Design, Synthesis and Fungicidal Activity of N-(thiophen-2-yl) Nicotinamide Derivatives

Abstract

Based on the modification of natural products and the active substructure splicing method, a series of new N-(thiophen-2-yl) nicotinamide derivatives were designed and synthesized by splicing the nitrogen-containing heterocycle natural molecule nicotinic acid and the sulfur-containing heterocycle thiophene. The structures of the target compounds were identified through ¹H NMR, ¹³C NMR and HRMS spectra. The in vivo bioassay results of all the compounds against cucumber downy mildew (CDM, Pseudoperonospora cubensis (Berk.et Curt.) Rostov.) in a greenhouse indicated that compounds 4a (EC₅₀ = 4.69 mg/L) and 4f (EC₅₀ = 1.96 mg/L) exhibited excellent fungicidal activities which were higher than both diflumetorim (EC₅₀ = 21.44 mg/L) and flumorph (EC₅₀ = 7.55 mg/L). The bioassay results of the field trial against CDM demonstrated that the 10% EC formulation of compound 4f displayed excellent efficacies (70% and 79% control efficacies, respectively, each at 100 mg/L and 200 mg/L) which were superior to those of the two commercial fungicides flumorph (56% control efficacy at 200 mg/L) and mancozeb (76% control efficacy at 1000 mg/L). N-(thiophen-2-yl) nicotinamide derivatives are significant lead compounds that can be used for further structural optimization, and compound 4f is also a promising fungicide candidate against CDM that can be used for further development.

1. Introduction

The four main classes of fungal phytopathogens, including oomycetes, ascomycetes, basidiomycetes and deuteromycetes, severely threaten human health, food safety, and agriculture [1,2]. Fungicides are the main approaches to control plant diseases and play a critical role in modern agriculture by increasing both crop quality and yield. Nevertheless, with the widespread application of, especially overused, fungicides, the development of resistance is inevitable [3,4]. Therefore, there is an urgent demand to develop efficient, safe, and eco-friendly fungicides with innovative structures.

Natural products bring bioinspiration to laboratories for the discovery of new weeds, plant pathogens and insect pest control agents, and they play an important role in the advancement of crop protection research [5,6,7,8,9]. Heterocycles are present in a large proportion of natural molecules and often contribute significantly to their structural and physical properties as well as to their biological activity [10,11,12,13]. Approximately 70% of all the agrochemicals that have been launched within the last 20 years bear at least one heterocyclic ring [14]. The nitrogen-containing heterocycle in the natural molecule nicotinic acid, vitamin B3, is the first lipid-lowering drug used for dyslipidemia treatment, and it has been applied for more than five decades [15]. Nicotinic acid and its derivatives play crucial roles as multifunctional pharmacophores in governing many biological activities related to physiological functions and pharmacological activities [16]. In agriculture, nicotinic acid derivatives display extensive applications as well. Agrochemicals, such as diflufenican as a herbicide, flonicamid as an insecticide and boscalid as a fungicide, have already been widely used for crop protection [17,18,19]. Currently, a few new nicotinic acid derivatives have been discovered and are under development as agrochemical candidates, for example, aminopyrifen as a fungicide candidate, cyclobutrifluram as a fungicide–nematicide candidate and nicofluprole as an insecticide candidate (Figure 1) [20,21,22]. Multidisciplinary interest has been focused on the study of nicotinic acid and its derivatives.

Meanwhile, thiophene, a widely researched five-membered sulfur heterocycle, exists in commercialized agricultural fungicides, including silthiofam, ethaboxam, penthiopyrad and isofetamid (Figure 2) [23,24,25,26]. The extensive literature on thiophenes is indicative of the research on and the commercial interest in the heterocycle. Some hundreds of patents appear each year, many applying thiophene compounds as alternatives to benzenoid products, and, in many cases, the thiophene-containing molecule shows a higher activity than the benzene-containing one [27]. As an attractive small heterocycle molecule, thiophene has been widely studied for the development of novel fungicides because of its wide and satisfactory antifungal activity [28,29,30,31,32].

Obviously, based on the modification of natural products and the active substructure splicing method, combinations of the two active substructures of nicotinic acid and thiophene are significant for the discovery of agricultural fungicides with novel molecular structures. A series of N-(thiophen-2-yl) nicotinamide derivatives were designed to generate novel compounds with excellent fungicidal activity (Scheme 1).

2. Results and Discussion

2.1. Chemistry

The synthetic pathways used to prepare target compounds 4a~4s are shown in Scheme 2. The substituted nicotinic acid 1 was acyl chlorinated with oxalyl chloride to obtain acyl chloride 2. The substituted thiophen-2-amine 3 was converted into the desired N-(thiophen-2-yl) nicotinamide derivatives 4a~4s through acylation with the obtained acyl chloride 2 under basic conditions. Their structures were confirmed with ¹H NMR, ¹³C NMR and HRMS spectra. Meanwhile, compound 4f was confirmed with IR spectra (supplementary materials).

