8.32	7.59~9.12
	S. s.	y = 2.8079 + 1.3590x	0.9264	41.03	27.86~64.46

A. s, Alternaria solani; B. c, Botrytis cinerea; C. a, Cercospora arachidicola; F.g, Fusarium graminearum; P. p, Physalospora piricola; R. s, Rhizoctonia solani; S. s, Sclerotinia sclerotiorum. * Data from [1]. The drugs used for testing are pure drugs.

, compound 2ai displayed higher fungicidal activity against A. solani, with an EC₅₀ value of 3.20 μg/mL, being more potent than compound 1a (EC₅₀ = 6.24 μg/mL). For B. cinerea, compound 2ai showed slightly weaker activity than compound 1a. Compound 2ai was more potent against C. arachidicola (EC₅₀ = 3.82 μg/mL) than compound 2aj (EC₅₀ = 5.08 μg/mL) and the positive control compound 1a (EC₅₀ = 5.18 μg/mL). For P. piricola, compound 2ai (with an EC₅₀ value of 3.65 μg/mL) showed 2.5 times more potency than compound 1a (with an EC₅₀ value of 9.37 μg/mL). For S. sclerotiorum, compound 2bg (EC₅₀ = 5.71 μg/mL) displayed higher fungicidal activity than compound 1b (EC₅₀ = 9.78 μg/mL); therefore, some of the esters could exhibit better fungicidal activity towards certain pathogens than the original hydroxyl derivatives.

2.4. In Vivo Fungicidal Activity

The in vivo fungicidal activity of the target compounds was tested against Pseudoperonospora cubensis. The results are shown in

Table 4. In vivo fungicidal activity of the target compounds against CDM * (%).

Compd.	200 μg/mL	100 μg/mL	50 μg/mL 2 L	25 μg/mL 10 μg/mL
2ai	100	100	30 40	0 nd
2aj	100	100	0 75	0 nd
2ar	100	100	80 0	75 nd
2bg	100	100	88 20	65 nd
1a **	100	100	88 nd	65 nd
1b **	100	100	75 100	20 100
Osthole	100	0	// 100	// nd
Cyazofamid	100	100	100	100

* CDM: P. cubensis (Berk. & Curt.) Rostov. // = not detected. ** Data from [1]. The drugs used for testing are pure drugs.

. Compounds 2ai, 2aj, 2ar and 2bg showed excellent activity against P. cubensis at 200 and 100 μg/mL, results which were consistent with compounds 1a and 1b. When the concentration was reduced to 50 μg/mL and 25 μg/mL, the compounds 2ar maintained 80% and 75% inhibition rates, and compound 2bg maintained 88% and 65% inhibition rates, being equivalent to the positive control compound 1a. In short, 2ar and 2bg showed excellent in vivo activity against P. cubensis, and they could be potential candidates for further research.

2.5. 3D-QSAR Analysis

The analysis of the statistical parameters of the established 3D-QSAR model was carried out on the basis of the optimal molecular conformation of fungicidal activity, and the statistical results are shown in

Table 5. Statistical parameters of the CoMFA.

Statistical Parameter	CoMFA
q2	0.797
r2	0.979
ONC	5
SEE	0.096
F	166.514
Steric	0.754
Electrostatic	0.246

. The CoMFA model includes the effects of the steric field and electrostatic field. When the optimum component was five, the parameters (Q² = 0.797, R² = 0.979, SEE = 0.096, F = 166.514) were obtained. For CoMFA models, when Q² is greater than 0.5 and R² is greater than 0.8, this suggests that the models have high reliability and a good prediction ability. The predicted versus experimental values (Figure 4) obtained using this 3D-QSAR model indicate that the predictive capability of this model is robust.

In the steric field equipotential diagram of the CoMFA model, the green area indicates that the large-volume group is beneficial for improving the molecular activity, and the yellow area indicates that the large-volume group is not beneficial for improving the molecular activity. Figure 5A indicates that increasing the group volume of the 3-position and 7-position on the coumarin ring is conducive to improving the inhibitory activity of the target compounds against P. piricola. In the electrostatic field equipotential diagram of the CoMFA model, the blue area indicates that the positively charged group is beneficial for improving the molecular activity, and the red area indicates that the negatively charged group is beneficial for improving the molecular activity. Figure 5B indicates that increasing the positively charged group in the 7-position on the coumarin ring is conducive to improving the inhibitory activity of the target compounds against P. piricola.

As mentioned above, a larger group and positively charged group at the 7-position of the coumarin ring and a larger group at the 3-position of the coumarin ring are beneficial for improving fungicidal activity against P. piricola.

2.6. DFT Calculations of 2ai, 2aq and 1a

The molecular total energy (MTE), frontier orbital energy (FMO), energy gaps between the HOMO and LUMO of compounds with high (2ai) or low (2aq) fungicidal potency and the positive control 1a are listed in

Table 6. Frontier orbital energy.

Compd.	EHOMU (Hartree)	ELUMU (Hartree)	ΔE (Hartree/eV)
2ai	−0.24774	−0.09462	0.15312/4.17
2aq	−0.23359	−0.09296	0.14063/3.83
1a	−0.23646	−0.08930	0.14716/4.00

. The energy gaps between the HOMO and LUMO were 4.17, 3.83 and 4.00 eV, respectively (Table 6).

LUMO and HOMO affect electron transition by accepting and providing electrons, respectively, according to the FMO theory. In Figure 6, the HOMO of 2ai and 1a is mainly located on the benzopyranone ring, while the LUMO is mainly located on the benzopyranone ring and the isothiazole ring, suggesting that the electrons of both compounds were transferred from the benzopyranone ring to the isothiazole ring. However, the HOMO of 2aq with low fungicidal potency is primarily distributed in the naphthalene ring, and the LUMO is distributed in the ester bond and benzopyranone and isothiazole rings, indicating that the direction of electron transition is different from that of 2ai with high fungicidal potency and the positive control 1a.

The aforementioned results regarding the energy and electron transition of 2ai are in approximate agreement with those of 1a, whereas that for 2aq was different, explaining their difference in fungicidal potency on the basis of their chemical structures.

