Design, Synthesis, and Bioactivity Evaluation of New Thiochromanone Derivatives Containing a Carboxamide Moiety

Abstract

In this study, using the botanical active component thiochromanone as the lead compound, a total of 32 new thiochromanone derivatives containing a carboxamide moiety were designed and synthesized and their in vitro antibacterial activities against Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicolaby (Xoc), and Xanthomonas axonopodis pv. citri (Xac) were determined, as well as their in vitro antifungal activities against Botryosphaeria dothidea (B. dothidea), Phomopsis sp., and Botrytis cinerea (B. cinerea). Bioassay results demonstrated that some of the target compounds exhibited moderate to good in vitro antibacterial and antifungal activities. In particular, compound 4e revealed excellent in vitro antibacterial activity against Xoo, Xoc, and Xac, and its EC₅₀ values of 15, 19, and 23 μg/mL, respectively, were superior to those of Bismerthiazol and Thiodiazole copper. Meanwhile, compound 3b revealed moderate in vitro antifungal activity against B. dothidea at 50 μg/mL, and the inhibition rate reached 88%, which was even better than that of Pyrimethanil, however, lower than that of Carbendazim. To the best of our knowledge, this is the first report on the antibacterial and antifungal activities of this series of novel thiochromanone derivatives containing a carboxamide moiety.

1. Introduction

Plant bacterial and fungal diseases have posed serious threats in agricultural production and in spite of the best control efforts of plant pathologists, continue to contribute to heavy crop losses worldwide each year [1,2]. In recent years, the irrational use of traditional pesticides for plant bacterial and fungal disease control have posed a danger to living systems, killing not only target bacteria and fungi, but also affecting beneficial living systems [3]. Therefore, the resistance of plant bacterial and fungal diseases against pesticides is rapidly becoming a serious problem, and in pesticide research the development of novel antibacterial and antifungal agents is still a major challenge to be tackled [4].

Chromone, a kind of botanical active component with extensive biological activities, is widely found in the secondary metabolites of flowers, roots, stems, and pericarp of many plants [5,6]. Thiochromanone, a kind of chromone compound, is an important botanical active component with extensive biological activities, including antiviral [7], antibacterial [8,9], antifungal [8,10,11,12], herbicidal [13,14], and insecticidal [15] activity. Therefore, using thiochromanone as the leading compound to develop promising agrochemical candidates will become a reality. In our previous study, we reported a series of novel thiochromanone derivatives containing a sulfonyl hydrazone moiety (Figure 1) with moderate to good antibacterial activities against Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicolaby (Xoc), and Xanthomonas axonopodis pv. citri (Xac) [16]. Meanwhile, carboxamides, as important nitrogen-containing compounds in organic synthesis, have attracted considerable attention due to their broad range of biological activities, including antiviral [17], antibacterial [18,19], antifungal [20,21,22], herbicidal [23], and insecticidal [24,25] activity. Therefore, carboxamide could reasonably be considered as a potential active group in the design of new lead compounds.

In this study, using the botanical active component thiochromanone as the lead compound, a series of new thiochromanone derivatives containing a carboxamide moiety were designed and synthesized. We then determined the in vitro antibacterial activities of the derivatives against Xoo, Xoc, and Xac as well as their in vitro antifungal activities against Botryosphaeria dothidea (B. dothidea), Phomopsis sp., and Botrytis cinerea (B. cinerea).

2. Results and Discussion

2.1. Chemistry

The synthetic route to the target compounds 3a–3h and 4a–4x was carried out in three consecutive steps as shown in Scheme 1. Using a 4-substituted thiophenol as the starting material, the target compounds 3a–3h and 4a–4x were prepared with yields of 68–88% and their structures were determined by ¹H NMR, ¹³C NMR, and HRMS. The ¹H NMR, ¹³C NMR, and HRMS spectra for all the target compounds are shown in Supplementary Materials.

In the ¹H NMR spectra for compound 4d, two singlets at δ 11.73 and 10.36 ppm indicated the presence of –OH and –NH– groups, respectively; a chemical shift at 7.85–7.14 ppm indicated the presence of hydrogen atoms of the benzene ring in the thiochromanone group; two doublet-doublets at 3.32–3.12 ppm indicated the presence of CH₂ in the thiochromanone group. Meanwhile, in the ¹³C NMR spectra for compound 4d, a singlet at 168.30 ppm indicated the presence of C=O in the thiochromanone group; a doublet at 159.79 and 157.41 ppm indicated the presence of C=O; a singlet at 149.88 ppm indicated the presence of C=N in the thiochromanone group.

2.2. Biological Evaluations

The in vitro antibacterial activities of the racemic target compounds 3a–3h and 4a–4x against Xoo, Xoc, and Xac were determined by turbidimeter tests [26,27] and the bioassay results are listed in

Table 1. In vitro antibacterial activities of the target compounds 3a–3h and 4a–4x against Xoo, Xoc, and Xac at 200 and 100 μg/mL.

Compounds	Inhibition Rate (%) a
	Xoo		Xoc		Xac
	200 (μg/mL)	100 (μg/mL)	200 (μg/mL)	100 (μg/mL)	200 (μg/mL)	100 (μg/mL)
3a	56 ± 2.2	43 ± 1.7	50 ± 0.6	38 ± 1.9	42 ± 1.7	30 ± 2.3
3b	72 ± 1.6	54 ± 1.9	64 ± 1.1	49 ± 1.3	51 ± 2.2	41 ± 1.7
3c	65 ± 1.6	47 ± 1.5	57 ± 1.1	40 ± 2.2	48 ± 1.5	35 ± 2.2
3d	45 ± 2.1	34 ± 1.2	42 ± 2.0	32 ± 1.3	39 ± 1.1	26 ± 1.3
3e	42 ± 0.9	30 ± 1.2	35 ± 1.3	24 ± 1.5	31 ± 1.5	21 ± 1.1
3f	58 ± 1.5	41 ± 2.2	45 ± 1.5	33 ± 1.3	40 ± 1.5	28 ± 1.6
3g	50 ± 1.5	35 ± 1.5	40 ± 0.9	28 ± 1.3	36 ± 1.5	24 ± 3.0
3h	30 ± 0.6	15 ± 1.6	24 ± 1.6	15 ± 1.5	18 ± 1.6	10 ± 1.0
4a	78 ± 1.8	60 ± 1.4	70 ± 2.0	52 ± 0.9	53 ± 2.2	43 ± 1.4
4b	89 ± 1.8	72 ± 3.1	82 ± 2.1	61 ± 2.3	71 ± 1.9	56 ± 2.3
4c	82 ± 1.1	65 ± 2.1	75 ± 1.9	56 ± 1.8	65 ± 2.3	50 ± 1.8
4d	90 ± 1.1	80 ± 1.9	84 ± 2.3	69 ± 2.2	75 ± 1.1	59 ± 1.2
4e	100 ± 0.8	92 ± 1.3	100 ± 0.7	88 ± 2.3	94 ± 1.8	80 ± 2.3
4f	96 ± 0.8	84 ± 1.2	90 ± 1.2	75 ± 3.0	87 ± 2.1	78 ± 1.2
4g	85 ± 1.2	70 ± 2.3	77 ± 2.9	61 ± 1.1	68 ± 2.0	50 ± 1.9
4h	93 ± 2.0	80 ± 1.9	86 ± 1.8	74 ± 1.9	84 ± 2.1	71 ± 1.9
4i	90 ± 2.0	72 ± 1.7	80 ± 2.0	69 ± 1.7	72 ± 2.3	65 ± 1.5
4j	70 ± 1.2	52 ± 1.2	62 ± 1.7	48 ± 1.3	50 ± 1.9	40 ± 2.0
4k	80 ± 2.1	64 ± 1.8	74 ± 1.9	56 ± 1.7	63 ± 1.8	50 ± 1.7
4l	74 ± 1.8	56 ± 1.9	70 ± 1.8	50 ± 1.2	56 ± 1.1	43 ± 1.8
4m	59 ± 2.3	42 ± 0.6	51 ± 1.5	38 ± 2.5	45 ± 1.5	38 ± 2.2
4n	71 ± 1.5	58 ± 2.2	66 ± 1.5	47 ± 1.6	55 ± 1.4	41 ± 1.6
4o	65 ± 2.1	50 ± 1.4	60 ± 0.5	42 ± 1.5	51 ± 1.3	38 ± 1.2
4p	72 ± 1.8	54 ± 1.8	65 ± 1.2	48 ± 1.1	53 ± 1.7	41 ± 1.2
4q	83 ± 1.2	68 ± 1.9	77 ± 0.6	60 ± 1.6	67 ± 2.2	52 ± 1.1
4r	77 ± 2.0	60 ± 1.0	70 ± 1.9	53 ± 1.4	60 ± 1.6	48 ± 1.9
4s	65 ± 1.3	46 ± 1.7	54 ± 1.8	42 ± 1.4	50 ± 1.5	40 ± 1.5
4t	75 ± 3.0	60 ± 2.2	70 ± 1.5	55 ± 2.1	61 ± 1.5	46 ± 2.3
4u	71 ± 2.0	52 ± 1.3	64 ± 2.5	47 ± 1.6	55 ± 0.4	40 ± 1.6
4v	41 ± 1.5	20 ± 1.6	35 ± 1.5	18 ± 1.3	30 ± 1.5	13 ± 1.6
4w	52 ± 1.5	28 ± 1.3	41 ± 2.2	24 ± 1.6	38 ± 2.2	25 ± 1.5
4x	34 ± 1.6	24 ± 1.4	38 ± 1.5	20 ± 1.2	32 ± 1.6	20 ± 1.2
Bismerthiazol	70 ± 0.9	52 ± 1.6	57 ± 5.6	35 ± 6.8	55 ± 2.4	32 ± 3.3
Thiodiazole copper	63 ± 2.7	45 ± 2.7	35 ± 4.3	15 ± 2.1	36 ± 1.6	16 ± 2.2