Compound 4f (R_n = 5, 6-Cl₂; R¹ = OC₂H₅; R² = CH₃; R³ = CN) exhibited excellent fungicidal activities and was taken as an example to analyze the ¹H NMR spectra data (Figure 3). The chemical shift as triplet was observed at δ 1.31 ppm with J = 7.2 Hz due to the protons of the CH₃ of OC₂H₅. A singlet at δ 2.54 ppm was observed due to the protons of the CH₃ on the thiophene ring. The chemical shift as quartet was observed at δ 4.29 ppm with J = 7.2 Hz due to the protons of the CH₂ of OC₂H₅. The chemical shifts as doublet were observed at δ 8.64 ppm with J = 1.8 Hz and at δ 8.86 ppm with J = 1.8 Hz due to the protons at the fourth position and second position of pyridine, respectively. The spectrum showed a broad singlet at δ 12.67 ppm due to the proton of the CONH.

2.2. Fungicidal Activities In Vivo in a Greenhouse

The in vivo bioassay results shown in

Table 1. The in vivo fungicidal activities of target compounds 4a~4s in green house at 400 mg/L.

Compd.	Rn	R1	R2	R3	WPM	SCR	CA	CDM
4a	2-CH3-5-CN-6-Cl	OC2H5	CH3	CN	0	0	0	100
4b	2-CH3-5-Cl-6-Br	OC2H5	CH3	CN	0	50	0	100
4c	5,6-Br2	OC2H5	CH3	CN	0	0	100	100
4d	5-F-6-Br	OC2H5	CH3	CN	0	0	0	100
4e	5-F-6-Cl	OC2H5	CH3	CN	0	0	0	100
4f	5,6-Cl2	OC2H5	CH3	CN	0	0	0	100
4g	5-Br-6-Cl	OCH3	CH3	CN	0	0	100	100
4h	6-Br	OCH3	CH3	CN	0	0	100	100
4i	5,6-Cl2	OCH3	CH3	CN	0	0	0	98
4j	5,6-Cl2	OC3H7-i	CH3	CN	0	0	40	100
4k	5,6-Cl2	OC3H7-n	CH3	CN	0	0	30	100
4l	5,6-Cl2	OC4H9-n	CH3	CN	0	0	0	100
4m	5,6-Cl2	OC2H4OCH3	CH3	CN	0	0	0	50
4n	5,6-Cl2	OCH2C6H5	CH3	CN	0	0	0	85
4o	5,6-Cl2	NHCH3	CH3	CN	0	0	30	0
4p	5,6-Cl2	Cyclopropylamino	CH3	CN	0	0	0	0
4q	5,6-Cl2	NHPh	CH3	CN	50	100	85	98
4r	5,6-Cl2	OC2H5	C2H5	CN	0	0	0	100
4s	5,6-Cl2	OC2H5	CH3	H	0	0	0	100
Azoxystrobin					100	100	100	/ a
Diflumetorim					/ a	/ a	/ a	100
Flumorph					/ a	/ a	/ a	100

^a: Not tested.

indicated that, at 400 mg/L, a few compounds displayed fungicidal activities against wheat powdery mildew (WPM, Blumeria graminis (DC.) Speer) and southern corn rust (SCR, Puccinia sorghi); a few compounds had good fungicidal activities against cucumber anthracnose (CA, Colletotrichum orbiculare); and most compounds exhibited excellent fungicidal activities against cucumber downy mildew (CDM, Pseudoperonospora cubensis (Berk.et Curt.) Rostov.), which is one of the most destructive oomycete diseases. Further bioassay results in a greenhouse (

Table 2. EC₅₀ values of target compounds 4a~4s against cucumber downy mildew in green house.

Compd.	Y = ax + b	EC50 (mg/L)	95% CI a	r
4a	y = 4.14x + 1.28	4.69	3.27–6.71	0.98
4b	y = 1.71x + 2.92	16.52	13.27–20.58	0.94
4c	y = 1.87x + 2.57	19.89	16.10–24.56	0.95
4d	y = 1.62x + 2.55	32.44	26.12–40.29	0.95
4e	y = 1.58x + 2.78	25.61	20.83–31.48	0.97
4f	y = 1.15x + 4.66	1.96	1.17–3.29	0.95
4g	y = 1.62x + 2.52	34.29	27.46–42.82	0.94
4h	y = 1.62x + 2.72	25.54	20.80–31.35	0.97
4i	y = 1.48x + 3.64	8.31	6.37–10.84	0.97
4j	y = 1.58x + 2.88	21.94	17.58–27.36	0.93
4k	y = 1.58x + 2.66	30.41	24.51–37.73	0.95
4l	/ b	100–400	/ b	/ b
4m	/ b	>400	/ b	/ b
4n	/ b	100–400	/ b	/ b
4o	/ b	> 400	/ b	/ b
4p	/ b	>400	/ b	/ b
4q	/ b	100–400	/ b	/ b
4r	y = 1.48x + 3.70	7.53	5.71–9.94	0.98
4s	y = 1.26x + 2.72	63.81	47.23–86.22	0.92
Diflumetorim	y = 1.43x + 3.10	21.44	17.21–26.70	0.96
Flumorph	y = 1.25x + 3.91	7.55	5.57–10.22	0.98

^a: Confidence interval. ^b: The value could not be measured accurately.

