Synthesis,Characterization and Antifungal Activities of Amide and Acylthiourea of Substituted 2-Amino-1,3-Benzothiazole

Abstract

Objective: To search new antifungal compounds for effectively controling vital plant diseases, we designed and synthesized series of N-acyl and acylthiourea of substituted 2-amino-1,3-benzothiazole. Methods: The in vitro antifungal activities of the target compounds were screened against the mycelium growth rate of Valsa mali and Botrytis cinerea. The in vivo antifungal activities were evaluated on grape fruit against B. cinerea. Microscopic inspection and electrical conductivity detection were used to explore the effects of screened compound on the mycelium growth of B. cinerea. Results: The results showed that the most active compounds against V. mali were A12 and B9, while the most active compounds against B. cinerea were B9 and B10. Structure activity relationship (SAR) indicates that 6-NO₂ on benzothiazole ring and electron withdrawing substitutes (-NO₂, -CF₃) on the aryl ring of acyl group are benefical to the activity of benzothiazole acylthiourea. Meanwhile, compounds B9 and B10 exhibited 80%–95% control effects on grape fruit against B. cinerea at 200 μg/mL. Compound B9 could destroy the structure of mycelium by increasing the permeability of cell membrane to inhibit the growth of B. cinerea. Conclusions: The structure activity relationship and the action mechanism of the title compounds are helpful to design new fungicide.

Introduction

Benzothiazole skeleton is an important heterocyclic nuclei of many natural and synthetic active compounds. ¹ Benzothiazole has been identified as a volatile constituent from several kinds of microorganisms including fungi Aspergillus clavatus, ² Polyporus frondosus, ligninolytic basidiomycetes Armillaria astoyae, ³ and bacteria Pseudomonas chlororaphis. ⁴ It is demonstrated that this compound is used as a fumigant to control fungi Sclerotinia sclerotiorum, ⁴ nematode Ditylenchus destructor, ⁵ and insect Tribolium castaneum, ⁶ Bradysia odoriphaga ⁷ in nature. In fact, benzothiazole analogues have been found to exhibit a wide range of pharmacological actions including anticancer, antibacterial, antifungal, antiviral, anthelmintic, anti-inflammatory, analgesic, anticonvulsant, antidiabetic, anti-oxidant and neuroprotective properties. ⁸ Most of the reported antibacterial and antifungal benzothiazole compounds are 2-substituented derivatives, in which 2-N and 2-S substituents are notable. They displayed favourable antibacterial activities against Staphylococcus aureus, Escherichia coli, and antifungal activities against Candida albicans, Candida parapsilosis, Candida tropicalis. Research sugests that the antibacterial target of the benzothiazines is dihydroorotase, ⁹ while the antifungal target is lanosterol 14a-demethylase (CYP51). ¹⁰ Some benzothiazole derivatives have been developed as commercial fungicides and bactericides (Benthiazole, Benthiavalicarb-isopropyl), or herbicides (Benzthiazuron, Mefenacet, Figure 1).

Figure 1.

Natural or synthesized active benzothiazole and acylthiourea compounds.

Acylthiourea is a valuble structure unit of fungicides. Such as thiophenate and thiophanate-methyl have been using as agricultural fungicide since 1960s. In plants, thiophenate and thiophanate-methyl are first converted to ethyl carbendazim and carbendazim respectively, which could affect the division of fungal cells, damage the germ tubes and thereby kill the fungi. ¹¹ Though being used for several decades, these fungicides are still widely used today due to their broad-spectrum and highly effective. Acylthiourea compounds also exist in nature source. For example compound ethyl 4-(o-nitrophenyl)-3-thio-allophanate (Figure 1) was once identified from the resistant pyricularia oryzae Cav. Rive variety, which could strongly inhibit the growth of microorganism Agrobacterium tumefaciens and suppress the gene expression of virulence region. ¹² Further research found that benzothiazole thio-allophanate exhibited strong inhibition on the growth of agrobacterium. ¹³ Subsequently, some 5-substituted (-NO₂, -NH₂ or Br) benzothiazol-2-yl acylthiourea compounds are found to have antimicrobial activities, in which compounds 2b and 5b with nitro group on position 5 of benzothiazole ring exhibited high antimicrobial activity against bacteria and fungi Candida. ¹⁴ This may be attributed to that nitro, as electron withdrawing group, can decrease the electron density on benzene ring through resonance effect.