3. Materials and Methods

3.1. Instruments and Materials

All solvents and reagents used were obtained from commercial sources and were not further purified. The melting points were measured using an X-4 digital display micro-melting point apparatus (Gongyi, China) and were not corrected. ¹H NMR (400 MHz), ¹³C NMR (101 MHz) or ¹⁹F NMR (376 MHz) spectra were measured using TMS as the internal standard with a Bruker AV 400 spectrometer in CDCl₃. High-resolution mass spectrometry (HRMS) data were measured using a Varian QFT-ESI instrument. Crystal structures were determined using a Rigaku 007 Saturn 70 diffractometer.

3.2. General Procedures for Preparation of the Target Compounds 2aa–2bv

According to the reported method, intermediates 1a and 1b were synthesized [1]. The compounds 2aa–2bv were synthesized according to the reported method with minor modifications [28].

To a mixture of compound 1 (0.25 mmol), 4-DMAP (0.50 mmol) in dichloromethane (15 mL) and triethylamine (0.50 mmol), acyl chloride (0.75 mmol) was slowly added under stirring, and the reaction mixture was then stirred overnight at room temperature. After the reaction was complete, the mixture was extracted with dichloromethane (3 × 15 mL). The organic phase was sequentially washed with saturated NaHCO₃ (20 mL) and brine (20 mL) and dried over anhydrous Na₂SO₄. The solution was then filtered and concentrated under reduced pressure. The crude product was purified using column chromatography with a mobile phase of dichloromethane/methanol (500:1–200:1, v/v) to yield compounds 2aa–2bv.

2aa. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl benzoate, white solid; yield: 95%; m.p.: 186–188 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J = 7.2 Hz, 2H), 7.68 (t, J = 7.5 Hz, 1H), 7.54 (t, J = 7.8 Hz, 2H), 7.18 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 8.7 Hz, 1H), 2.39 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 164.23, 155.84, 152.77, 151.95, 150.91, 149.55, 139.00, 134.32, 130.34, 128.87, 128.35, 123.79, 123.27, 123.07, 120.53, 119.89, 115.74, 9.44. HRMS (ESI) m/z calcd for C₂₀H₁₁Cl₃NO₄S⁺ (M+H)⁺: 465.9474, found: 465.9467.

2ab. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 2-phenylacetate, pale yellow solid; yield: 90%; m.p.: 55–56 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.42–7.31 (m, 5H), 7.01 (d, J = 8.7 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H), 3.92 (s, 2H), 2.18 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 169.06, 155.78, 152.41, 151.87, 150.81, 149.59, 138.89, 132.71, 129.31, 128.92, 127.75, 123.62, 123.26, 123.12, 120.33, 119.58, 115.68, 41.30, 9.04. HRMS (ESI) m/z calcd for C₂₁H₁₃Cl₃NO₄S⁺ (M+H)⁺: 479.9631, found: 479.9623.

2ac. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 4-methoxybenzoate, white solid; yield: 54%; m.p.: 196–198 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.16 (d, J = 8.8 Hz, 2H), 7.16 (d, J = 8.7 Hz, 1H), 7.01 (d, J = 8.7 Hz, 3H), 3.91 (s, 3H), 2.39 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 164.45, 163.97, 155.97, 152.97, 151.96, 150.91, 149.60, 139.05, 132.61, 123.70, 123.29, 122.96, 120.61, 120.50, 120.03, 115.61, 114.14, 55.65, 9.44. HRMS (ESI) m/z calcd for C₂₁H₁₃Cl₃NO₅S⁺ (M+H)⁺: 495.9580, found: 495.9571.

2ad. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 2,4-difluorobenzoate, pale yellow solid; yield: 92%; m.p.: 192–194 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.19 (q, J = 7.9 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 7.11–6.97 (m, 3H), 2.43 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 166.68 (dd, J = 259.1, 12.2 Hz), 163.44 (dd, J = 265.3, 12.8 Hz), 161.08 (d, J = 4.6 Hz), 155.85, 152.21, 151.86, 150.90, 149.61, 138.96, 134.66 (t, J = 8.0 Hz), 123.81, 123.30, 123.28, 120.45, 119.73, 115.89, 113.43 (dd, J = 9.6, 3.6 Hz), 112.31 (dd, J = 21.6, 3.7 Hz), 105.82 (t, J = 25.3 Hz), 9.45. ¹⁹F NMR (376 MHz, Chloroform-d) δ −98.74 (d, J = 13.3 Hz), −101.46 (d, J = 14.3 Hz). HRMS (ESI) m/z calcd for C₂₀H₉Cl₃F₂NO₄S⁺ (M+H)⁺: 501.9286, found: 501.9277.

2ae. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 3-bromobenzoate, white solid; yield: 86%; m.p.: 191–193 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.35 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 8.7 Hz, 1H), 2.39 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 163.00, 155.80, 152.42, 151.85, 150.91, 149.65, 138.94, 137.27, 133.24, 130.43, 130.26, 128.90, 123.86, 123.32, 122.93, 120.52, 119.69, 115.93, 9.48. HRMS (ESI) m/z calcd for C₂₀H₁₀BrCl₃NO₄S⁺ (M+H)⁺: 543.8579, found: 543.8569.

2af. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 4-chlorobenzoate, white solid; yield: 98%; m.p.: 184–186 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.19 (d, J = 8.7 Hz, 1H), 7.05 (d, J = 8.7 Hz, 1H), 2.41 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 163.42, 155.79, 152.50, 151.87, 150.91, 149.61, 140.99, 138.94, 131.69, 129.27, 126.78, 123.82, 123.30, 123.24, 120.48, 119.74, 115.85, 9.44. HRMS (ESI) m/z calcd for C₂₀H₁₀Cl₄NO₄S⁺ (M+H)⁺: 499.9084, found: 499.9076.

2ag. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 4-fluorobenzoate, white solid; yield: 96%; m.p.: 168–169 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.31–8.23 (m, 2H), 7.27–7.17 (m, 3H), 7.05 (d, J = 8.7 Hz, 1H), 2.41 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 166.56 (d, J = 256.6 Hz), 163.31, 155.88, 152.57, 151.87, 150.91, 149.64, 138.98, 133.07 (d, J = 9.7 Hz), 124.57 (d, J = 3.0 Hz), 123.82, 123.26 (d, J = 10.5 Hz), 120.55, 119.82, 116.30, 116.08, 115.83, 9.45. ¹⁹F NMR (376 MHz, Chloroform-d) δ −102.78. HRMS (ESI) m/z calcd for C₂₀H₁₀Cl₃FNO₄S⁺ (M+H)⁺: 483.9380, found: 483.9377.