) indicated that more than half of these compounds showed moderate to significant fungicidal activity against CDM, and there were six compounds (4a, 4b, 4c, 4f, 4i and 4r) that displayed higher activities than diflumetorim (EC₅₀ = 21.44 mg/L). Compounds 4a (EC₅₀ = 4.69 mg/L) and 4f (EC₅₀ = 1.96 mg/L) exhibited especially excellent activities, which were higher than flumorph (EC₅₀ = 7.55 mg/L). The structure–activity relationships (SAR) were unfolded as follows.

Initially, the structural modification was mostly around the pyridine ring due to the use of the substituted nicotinic acids that were available at hand in compounds 4a~4h. The testing results shown in Table 2 illustrated that compound 4f, with a chloro at both the fifth position and sixth position of the pyridine ring, significantly indicated the highest fungicidal activity against CDM with an EC₅₀ value of 1.96 mg/L. The other 5,6-dihalosubstitued pyridine compounds, 4c (EC₅₀ = 19.89 mg/L), 4d (EC₅₀ = 32.44 mg/L), 4e (EC₅₀ = 25.61 mg/L) and 4g (EC₅₀ = 34.29 mg/L), also showed good fungicidal activities, but they were much lower than those of 4f. Additionally, 4a (R_n = 2-CH₃-5-CN-6-Cl), with a trisubstituted pyridine, had excellent fungicidal activities next to those of 4f. It was beneficial to the increase of the fungicidal activity to have a chloro at the sixth position because the two best compounds, 4a and 4f, had a chloro at the sixth position.

Next, the 5,6-dichloro on the pyridine ring of compound 4f was maintained, and the substitutions on the thiophene ring were changed to generate compounds 4i~4s. Table 2 also showed that the EC₅₀ values of the compounds 4i (R¹ = OCH₃), 4f (R¹ = OC₂H₅), 4j (R¹ = OC₃H₇-i), 4k (R¹ = OC₃H₇-n) and 4l (R¹ = OC₄H₉-n) were 8.31 mg/L, 1.96 mg/L, 21.94 mg/L, 30.41 mg/L and 100–400 mg/L, respectively, which meant that the fungicidal activity first increased and then decreased dramatically with the increase of the carbon chain length of the alkyloxy in the R¹ moiety and that 4f (R¹ = OC₂H₅) had the highest fungicidal activity. The fungicidal activity got much worse when R¹ had an alkylamine instead of an alkyloxy; for example, the EC₅₀ value of compound 4o (R¹ = NHCH₃) was over 400 mg/L, which was much larger than that of compound 4i (R¹ = OCH₃). Furthermore, when the methyl (R² = CH₃) of compound 4f was replaced with an ethyl to generate compound 4r (EC₅₀ = 7.53 mg/L), the fungicidal activity reduced. When compound 4f (R³ = CN) was decyanated to obtain compound 4s (R³ = H), the fungicidal activity decreased significantly. Compound 4f (R_n = 5,6-Cl₂; R¹ = OC₂H₅; R² = CH₃; R³ = CN) was still the most active one in all nineteen compounds.

2.3. Field Trials against CDM

A field trial of compound 4f was conducted to see its fungicidal activity in the field environment (

Table 3. The results of compound 4f against CDM in field (2021, Shenyang, open field).

Compd.	Concentration (mg/L)	Control Efficacy (%)
		r1	r2	r3	Mean
10% 4f EC	50	55	53	50	53
	100	74	66	69	70
	200	82	75	79	79
10% Flumorph EC	200	64	52	51	56
80% Mancozeb WP	1000	79	76	72	76
10% Cyazofamid SC	100	91	91	90	91
CK	(disease index)	69	61	60	63

), which was a critical assessment indicator for the demonstration of whether the compound was worth further optimization or even commercial development as a fungicide candidate. The results of the field trial in 2021 against CDM demonstrated that compound 4f displayed better control efficacies than the two commercial fungicides flumorph and mancozeb. Compound 4f exhibited 70% and 79% control efficacies, respectively, each at a concentration of 100 mg/L and 200 mg/L, whereas flumorph at 200 mg/L and mancozeb at 1000 mg/L showed 56% and 76% control efficacies, respectively. However, the control efficacy of compound 4f was inferior to that of the commercial fungicide cyazofamid, with a 91% control efficacy at 100 mg/L. Compared with flumorph, compound 4f, with a much lower EC₅₀ value against CDM in the greenhouse tests, was more active against CDM in the field trial.