^a Average of three replicates (mean ± SD).

and

Table 2. The EC₅₀ values of some of the target compounds against Xoo, Xoc, and Xac.

Compounds	EC50 (μg/mL) a
	Xoo	Xoc	Xac
4d	29 ± 1.2	34 ± 1.6	41 ± 2.2
4e	15 ± 1.2	19 ± 1.5	23 ± 1.3
4f	20 ± 1.5	28 ± 1.7	35 ± 1.7
4h	18 ± 1.7	25 ± 1.5	29 ± 1.7
4i	25 ± 1.6	30 ± 2.1	36 ± 1.6
Bismerthiazol	84 ± 2.9	151 ± 6.0	145 ± 2.7
Thiodiazole copper	109 ± 3.0	269 ± 7.1	230 ± 2.5

^a Average of three replicates (mean ± SD).

. As shown in Table 1, at 200 and 100 μg/mL, some of the target compounds exhibited moderate to good antibacterial activities against Xoo, Xoc, and Xac. Among of them, compound 4e at 200 μg/m, exhibited excellent in vitro antibacterial activity (100%) against Xoo, which was even better that that of Bismerthiazol and Thiodiazole copper. Meanwhile, as shown in Table 2, compounds 4d, 4e, 4f, 4h, and 4i displayed in vitro antibacterial activities against Xoo, Xoc, and Xac, with EC₅₀ values in the range of 15–29, 19–34, and 23–41 μg/mL, respectively, and their antibacterial activities were better than those of Bismerthiazol and Thiodiazole copper. In particular, compound 4d revealed the best in vitro antibacterial activity against Xoo, Xoc, and Xac, and its EC₅₀ values of 15, 19, and 23 μg/mL, respectively, were even better than those of Bismerthiazol and Thiodiazole copper as well as the other target compounds; however, lower than those of compound methyl 6-chloro-4-(2-((4-fluorophenyl)sulfonyl)hydrazineylidene)thiochromane-2-carboxylate [16].

Meanwhile, the in vitro antifungal activities of the racemic target compounds 3a–3h and 4a–4x against B. dothidea, Phomopsis sp., and B. cinerea were tested at 50 μg/mL by the mycelial growth rate method [28] and the results are listed in

Table 3. In vitro antifungal activities of the target compounds 3a–3h and 4a–4x against B. dothidea, Phomopsis sp., and B. cinerea at 50 μg/mL.

Compounds	Inhibition Rate (%) a
	B. dothidea	Phomopsis sp.	B. cinerea
3a	12 ± 1.3	34 ± 1.6	65 ± 1.6
3b	22 ± 2.2	60 ± 1.6	88 ± 1.5
3c	16 ± 2.2	42 ± 2.2	72 ± 1.4
3d	10 ± 2.2	29 ± 2.7	59 ± 1.5
3e	8 ± 2.1	30 ± 1.7	61 ± 1.3
3f	11 ± 1.1	35 ± 1.6	64 ± 4.3
3g	13 ± 2.5	48 ± 1.5	81 ± 3.5
3h	6 ± 1.2	20 ± 2.7	56 ± 1.6
4a	0	24 ± 2.3	50 ± 2.3
4b	4 ± 1.1	32 ± 2.3	61 ± 1.6
4c	0	17 ± 1.2	42 ± 1.7
4d	0	30 ± 1.6	60 ± 1.3
4e	8 ± 1.5	35 ± 2.7	69 ± 3.1
4f	0	21 ± 2.3	51 ± 2.3
4g	0	17 ± 2.0	41 ± 2.3
4h	0	22 ± 1.9	51 ± 1.0
4i	0	14 ± 3.0	35 ± 1.3
4j	0	8 ± 2.7	40 ± 1.3
4k	0	11 ± 1.3	46 ± 2.0
4l	0	4 ± 1.3	30 ± 2.2
4m	0	0	20 ± 1.3
4n	0	8 ± 2.2	25 ± 1.0
4o	0	0	14 ± 1.2
4p	0	0	10 ± 2.4
4q	0	4 ± 1.3	15 ± 2.1
4r	0	0	7 ± 1.3
4s	0	0	6 ± 1.3
4t	0	2 ± 1.1	10 ± 2.2
4u	0	0	3 ± 2.2
4v	0	0	4 ± 1.0
4w	0	0	6 ± 1.1
4x	0	0	2 ± 1.4
Pyrimethanil	80 ± 1.3	84 ± 1.3	81 ± 2.4
Carbendazim	86 ± 2.2	100 ± 0.3	100 ± 0.5

^a Average of three replicates (mean ± SD).

. As shown in Table 3, the target compounds revealed certain antifungal activities against B. dothidea, Phomopsis sp., and B. cinerea at 50 μg/mL with inhibition rate ranges of 0–22%, 0–60%, and 2–88%, respectively. In particular, compound 3b revealed moderate antifungal activity against B. dothidea at 50 μg/mL, and the inhibition rate reached 88%, which was even better than that of Pyrimethanil, however, lower than that of Carbendazim.