2ah. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 4-(trifluoromethyl)benzoate, white solid; yield: 60%; m.p.: 179–180 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.34 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.3 Hz, 2H), 7.19 (d, J = 8.7 Hz, 1H), 7.05 (d, J = 8.7 Hz, 1H), 2.39 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 163.08, 155.72, 152.31, 151.83, 150.92, 149.61, 138.90, 135.68 (d, J = 32.8 Hz), 131.62, 130.76, 125.91 (q, J = 3.3 Hz), 123.91, 123.41 (q, J = 273.1 Hz), 123.39, 123.31, 120.43, 119.62, 116.00, 9.43. ¹⁹F NMR (376 MHz, Chloroform-d) δ −63.24. HRMS (ESI) m/z calcd for C₂₁H₁₀Cl₃F₃NO₄S⁺ (M+H)⁺: 533.9348, found: 533.9346.

2ai. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl acetate, white solid; yield: 97%; m.p.: 140–142 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.05 (d, J = 8.7 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 2.39 (s, 3H), 2.33 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 168.45, 155.80, 152.44, 151.90, 150.82, 149.49, 138.95, 123.73, 123.22, 123.02, 120.23, 119.71, 115.68, 20.76, 9.30. HRMS (ESI) m/z calcd for C₁₅H₉Cl₃NO₄S⁺ (M+H)⁺: 403.9318, found: 403.9308.

2aj. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl propionate, white solid; yield: 80%; m.p.: 125–127 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.05 (d, J = 8.7 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 2.69 (q, J = 7.5 Hz, 2H), 2.34 (s, 3H), 1.32 (t, J = 7.5 Hz, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 171.98, 155.83, 152.56, 151.93, 150.85, 149.52, 138.96, 123.68, 123.23, 122.97, 120.23, 119.72, 115.60, 27.55, 9.26, 9.08. HRMS (ESI) m/z calcd for C₁₆H₁₁Cl₃NO₄S⁺ (M+H)⁺: 417.9474, found: 417.9464.

2ak. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl cyclopropanecarboxylate, white solid; yield: 94%; m.p.: 138–139 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.05 (d, J = 8.7 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 2.34 (s, 3H), 1.98–1.85 (m, 1H), 1.24–1.19 (m, 2H), 1.14–1.08 (m, 2H). ¹³C NMR (101 MHz, Chloroform-d) δ 172.53, 155.85, 152.58, 151.95, 150.81, 149.48, 138.97, 123.65, 123.20, 122.90, 120.32, 119.75, 115.57, 12.80, 9.70, 9.24. HRMS (ESI) m/z calcd for C₁₇H₁₁Cl₃NO₄S⁺ (M+H)⁺: 429.9474, found: 429.9465.

2al. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl isobutyrate, white solid; yield: 91%; m.p.: 155–157 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.00 (d, J = 8.7 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 2.88 (hept, J = 7.0 Hz, 1H), 2.31 (s, 3H), 1.35 (d, J = 7.0 Hz, 6H). ¹³C NMR (101 MHz, Chloroform-d) δ 174.59, 155.84, 152.65, 151.94, 150.83, 149.50, 138.97, 123.67, 123.22, 122.93, 120.23, 119.68, 115.57, 34.20, 18.95, 9.18. HRMS (ESI) m/z calcd for C₁₇H₁₃Cl₃NO₄S⁺ (M+H)⁺: 431.9631, found: 431.9624.

2am. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl pivalate, white solid; yield: 88%; m.p.:166–168 °C; ¹H NMR (400 MHz, Chloroform-d) δ 6.98 (d, J = 8.8 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 2.30 (s, 3H), 1.39 (s, 9H). ¹³C NMR (101 MHz, Chloroform-d) δ 176.10, 155.86, 152.89, 151.95, 150.82, 149.49, 139.00, 123.66, 123.21, 122.88, 120.25, 119.65, 115.51, 39.41, 27.14, 9.13. HRMS (ESI) m/z calcd for C₁₈H₁₅Cl₃NO₄S⁺ (M+H)⁺: 445.9787, found: 445.9778.

2an. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl thiophene-2-carboxylate, white solid; yield: 77%; m.p.: 210–211 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.03 (d, J = 3.8 Hz, 1H), 7.74 (d, J = 5.0 Hz, 1H), 7.25–7.16 (m, 2H), 7.01 (d, J = 8.7 Hz, 1H), 2.41 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 159.59, 155.90, 152.25, 151.88, 150.88, 149.63, 138.99, 135.64, 134.64, 131.40, 128.44, 123.75, 123.31, 123.18, 120.61, 119.84, 115.80, 9.45. HRMS (ESI) m/z calcd for C₁₈H₉Cl₃NO₄S₂⁺ (M+H)⁺: 471.9038, found: 471.9032.

2ao. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl furan-2-carboxylate, white solid; yield: 71%; m.p.: 203–204 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.72 (s, 1H), 7.46 (d, J = 3.5 Hz, 1H), 7.17 (d, J = 8.7 Hz, 1H), 7.01 (d, J = 8.7 Hz, 1H), 6.64 (dd, J = 3.5, 1.7 Hz, 1H), 2.39 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 155.81, 151.85, 150.88, 149.59, 147.95, 142.93, 138.94, 123.78, 123.29, 123.23, 120.59, 119.73, 115.87, 112.54, 9.40. HRMS (ESI) m/z calcd for C₁₈H₉Cl₃NO₅S⁺ (M+H)⁺: 455.9267, found: 455.9258.