3. Materials and Methods

3.1. Chemicals and Target Compounds

All starting materials and reagents were commercially available and used without further purification except as indicated. (The starting materials 1 and 3 were from Taizhou Jiakang Chemical Co., Ltd., a custom chemicals supplier in Zhejiang in China, and the reagents were from Sinopharm Chemical Reagent Co., Ltd., in Shanghai in China.) ¹H nuclear magnetic resonance (NMR) spectra were obtained at 300 MHz using a Varian Mercury 300 spectrometer (Varian, Palo Alto, CA, USA), at 600 MHz using a Varian Unity Plus 600 spectrometer (Varian, Palo Alto, CA, USA) or at 600 MHz using a JEOL JNM-ECZ600R spectrometer (JEOL RESONANCE Inc., Akishima, Tokyo, Japan) with DMSO-d₆ as the solvent and tetramethylsilane (TMS) as the internal standard. ¹³C nuclear magnetic resonance (NMR) spectra were obtained at 150 MHz using a Varian Unity Plus 600 spectrometer (Varian, Palo Alto, CA, USA) or at 150 MHz using a JEOL JNM-ECZ600R spectrometer (JEOL RESONANCE Inc., Akishima, Tokyo, Japan) with DMSO-d₆ as the solvent and tetramethylsilane (TMS) as the internal standard. Chemical-shift values (δ) were given in parts per million (ppm). Mass spectra were acquired with a Thermo Scientific Q Exactive Focus mass spectrometer system (Thermo Fisher Scientific Inc., Waltham, MA, USA). Melting points were determined on an X-4 precision microscope melting point tester and were uncorrected. Petroleum ether used for column chromatography had a boiling range of 60~90 °C. Chemical names were generated using ChemDraw (Cambridge Soft, version 15.0). Yields were not optimized. An overview synthesis of N-(thiophen-2-yl)nicotinamide derivatives 4a~4s is shown in Scheme 2.

3.1.1. General Synthetic Procedures

General procedure for the preparation of compounds 4a~4s: To a solution of acid 1 (2.3 mmol) in CH₂Cl₂ (20 mL), oxalyl chloride (6.9 mmol) was added dropwise, and then a drop of DMF was added. The mixture was stirred at room temperature for 6 h and concentrated under reduced pressure to obtain acyl chloride 2. To a mixture of amine 3 (2.0 mmol) and triethylamine (2.4 mmol) in CH₂Cl₂ (20 mL), the solution of acyl chloride 2 in CH₂Cl₂ (10 mL) was added dropwise in an ice-water bath. The resulting mixture was stirred at room temperature until TLC indicated that the reaction was complete. To the mixture, water (10 mL) was added, and then the organic phase was washed with brine, dried over anhydrous MgSO₄ and concentrated under reduced pressure to give a crude product. The crude product was purified through chromatography on a column of silica gel with petroleum ether and ethyl acetate to obtain the product amide 4.

3.1.2. Chemical Property of the Compounds

Ethyl 5-(6-chloro-5-cyano-2-methylnicotinamido)-4-cyano-3-methylthiophene-2-carboxylate (4a): White solid (0.58 g, yield 74%). Decomposition temperature > 175 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 12.78 (bs, 1H, CONH), 8.77 (s, 1H, pyridine-4-H), 4.29 (q, J = 7.2 Hz, 2H, OCH₂CH₃), 2.63 (s, 3H, CH₃), 2.56 (s, 3H, CH₃) and 1.31 (t, J = 7.2 Hz, 3H, OCH₂CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 164.7, 162.3, 161.8, 151.8, 144.2, 144.1, 128.9, 118.1, 118.0, 115.1, 113.8, 106.8, 98.6, 61.4, 23.4, 14.7 and 14.5. HR-MS (m/z): calcd. for C₁₇H₁₂ClN₄O₃S [M-H]⁻ 387.0324; found, 387.0327.

Ethyl 5-(6-bromo-5-chloro-2-methylnicotinamido)-4-cyano-3-methylthiophene-2-carboxylate (4b): White solid (0.66 g, yield 74%). Decomposition temperature > 196 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 12.71 (bs, 1H, CONH), 8.44 (s, 1H, pyridine-4-H), 4.24 (q, J = 7.2 Hz, 2H, OCH₂CH₃), 2.49 (s, 3H, CH₃), 2.45 (s, 3H, CH₃) and 1.27 (t, J = 7.2 Hz, 3H, OCH₂CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 165.0, 162.0, 156.6, 151.4, 150.3, 144.3, 143.2, 130.0, 118.2, 116.2, 113.9, 98.6, 61.5, 22.4, 14.8 and 14.7. HR-MS (m/z): calcd. for C₁₆H₁₃BrClN₃NaO₃S [M + Na]⁺ 465.9422; found, 465.9421.

Ethyl 4-cyano-5-(5,6-dibromonicotinamido)-3-methylthiophene-2-carboxylate (4c): White solid (0.55g, yield 57%). Decomposition temperature > 231 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 12.56 (bs, 1H, CONH), 8.86 (d, J = 2.1 Hz, 1H, pyridine-2-H), 8.69 (d, J = 2.1 Hz, 1H, pyridine-4-H), 4.29 (q, J = 6.9 Hz, 2H, OCH₂CH₃), 2.54 (s, 3H, CH₃) and 1.34 (t, J = 6.9 Hz, 3H, OCH₂CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.3, 162.0, 153.2, 151.4, 149.0, 144.3, 143.1, 129.5, 119.1, 118.5, 114.0, 99.0, 61.5, 14.9 and 14.7. HR-MS (m/z): calcd. for C₁₅H₁₀Br₂N₃O₃S [M-H]⁻ 471.8800; found, 471.8795.