As an ongoing work to search for new agricultural fungicidal compounds, we focused our interest in benzothiazole and acylthiourea moiety. To understand how the electrostatic effect of substitutes on benzothiazole ring affect the fungicidal activity of benzothiazole acylthiourea, we would plan to design and synthesize a series of target compounds with -NO₂ or -OCH₃ at different site of benzothiazole ring and evaluate their antifungal activities. Using o-, m-, or p-nitrophenylamine as starting material, 4-, 5- and 6-NO₂ benzothiazol-2-yl arylacylthiourea were successfully obtained accordingly by our synthetic scheme. But only 6-OCH₃ benzothiazol-2-yl carboxamides instead of arylacylthiourea were produced from p-anisidine no matter with alkyl or aryl chlorides by the same synthetic method.

Since 6-OCH₃ benzothiazol-2-yl carboxamides have similar core structure as commercial fungicide Carbendazim, the antifungal activities of these compounds should be screened. Aditionally, some 6-substituted benzothiazol-2-yl cyclohexanecarboxamides and cyclopropanecarboxamides are reported to exhibit antimicrobial activities,^15,16 in which 6-MeO benzothiazole derivative 2b are more active than 6-NO₂ benzothiazole derivative 2f against bacteria and fungi Aspergillus niger. It seems that electron donating group may be better than the electron withdrawing group to the antimicrobial activities.

In summary, the NO₂ substituted benzothiazole arylacylthiourea and 6-OCH₃ benzothiazole carboxamides were synthesized and their in vitro antifungal activities against 2 kinds of phytopathogens fungi were screened. The in vivo antifungal activities, mycelium morphology and permeability of cell membrane of the candidate compounds on B. cinerea were also investigated.

Materials and Methods

Instruments and Chemicals

All of the chemicals were purchased from commercial suppliers and used without further purification. Reactions were monitored by TLC and visualized under UV light at 254 nm. Melting points (uncorrected) of compounds were measured using an X-4B micro melting point instrument (Yidian Physical Optical Instrument Co. Ltd Shanghai, China). The ¹H and ¹³C NMR spectra were displayed on a BRUKER AVANCE III HD 500 MHz (Bruker, Switzerland) using deuterated chloroform (DCCl₃) or deuterated dimethyl sulfoxide (DMSO-d₆) as solvent. High-resolution mass spectra (ESI TOF (+)) were recorded on the Solari X70FT-ICR-MS (Bruker, Germany). The x-ray diffraction was measured by Bruker D8 QUEST-X (Bruker, Germany).

General Procedure for Synthesis of Intermediates 1-4

Substituted 2-amino-benzothiazoles (1-4) were prepared according to the reported method. ¹⁷ A mixture of 20 mmol of substituted aniline and 1.94 g (20 mmol) of potassium thiocyanate (KSCN) in 20 mL glacial acetic acid (AcOH) was cooled in ice bath and stirred for 10-20 min. Then 3.20 g (20 mmol) bromine in AcOH was added drop wise to keep the temperature below 10 °C. After that, the reaction mixture was stirred at room temperature for 7 h till the end monitored by TLC. The resulted hydrobromide (HBr) salt of product was filtered, washed with AcOH and dried successively. The obtained crude product was dissolved in hot water, and the pH was adjusted to alkaline with ammonia solution. The precipitate was filtered, washed with water, and dried to get the desired product.