2ap. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl cinnamate, white solid; yield: 70%; m.p.: 168–169 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.72 (d, J = 16.0 Hz, 1H), 7.43–7.36 (m, 2H), 7.26–7.19 (m, 3H), 6.92 (d, J = 8.7 Hz, 1H), 6.79 (d, J = 8.7 Hz, 1H), 6.46 (d, J = 16.0 Hz, 1H), 2.17 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 164.43, 155.87, 152.66, 151.95, 150.90, 149.57, 148.16, 138.98, 133.74, 131.24, 129.12, 128.50, 123.71, 123.27, 122.99, 120.47, 119.82, 115.84, 115.65, 29.71, 9.43. HRMS (ESI) m/z calcd for C₂₂H₁₃Cl₃NO₄S⁺ (M+H)⁺: 491.9631, found: 491.9622.

2aq. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 1-naphthoate, pale yellow solid; yield: 78%; m.p.: 213–215 °C; ¹H NMR (400 MHz, Chloroform-d) δ 9.02 (d, J = 8.6 Hz, 1H), 8.56 (d, J = 7.3 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.72–7.65 (m, 1H), 7.65–7.56 (m, 2H), 7.24 (d, J = 8.7 Hz, 1H), 7.05 (d, J = 8.7 Hz, 1H), 2.46 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 164.34, 155.67, 153.01, 151.92, 150.19, 147.36, 139.83, 134.92, 133.51, 131.60, 130.76, 129.00, 128.51, 126.70, 125.03, 124.80, 124.64, 124.22, 121.78, 121.76, 120.04, 119.11, 116.27, 9.06. HRMS (ESI) m/z calcd for C₂₄H₁₃Cl₃NO₄S⁺ (M+H)⁺: 515.9631, found: 515.9625.

2ar. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl acrylate, white solid; yield: 54%; m.p.: 118–119 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.08 (d, J = 8.7 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 6.67 (d, J = 17.3 Hz, 1H), 6.36 (dd, J = 17.3, 10.5 Hz, 1H), 6.12 (d, J = 10.5 Hz, 1H), 2.33 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 163.50, 155.82, 152.33, 151.89, 150.86, 149.56, 138.95, 134.17, 126.83, 123.73, 123.26, 123.09, 120.37, 119.67, 115.73, 9.32. HRMS (ESI) m/z calcd for C₁₆H₉Cl₃NO₄S⁺ (M+H)⁺: 415.9318, found: 415.9310.

2as. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylate, white solid; yield: 69%; m.p.: 202–204 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.17 (s, 1H), 7.15 (d, J = 8.7 Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H), 4.06 (s, 3H), 2.38 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 159.05, 155.79, 151.85, 150.86, 149.56, 146.95 (t, J = 25.6 Hz), 138.95, 136.23, 123.77, 123.26, 123.19, 120.55, 119.80, 115.86, 111.04 (t, J = 2.2 Hz), 109.23 (t, J = 237.4 Hz), 39.97, 9.33. ¹⁹F NMR (376 MHz, Chloroform-d) δ -115.63. ¹⁹F NMR (376 MHz, Chloroform-d) δ −115.63. HRMS (ESI) m/z calcd for C₁₉H₁₁Cl₃F₂N₃O₄S⁺ (M+H)⁺: 519.9504, found: 519.9496.

2at. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylate, white solid; yield: 77%; m.p.: 220–221 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.23 (s, 1H), 7.14 (d, J = 8.7 Hz, 1H), 7.01 (d, J = 8.7 Hz, 1H), 4.07 (s, 3H), 2.37 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 158.03, 155.79, 151.85, 150.85, 149.54, 142.21 (q, J = 38.7 Hz), 138.97, 137.44, 123.78, 123.25, 123.17, 120.51, 120.12 (q, J = 269.6 Hz), 119.73, 115.86, 111.09, 40.11, 9.26. ¹⁹F NMR (376 MHz, Chloroform-d) δ −62.06. HRMS (ESI) m/z calcd for C₁₉H₁₀Cl₃F₃N₃O₄S⁺ (M+H)⁺: 537.9409, found: 537.9401.

2au. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 2-methylfuran-3-carboxylate, yellow solid; yield: 67%; m.p.: 159–160 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.33 (d, J = 1.8 Hz, 1H), 7.14 (d, J = 8.7 Hz, 1H), 7.00 (d, J = 8.7 Hz, 1H), 6.79 (d, J = 1.7 Hz, 1H), 2.64 (s, 3H), 2.36 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 161.70, 161.35, 155.90, 152.39, 151.95, 150.90, 149.54, 139.02, 123.71, 123.25, 122.98, 120.54, 120.06, 115.64, 111.90, 110.67, 110.59, 14.09, 9.44. HRMS (ESI) m/z calcd for C₁₉H₁₁Cl₃NO₅S⁺ (M+H)⁺: 469.9423, found: 469.9416.

2av. 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 3-methylthiophene-2-carboxylate, white solid; yield: 91%; m.p.: 180–182 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.59–7.50 (m, 1H), 7.19 (d, J = 8.7 Hz, 1H), 7.07–6.94 (m, 2H), 2.59 (s, 3H), 2.39 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 159.95, 155.89, 152.30, 151.96, 150.88, 149.52, 149.33, 139.04, 132.29, 132.20, 124.19, 123.70, 123.25, 122.99, 120.59, 120.06, 115.67, 16.22, 9.49. HRMS (ESI) m/z calcd for C₁₉H₁₁Cl₃NO₄S₂⁺ (M+H)⁺: 485.9195, found: 485.9188.

2ba. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 2-chloronicotinate, pale yellow solid; yield 97%; m.p. 197–198 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.71 (dd, J = 4.7, 1.8 Hz, 1H), 8.65 (dd, J = 7.7, 1.8 Hz, 1H), 7.70 (dd, J = 7.7, 4.8 Hz, 1H), 7.44 (d, J = 8.7 Hz, 1H), 7.39 (d, J = 8.7 Hz, 1H), 6.83 (s, 1H), 2.32 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.83, 158.42, 154.60, 153.27, 152.12, 151.41, 148.67, 147.12, 142.72, 141.53, 125.03, 124.75, 123.39, 121.42, 119.04, 118.89, 118.05, 115.23, 8.99. HRMS (ESI) m/z calcd for C₁₉H₁₀Cl₃N₂O₄S⁺ (M+H)⁺: 466.9427, found: 466.9421.