Ethyl 5-(6-bromo-5-fluoronicotinamido)-4-cyano-3-methylthiophene-2-carboxylate (4d): White solid (0.53 g, yield 64%). Decomposition temperature > 220 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 12.66 (bs, 1H, CONH), 8.75 (s, 1H, pyridine-2-H), 8.42 (d, J = 9.0 Hz, 1H, pyridine-4-H), 4.24 (q, J = 7.2 Hz, 2H, OCH₂CH₃), 2.50 (s, 3H, CH₃) and 1.27 (t, J = 7.2 Hz, 3H, OCH₂CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.4, 162.0, 153.9 (d, ¹J_CF = 258.6 Hz), 151.3, 145.9 (d, ³J_CF = 6.3 Hz), 141.4 (d, ²J_CF = 19.4 Hz), 144.4, 130.0, 120.2 (d, ²J_CF = 20.9 Hz), 118.6, 113.9, 99.1, 61.5, 14.9 and 14.7. HR-MS (m/z): calcd. for C₁₅H₁₀BrFN₃O₃S [M-H]⁻ 409.9623; found, 409.9616.

Ethyl 5-(6-chloro-5-fluoronicotinamido)-4-cyano-3-methylthiophene-2-carboxylate (4e): White solid (0.52 g, yield 70%). Decomposition temperature > 275 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 12.59 (bs, 1H, CONH), 8.80 (d, J = 2.1 Hz, 1H, pyridine-2-H), 8.43 (dd, J = 9.0, 2.1 Hz, 1H, pyridine-4-H), 4.30 (q, J = 7.2 Hz, 2H, OCH₂CH₃), 2.57 (s, 3H, CH₃) and 1.34 (t, J = 7.2 Hz, 3H, OCH₂CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.5, 162.0, 153.9 (d, ¹J_CF = 258.6 Hz), 151.5, 145.9 (d, ³J_CF = 6.6 Hz), 144.4, 141.3 (d, ²J_CF = 19.7 Hz), 130.1, 126.2 (d, ²J_CF = 21.6 Hz), 118.5, 114.0, 99.1, 61.5, 14.9 and 14.7. HR-MS (m/z): calcd. for C₁₅H₁₀ClFN₃O₃S [M-H]⁻ 366.0120; found, 366.0120.

Ethyl 4-cyano-5-(5,6-dichloronicotinamido)-3-methylthiophene-2-carboxylate (4f): White solid (0.58 g, yield 75%). Decomposition temperature > 262 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 12.67 (bs, 1H, CONH), 8.86 (d, J = 1.8 Hz, 1H, pyridine-2-H), 8.64 (d, J = 1.8 Hz, 1H, pyridine-4-H), 4.29 (q, J = 7.2 Hz, 2H, OCH₂CH₃), 2.54 (s, 3H, CH₃) and 1.31 (t, J = 7.2 Hz, 3H, OCH₂CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.2, 161.8, 151.3, 151.2, 148.3, 144.2, 139.7, 129.3, 129.2, 118.3, 113.8, 98.9, 61.3, 14.7 and 14.5. HR-MS (m/z): calcd. for C₁₅H₁₁Cl₂N₃NaO₃S [M + Na]⁺ 405.9791; found, 405.9793.

Methyl 5-(5-bromo-6-chloronicotinamido)-4-cyano-3-methylthiophene-2-carboxylate (4g): White solid (0.54 g, yield 64%). Decomposition temperature > 238 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 12.62 (bs, 1H, CONH), 8.82 (d, J = 1.8 Hz, 1H, pyridine-2-H), 8.68 (d, J = 1.8 Hz, 1H, pyridine-4-H), 3.78 (s, 3H, OCH₃) and 2.49 (s, 3H, CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.3, 162.4, 153.2, 151.5, 149.0, 144.5, 143.1, 129.1, 119.5, 118.2, 113.9, 99.0, 52.7 and 14.9. HR-MS (m/z): calcd. for C₁₄H₁₀BrClN₃O₃S [M + H]⁺ 415.9284; found, 415.9289.

Methyl 5-(6-bromonicotinamido)-4-cyano-3-methylthiophene-2-carboxylate (4h): White solid (0.55 g, yield 72%). Decomposition temperature > 261 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 12.60 (bs, 1H, CONH), 8.91 (s, 1H, pyridine-2-H), 8.34 (d, J = 8.1 Hz, 1H, pyridine-4-H), 7.67 (d, J = 8.1 Hz, 1H, pyridine-5-H), 3.83 (s, 3H, OCH₃) and 2.56 (s, 3H, CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 164.7, 162.5, 154.2, 150.7, 149.8, 144.6, 140.5, 128.0, 124.6, 117.9, 114.0, 99.0, 52.7 and 14.9. HR-MS (m/z): calcd. for C₁₄H₁₁BrN₃O₃S [M + H]⁺ 381.9676; found, 381.9679.