General Procedure for Synthesis of Title Compounds A1-A12 and B1-B12

Target compounds A1-A12 and B1-B12 were synthesized according to the reported method ¹⁸ with small modification. To a solution of 0.16 g (2 mmol) of ammonium thiocyanate (NH₄SCN) in toluene, 2 mmol acyl chloride was added drop wise. The reaction mixture was heated to 60 °C for 30 min to obtain the intermediate acyl isothiocyanate. Then, 1.5 mmol of 2-amino-benzothiazole derivative (prepared above) was added in multiple times and refluxed for 8 h. The solid precipitate of the product was collected by filtration. The crude product was purified by silica column chromatography (ethyl acetate: petroleum ether =1:3).

N-(6-Methoxybenzo[d]Thiazol-2-yl)Butyramide (A1)

Yellow solid; yield 72.3%; mp: 174-178 °C; ¹H NMR (500 MHz, CDCl₃) δ 7.57 (d, J = 8.9 Hz, 1H), 7.23 (d, J = 2.5 Hz, 1H), 7.00 (dd, J₁= 8.9 Hz, J₂= 2.5 Hz, 1H), 3.81 (s, 3H), 2.46 (t, J = 7.5 Hz, 2H), 1.75-1.67 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 171.85, 158.17, 156.97, 141.11, 132.84, 120.65, 115.49, 104.50, 55.88, 38.27, 18.54, 13.62. HRMS (ESI): calcd for C₁₂H₁₅N₂O₂S ([M + H]⁺), 251.0848, found, 251.0850.

N-((6-Nitrobenzo[d]Thiazol-2-yl)Carbamothioyl)-3-(Trifluoromethyl)Benzamide (B1)

Yellow solid; yield 73.2%; mp: 196-198 °C; ¹H NMR (500 MHz, CDCl₃) δ 11.31 (s, 1H), 8.82 (s, 1H), 8.33-8.23 (m, 3H), 7.92 (d, J = 7.7 Hz, 1H), 7.72 (t, J = 7.8 Hz, 1H), 7.61 (d, J = 8.9 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 164.32, 163.31, 152.24, 144.32, 132.41, 132.06, 131.97(q, J = 32.7 Hz), 131.14, 130.28 (q, J = 3.6 Hz), 130.07, 128.39 (q, J = 230.1 Hz), 124.91 (q, J = 4.1 Hz), 124.37, 122.19, 120.74, 118.37. HRMS (ESI): calcd for C₁₆H₁₀F₃N₄O₃S₂ ([M + H]⁺), 427.0140, found, 427.0141.

The data of the other compounds and their NMR and HRMS spectral details can be found in the Supplemental Material.

Bioassay

Mycelium growth rate method was used to evaluate the in vitro antifungal activities of title compounds against 2 kinds of vital plant pathogens V. mali and B. cinerea. ¹⁹ Each treatment was conducted in triplicate. And commercial fungicide Hymexazol was used as a reference drug. The in vivo curative and protective effects of compounds B9 and B10 on grape fruit against B. cinerea at 200 μg/mL were evaluated according to our previously reported method. ²⁰ Each treatment was repeated eight times. And solvent 0.5% DMSO was used as blank control. The effect of compound B9 on mycelial morphology of B. cinerea was observed under a microscope according to the literaure method. ²¹ The effect of compound B9 on the permeability of mycelial cell membrane was evaluated according to the reported method. ²² The detailed procedures refer to the Supplemental Material.

Statistical Processing

Values are expressed as mean ± standard error of the mean. Analyses were performed using Excel Statistics. Toxicity regression equations with a correlation coefficient and effective concentration that inhibited mycelium growth by 50% (EC₅₀) were expressed as the mean of values obtained using toxicity regression equation software. ²³

Results

In reference to Arpana's method, ¹⁸ we used less toxic toluene instead of benzene in the second and 3^ird step of the Scheme to prepare the products (follow Scheme 1, series B). To our surprise, when 6-methoxyl-2-amino-benzothiazole reacted with 11 kinds of different acyl isothiocyanate including alkanoyl, cycloalkyl formyl and aryl formyl, carboxamides A1-A11 were obtained as the main products. 6-Nitro-2-amino-benzothiazole and cyclopropyl formyl isothiocyanate also occurred through a similar reaction to give A12. Whereas, other nitro substituted 2-amino-benzothiazole and aryl isothiocyanate containing electron-withdrawing substituent on aryl ring reacted smoothly to give products B1-B12. All of the target compounds were fully characterized by ¹H NMR, ¹³C NMR, and HR-MS spectroscopic data. The single crystal x-ray analysis structure of target compound A3 was also determined and is shown in Figure 2. The structure is deposited with the Cambridge Crystallographic Data Centre (CCDC). The deposition number is 2034811.