2.3. Structure–Activity Relationship Analysis

The structure–activity relationship (SAR) analysis was deduced on the basis of the antibacterial and antifungal activity values listed in Table 1, Table 2 and Table 3. First, the introduction of an oxime ether or oxime fragment to the 4-position of thiochromanone can increase the antibacterial activity against Xoo, Xoc, and Xac (4a > 3a and 4d > 3b); to the contrary, it can decrease the antifungal activity against B. dothidea, Phomopsis sp., and B. cinerea (3a > 4a and 3b > 4d). Second, on comparing the same substituent at the R₂ and R₃ substituent groups, with the presence of a −Cl group at the R₁ substituent group, the corresponding compounds presented better in vitro antibacterial and antifungal activities which followed the order 3a (R₁ = −Cl) > 3e (R₁ = −CH₃) and 4a (R₁ = −Cl) > 4m (R₁ = −CH₃). Third, compared with the same substituent at the R₁ and R₃ substituent groups, a smaller electron drawing group at the R₂ substituent group could cause an increase in the antibacterial and antifungal activities which followed the order 3b (R₂ = −F) > 3c (R₂ = −Cl) > 3a (R₂ = −H) > 3d (R₂ = −CH₃) and 4d (R₂ = −F) > 4g (R₂ = −Cl) > 4a (R₂ = −H) > 4j (R₂ = −CH₃). Forth, compared with the same substituent at the R₁ and R₂ substituent groups, a –CH₃ at the R₃ substituent group could cause an increase in the antibacterial and antifungal activities which followed the order 4b (R₃ = −CH₃) > 4c (R₃ = −C₂H₅) > 4a (R₃ = −H) and 4n (R₃ = −CH₃) > 4o (R₃ = −C₂H₅) > 4m (R₃ = −H).

3. Materials and Methods

3.1. General Information

The melting points were determined by an uncorrected WRX-4 binocular microscope (Shanghai Yice Tech. Instrument Co., Shanghai, China). ¹H NMR and ¹³C NMR spectral analyses were performed on a Bruker DRX-400 NMR spectrometer (Bruker, Rheinstetten, Germany). HRMS data were measured on a Waters Xevo G2-S QTOF mass spectrometer (Waters, Milford, MA, USA).

3.2. Chemical Synthesis

3.2.1. Preparation Procedure of Intermediate 2

As shown in Scheme 1, intermediate 2 was prepared according to our previously reported method [16].

3.2.2. Preparation Procedure for the Target Compounds 3a–3h

To a 50 mL round bottom flask equipped with a magnetic stirrer, intermediate 2 (0.02 mol) was dissolved in DMF (10 mL), and then substituted phenylamine (0.02 mol), dimethylaminopyridine (DMAP, 0.0002 mol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.03 mol) were added. The reactions were performed overnight at room temperature. Upon completion of the reaction (determined by TLC), the mixture was quenched with distilled water (50 mL) and the precipitated residues were filtered, dried under vacuum, and recrystallized from methanol to give the pure racemic target compounds 3a–3h. The physical characteristics, ¹H NMR, ¹³C NMR, and HRMS data for the target compounds 3a–3h are shown below. The ¹H NMR, ¹³C NMR, and HRMS spectra for the target compounds 3a–3h are shown in Supplementary Materials.

Data for 6-chloro-4-oxo-N-phenylthiochromane-2-carboxamide (3a). Yellow solid; mp 121–123 °C; Yield 76%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.33 (s, 1H, CONH), 7.88 (d, J = 2.4 Hz, 1H, Ph-H), 7.54–7.48 (m, 3H, Ph-H), 7.38 (d, J = 8.4 Hz, Ph-H), 7.28 (t, J = 8.0 Hz, 2H, Ph-H), 7.04 (t, J = 7.2 Hz, 1H, Ph-H), 4.36 (t, J = 4.4 Hz, 1H, SCH), 3.22 (dd, ¹J = 4.4 Hz, ²J= 17.2 Hz, 1H, CH₂), 3.13 (dd, ¹J = 4.8 Hz, ²J = 16.8 Hz, 1H, CH₂); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 191.56, 169.01, 139.05, 137.29, 133.56, 132.07, 130.52, 129.77, 129.33, 127.09, 124.10, 119.48, 42.62, 40.41; HRMS (ESI) [M + Na]⁺ calcd.. for C₁₆H₁₂ClNO₂S: 340.01695, found 340.01728.

Data for 6-chloro-N-(4-fluorophenyl)-4-oxothiochromane-2-carboxamide (3b). Brown solid; mp 211–213 °C; Yield 72%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.41 (s, 1H, CONH), 7.54–7.49 (m, 3H, Ph-H), 7.38 (d, J = 8.8 Hz, 1H, Ph-H), 7.16–7.09 (m, 2H, Ph-H), 4.35 (t, J = 4.8 Hz, 1H, SCH), 3.22 (dd, ¹J = 4.0 Hz, ²J = 16.8 Hz, 1H, CH₂), 3.13 (dd, ¹J = 4.8 Hz, ²J = 16.8 Hz, 1H, CH₂); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 191.53, 168.91, 158.60 (d, J = 239.0 Hz), 137.24, 135.45, 135.45, 135.43, 133.57, 132.07, 130.55, 129.76, 127.11, 121.33, 121.25, 116.04, 115.81, 42.58, 40.42; HRMS (ESI) [M + Na]⁺ calcd.. for C₁₆H₁₁ClFNO₂S: 358.00753, found 358.00755.

Data for 6-chloro-N-(4-chlorophenyl)-4-oxothiochromane-2-carboxamide (3c). Brown solid; mp 234–235 °C; Yield 81%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.43 (s, 1H, CONH), 7.88 (d, J = 2.4 Hz, 1H, Ph-H), 7.54–7.49 (m, 3H, Ph-H), 7.38 (d, J = 8.4 Hz, 1H, Ph-H), 7.16–7.11 (m, 2H, Ph-H), 4.35 (t, J = 4.4 Hz, 1H, SCH), 3.22 (dd, ¹J = 4.4 Hz, ²J = 16.8 Hz, 1H, CH₂), 3.13 (dd, ¹J = 4.4 Hz, ²J = 16.8 Hz, 1H, CH₂); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 191.53, 168.91, 159.80, 157.41, 137.23, 135.42, 133.56, 132.06, 130.55, 129.76, 127.11, 121.32, 116.03, 42.58, 40.41; HRMS (ESI) [M + Na]⁺ calcd. for C₁₆H₁₁Cl₂NO₂S: 373.97798, found 373.98073.

Data for 6-chloro-4-oxo-N-(p-tolyl)thiochromane-2-carboxamide (3d). Brown solid; mp 216–218 °C; Yield 74%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.24 (s, 1H, CONH), 7.88 (d, J = 2.8 Hz, 1H, Ph-H), 7.52 (dd, ¹J = 2.4 Hz, ²J = 8.4 Hz, 1H, Ph-H), 7.38 (d, J = 8.8 Hz, 3H, Ph-H), 7.08 (d, J = 8.0 Hz, 2H, Ph-H), 4.33 (t, J = 4.4 Hz, 1H, SCH), 3.21 (dd, ¹J = 4.4 Hz, ²J = 16.8 Hz, 1H, CH₂), 3.12 (dd, ¹J = 4.8 Hz, ²J = 16.8 Hz, 1H, CH₂), 2.23 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 191.57, 168.75, 137.35, 136.54, 133.53, 133.06, 132.09, 130.49, 129.74, 129.69, 127.08, 119.48, 40.60, 40.42, 20.88; HRMS (ESI) [M + Na]⁺ calcd. for C₁₇H₁₄ClNO₂S: 354.03260, found 354.03258.

