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Abstract

Twenty-one novel luotonin derivatives were synthesized from 2-aminobenzamide in excellent yields. The prepared compounds, when tested against 8 strains of bacteria and many plant pathogen fungi showed diverse and promising antimicrobial activities. Notably, compounds a5, a9, b4, and b5 demonstrated good activities and might be potential lead compounds for further development as antifungal agents. The relationship between structure and biological activity is also discussed.

Keywords

luotonin derivatives synthesis antimicrobial agrochemicals bioactivity

In nature, tricyclic heteroaromatic skeletons are very important moieties that are widespread in a large family of natural products with a broad range of attractive bioactivities.¹ Luotonin A (Figure 1) and derivatives, which possess a novel kind of tricyclic heteroaromatic scaffold, have displayed important biological activities such as anticancer, cytotoxic, and antiproliferative activity.^2,3 Because of this wide scope of biological activities, a great deal of study toward the synthesis and antifungal activities of luotonin A has been reported.^{4

-17} Therefore, we focused on structural modification of luotonin A in the hope that a lead compound could be found for the discovery of novel antimicrobial agents. Herein, we report the synthesis and bioactivity of a series of novel luotonin analogs using 2-aminobenzamide as the starting material.

Figure 1

Structure of luotonin A.

To the best of our knowledge, the antimicrobial activities of the synthetic compounds are reported for the first time.

Materials and Methods

Instruments and Chemicals

All chemicals were purchased from commercial sources and purified based on standard procedures before use. Analytical thin-layer chromatography was performed with silica gel plates using silica gel 60 GF₂₅₄ (Qingdao Haiyang Chemical Co., Ltd., China). The ¹H nuclear magnetic resonance (NMR; 500 MHz) and ¹³C NMR (125 MHz) spectra were obtained on an AM-500 FT-NMR spectrometer (Bruker Corporation, Switzerland) with deuterated chloroform (CDCl₃) as the solvent and trimethylsilane as the internal standard. Melting points were taken on an electrothermal digital apparatus (Beijing, China) and were uncorrected. Mass spectra (MS) were recorded under electrospray ionization (ESI) conditions using an LCQ Fleet instrument (Thermo Fisher, Waltham, MA, USA). The title compounds were synthesized under a nitrogen atmosphere. Yields were not optimized.

Synthesis

The general synthetic methods for luotonin derivatives are depicted in Scheme 1.

Scheme 1

Synthetic route for the title compounds.

Synthesis of compound 3

A mixture of 2-aminobenzamide (2.72 g, 20 mmol) and diethyl oxalate (20 mL) was refluxed at 185.5 °C for 5 hours. The reaction mixture was then allowed to cool to room temperature (r.t). The precipitate was collected by filtration using a Hirsch funnel to give compound 3 as a white solid (3.55 g, 16.4 mmol, 82%).

Synthesis of compound 4

Compound 3 (3.5 g 1.60 mmol) was added to ammonium hydroxide (30 mL) and stirred overnight at r.t. The precipitate was filtered and dried in a low-temperature drying oven to give compound 4 (2.76 g, 14.6 mmol, 91%).

Synthesis of compound 5

To a stirred solution of compound 4 (2.70 g, 1.43 mmol) and potassium carbonate (1.97 g, 2.86 mmol) in N, N-dimethylformamide (20 mL) was added propargyl bromide (2.15 mL, 2.86 mmol) at r.t. for 30 minutes. The solution was allowed to stir for 1 hour at r.t. and then extracted with dichloromethane (3 × 40 mL). The combined organic extracts were dried over anhydrous sodium sulfate and evaporated under reduced pressure yielding compound 5 as a white solid (2.64 g, 11.6 mmol, 83%).

Synthesis of compound 7

A mixture of crude residue 5 (4.5 g) and mercuric acetate as a catalyst in formic acid (30 mL) was heated to 50-55 °C with magnetic stirring for 1 hour. The reaction mixture was extracted with dichloromethane. The combined extracts were washed with brine and then dried with sodium sulfate. All the crude residues were purified by silica-gel column chromatography and evaporated under reduced pressure to provide the desired compound 7 as a yellow solid (2.52 g, 11.7 mmol, 82%).