2bb. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylate, pale yellow solid; yield 89%; m.p. 196–198 °C. ¹H NMR (400 MHz, Chloroform-d) δ 8.16 (s, 1H), 7.29–7.23 (m, 1H), 7.15 (d, J = 8.7 Hz, 1H), 7.09 (t, J = 53.8 Hz, 1H), 6.52 (s, 1H), 4.07 (s, 3H), 2.39 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 159.12, 158.84, 153.76, 153.05, 152.06, 149.57, 147.01 (t, J = 25.5 Hz), 142.78, 136.13, 123.99, 122.68, 120.65, 119.06, 117.67, 114.69, 111.21 (t, J = 2.4 Hz), 109.20 (t, J = 237.4 Hz), 39.98, 9.35. ¹⁹F NMR (376 MHz, Chloroform-d) δ −115.64, −115.78. ¹⁹F NMR (376 MHz, Chloroform-d) δ −115.71. HRMS (ESI) m/z calcd for C₁₉H₁₂Cl₂F₂N₃O₄S⁺ (M+H)⁺: 485.9893, found: 485.9885.

2bc. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylate, white solid; yield 58%; m.p. 163–164 °C. ¹H NMR (400 MHz, Chloroform-d) δ 8.20 (s, 1H), 7.23 (d, J = 8.7 Hz, 1H), 7.13 (d, J = 8.7 Hz, 1H), 6.50 (s, 1H), 4.06 (s, 3H), 2.37 (s, 3H).¹³C NMR (101 MHz, Chloroform-d) δ 158.84, 158.11, 153.77, 153.06, 152.07, 149.57, 142.78, 142.31 (q, J = 38.8 Hz), 137.33, 123.98, 122.68, 120.63, 120.12 (q, J = 269.7 Hz), 118.97, 117.67, 114.70, 111.32. ¹⁹F NMR (376 MHz, Chloroform-d) δ −62.06. HRMS (ESI) m/z calcd for C₁₉H₁₁Cl₂F₃N₃O₄S⁺ (M+H)⁺: 503.9799, found: 503.9791.

2bd. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 2-methylfuran-3-carboxylate, brown red solid; yield 82%; m.p. 186–188 °C. ¹H NMR (400 MHz, Chloroform-d) δ 7.37 (d, J = 1.9 Hz, 1H), 7.25 (d, J = 8.7 Hz, 1H), 7.15 (d, J = 8.7 Hz, 1H), 6.83 (d, J = 1.9 Hz, 1H), 6.51 (s, 1H), 2.68 (s, 3H), 2.40 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 161.60, 161.39, 158.93, 153.88, 153.09, 152.62, 149.54, 142.83, 141.02, 123.86, 122.65, 120.62, 119.28, 117.46, 114.46, 112.00, 110.65, 14.02, 9.40. HRMS (ESI) m/z calcd for C₁₉H₁₂Cl₂NO₅S⁺ (M+H)⁺: 435.9813, found: 435.9804.

2be. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 3-methylthiophene-2-carboxylate, pale yellow solid; yield 90%; m.p. 188–189 °C. ¹H NMR (400 MHz, Chloroform-d) δ 7.56 (d, J = 5.0 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 7.18 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 5.0 Hz, 1H), 6.49 (s, 1H), 2.62 (s, 3H), 2.40 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 160.03, 158.93, 153.88, 153.07, 152.53, 149.55, 149.20, 142.83, 132.22, 132.05, 124.37, 123.83, 122.66, 120.69, 119.28, 117.49, 114.48, 16.19, 9.44. HRMS (ESI) m/z calcd for C₁₉H₁₂Cl₂NO₄S₂⁺ (M+H)⁺: 451.9585, found: 451.9577.

2bf. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl benzoate, white solid; yield 93%; m.p. 172–173 °C. ¹H NMR (400 MHz, Chloroform-d) δ 8.25 (d, J = 7.5 Hz, 2H), 7.72 (t, J = 7.3 Hz, 1H), 7.58 (t, J = 7.5 Hz, 2H), 7.28 (d, J = 7.5 Hz, 1H), 7.20 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 2.42 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 164.29, 158.89, 153.87, 153.10, 153.01, 149.56, 142.83, 134.24, 130.34, 128.84, 128.48, 123.97, 122.67, 120.62, 119.13, 117.53, 114.56, 9.41. HRMS (ESI) m/z calcd for C₂₀H₁₂Cl₂NO₄S⁺ (M+H)⁺: 431.9864, found: 431.9854.

2bg. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl propionate, white solid; yield 98%; m.p. 111–112 °C. ¹H NMR (400 MHz, Chloroform-d) δ 7.19 (d, J = 8.7 Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H), 6.47 (s, 1H), 2.68 (q, J = 7.5 Hz, 2H), 2.32 (s, 3H), 1.31 (t, J = 7.5 Hz, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 172.07, 158.93, 153.85, 153.03, 152.79, 149.54, 142.81, 123.89, 122.65, 120.35, 119.00, 117.45, 114.44, 27.59, 9.27, 9.11. HRMS (ESI) m/z calcd for C₁₆H₁₂Cl₂NO₄S⁺ (M+H)⁺: 383.9864, found: 383.9854.

2bh. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl cyclopropane carboxylate, yellow solid; yield 94%; m.p. 102–104 °C. ¹H NMR (400 MHz, Chloroform-d) δ 7.17 (d, J = 8.7 Hz, 1H), 7.01 (d, J = 8.7 Hz, 1H), 6.43 (s, 1H), 2.30 (s, 3H), 1.93–1.85 (m, 1H), 1.21–1.15 (m, 2H), 1.11–1.04 (m, 2H). ¹³C NMR (101 MHz, Chloroform-d) δ 172.51, 158.84, 153.90, 152.97, 152.79, 149.37, 142.75, 123.84, 122.55, 120.35, 118.99, 117.39, 114.38, 12.81, 9.62, 9.22. HRMS (ESI) m/z calcd for C₁₇H₁₂Cl₂NO₄S⁺ (M+H)⁺: 395.9864, found: 395.9854.