Data for 6-methyl-4-oxo-N-phenylthiochromane-2-carboxamide (3e). Yellow solid; mp 178–179 °C; Yield 75%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.32 (s, 1H, CONH), 7.79 (d, J = 0.8 Hz, 1H, Ph-H), 7.51 (dd, ¹J = 0.8 Hz, ²J = 8.4 Hz, 2H, Ph-H), 7.30–7.26 (m, 3H, Ph-H), 7.20 (d, J = 8.0 Hz, 1H, Ph-H), 7.04 (t, J = 7.6 Hz, 1H, Ph-H), 4.32 (t, J = 4.8 Hz, 1H, SCH), 3.16 (dd, ¹J = 4.0 Hz, ²J = 16.4 Hz, 1H, CH₂), 3.09 (dd, ¹J = 5.2 Hz, ²J = 16.8 Hz, 1H, CH₂), 2.29 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 192.57, 169.25, 138.13, 135.29, 134.85, 134.78, 130.57, 129.21, 128.24, 127.59, 127.55, 121.02, 42.79, 40.98, 20.83; HRMS (ESI) [M + Na]⁺ calcd. for C₁₇H₁₅NO₂S: 320.07157, found 320.07151.

Data for N-(4-fluorophenyl)-6-methyl-4-oxothiochromane-2-carboxamide (3f). Yellow solid; mp 206–207 °C; Yield 79%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.39 (s, 1H, CONH), 7.79 (d, J = 1.2 Hz, 1H, Ph-H), 7.29 (dd, ¹J = 1.6 Hz, ²J = 8.0 Hz, 1H, Ph-H), 7.20 (d, J = 8.0 Hz, 1H, Ph-H), 7.16–7.10 (m, 2H, Ph-H), 4.31 (t, J = 4.4 Hz, 1H, SCH), 3.17 (dd, ¹J = 4.4 Hz, ²J = 16.8 Hz, 1H, CH₂), 3.09 (dd, ¹J = 4.8 Hz, ²J = 16.8 Hz, 1H, CH₂), 2.29 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 192.62, 168.99, 158.55 (d, J = 239.0 Hz), 135.58, 135.56, 135.25, 134.91, 134.83, 130.58, 128.24, 127.58, 121.25, 121.18, 115.99, 115.77, 42.79, 41.06, 20.82; HRMS (ESI) [M + Na]⁺ calcd. for C₁₇H₁₄FNO₂S: 338.06215, found 338.06226.

Data for N-(4-chlorophenyl)-6-methyl-4-oxothiochromane-2-carboxamide (3g). Yellow solid; mp 209–210 °C; Yield 70%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.46 (s, 1H, CONH), 7.78 (s, 1H, Ph-H), 7.53 (d, J = 8.8 Hz, 2H, Ph-H), 7.34 (d, J = 8.8 Hz, 2H, Ph-H), 7.29 (d, J = 8.0 Hz, 1H, Ph-H), 7.20 (d, J = 8.0 Hz, 1H, Ph-H), 4.31 (t, J = 4.8 Hz, 1H, SCH), 3.16 (dd, ¹J = 4.0 Hz, ²J = 16.8 Hz, 1H, CH₂), 3.09 (dd, ¹J = 4.8 Hz, ²J = 16.8 Hz, 1H, CH₂), 2.29 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 192.57, 169.25, 138.13, 135.29, 134.85, 134.78, 130.57, 129.21, 128.24, 127.59, 121.02, 42.79, 40.98, 20.83; HRMS (ESI) [M + Na]⁺ calcd. for C₁₇H₁₄ClNO₂S: 354.03260, found 354.03244.

Data for 6-methyl-4-oxo-N-(p-tolyl)thiochromane-2-carboxamide (3h). Yellow solid; mp 199–200 °C; Yield 68%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.22 (s, 1H, CONH), 7.78 (d, J = 1.2 Hz, 1H, Ph-H), 7.39 (d, J = 8.4 Hz, 2H, Ph-H), 7.28 (dd, ¹J = 1.6 Hz, ²J = 8.0 Hz, 1H, Ph-H), 7.19 (d, J = 8.0 Hz, 1H, Ph-H), 7.08 (d, J = 8.0 Hz, 2H, Ph-H), 4.29 (t, J = 4.8 Hz, 1H, SCH), 3.15 (d, J = 4.0 Hz, ²J = 16.8 Hz, 1H, CH₂), 3.07 (dd, ¹J = 4.2 Hz, ²J = 16.8 Hz, 1H, CH₂), 2.29 (s, 3H, CH₃), 2.23 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 192.66, 168.83, 136.68, 135.18, 135.03, 134.81, 132.93, 130.60, 129.65, 128.21, 127.56, 119.43, 42.82, 41.11, 20.88, 20.83; HRMS (ESI) [M + Na]⁺ calcd. for C₁₈H₁₇NO₂S: 334.08722, found 334.08714.

3.2.3. Preparation Procedure for the Target Compounds 4a–4x

To a 50 mL round bottom flask equipped with a magnetic stirrer, a mixture of compound 3 (10 mmol), R₃ONH₂·HCl (15 mmol), pyridine (10 mL), and ethanol (10 mL) were added and reacted under a reflux temperature for 3–5 h. Upon completion of the reaction (determined by TLC), the mixture was cooled to room temperature and the precipitated residues were dried under vacuum and recrystallized from ethanol to give the pure racemic target compounds 4a–4x. The physical characteristics, ¹H NMR, ¹³C NMR, and HRMS data for the target compounds 4a–4x are shown below. The ¹H NMR, ¹³C NMR, and HRMS spectra for the target compounds 4a–4x are shown in Supplementary Materials.

Data for 6-chloro-4-(hydroxyimino)-N-phenylthiochromane-2-carboxamide (4a). White solid; mp 235–237 °C; Yield 80%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 11.73 (s, 1H, OH), 10.29 (s, 1H, CONH), 7.86 (t, J = 1.6 Hz, 1H, Ph-H), 7.54 (d, J = 1.2 Hz, 1H, Ph-H), 7.52 (s, 1H, Ph-H), 7.31 (d, J = 1.2 Hz, 2H, Ph-H), 7.29 (d, J = 8.0 Hz, 2H, Ph-H), 7.05 (t, J = 7.2 Hz, 1H, Ph-H), 4.18 (dd, ¹J = 4.8 Hz, ²J = 7.2 Hz, 1H, SCH), 3.29 (dd, ¹J = 7.2 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.15 (dd, ¹J = 4.4 Hz, ²J = 18.0 Hz, 1H, CH₂); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.38, 149.92, 139.16, 132.55, 131.57, 130.63, 130.26, 129.28, 129.05, 129.45, 124.05, 119.60, 42.67, 28.32; HRMS (ESI) [M + Na]⁺ calcd. for C₁₆H₁₃ClN₂O₂S: 355.02785, found 355.02769.

Data for 6-chloro-4-(methoxyimino)-N-phenylthiochromane-2-carboxamide (4b). White solid; mp 197–198 °C; Yield 85%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.45 (s, 1H, CONH), 7.86 (d, J = 1.6 Hz, 1H, Ph-H), 7.53 (d, J = 8.0 Hz, 2H, Ph-H), 7.36–7.27 (m, 4H, Ph-H), 7.05 (t, J = 7.2 Hz, 1H, Ph-H), 4.24 (t, J = 5.6 Hz, 1H, SCH), 4.00 (s, 3H, CH₃), 3.33 (dd, ¹J = 6.4 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.09 (dd, ¹J = 4.4 Hz, ²J = 18.0 Hz, 1H, CH₂); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.37, 150.63, 143.08, 139.19, 131.91, 131.39, 130.66, 130.36, 129.62, 129.25, 127.44, 124.68, 124.02, 119.57, 62.79, 40.03, 28.54; HRMS (ESI) [M + Na]⁺ calcd. for C₁₇H₁₅ClN₂O₂S: 369.04350, found 369.04279.