Figure 2.

The X-ray diffraction structure of target compound A3.

Scheme 1.

Synthesis of target compounds A1-A12 and B1-B12.

The inhibitory rates of target compounds against the mycelium growth of V. mali and B. cinerea were listed in Table 1. In series A, 6-MeO benzothiazol-2-yl cyclohexylcarboxamide A3, 6-MeO benzothiazol-2-yl naphthylcarboxamide A10 and 6-NO₂ benzothiazol-2-yl cyclopropylcarboxamide A12 exhibited the highest antifungal activities, which were more active than commercial fungicide Hymexazol except the activity of A10 against B. cinerea. This was followed by the activity of compound 6-MeO benzothiazol-2-yl 3-chlorophenylcarboxamide A7. For series B, compound 4-NO₂ benzothiazol-2-yl 3-nitrophenyl acylthiourea B9, 4-NO₂ benzothiazol-2-yl 3-trifluoromethylphenyl acylthiourea B10 and 4-NO₂ benzothiazol-2-yl naphthyl-1-acylthiourea B11 exhibited the highest antifungal activities, which were more active than commercial fungicide Hymexazol except the activity of B11 against V. mali. This was followed by the activity of compound 4-NO₂ benzothiazol-2-yl 3-chlorophenyl acylthiourea B8.

Table 1.

In Vitro Antifungal Activities of the Target Compounds (100 μg/mL; Inhibition Rate, %).

Compd.	V. mali	B. cinerea	Compd.	V. mali	B. cinerea
A1	33.3 ± 0.6	61.7 ± 2.4	B1	60.5 ± 2.3	52.3 ± 3.6
A2	56.7 ± 2.8	63.1 ± 0.4	B2	51.3 ± 1.4	46.3 ± 3.2
A3	91.8 ± 1.5	98.6 ± 0.5	B3	46.7 ± 3.1	30.7 ± 1.8
A4	40.0 ± 2.6	66.5 ± 3.4	B4	68.0 ± 1.1	67.9 ± 1.1
A5	63.3 ± 1.2	79.1 ± 1.1	B5	46.7 ± 2.1	50.5 ± 3.1
A6	50.0 ± 3.2	50.1 ± 2.6	B6	34.2 ± 2.4	49.5 ± 1.6
A7	75.0 ± 0.8	89.7 ± 1.1	B7	40.1 ± 2.1	70.9 ± 1.6
A8	53.3 ± 1.4	50.5 ± 1.6	B8	72.1 ± 3.5	72.6 ± 2.4
A9	63.3 ± 0.9	29.6 ± 3.5	B9	97.7 ± 0.6	96.5 ± 1.2
A10	93.7 ± 1.1	64.1 ± 3.5	B10	97.9 ± 0.3	99.3 ± 0.2
A11	38.3 ± 0.6	14.1 ± 2.6	B11	71.2 ± 1.2	98.8 ± 0.5
A12	98.3 ± 0.3	96.5 ± 0.6	B12	59.6 ± 1.8	62.4 ± 1.1
Hymexazol	77.1 ± 0.7	81.3 ± 1.5

To understand the potential of the high active compounds screened above, the toxic regression equation and EC₅₀ of A3, A10, A12, B9, B10 and B11 were further investigated and listed in Table 2. The results showed that the most active compound was B9 with EC₅₀ of 13-14 μg/mL against the mycelium growth of V. mali and B. cinerea. The less active compound was A12 with EC₅₀ of about 9 μg/mL against V. mali and EC₅₀ of about 19 μg/mL against B. cinerea. This was followed by the activity of compound B10 with EC₅₀ of 12 μg/mL against B. cinerea and 20 μg/mL against V. mali. The EC₅₀ of the other 3 compounds against the 2 fungi were 19-27 μg/mL.