Methyl 4-cyano-5-(5,6-dichloronicotinamido)-3-methylthiophene-2-carboxylate (4i): White solid (0.56 g, yield 75%). Decomposition temperature > 263 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 12.55 (bs, 1H, CONH), 8.85 (d, J = 2.1 Hz, 1H, pyridine-2-H), 8.59 (d, J = 2.1 Hz, 1H, pyridine-4-H), 3.84 (s, 3H, OCH₃) and 2.57 (s, 3H, CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.4, 162.4, 151.6, 151.4, 148.5, 144.6, 139.9, 129.4, 129.3, 118.1, 114.0, 99.0, 52.7 and 14.9. HR-MS (m/z): calcd. for C₁₄H₈Cl₂N₃O₃S [M-H]⁻ 367.9668; found, 367.9671.

Isopropyl 4-cyano-5-(5,6-dichloronicotinamido)-3-methylthiophene-2-carboxylate (4j): White solid (0.57 g, yield 71%). An m.p. of 239~240 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 12.58 (bs, 1H, CONH), 8.86 (d, J = 2.1 Hz, 1H, pyridine-2-H), 8.61 (d, J = 2.1 Hz, 1H, pyridine-4-H), 5.11 (hept, J = 6.3 Hz, 1H, OCH(CH₃)₂), 2.56 (s, 3H, CH₃) and 1.34 (d, J = 6.3 Hz, 6H, OCH(CH₃)₂). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.4, 161.6, 151.3 (2 C), 148.5, 144.2, 139.9, 129.4 (2 C), 118.8, 114.1, 99.4, 69.2, 22.2 (2 C) and 14.9. HR-MS (m/z): calcd. for C₁₆H₁₂Cl₂N₃O₃S [M-H]⁻ 395.9981; found, 395.9986.

Propyl 4-cyano-5-(5,6-dichloronicotinamido)-3-methylthiophene-2-carboxylate (4k): White solid (0.56 g, yield 70%). An m.p. of 229~230 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 12.61 (bs, 1H, CONH), 8.87 (d, J = 2.1 Hz, 1H, pyridine-2-H), 8.62 (d, J = 2.1 Hz, 1H, pyridine-4-H), 4.21 (t, J = 6.6 Hz, 1H, OCH₂CH₂CH₃), 2.57 (s, 3H, CH₃), 1.69–1.81 (m, 2H, OCH₂CH₂CH₃) and 1.01 (t, J = 6.9 Hz, 3H, OCH₂CH₂CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.5, 162.1, 151.3 (2 C), 148.5, 144.3, 139.9, 129.4 (2 C), 118.4, 114.1, 99.4, 66.8, 22.1, 14.9 and 10.9. HR-MS (m/z): calcd. for C₁₆H₁₂Cl₂N₃O₃S [M-H]⁻ 395.9981; found, 395.9986.

Butyl 4-cyano-5-(5,6-dichloronicotinamido)-3-methylthiophene-2-carboxylate (4l): White solid (0.55 g, yield 66%). An m.p. of 215~216 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 12.66 (bs, 1H, CONH), 8.83 (d, J = 2.1 Hz, 1H, pyridine-2-H), 8.62 (d, J = 2.1 Hz, 1H, pyridine-4-H), 4.21 (t, J = 6.6 Hz, 1H, OCH₂CH₂CH₂CH₃), 2.51 (s, 3H, CH₃), 1.62–1.64 (m, 2H, OCH₂CH₂CH₂CH₃), 1.36–1.39 (m, 2H, OCH₂CH₂CH₂CH₃) and 0.89 (t, J = 7.2 Hz, 3H, OCH₂CH₂CH₂CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.5, 162.1, 151.3 (2 C), 148.5, 144.4, 139.9, 129.4 (2 C), 120.6, 114.1, 99.1, 65.1, 30.7, 19.3, 14.9 and 14.1. HR-MS (m/z): calcd. for C₁₇H₁₄Cl₂N₃O₃S [M-H]⁻ 410.0138; found, 410.0145.

2-methoxyethyl 4-cyano-5-(5,6-dichloronicotinamido)-3-methylthiophene-2-carboxylate (4m): White solid (0.58 g, yield 69%). An m.p. of 213~214 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 12.62 (bs, 1H, CONH), 8.86 (d, J = 2.1 Hz, 1H, pyridine-2-H), 8.62 (d, J = 2.1 Hz, 1H, pyridine-4-H), 4.34–4.38 (m, 2H, OCH₂CH₂OCH₃), 3.62–3.65 (m, 2H, OCH₂CH₂OCH₃), 3.33 (s, 3H, OCH₂CH₂OCH₃) and 2.57 (s, 3H, CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.4, 161.9, 151.7, 151.4, 148.5, 144.7, 139.9, 129.5, 129.3, 118.2, 114.0, 99.1, 70.2, 64.4, 58.7 and 14.9. HR-MS (m/z): calcd. for C₁₆H₁₂Cl₂N₃O₄S [M-H]⁻ 411.9931; found, 411.9936.