Data for 6-chloro-4-(ethoxyimino)-N-phenylthiochromane-2-carboxamide (4c). White solid; mp 191–192 °C; Yield 88%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.31 (s, 1H, CONH), 7.87 (d, J = 1.2 Hz, 1H, Ph-H), 7.52 (d, J = 7.6 Hz, 2H, Ph-H), 7.36–7.27 (m, 4H, Ph-H), 7.05 (t, J = 7.2 Hz, 1H, Ph-H), 4.25 (q, J = 7.2 Hz, 2H, CH₂CH₃), 4.18 (dd, ¹J = 4.8 Hz, ²J = 6.8 Hz, 1H, SCH), 3.32 (dd, ¹J = 6.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.11 (dd, ¹J = 4.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 1.29 (t, J = 7.2 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.29, 150.34, 139.13, 131.84, 131.71, 130.71, 130.38, 129.54, 129.28, 124.70, 119.57, 70.35, 42.26, 28.78, 15.14; HRMS (ESI) [M + Na]⁺ calcd. for C₁₈H₁₇ClN₂O₂S: 383.05915, found 383.05883.

Data for 6-chloro-N-(4-fluorophenyl)-4-(hydroxyimino)thiochromane-2-carboxamide (4d). Light yellow solid; mp 225–227 °C; Yield 74%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 11.73 (s, 1H, OH), 10.36 (s, 1H, CONH), 7.85 (d, J = 1.2 Hz, 1H, Ph-H), 7.57–7.53 (m, 2H, Ph-H), 7.31 (d, J = 0.8 Hz, 2H, Ph-H), 7.14 (t, J = 8.8 Hz, 2H, Ph-H), 4.16 (dd, ¹J = 4.4 Hz, ²J = 7.2 Hz, 1H, SCH), 3.28 (dd, ¹J = 7.2 Hz, ²J = 17.6 Hz, 1H, CH₂), 3.15 (dd, ¹J = 4.4 Hz, ²J = 18.0 Hz, 1H, CH₂); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.30, 158.60 (d, J = 239.0 Hz), 149.88, 135.55, 132.55, 131.51, 130.65, 130.26, 129.19, 129.06, 124.49, 121.44, 121.36, 115.98, 115.76, 42.61, 28.31; HRMS (ESI) [M + Na]⁺ calcd. for C₁₆H₁₂ClFN₂O₂S: 373.01843, found 373.01799.

Data for 6-chloro-N-(4-fluorophenyl)-4-(methoxyimino)thiochromane-2-carboxamide (4e). White solid; mp 195–196 °C; Yield 68%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.35 (s, 1H, CONH), 7.86 (d, J = 1.6 Hz, 1H, Ph-H), 7.55–7.51 (m, 2H, Ph-H), 7.36–7.31 (m, 2H, Ph-H), 7.15–7.11 (m, 2H, Ph-H), 4.16 (dd, ¹J = 4.8 Hz, ²J = 6.8 Hz, 1H, SCH), 4.00 (s, 3H, CH₃), 3.32 (dd, ¹J = 6.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.10 (dd, ¹J = 4.8 Hz, ²J = 18.0 Hz, 1H, CH₂); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.22, 158.60 (d, J = 239.0 Hz), 150.59, 135.50, 131.79, 131.40, 130.72, 130.37, 129.64, 124.70, 121.3841, 121.34, 115.98, 115.76, 62.78, 41.99, 28.52; HRMS (ESI) [M + Na]⁺ calcd. for C₁₇H₁₄ClFN₂O₂S: 387.03408, found 387.03343.

Data for 6-chloro-4-(ethoxyimino)-N-(4-fluorophenyl)thiochromane-2-carboxamide (4f). White solid; mp 200–201 °C; Yield 78%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.37 (s, 1H, CONH), 7.87 (d, J = 1.2 Hz, 1H, Ph-H), 7.56–7.52 (m, 2H, Ph-H), 7.36–7.31 (m, 2H, Ph-H), 7.16–7.11 (m, 2H, Ph-H), 4.25 (dd, J = 7.2 Hz, 2H, CH₂CH₃), 4.17 (dd, ¹J = 4.8 Hz, ²J = 7.2 Hz, 1H, SCH), 3.31 (dd, ¹J = 7.2 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.12 (dd, ¹J = 4.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 1.29 (t, J = 7.2 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.21, 158.60 (d, J = 239.0 Hz), 150.31, 135.53, 131.78, 131.71, 130.74, 130.78, 129.55, 124.71, 121.38 (d, J = 8.0 Hz), 115.98, 115.76, 70.35, 42.20, 28.77, 15.14; HRMS (ESI) [M + Na]⁺ calcd. for C₁₈H₁₆CLFN₂O₂S: 401.04973, found 401.04886.

Data for 6-chloro-N-(4-chlorophenyl)-4-(hydroxyimino)thiochromane-2-carboxamide (4g). Light yellow solid; mp 239–240 °C; Yield 79%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 11.73 (s, 1H, OH), 10.44 (s, 1H, CONH), 7.85 (s, 1H, Ph-H), 7.56 (d, J = 8.8 Hz, 2H, Ph-H), 7.35 (d, J = 8.8 Hz, 1H, Ph-H), 7.31 (d, J = 1.2 Hz, 3H, Ph-H), 4.17 (dd, ¹J = 4.8 Hz, ²J = 7.2 Hz, 1H, SCH), 3.30 (dd, ¹J = 6.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.13 (dd, ¹J = 4.4 Hz, ²J = 18.0 Hz, 1H, CH₂); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.57, 149.83, 138.11, 132.56, 131.35, 130.86, 130.27 129.19, 129.06, 127.60, 124.44, 121.16, 42.51, 28.20; HRMS (ESI) [M + Na]⁺ calcd. for C₁₆H₁₂Cl₂N₂O₂S: 388.98888, found 388.98813.

Data for 6-chloro-N-(4-chlorophenyl)-4-(methoxyimino)thiochromane-2-carboxamide (4h). Light pink solid; mp 216–218 °C; Yield 76%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.45 (s, 1H, CONH), 7.86 (d, J = 1.6 Hz, 1H, Ph-H), 7.54 (dd, ¹J = 2.4 Hz, ²J = 7.2 Hz, 2H, Ph-H), 7.36–7.33 (m, 4H, Ph-H), 4.17 (dd, ¹J = 1.6 Hz, ²J = 5.6 Hz, 1H, SCH), 4.00 (s, 3H, CH₃), 3.34 (dd, ¹J = 6.4 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.07 (dd, ¹J = 4.4 Hz, ²J = 18.0 Hz, 1H, CH₂); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.50, 150.54, 138.08, 131.63, 131.40, 130.75, 129.65, 129.20, 127.60, 124.69, 121.14, 62.79, 41.90, 28.41; HRMS (ESI) [M + Na]⁺ calcd. for C₁₆H₁₂Cl₂N₂O₂S: 403.00453, found 403.00421.