2bi. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl furan-2-carboxylate, white solid; yield 75%; m.p. 195–197 °C. ¹H NMR (400 MHz, Chloroform-d) δ 7.75–7.70 (m, 1H), 7.46 (d, J = 3.5 Hz, 1H), 7.24 (d, J = 8.8 Hz, 1H), 7.17 (d, J = 8.7 Hz, 1H), 6.64 (dd, J = 3.5, 1.7 Hz, 1H), 6.50 (s, 1H), 2.39 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 158.83, 155.87, 153.79, 153.07, 152.09, 149.56, 147.85, 143.06, 142.78, 123.98, 122.68, 120.65, 120.45, 118.98, 117.65, 114.70, 112.49, 9.38. HRMS (ESI) m/z calcd for C₁₈H₁₀Cl₂NO₅S⁺ (M+H)⁺: 421.9656, found: 421.9647.

2bj. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl thiophene-2-carboxylate, white solid; yield 84%; m.p. 178–179 °C. ¹H NMR (400 MHz, Chloroform-d) δ 8.04 (dd, J = 3.8, 1.1 Hz, 1H), 7.74 (dd, J = 5.0, 1.1 Hz, 1H), 7.27–7.22 (m, 2H), 7.19 (d, J = 8.7 Hz, 1H), 6.50 (s, 1H), 2.40 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 159.66, 158.95, 153.82, 153.05, 152.48, 149.58, 142.84, 135.50, 134.47, 131.54, 128.39, 123.97, 122.69, 120.67, 119.11, 117.59, 114.62, 9.44. HRMS (ESI) m/z calcd for C₁₈H₁₀Cl₂NO₄S₂⁺ (M+H)⁺: 437.9428, found: 437.9426.

2bk. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 2-phenylacetate, yellow solid; yield 59%; m.p. 82–83 °C. ¹H NMR (400 MHz, Chloroform-d) δ 7.42–7.30 (m, 5H), 7.17 (d, J = 8.7 Hz, 1H), 7.00 (d, J = 8.7 Hz, 1H), 6.44 (s, 1H), 3.93 (s, 2H), 2.18 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 169.12, 158.81, 153.85, 152.97, 152.64, 149.45, 142.71, 132.84, 129.35, 128.90, 127.70, 123.88, 122.60, 120.35, 118.87, 117.53, 114.51, 41.28, 9.06. HRMS (ESI) m/z calcd for C₂₁H₁₄Cl₂NO₄S⁺ (M+H)⁺: 446.0020, found: 446.0013.

2bl. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 4-methoxybenzoate, yellow solid; yield 53%; m.p. 192–194 °C. ¹H NMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 8.9 Hz, 2H), 7.24 (d, J = 8.7 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.01 (d, J = 8.9 Hz, 2H), 6.49 (s, 1H), 3.91 (s, 3H), 2.38 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 164.41, 163.99, 158.97, 153.91, 153.19, 153.09, 149.54, 142.86, 132.83, 132.52, 123.88, 122.65, 120.66, 119.26, 117.40, 114.42, 114.12, 55.62, 9.39. HRMS (ESI) m/z calcd for C₂₁H₁₄Cl₂NO₅S⁺ (M+H)⁺: 461.9969, found: 461.9959.

2bm. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl isobutyrate, white solid; yield 75%; m.p. 127–129 °C. ¹H NMR (400 MHz, Chloroform-d) δ 7.19 (d, J = 8.7 Hz, 1H), 7.00 (d, J = 8.7 Hz, 1H), 6.47 (s, 1H), 2.89 (hept, J = 7.0 Hz, 1H), 2.32 (s, 3H), 1.37 (d, J = 7.0 Hz, 6H). ¹³C NMR (101 MHz, Chloroform-d) δ 174.66, 158.92, 153.86, 153.02, 152.89, 149.53, 142.81, 123.86, 122.63, 120.34, 118.95, 117.41, 114.39, 34.22, 18.96, 9.17. HRMS (ESI) m/z calcd for C₁₇H₁₄Cl₂NO₄S⁺ (M+H)⁺: 398.0020, found: 398.0010.

2bn. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 4-fluorobenzoate, yellow solid; yield 82%; m.p. 173–174 °C. ¹H NMR (400 MHz, Chloroform-d) δ 8.31–8.24 (m, 2H), 7.30–7.23 (m, 3H), 7.18 (d, J = 8.7 Hz, 1H), 6.53 (s, 1H), 2.41 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 166.52 (d, J = 256.6 Hz), 163.32, 158.87, 153.81, 153.09, 152.82, 149.58, 142.81, 133.02 (d, J = 9.5 Hz), 124.71 (d, J = 2.6 Hz), 124.01, 122.68, 120.58, 119.05, 117.60, 116.14 (d, J = 22.1 Hz), 114.64, 9.41. ¹⁹F NMR (376 MHz, Chloroform-d) δ −102.94. HRMS (ESI) m/z calcd for C₂₀H₁₁Cl₂FNO₄S⁺ (M+H)⁺: 449.9770, found: 449.9763.

2bo. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 4-(trifluoromethyl)-benzoate, yellow solid; yield: 98%; m.p. 175–177 °C. ¹H NMR (400 MHz, Chloroform-d) δ 8.39 (d, J = 8.1 Hz, 2H), 7.86 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 9.0 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 6.55 (s, 1H), 2.42 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 163.15, 158.78, 153.75, 153.10, 152.55, 149.61, 142.78, 135.66 (q, J = 32.8 Hz), 131.71, 130.76, 125.91 (q, J = 4.0 Hz), 123.42 (q, J = 271.3 Hz), 124.13, 122.72, 120.52, 118.89, 117.78, 114.82, 9.45. ¹⁹F NMR (376 MHz, Chloroform-d) δ −63.23. HRMS (ESI) m/z calcd for C₂₁H₁₁Cl₂F₃NO₄S⁺ (M+H)⁺: 499.9738, found: 499.9729.

2bp. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl pivalate, yellow solid; yield 89%; m.p. 156–158 °C. ¹H NMR (400 MHz, Chloroform-d) δ 7.19 (d, J = 8.7 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 6.47 (s, 1H), 2.32 (s, 3H), 1.42 (s, 9H). ¹³C NMR (101 MHz, Chloroform-d) δ 176.16, 158.94, 153.88, 153.14, 153.01, 149.54, 142.82, 123.83, 122.63, 120.36, 118.91, 117.37, 114.32, 39.42, 27.16, 9.11. HRMS (ESI) m/z calcd for C₁₈H₁₆Cl₂NO₄S⁺ (M+H)⁺: 412.0177, found: 412.0170.