Benzyl 4-cyano-5-(5,6-dichloronicotinamido)-3-methylthiophene-2-carboxylate (4n): White solid (0.57 g, yield 63%). Decomposition temperature > 213 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 12.68 (bs, 1H, CONH), 8.85 (d, J = 2.1 Hz, 1H, pyridine-2-H), 8.63 (d, J = 2.1 Hz, 1H, pyridine-4-H), 7.36–7.45 (m, 5H, Ph-2,3,4,5,6-5H), 5.32 (s, 2H, OCH₂) and 2.57 (s, 3H, CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.5, 161.8, 151.7, 151.4, 148.5, 144.9, 139.9, 136.3, 129.5, 129.3, 129.1 (2 C), 128.8, 128.5 (2 C), 118.0, 113.9, 99.1, 66.8 and 14.9. HR-MS (m/z): calcd. for C₂₀H₁₂Cl₂N₃O₃S [M-H]⁻ 443.9987; found, 443.9981.

5,6-dichloro-N-(3-cyano-4-methyl-5-(methylcarbamoyl)thiophen-2-yl)nicotinamide (4o): White solid (0.51 g, yield 68%). Decomposition temperature > 265 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 12.44 (bs, 1H, CONH), 8.86 (d, J = 2.1 Hz, 1H, pyridine-2-H), 8.64 (d, J = 2.1 Hz, 1H, pyridine-4-H), 8.05 (q, J = 4.5 Hz, 3H, CONHCH₃), 2.75 (d, J = 4.5 Hz, 3H, CONHCH₃) and 2.47 (s, 3H, CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.0, 162.4, 151.3, 148.7, 148.4, 139.8, 137.5, 129.5, 129.4, 124.8, 114.3, 98.7, 26.9 and 14.7. HR-MS (m/z): calcd. for C₁₄H₉Cl₂N₄O₂S [M-H]⁻ 366.9828; found, 366.9830.

5,6-dichloro-N-(3-cyano-5-(cyclopropylcarbamoyl)-4-methylthiophen-2-yl)nicotinamide (4p): White solid (0.55 g, yield 69%). Decomposition temperature > 258 °C; ¹H NMR (300 MHz, DMSO-d₆): δ 12.41 (bs, 1H, CONH), 8.86 (d, J = 2.1 Hz, 1H, pyridine-2-H), 8.63 (d, J = 2.1 Hz, 1H, pyridine-4-H), 8.19 (d, J = 6.9 Hz, 1H, CONHCH), 2.74–2.83 (m, 1H, cyclopropane-H), 2.44 (s, 3H, CH₃) and 0.55–0.72 (m, 4H, cyclopropane-H). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.3, 163.0, 151.3, 148.6, 148.4, 139.8, 137.7, 129.5 (2 C), 124.6, 114.3, 98.6, 23.6, 14.7 and 6.4. HR-MS (m/z): calcd. for C₁₆H₁₃Cl₂N₄O₂S [M + H]⁺ 395.0131; found, 395.0123.

5,6-dichloro-N-(3-cyano-4-methyl-5-(phenylcarbamoyl)thiophen-2-yl)nicotinamide (4q): White solid (0.57 g, yield 65%). Decomposition temperature > 243 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 12.56 (bs, 1H, CONH), 10.11 (s, 1H, CONH), 8.86 (d, J = 2.1 Hz, 1H, pyridine-2-H), 8.64 (d, J = 2.1 Hz, 1H, pyridine-4-H), 7.62–7.64 (m, 2H, Ph-H), 7.29–7.32 (m, 2H, Ph-H), 7.06–7.09 (m, 1H, Ph-H) and 2.47 (s, 3H, CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.1, 160.8, 151.3, 148.5, 139.9, 139.2, 138.7, 129.5, 129.2 (2 C), 129.1, 124.8, 124.5, 121.0 (2 C), 120.8, 114.3, 98.6 and 14.9. HR-MS (m/z): calcd. for C₁₉H₁₁Cl₂N₄O₂S [M-H]⁻ 428.9985; found, 428.9988.

Ethyl 4-cyano-5-(5,6-dichloronicotinamido)-3-ethylthiophene-2-carboxylate (4r): White solid (0.55 g, yield 68%). Decomposition temperature > 205 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 12.63 (bs, 1H, CONH), 8.80 (d, J = 2.1 Hz, 1H, pyridine-2-H), 8.58 (d, J = 2.1 Hz, 1H, pyridine-4-H), 4.24 (q, J = 7.2 Hz, 2H, OCH₂CH₃), 2.95 (q, J = 7.2 Hz, 2H, CH₂CH₃), 1.26 (t, J = 7.2 Hz, 3H, OCH₂CH₃) and 1.12 (t, J = 7.2 Hz, 3H, CH₂CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.3, 161.6, 151.7, 151.4, 150.5, 148.5, 139.8, 129.5, 129.2, 118.1, 113.8, 98.1, 61.5, 22.0, 14.8 and 14.6. HR-MS (m/z): calcd. for C₁₆H₁₄Cl₂N₃O₃S [M + H]⁺ 398.0128; found, 398.0120.