Data for 6-chloro-N-(4-chlorophenyl)-4-(ethoxyimino)thiochromane-2-carboxamide (4i). White solid; mp 200–202 °C; Yield 79%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.45 (s, 1H, CONH), 7.87 (d, J = 1.2 Hz, 1H, Ph-H), 7.55 (dd, ¹J = 2.0 Hz, ²J = 6.4 Hz, 2H, Ph-H), 7.34 (dd, ¹J = 2.0 Hz, ²J = 9.2 Hz, 4H, Ph-H), 4.25 (q, J = 7.2 Hz, 2H, CH₂CH₃), 4.17 (dd, ¹J = 4.8 Hz, ²J = 6.8 Hz, 1H, SCH), 3.33 (dd, ¹J = 6.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.10 (dd, ¹J = 4.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 1.28 (t, J = 7.2 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.48, 150.26, 138.09, 131.71, 131.62, 130.77, 130.39, 129.55, 129.20, 127.60, 124.70, 121.14, 70.36, 42.11, 28.66, 15.15; HRMS (ESI) [M + Na]⁺ calcd. for C₁₈H₁₆Cl₂N₂O₂S: 417.02018, found 417.01923.

Data for 6-chloro-4-(hydroxyimino)-N-(p-tolyl)thiochromane-2-carboxamide (4j). While solid; mp 249–250 °C; Yield 73%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 11.72 (s, 1H, OH), 10.20 (s, 1H, CONH), 7.85 (t, J = 1.6 Hz, 1H, Ph-H), 7.41 (d, J = 8.4 Hz, Ph-H), 7.31 (d, J = 1.2 Hz, 2H, Ph-H), 7.10 (d, J = 8.0 Hz, 2H, Ph-H), 4.15 (dd, ¹J = 4.4 Hz, ²J = 7.2 Hz, 1H, SCH), 3.26 (dd, ¹J = 7.2 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.14 (dd, ¹J = 4.4 Hz, ²J = 18.0 Hz, 1H, CH₂), 2.24 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.10, 149.94, 136.64, 133.01, 132.53, 131.67, 130.60, 129.64, 129.04, 124.45, 119.61, 42.73, 28.37, 20.90; HRMS (ESI) [M + Na]⁺ calcd. for C₁₇H₁₅ClN₂O₂S: 369.04350, found 369.04307.

Data for 6-chloro-4-(methoxyimino)-N-(p-tolyl)thiochromane-2-carboxamide (4k). While solid; mp 215–216 °C; Yield 79%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.21 (s, 1H, CONH), 7.86 (d, J = 1.6 Hz, 1H, Ph-H), 7.40 (d, J = 8.8 Hz, 2H, Ph-H), 7.36–7.30 (m, 2H, Ph-H), 7.09 (d, J = 8.4 Hz, 2H, Ph-H), 4.15 (dd, ¹J = 4.8 Hz, ²J = 6.8 Hz, 1H, SCH), 3.98 (s, 3H, CH₃), 3.31 (dd, ¹J = 6.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.09 (dd, ¹J = 4.4 Hz, ²J = 18.0 Hz, CH₂), 2.23 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.03, 150.65, 136.62, 133.01, 131.95, 131.38, 130.66, 130.35, 129.64, 124.69, 119.58, 62.78, 42.10, 28.57, 20.89; HRMS (ESI) [M + Na]⁺ calcd. for C₁₈H₁₇ClN₂O₂S: 383.05915, found 383.05886.

Data for 6-chloro-4-(ethoxyimino)-N-(p-tolyl)thiochromane-2-carboxamide (4l). Light yellow solid; mp 196–198 °C; Yield 72%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.21 (s, 1H, CONH), 7.86 (d, J = 2.0 Hz, 1H, Ph-H), 7.40 (d, J = 8.4 Hz, 2H, Ph-H), 7.35–7.32 (m, 2H, Ph-H), 7.09 (d, J = 8.4 Hz, 2H, Ph-H), 4.24 (q, J = 6.8 Hz, 2H, CH₂CH₃), 4.16 (dd, ¹J = 4.8 Hz, ²J = 7.2 Hz, 1H, SCH), 3.29 (dd, ¹J = 7.2 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.11 (dd, ¹J = 4.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 2.25 (s, 3H, CH₃), 1.28 (t, J = 7.2 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.01, 150.37, 136.62, 133.01, 131.95, 131.69, 130.68, 130.36, 129.64, 129.53, 124.70, 119.59, 70.34, 42.32, 28.83, 20.90, 15.14; HRMS (ESI) [M + Na]⁺ calcd. for C₁₉H₁₉ClN₂O₂S: 397.07480, found 397.07433.

Data for 4-(hydroxyimino)-6-methyl-N-phenylthiochromane-2-carboxamide (4m). Light pink solid; mp 237–238 °C; Yield 79%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 11.46 (s, 1H, OH), 10.26 (s, 1H, CONH), 7.71 (s, 1H, Ph-H), 7.54 (d, J = 8.0 Hz, 2H, Ph-H), 7.30 (t, J = 8.0 Hz, 2H, Ph-H), 7.15 (d, J = 8.0 Hz, 1H, Ph-H), 7.09–7.03 (m, 2H, Ph-H), 4.10 (t, J = 6.4 Hz, 1H, SCH), 3.20 (d, J = 1.6 Hz, 2H, CH₂), 2.28 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.48, 150.85, 139.22, 135.40, 130.73, 130.26, 129.42, 129.26, 128.41, 125.74, 124.00, 119.60, 43.42, 29.13, 21.19; HRMS (ESI) [M + Na]⁺ calcd. for C₁₇H₁₆N₂O₂S: 335.08247, found 335.08237.

Data for 4-(methoxyimino)-6-methyl-N-phenylthiochromane-2-carboxamide (4n). Yellow solid; mp 142–144 °C; Yield 82%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.26 (s, 1H, CONH), 7.71 (s, 1H, Ph-H), 7.53 (d, J = 7.6 Hz, 2H, Ph-H), 7.29 (t, J = 7.6 Hz, 2H, Ph-H), 7.17 (d, J = 8.0 Hz, 1H, Ph-H), 7.11 (dd, ¹J = 1.2 Hz, ²J = 8.0 Hz, 1H, Ph-H), 7.05 (t, J = 7.2 Hz, 1H, Ph-H), 4.11 (dd, ¹J = 4.8 Hz, ²J = 7.2 Hz, 1H, SCH), 3.96 (s, 3H, CH₃), 3.26 (dd, ¹J = 7.6 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.15 (dd, ¹J = 4.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 2.28 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.38, 151.69, 139.21, 135.56, 130.86, 129.70, 129.63, 129.26, 128.53, 125.93, 124.00, 119.57, 62.49, 42.77, 29.37, 21.10; HRMS (ESI) [M + Na]⁺ calcd. for C₁₈H₁₈N₂O₂S: 349.09812, found 349.09763.

Data for 4-(ethoxyimino)-6-methyl-N-phenylthiochromane-2-carboxamide (4o). Yellow solid; mp 178–180 °C; Yield 80%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.27 (s, 1H, CONH), 7.71 (s, 1H, Ph-H), 7.53 (d, J = 7.6 Hz, 2H, Ph-H), 7.29 (t, J = 7.2 Hz, 2H, Ph-H), 7.17 (d, J = 8.0 Hz, 1H, Ph-H), 7.11 (dd, ¹J = 1.6 Hz, ²J = 8.0 Hz, 1H, Ph-H), 7.05 (t, J = 7.2 Hz, 1H, Ph-H), 4.22 (q, J = 7.2 Hz, 2H, CH₂CH₃), 4.11 (dd, ¹J = 4.8 Hz, ²J = 7.6 Hz, 1H, SCH), 3.24 (dd, ¹J = 8.0 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.16 (dd, ¹J = 4.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 2.28 (s, 3H, CH₃), 1.28 (t, J = 6.8 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.37, 151.40, 139.20, 135.58, 130.77, 129.94, 129.70, 129.26, 128.55, 125.96, 124.01, 119.57, 69.99, 43.02, 29.64, 21.13, 15.19; HRMS (ESI) [M + Na]⁺ calcd. for C₁₉H₂₀N₂O₂S: 363.11377, found 363.11337.