Table 2.

EC₅₀ of Target Compounds Against V. mali and B. cinerea.

Compd.	Toxic regression equation	Correlation coefficient (r)	EC50 (μg/mL)	95% d a
V. mali
A3	Y = 2.34x + 2.07	0.994	19.1	7.6-48.1
A10	Y = 1.98x + 2.23	0.996	22.6	10.3-49.8
A12	Y = 3.17x + 1.93	0.994	8.9	1.9-43.2
B9	Y = 2.37x + 2.30	0.993	13.9	5.1-37.6
B10	Y = 1.46x + 2.72	0.996	20.1	10.1-40.3
B. cinerea
A3	Y = 1.16x + 2.92	0.992	20.6	10.6-39.9
A12	Y = 2.01x + 2.33	0.992	19.2	8.6-42.7
B9	Y = 2.91x + 1.88	0.992	13.0	3.9-43.4
B10	Y = 2.18x + 2.58	0.992	12.4	4.6-33.1
B11	Y = 0.01x + 3.51	0.992	26.5	13.4-52.5

^a

95% confidence limit.

The most active and easily obtained compounds B10 and B9 against B. cinerea were further evaluated for their in vivo antifungal activities on grape fruit. The results (Table 3 and Figure 3) showed that the curative and protective activities of compound B9 were higher than that of compound B10 at the same concentration. And for the 2 compounds, the protective activities were both higher than the curative activities at the set concentrations.

Figure 3.

Protective effect of compound B9 against B. cinerea on grape fruit.

Table 3.

Curative and Protective Effects of Compounds B9 and B10 Against B. cinerea on Grape Fruit (%).

Compd(μg/mL)	B9		B10
	Curative effects	Protective effects	Curative effects	Protective effects
100	70.0 ± 1.1	73.7 ± 1.0	65.0 ± 1.2	68.4 ± 1.6
200	80.0 ± 0.6	94.7 ± 1.7	80.0 ± 0.7	84.2 ± 0.9

When the mycelium of B. cinerea cultivated with compound B9 for 48 h, the mycelium morphology changed significantly compared to the blank control. The mycelium of blank (Figure 4, left) is full, and the separation between the mycelium cells are obvious. The intracellular material is evenly distributed. And there are more bifurcations of mycelium, and the cell wall thickness are uniform. However, after exposed to compound B9 (Figure 4, right), some mycelium shriveled, and there is no obvious cell separation in some mycelium. The number of mycelium bifurcation decreased, and the thickness of cell wall is uneven. Furthermore, the intracellular material accumulated into blocks or the intracellular material disappeared in some mycelium.

Figure 4.

The effect of compound B9 on the mycelium morphology of B. cinerea (250 μg/mL).

To understand the effect of compound B9 on the permeability of mycelial cell membrane of B. cinerea, the mycelium of B. cinerea was cultivated with different concentration solutions of compound B9 for 4 h, and the conductivity of the solutions at different cultivation time were recorded. The curves of conductivity increased with exposure time in Figure 5 showed that the conductivity of all treatments increased as cultivation time went on. There is not distinct difference between the blank and the lower 2 concentrations of treatments, but the curves of the higher 2 concentrations are much different. Though the conductivities increased of the higher 2 concentrations at 15 min are much different, they are close after 30 min. This may mean that the 250 μg/mL may be the lowest concentration that could destroy the cell membrane of mycelium completely.

Figure 5.

Effect of B9 on the permeability of the mycelial cell membrane of B. cinerea.