Ethyl 5-(5,6-dichloronicotinamido)-3-methylthiophene-2-carboxylate (4s): White solid (0.53 g, yield 73%). Decomposition temperature > 211 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 12.04 (bs, 1H, CONH), 8.85 (d, J = 2.4 Hz, 1H, pyridine-2-H), 8.56 (d, J = 2.4 Hz, 1H, pyridine-4-H), 6.73 (s, 1H, thiophene-H), 4.18 (q, J = 7.2 Hz, 2H, OCH₂CH₃), 2.40 (s, 3H, CH₃) and 1.25 (t, J = 7.2 Hz, 3H, OCH₂CH₃). ¹³C NMR (150 MHz, DMSO-d₆): δ 163.1, 160.9, 151.2, 147.9, 144.5, 143.9, 138.9, 129.9, 129.5, 117.7, 117.5, 60.6, 16.3 and 14.8. HR-MS (m/z): calcd. for C₁₄H₁₃Cl₂N₂O₃S [M + H]⁺ 359.0019; found, 359.0012.

3.2. Fungicidal Activities In Vivo in a Greenhouse

Each of the test compounds (4 mg) were first dissolved in 5 mL of dimethyl sulfoxide, and then 5 mL of water containing 0.1% Tween 80 was added to generate 10 mL stock solutions at a concentration of 400 mg/L. Serial test solutions were prepared by diluting the above solution (testing range of 1.5625~400 mg/L). Evaluations of the in vivo fungicidal activity of the synthesized compounds against Blumeria graminis (DC.) Speer, Puccinia sorghi, Colletotrichum orbiculare and Pseudoperonospora cubensis (Berk.et Curt.) Rostov. Were performed as follows: Seeds (wheat: Triticum aestivum L., maize: Zea mays L., and cucumber: Cucumis sativus L.) were grown to the one-leaf, two-leaf, and two- to three-leaf stages, and then the test solutions were sprayed on the host plants with a homemade sprayer. After 24 h, the leaves of the host plants were inoculated with sporangial suspensions of the fungi which were cultured by Shenyang Sinochem Agrochemicals R&D Company Ltd. (Shenyang, China), each at a concentration of 5 × 10⁵ spores/mL, using a PS289 Procon Boy WA double action 0.3 mm airbrush (GSI, Tokyo, Japan), but the fungi Blumeria graminis (DC.) Speer was inoculated by shaking off the spores directly onto the wheat leaves. The plants were stored in a humidity chamber (24 ± 1 °C, RH > 90%, dark) and then were transferred to a greenhouse (18~30 °C, RH > 50~60%) 24 h after infection. Three replicates were carried out. The activity of each compound was estimated through visual inspection after 7 d, and the screening results were reported in the range of 0% (no control of the fungus) to 100% (complete control of the fungus). The inhibitory activity (%) was estimated as:

Inhibitory activity (%) = [(viability of the blank control − viability of the treated plant)/viability of the blank control] × 100%

The EC₅₀ values were calculated with Duncan’s new multiple-range test (DMRT) using DPS version 14.5.

3.3. Field Trials against CDM

Field trials were conducted in the open field owned by Shenyang Sinochem Agrochemicals R&D Company Ltd. in Shenyang, Liaoning province (25 m², at the five-leaf stage). The test solution was sprayed on the host plant (Cucumis sativus L.) with a WS-15D knapsack electric sprayer (WishSprayer, Shandong, China). Two spays were carried out with an interval period of 6~7 d. Seven days after the second treatment, the incidence of disease spots in each plot was investigated by randomly selecting 4 samples per plot and 8 plants per sample. The incidence of the whole plant was recorded by counting the number of diseased leaves and by determining the incidence grade [33]. The grade scales were divided into six levels (ratio of leaf-spot area to leaf area): level 0 (no disease), level 1 (<5%), level 3 (6~10%), level 5 (11~25%), level 7 (26~50%), and level 9 (> 51%). The disease index (DI, %) was calculated as

DI (%) = [∑ (number of diseased leaves × relative level)/(total number of investigated leaves × the highest level)] × 100%

The inhibitory activity (%) was calculated as

Inhibitory activity (%) = [(DI of the blank control − DI of the treated plot)/DI of the blank control] × 100%

4. Conclusions

In summary, nineteen N-(thiophen-2-yl) nicotinamide derivatives were designed and synthesized. The in vivo bioassay results of all nineteen compounds against WPM, SCR, CA and CDM in a greenhouse indicated that over half of these compounds showed moderate to significant fungicidal activity against CDM, and there were six compounds (4a, 4b, 4c, 4f, 4i and 4r) that displayed higher activities than diflumetorim (EC₅₀ = 21.44 mg/L); compounds 4a (EC₅₀ = 4.69 mg/L) and 4f (EC₅₀ = 1.96 mg/L) exhibited especially excellent activities, which were higher than those of flumorph (EC₅₀ = 7.55 mg/L). The bioassay results of the field trial against CDM demonstrated that compound 4f displayed excellent control efficacies (70% and 79% control efficacies, respectively, each at 100 mg/L and 200 mg/L), which were superior to those of the two commercial fungicides flumorph (56% control efficacy at 200 mg/L) and mancozeb (76% control efficacy at 1000 mg/L). However, the control efficacy of compound 4f was inferior to that of the commercial fungicide cyazofamid (91% control efficacy at 100 mg/L). This study illustrated that N-(thiophen-2-yl) nicotinamide derivatives are significant lead compounds that can be used for the further discovery of new compounds to control the oomycete disease CDM, and it illustrated that compound 4f is also a promising fungicide candidate against CDM that can be used for further development.