Synthesis of compounds a1-a11

Compound 7 (0.20 g, 0.9 mmol) and potassium carbonate (0.25 g, 1.8 mmol) in N, N-dimethylformamide (10 mL) was stirred at 0 °C for 0.5 hours. The solution was allowed to stir for 2-3 hours at r.t. The corresponding desired reagent was added to the flask and then extracted with dichloromethane (3 × 15 mL). The combined organic extracts were dried over anhydrous sodium sulfate and evaporated under reduced pressure yielding a white solid. All the crude residues were purified by silica-gel column chromatography and evaporated under reduced pressure to provide the desired compounds a1-a11.

Synthesis of compounds b1-b5

A solution of compound 7 (0.20 g, 0.9 mmol) and triethylamine (0.20 mL) in dichloromethane was added dropwise over 30 minutes to the corresponding desired reagent solution in an ice-salt bath. The mixture was stirred for 1 hour. Then the reaction mixture was allowed to warm up to r.t., and water was added. The mixture was extracted with dichloromethane (3 × 20 mL) and the combined organic extracts were dried over anhydrous sodium sulfate and evaporated under reduced pressure yielding a white solid. The crude residues were purified by silica-gel column chromatography and evaporated under reduced pressure to provide the desired compounds b1-b5.

Synthesis of compounds c1-c5

A solution of compound 7 (0.20 g, 0.9 mmol) and potassium carbonate (0.25 g, 1.8 mmol) in N, N-dimethylformamide (10 mL) was stirred at 0 °C for 0.5 hours. The solution was allowed to stir for 2-3 hours at r.t. The corresponding desired reagent was added to the flask and then extracted with dichloromethane (3 × 15 mL). The combined organic extracts were dried over anhydrous sodium sulfate and evaporated under reduced pressure yielding a white solid. All the crude residues were purified by silica-gel column chromatography and evaporated under reduced pressure to provide the desired compounds c1-c5.

3-Methyl-2-(4-methylbenzyl)-2H-pyrazino[2,1-b]quinazoline-1,6-dione (a1)

Yellow solid, m.p., 231-233 °C, Formula: C₂₀H₁₇N₃O₂ (81% yield), ¹H NMR (500 MHz, CDCl₃), δ (ppm): 8.38 (dd, J = 1.0 Hz, 8.0 Hz, 1 H, 7 H), 8.07 (d, J = 9.0 Hz, 1 H, 10 H), 7.86 (m, 1 H, 9 H), 7.60 (m, 1 H, 8 H), 7.52 (m, 1 H, 4 H), 7.18 (m, 2 H, 15-CH × 2), 7.12 (m, 2 H, 14-CH × 2), 5.27 (s, 2 H,12-CH₂), 2.31 (s, 3 H, 17-CH₃), 2,28 (s, 3 H, 3-CH₃).¹³C NMR (125 MHz, CDCl₃), δ (ppm): 157.98 (C = O), 157.06 (C = O), 146.91 (10a-C), 139.83 (11a-C), 137.70 (13 C), 134.94 (9-CH), 132.75 (16 C), 129.62 (15-CH × 2), 129.51 (8-CH, 128.28 (10-CH), 127.02 (3 C), 126.99 (14-CH × 2), 125.53 (7-CH), 119.77 (6a-C), 101.60 (4-CH), 47.45 (12-CH₂), 21.07 (17-CH₃), 17.45 (3-CH₃). ESI-MS: [M + H]⁺ 332.1399 ; found 332.1387.

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

Biological Activity

The antimicrobial activity of the N-protected luotonin derivatives was measured according to the previously reported method.^18,19

Minimal inhibitory concentration

Antibacterial activities were evaluated by the micro broth dilution method in 96-well culture plates using Mueller-Hinton broth, according to the National Committee for Clinical Laboratory Standards.^20,21 All compounds were tested in triplicate at each concentration.

Inhibition of spore germination method

All experiments were conducted in triplicate. The half-maximal effective concentration values were determined by performing the bioassay as described above with concentrations of 100, 75, 50, 37.5, and 25 µg/mL, respectively.

Results and Discussion

Design and Synthesis of Luotonin Analogs

The synthetic route is outlined in Scheme 1. Twenty-one luotonin analogs, including the previously prepared c1-c5, have been synthesized by this efficient method. The luotonin analogs were synthesized using 2-aminobenzamide as the starting material via acylation or alkylation at the N-position. The structures of the synthesized compounds were confirmed by ¹H NMR and ¹³C NMR spectroscopy, and high-resolution mass spectrometry.