Discussion

For benzothiazol-2-yl carboxamides (series A), the most active compounds against phytopathogens V. mali and B. cinerea are A3 and A12 which are identical to the antimicrobial activities in the literatures^14,15 though in different compounds clusters and to different fungi. In series of A1-A11, cyclohexylacyl is the most effective group in alkyl, cycloalkyl and aryl groups. Compound A12 show the strongest activity while the activity of compound A2 is middle though they have the same cyclopropylcarboxamide group. The positive effect of A12 may be attribute to the electron withdrawing group NO₂.

It is reported, for the 6-substituted 2-aminobenzothiazole derivatives, the electron-withdrawing groups (Cl, F and CF₃) at the 6-position of the benzothiazole moiety were better than small electron-donating groups (CH₃O, C₂H₅O) against all Candida spp. tested. ¹⁰ Our above conclusion is agree with this result. However, more 6-NO₂ benzothiazol-2-yl carboxamides should be designed and evaluated for their antifungal activities to illustrate the SAR of series A conpounds in the article.

In NO₂ substituted benzothiazol-2-yl acylthioureas (series B), the antifungal activities follow the order 4-NO₂ (B8-B12), 6-NO₂ (B1-B2) and 5-NO₂ (B3-B7). This order is identical to the intensity of electrostatic effect of NO₂ on the N atom in benzothiazole ring. Additionally, the compounds with strong electron withdrawing group (-NO₂, -CF₃) on the aryl ring of acyl group exhibit higher activity in their own subgroups, such as B9 and B10 in B8-B12. Furthermore, the larger aryl substitute (naphyl) of acyl group is beneficial to the antifungal activity of the target compounds, for example A10 and B11 which are high antifungal against one kind of the test fungi. Similarly, the m-Cl-ph (in A7 and B8) of acyl group should be worth noting. Moreover, other 6- electron-withdrawing groups (Cl, F, CF₃ and CN) substituted benzothiazol-2-yl acylthioureas should be designed to search for more potential antifungal conpounds.

It is reported that benzothiazole fungicide Benthiavalicarb-isopropyl could inhibit mycelia growth, zoosporangia and cystospore germination, and also inhibit the sporulation of Phytophthora infestans at a very low concentration. It is presumed that benthiavalicarb-isopropyl inhibits the fibrillization of cellulose, which is involved in the biosynthesis of the cell wall. ²⁴ As mentioned above, acylthiourea fungicide Thiophenate mainly affect the division of fungal cells, damaging the germ tubes and thereby killing the fungi. ¹¹ However, there is no report about the mechanism of the hybird compound benzothiazole acylthiourea. Our preliminary research found that our target compound did affect the mycelial cell wall and cell membrane of B. cinerea. But whether our compound inhibits the fibrillization of cellulose, there need more research to verify. And we still do not know whether the selected compound affect the division of fungal cells.

Conclusions

In conclusion, target compounds A1-A12 and B1-B12 were designed and synthesized according to the principle of bioactive substructure combination. The evaluation of antifungal activities showed that compounds A3, A10, A12, B9 and B10 had strong inhibition on V. mali, and compounds A3, A12, B9, B10 and B11 had strong inhibition on B. cinerea. Structure activity relationship (SAR) show that 6-NO₂ on benzothiazole ring and electron withdrawing substitutes (-NO₂, -CF₃) on the aryl ring of acyl group are benefical to the activity of benzothiazole acylthiourea. Further studies showed that compound B9 had better protective effect against B. cinerea on grape fruit. In addition, the hyphae treated with B9 had obvious morphological changes, and the cell membrane permeability increased significantly.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X241311738 - Supplemental material for Synthesis, Characterization and Antifungal Activities of Amide and Acylthiourea of Substituted 2-Amino-1,3-Benzothiazole

Supplemental material, sj-docx-1-npx-10.1177_1934578X241311738 for Synthesis, Characterization and Antifungal Activities of Amide and Acylthiourea of Substituted 2-Amino-1,3-Benzothiazole by Junping Wang, Chuanping Wang, Hongmei Wang, Yan Wei, Jin Lin and Shuanghong Hao in Natural Product Communications