2bq. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 3-bromobenzoate, yellow solid; yield 84%; m.p. 203–204 °C. ¹H NMR (400 MHz, Chloroform-d) δ 8.34 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.24 (d, J = 3.2 Hz, 1H), 7.14 (d, J = 8.7 Hz, 1H), 6.50 (s, 1H), 2.37 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 163.06, 158.87, 153.77, 153.08, 152.64, 149.59, 142.81, 137.22, 133.24, 130.43, 130.35, 128.91, 124.10, 122.91, 122.71, 120.59, 118.97, 117.70, 114.75, 9.48. HRMS (ESI) m/z calcd for C₂₀H₁₁BrCl₂NO₄S⁺ (M+H)⁺: 509.8969, found: 509.8962.

2br. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 2,4-difluorobenzoate, yellow solid; yield 84%; m.p. 200–202 °C. ¹H NMR (400 MHz, Chloroform-d) δ 8.20 (q, J = 8.4 Hz, 1H), 7.28 (d, J = 7.9 Hz, 1H), 7.20 (d, J = 8.7 Hz, 1H), 7.12–6.97 (m, 2H), 6.53 (s, 1H), 2.42 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 166.64 (dd, J = 259.0, 12.0 Hz), 163.43 (dd, J = 265.2, 12.8 Hz), 161.10 (d, J = 4.4 Hz), 158.83, 153.80, 153.09, 152.46, 149.57, 142.78, 134.60 (d, J = 10.7 Hz), 123.99, 122.68, 120.50, 118.96, 117.68, 114.71, 113.57 (dd, J = 9.7, 3.6 Hz), 112.24 (dd, J = 21.7, 3.8 Hz), 105.77 (t, J = 25.6 Hz), 9.41. ¹⁹F NMR (376 MHz, Chloroform-d) δ −98.91 (d, J = 12.9 Hz), −101.54 (d, J = 13.6 Hz). HRMS (ESI) m/z calcd for C₂₀H₁₀Cl₂F₂NO₄S⁺ (M+H)⁺: 467.9675, found: 467.9667.

2bs. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 1-naphthoate, yellow solid; yield 77%; m.p. 201–203 °C. ¹H NMR (400 MHz, Chloroform-d) δ 9.03 (d, J = 8.6 Hz, 1H), 8.56 (d, J = 7.3 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.63 (dq, J = 20.9, 6.7, 6.1 Hz, 3H), 7.33–7.14 (m, 2H), 6.50 (s, 1H), 2.45 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 164.68, 158.92, 153.89, 153.16, 153.11, 149.57, 142.84, 135.16, 134.00, 131.81, 131.67, 128.88, 128.61, 126.68, 125.50, 124.55, 124.49, 124.02, 122.69, 120.81, 119.32, 117.53, 114.59, 9.56. HRMS (ESI) m/z calcd for C₂₄H₁₄Cl₂NO₄S⁺ (M+H)⁺: 482.0020, found: 482.0011.

2bt. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl cinnamate, yellow solid; yield 58%; m.p. 174–175 °C. ¹H NMR (400 MHz, Chloroform-d) δ 7.93 (d, J = 16.0 Hz, 1H), 7.68–7.56 (m, 2H), 7.50–7.38 (m, 3H), 7.23 (d, J = 8.7 Hz, 1H), 7.12 (d, J = 8.7 Hz, 1H), 6.68 (d, J = 16.0 Hz, 1H), 6.48 (s, 1H), 2.38 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 164.51, 158.95, 153.88, 153.09, 152.89, 149.55, 148.03, 142.83, 133.80, 131.19, 129.12, 128.48, 123.92, 122.67, 120.57, 119.09, 117.46, 115.97, 114.50, 9.41. HRMS (ESI) m/z calcd for C₂₂H₁₄Cl₂NO₄S⁺ (M+H)⁺: 458.0020, found: 458.0010.

2bu. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl 4-chlorobenzoate, yellow solid; yield 77%; m.p. 185–187 °C. ¹H NMR (400 MHz, Chloroform-d) δ 8.19 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 5.8 Hz, 1H), 7.18 (d, J = 8.7 Hz, 1H), 6.53 (s, 1H), 2.40 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 163.48, 158.84, 153.80, 153.09, 152.74, 149.58, 142.80, 140.92, 131.68, 129.25, 126.89, 124.03, 122.69, 120.56, 119.00, 117.64, 114.68, 9.42. HRMS (ESI) m/z calcd for C₂₀H₁₁Cl₃NO₄S⁺ (M+H)⁺: 465.9474, found: 465.9468.

2bv. 4-(3,4-Dichloroisothiazol-5-yl)-8-methyl-2-oxo-2H-chromen-7-yl acrylate, white solid; yield 68%; m.p. 142–144 °C. ¹H NMR (400 MHz, Chloroform-d) δ 7.21 (d, J = 8.7 Hz, 1H), 7.07 (d, J = 8.7 Hz, 1H), 6.67 (d, J = 18.0 Hz, 1H), 6.47 (s, 1H), 6.36 (dd, J = 17.3, 10.5 Hz, 1H), 6.11 (d, J = 11.3 Hz, 1H), 2.32 (s, 3H). ¹³C NMR (101 MHz, Chloroform-d) δ 163.56, 158.85, 153.83, 153.03, 152.56, 149.49, 142.78, 134.00, 126.93, 123.94, 122.63, 120.43, 118.92, 117.53, 114.55, 9.30. HRMS (ESI) m/z calcd for C₁₆H₁₀Cl₂NO₄S⁺ (M+H)⁺: 381.9707, found: 381.9697.

3.3. X-ray Diffraction

The crystal structure of compound 2be was cultured using the fusion method. Using a hot stage to heat the compound until it melted, the temperature was then meticulously controlled just below the melting point during the slow crystal growth process. The crystal structure was determined using a Rigaku Saturn 724 CCD diffractometer equipped with graphite-monochromatic Mo Kα radiation (λ = 0.71073 Å) at 113 K. The atomic coordinates for 2be were deposited in the Cambridge Crystallographic Data Centre (CCDC).