Data for N-(4-fluorophenyl)-4-(hydroxyimino)-6-methylthiochromane-2-carboxamide (4p). White solid; mp 227–228 °C; Yield 75%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 11.46 (s, 1H, OH), 10.33 (s, 1H, CONH), 7.70 (s, 1H, Ph-H), 7.57–7.54 (m, 2H, Ph-H), 7.17–7.07 (m, 4H, Ph-H), 4.09 (t, J = 6.8 Hz, 1H, SCH), 3.20 (d, J = 6.8 Hz, 2H, CH₂), 2.28 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.40, 158.58 (d, J = 239.0 Hz), 150.82, 135.43, 130.73, 130.27, 129.36, 128.41, 125.74, 121.43, 121.35, 115.96, 115.74, 43.33, 29.11, 21.19; HRMS (ESI) [M + Na]⁺ calcd. for C₁₇H₁₅FN₂O₂S: 353.07305, found 353.07262.

Data for N-(4-fluorophenyl)-4-(methoxyimino)-6-methylthiochromane-2-carboxamide (4q). White solid; mp 184–185 °C; Yield 70%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.32 (s, 1H, CONH), 7.71 (s, 1H, Ph-H), 7.56–7.52 (m, 2H, Ph-H), 7.18–7.10 (m, 4H, Ph-H), 4.09 (dd, ¹J = 4.8 Hz, ²J = 7.6 Hz, 1H, SCH), 3.96 (s, 3H, CH₃), 3.25 (dd, ¹J = 7.6 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.14 (dd, ¹J = 4.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 2.28 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.30, 158.60 (d, J = 239.0 Hz), 151.66, 135.60, 130.87, 129.63, 128.54, 125.94, 12140, 121.32, 115.96, 115.74, 62.50, 42.68, 29.35, 21.10; HRMS (ESI) [M + Na]⁺ calcd. for C₁₈H₁₇FN₂O₂S: 367.08870, found 367.08810.

Data for 4-(ethoxyimino)-N-(4-fluorophenyl)-6-methylthiochromane-2-carboxamide (4r). White solid; mp 168–170 °C; Yield 77%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.33 (s, 1H, CONH), 7.71 (s, 1H, Ph-H), 7.56–7.53 (m, 2H, Ph-H), 7.18–7.10 (m, 4H, Ph-H), 4.22 (q, J = 7.2 Hz, 2H, CH₂CH₃), 4.09 (dd, ¹J = 4.8 Hz, ²J = 7.6 Hz, 1H, SCH), 3.24 (dd, ¹J = 7.6 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.16 (dd, ¹J = 4.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 2.28 (s, 3H, CH₃), 1.28 (t, J = 7.2 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.29, 158.58 (d, J = 240.0 Hz), 135.61, 130.78, 129.94, 128.55, 125.96, 121.41, 121.33, 115.96, 115.74, 70.00, 42.92, 29.61, 21.12, 15.19; HRMS (ESI) [M + Na]⁺ calcd. for C₁₉H₁₉FN₂O₂S: 381.10435, found 381.10381.

Data for N-(4-chlorophenyl)-4-(hydroxyimino)-6-methylthiochromane-2-carboxamide (4s). White solid; mp 235–236 °C; Yield 78%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 11.46 (s, 1H, OH), 10.41 (s, 1H, CONH), 7.71 (s, 1H, Ph-H), 7.58–7.55 (m, 2H, Ph-H), 7.37–7.32 (m, 2H, Ph-H), 7.15 (d, J = 8.0 Hz, 1H, Ph-H), 7.08 (dd, ¹J = 2.0 Hz, ²J = 8.0 Hz, 1H, Ph-H), 4.01 (dd, ¹J = 5.6 Hz, ²J = 7.2 Hz, 1H, SCH), 3.22 (dd, ¹J = 7.2 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.17 (dd, ¹J = 5.2 Hz, ²J = 18.0 Hz, 1H, CH₂), 2.28 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.68, 150.77, 138.18, 135.45, 130.75, 130.27, 129.18, 128.41, 127.55, 125.73, 121.16, 43.22, 29.00, 21.19; HRMS (ESI) [M + Na]⁺ calcd. for C₁₇H₁₅ClN₂O₂S: 369.04350, found 369.04330.

Data for N-(4-chlorophenyl)-4-(methoxyimino)-6-methylthiochromane-2-carboxamide (4t). White solid; mp 197–199 °C; Yield 70%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.41 (s, 1H, CONH), 7.71 (s, 1H, Ph-H), 7.55 (dd, ¹J = 2.0 Hz, ²J = 6.8 Hz, 2H, Ph-H), 7.34 (dd, ¹J = 2.0 Hz, ²J = 6.4 Hz, 2H, Ph-H), 7.16 (d, J = 8.0 Hz, 1H, Ph-H), 7.11 (dd, ¹J = 1.2 Hz, ²J = 8.0 Hz, 1H, Ph-H), 4.11 (dd, ¹J = 4.8 Hz, ²J = 7.2 Hz, 1H, SCH), 3.96 (s, 3H, CH₃), 3.27 (dd, ¹J = 7.2 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.13 (dd, ¹J = 4.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 2.28 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.59, 151.61, 138.16, 135.62, 130.88, 129.64, 129.46, 129.17, 128.53, 127.54, 125.92, 121.13, 62.50, 42.58, 29.23, 21.10; HRMS (ESI) [M + Na]⁺ calcd. for C₁₈H₁₇ClN₂O₂S: 383.05915, found 383.05863.

Data for N-(4-chlorophenyl)-4-(ethoxyimino)-6-methylthiochromane-2-carboxamide (4u). White solid; mp 169–170 °C; Yield 79%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.41 (s, 1H, CONH), 7.71 (s, 1H, Ph-H), 7.56 (dd, ¹J = 2.0 Hz, ²J = 7.2 Hz, 2H, Ph-H), 7.36 (d, J = 3.2 Hz, 1H, Ph-H), 7.34 (d, J = 2.0 Hz, 1H, Ph-H), 7.17 (d, J = 8.0 Hz, 1H, Ph-H), 7.10 (d, J = 8.4 Hz, 1H, Ph-H), 4.22 (q, J = 7.2 Hz, 2H, CH₂CH₃), 4.10 (dd, ¹J = 4.8 Hz, ²J = 7.6 Hz, 1H, SCH), 3.26 (dd, ¹J = 7.6 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.15 (dd, ¹J = 4.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 2.28 (s, 3H, CH₃), 1.28 (t, J = 7.2 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.57, 151.31, 138.16, 135.63, 130.78, 129.95, 129.46, 129.18, 128.54, 127.55, 125.95, 121.14, 70.00, 42.81, 29.49, 21.13, 15.19; HRMS (ESI) [M + Na]⁺ calcd. for C₁₉H₁₉ClN₂O₂S: 397.07480, found 397.07421.