3.4. In Vitro Fungicidal Activity

The fungicidal activity of compounds 2 against A. solani (A. s), B. cinerea (B. c), C. arachidicola (C. a), Fusarium graminearum (F. g), P. piricola (P. p), R. solani (R. s) and S. sclerotiorum (S. s) were evaluated in vitro at a concentration of 50 μg/mL according to the mycelium growth inhibition method. Pure drugs of osthole, isotianil and compounds 1a and 1b were chosen as positive controls. The median effective concentration (EC₅₀) of the active target compounds was also assessed according to the mycelium growth inhibition method. To prepare the test solution, a highly concentrated mother solution of the test substance was carefully created at a concentration of 25,000 μg/mL using N, N-dimethylformamide as the solvent. A precise amount of the mother solution was added to sterile water containing 0.1% Tween 80 to create a test solution with a concentration of 500 μg/mL. This solution was then mixed with potato dextrose agar (PDA) to create a culture with a controlled concentration of 50 μg/mL of the test compound. A blank control was also included using pure DMF without the target compound. To ensure accuracy, three replicates were included for each treatment. The EC₅₀ was determined using seven concentration gradients ranging from 1.56 to 100 μg/mL, and advanced DPS software was used to process the data.

3.5. In Vivo Fungicidal Activity

The in vivo activity of compounds 2 against Pseudoperonospora cubensis on cucumber was evaluated at Shenyang Sinochem Agrochemicals R&D Co., Ltd., Shenyang, China. Pure osthole and cyzofamid were used as a positive control. The compounds with an inhibition rate of 100% were chosen for further tests with a concentration gradient. To prepare the test solution, 10 mg of the compound to be tested was accurately weighed and dissolved in 0.5 mL of N, N-dimethylformamide. The resulting solution was then diluted with distilled water containing 1% Tween 80 to create a stock concentration of 500 μg/mL. Potted plants with uniform growth were carefully selected as test plants, and the stock solution was further diluted with distilled water to the desired test concentration. The test solution was then sprayed onto the surface of the plant and allowed to dry for 2 h to allow for solvent evaporation. After 24 h, the treated test colonies were properly inoculated on the leaves of the plant for three replicates. A blank control was also included, which involved spraying the plants with plain distilled water containing no test solution. The treated plants were then cultured normally in the greenhouse, and the antifungal efficacy was assessed after 7 d.

3.6. 3D-QSAR Molecular Modelling

Next, 3D-QSAR studies were carried out with 29 target compounds (listed in

Table 7. In vitro biological activity, the biological activity index and experimental and predicted pEC₅₀ values of target compounds against Physalospora piricola.

Compd.	Inhibition (%)	D	CoMFA
			Pred. *	Res. **
2ac	10	1.74	1.70	0.04
2ae	3	1.22	1.22	0.00
2af	10	1.74	1.70	0.04
2ah	4	1.35	1.45	−0.10
2ai	69	2.95	2.99	−0.03
2aj	78	3.17	3.20	−0.03
2al	77	3.16	3.07	0.09
2am	54	2.72	2.70	0.02
2ar	77	3.14	2.98	0.16
2as	23	2.19	2.24	−0.05
2at	23	2.21	2.20	0.01
2au	24	2.17	2.13	0.04
2av	17	2.00	1.95	0.05
2ba	17	1.98	1.97	0.01
2bc	9	1.70	1.71	−0.01
2bd	23	2.11	2.15	−0.04
2be	10	1.70	1.74	−0.04
2bi	14	1.84	1.77	0.07
2bm	63	2.83	2.69	0.14
2bo	10	1.74	1.69	0.05
2bp	35	2.35	2.54	−0.19
2bq	8	1.65	1.64	0.01
2br	8	1.61	1.62	−0.01
2bv	53	2.63	2.84	−0.21
2an ***	10	1.71	1.91	−0.20
2bg ***	56	2.68	2.85	−0.17
2bh ***	41	2.43	2.89	−0.46
2bs ***	18	2.02	1.72	0.30
2bu ***	5	1.38	1.55	−0.17

*: Predicted with CoMFA; **: relative error between the D and predicted pEC₅₀; ***: test set.

) with fungicidal activity against P. piricola. The construction of the 3D-QSAR model was completed in the CoMFA modules in SYBYL X 2.1.1. All the structures were constructed using the molecular fragment library of the SYBYL software. The Powell method was used in the optimization and conformation search process. The gradients were limited to 0.005 kJ·mol⁻¹, and the maximum number of iterations was 1000. The selected small-molecular force field was the Tripos force field, and the charge carried by the molecule was the Gasteiger–Hückel charge. The potent compound 2aj was chosen as a template molecule. The biological activity was expressed in terms of D (Table 7) using the following formula [29]:

$$\mathrm{D}={\log }_{10}\left[\frac{a}{1-a}\right]+{\log }_{10}{M}_w$$

where a is the percentage inhibition against P. piricola at 50 μg/mL in vitro, M_w is the molecular weight of the target compounds, and D is the biological activity index.

3.7. Density Functional Theory (DFT) Calculation

Based on their fungicidal activity, the highly active compound 2ai, the slightly active compound 2aq and the lead compound 1a were selected for molecular orbital calculations using Gaussian 09W and drawn in Gaussview 6.0. The structures were preprocessed and geometrically optimized, and frequency calculations were carried out to generate geometrically stable structures using the DFT-B3LYP/6-31G (d) method. Single-point energy calculations were then performed using the DFT-B3LYP/6-31G (d, p) method.

4. Conclusions

A series of new coumarin derivatives were designed and synthesized. The bioassay results indicated that compound 2ai exhibited broad-spectrum fungicidal in vitro activity, and compounds 2ar and 2bg showed good fungicidal activity against P. cubensis in vivo. Overall, we successfully adjusted the solubility of this series of compounds via esterification, and the activity was equivalent to that of the lead compound. The 3D-QSAR analysis showed that a larger group and positively charged group at the 7-position of the coumarin ring and a larger group at the 3-position of the coumarin ring were beneficial for improving fungicidal activity against P. piricola, the results of this study provide guidance for further coumarin lead based fungicide development.