Data for 4-(hydroxyimino)-6-methyl-N-(p-tolyl)thiochromane-2-carboxamide (4v). White solid; mp 237–239 °C; Yield 76%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 11.45 (s, 1H, OH), 10.17 (s, 1H, CONH), 7.70 (s, 1H, Ph-H), 7.42 (d, J = 8.4 Hz, 2H, Ph-H), 7.17–7.07 (m, 3H, Ph-H), 7.15 (d, J = 8.0 Hz, 1H, Ph-H), 7.17–7.07 (m, 4H, Ph-H), 4.08 (dd, ¹J = 5.6 Hz, ²J = 7.2 Hz, 1H, SCH), 3.22 (dd, ¹J = 5.6 Hz, ²J = 18.4 Hz, 1H, CH₂), 3.16 (dd, ¹J = 8.0 Hz, ²J = 18.0 Hz, 1H, CH₂), 2.27 (s, 3H, CH₃), 2.24 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.20, 150.88, 136.71, 135.37, 132.95, 130.72, 130.25, 129.62, 128.40, 125.74, 119.61, 43.50, 29.20, 21.19, 20.90; HRMS (ESI) [M + Na]⁺ calcd. for C₁₈H₁₈N₂O₂S: 349.09812, found 349.09779.

Data for 4-(methoxyimino)-6-methyl-N-(p-tolyl)thiochromane-2-carboxamide (4w). White solid; mp 205–207 °C; Yield 73%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.17 (s, 1H, CONH), 7.71 (s, 1H, Ph-H), 7.41 (d, J = 8.4 Hz, 2H, Ph-H), 7.16 (d, J = 8.0 Hz, 1H, Ph-H), 7.12 (d, J = 1.6 Hz, 1H, Ph-H), 7.09 (d, J = 8.0 Hz, 2H, Ph-H), 4.09 (dd, ¹J = 5.2 Hz, ²J = 8.0 Hz, 1H, SCH), 3.96 (s, 3H, CH₃), 3.24 (dd, ¹J = 7.6 Hz, ²J = 18.0 Hz, 1H, CH₂), 3.14 (dd, ¹J = 4.8 Hz, ²J = 18.0 Hz, 1H, CH₂), 2.28 (s, 3H, CH₃), 2.24 (s, 3H, CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.10, 151.72, 136.69, 135.53, 132.95, 130.86, 129.81, 129.62, 128.52, 125.94, 119.59, 62.49, 42.85, 29.43, 21.10, 20.90; HRMS (ESI) [M + Na]⁺ calcd. for C₁₉H₂₀N₂O₂S: 363.11377, found 363.11303.

Data for 4-(ethoxyimino)-6-methyl-N-(p-tolyl)thiochromane-2-carboxamide (4x). White solid; mp 198–200 °C; Yield 78%; ¹H NMR (400 MHz, DMSO-d₆, ppm) δ: 10.18 (s, 1H, CONH), 7.71 (s, 1H, Ph-H), 7.42 (d, J = 8.4 Hz, 2H, Ph-H), 7.16 (d, J = 8.0 Hz, 1H, Ph-H), 7.11 (d, J = 1.6 Hz, 1H, Ph-H), 7.09 (d, J = 8.4 Hz, 2H, Ph-H), 4.22 (q, J = 6.8 Hz, 2H, CH₂CH₃), 4.09 (dd, ¹J = 5.2 Hz, ²J = 7.6 Hz, 1H, SCH), 3.23 (dd, ¹J = 8.0 Hz, ²J = 18.4 Hz, 1H, CH₂), 3.16 (dd, ¹J = 5.2 Hz, 2J= 18.0 Hz, 1H, CH₂), 2.28 (s, 3H, CH₃), 2.24 (s, 3H, CH₃), 1.28 (t, J = 7.2 Hz, 3H, CH₂CH₃); ¹³C NMR (100 MHz, DMSO-d₆, ppm) δ: 168.08, 151.43, 136.69, 135.55, 132.96, 130.76, 129.92, 129.82, 129.62, 128.53, 125.96, 119.59, 69.99, 43.11, 29.70, 21.12, 20.90, 15.19; HRMS (ESI) [M + Na]⁺ calcd. for C₂₀H₂₂N₂O₂S: 377.12942, found 377.12911.

3.3. Bioactivity Evaluation

3.3.1. Bacterial and Fungal Strains

All bacteria used in this study were provided by Guizhou University and all fungal strains used in this study were provided by Guiyang University.

3.3.2. In Vitro Antibacterial Activity Test

Each target compound (7.5 mg) was dissolved in 150 μL DMSO and then 80 and 40 μL of the solution, respectively, was poured into two 15 mL centrifuge tubes each containing 4 mL 0.1% Twain aqueous solution. The solutions (1 mL) were then added into glass test tubes each containing 4 mL nutrient broth (NB) medium to prepare 5 mL test solutions with concentrations of 200 and 100 μg/mL, respectively. Then, 40 μL of the NB mediums containing Xoo, Xoc, and Xac, respectively, were added to the test tubes mentioned above. The inoculated test tubes were incubated at 30 °C and 180 rpm for 24–48 h until the OD₅₉₅ values of the negative control reached 0.6–0.8 (the logarithmic growth phase). DMSO served as the negative control, whereas Thiodiazole copper and Bismerthiazol served as positive controls. Three replicates were conducted for each treatment. The OD₅₉₅ values of the cultures were monitored on a Multiskan Sky 1530 spectrophotometer (Thermo Scientific, Poland). The inhibition rate I (%) was calculated by the following formula (1), where C is the corrected turbidity value of the untreated NB medium, and T is the corrected turbidity value of the treated NB medium.

Inhibition rate I (%) = (C–T)/C × 100

(1)

On the basis of the preliminary bioassay results, the antibacterial activities (expressed by EC₅₀) of some of the target compounds against Xoo, Xoc and Xac were evaluated and calculated using SPSS 17.0 software. Three replicates were conducted for each treatment.

3.3.3. In Vitro Antifungal Activity Test

Each target compound (5 mg) was dissolved in 1 mL DMSO and mixed with 90 mL potato dextrose agar (PDA) medium. The mixed PDA mediums were then poured into 6 dishes and cooled to room temperature to prepare the PDA plates with the test solution concentration of 50 μg/mL. Mycelia dishes of approximately 0.4 cm diameter were then cut from the culture medium and picked up with a germfree inoculation needle and placed into the middle of PDA plates aseptically. The inoculated PDA plates were fostered in an incubator at 28 °C for 3–4 days until the colony diameter of the negative control reached 5–6 cm. DMSO served as the negative control, whereas Pyrimethanil and Carbendazim acted as positive controls. Three replicates were conducted for each treatment. The inhibition rate I (%) was calculated by the following formula (2), where C (cm) represents the diameter of fungi growth on the untreated PDA plate, and T (cm) represents the diameter of fungi on the treated PDA plate.

Inhibition rate I (%) = [(C−T)/(C−0.4)] × 100

(2)

4. Conclusions

In this study, a total of 32 new thiochromanone derivatives containing a carboxamide moiety were designed and synthesized. The bioassay results demonstrated that compound 4e exhibited excellent in vitro antibacterial activity against Xoo, Xoc, and Xac which was superior to those of Bismerthiazol and Thiodiazole copper. Meanwhile, compound 3b revealed moderate in vitro antifungal activity against B. dothidea at 50 μg/mL which was even better than that of Pyrimethanil, nevertheless, lower than that of Carbendazim. For controlling plant bacterial and fungal diseases, this study provided a practical tool for guiding the design and synthesis of novel and more promising active small molecules of thiochromanone derivatives containing a carboxamide moiety.