Antimicrobial Activity

All the target compounds were screened for antibacterial activities against 5 Gram-negative (Pseudomonas solanacearum, Pseudomonas aeruginosa, Escherichia coli, Ralstonia solanacearum, and Pseudomonas syringae pv. actinidiae) and 3 Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and B. cereus). The minimum inhibitory concentrations (MICs) were determined by the double-dilution method using penicillin sodium and fosfomycin sodium as the positive controls. The MIC values are summarized in Table 1. The target compounds showed moderate antibacterial activities against S. aureus, E. coli, P. aeruginosa, B. cereus, P. syringae pv. actinidiae, P. solanacearum, and R. solanacearum. Compounds a1, a3, a6, a9, b2, and b5 manifested greater activity against P. solanacearum than fosfomycin sodium. Compound a6 showed a similar degree of activity as fosfomycin sodium against P. aeruginosa. Compounds a5 and b5 showed similar degrees of activity as fosfomycin sodium and penicillin sodium against P. syringae pv. actinidiae.

Table 1

Antibacterial Activity of Luotonin Derivatives Against B. c., B. s., S. a., E.c., P. a., P. s., P. s. a., and R. s.

Compounds	B. c.	B. s.	S. a.	E. c.	P. a.	P. s. a.	P. s.	R. s.
Compounds	2MIC (µg/mL)
P	7.81	7.81	3.91	0.98	62.5	1.95	7.81	3.91
F	3.91	7.81	3.91	0.98	7.81	>125	62.5	0.98
a1	125	>125	>125	>125	>125	>125	31.25	62.5
a2	>125	>125	>125	125	125	>125	62.5	62.5
a3	62.5	31.25	62.5	125	125	31.25	31.25	>125
a4	31.25	>125	15.62	>125	>125	125	125	15.62
a5	15.62	15.62	31.25	15.62	>125	7.81	125	31.25
a6	31.25	>125	31.25	125	7.81	31.25	15.62	62.5
a7	62.5	62.5	31.25	>125	>125	>125	125	15.62
a8	62.5	31.25	62.5	>125	15.62	31.25	125	>125
a9	125	62.5	62.5	>125	>125	15.62	15.62	62.5
a10	62.5	31.25	62.5	15.62	125	125	125	31.25
a11	125	62.5	62.5	>125	125	125	125	31.25
b1	62.5	62.5	62.5	>125	>125	>125	125	62.5
b2	62.5	31.25	62.5	125	15.62	125	15.62	31.25
b3	>125	62.5	62.5	>125	>125	>125	125	15.62
b4	>125	62.5	125	>125	>125	>125	125	62.5
b5	31.25	15.62	62.5	125	>125	15.62	15.62	31.25
c1	>125	>125	125	125	>125	>125	125	>125
c2	62.5	125	62.5	>125	125	>125	125	62.5
c3	62.5	>125	62.5	125	125	62.5	62.5	62.5
c4	62.5	31.25	62.5	125	62.5	>125	62.5	>125
c5	>125	31.25	>125	125	125	>125	62.5	62.5

Abbreviations: Abbreviations: B. c, Bacillus cereus; B. s., Bacillus subtilis; E. c., Escherichia coli; F, fosfomycin sodium; MIC, minimum inhibitory concentration; P, penicillin sodium; P. a., Pseudomonas aeruginosa; P. s., Pseudomonas solanacearum; P. s. a., Pseudomonas syringae pv. actinidiae; R. s., Ralstonia solanacearum.

Note. P and F were used as positive controls.

The activity of the synthesized compounds toward plant pathogen fungi was also screened, and the results are listed in Table 2. A mycelial growth inhibition assay was utilized, with carbendazim as the positive control, to evaluate the activity of the 21 synthesized N-protected luotonin derivatives against Alternaria alternata, Fusarium oxysporum f. sp. niveum, Colletotrichum gloesporioides, Sclerotinia sclerotiorum, Gibberella zeae, Botrytis cinerea, and Curvularia lunata (Walk) Boedijn at a concentration of 100 µg/mL. Among the synthesized compounds, a5 displayed great activity against C. gloesporioides, with 61.0% inhibition, and compound b5 showed 60.2% inhibition against G. zeae (Table 2). Compound a9 produced 57.3% inhibition of C. gloesporioides, compound b4 showed 52.4% inhibition of S. sclerotiorum, and compound a5 showed 50.5% against B. cinerea.

Table 2

Inhibitory Effects of Luotonin Derivatives Against A.a., F.O.N., C.g., S.s., G., B.c., and C.l.b.

Compounds	Inhibitory ratio at 100 µg/mL (MEANMEAN ± SD) (%)							Sporegermination
Compounds	A.a.	F.O.N.	C. g.	S.s.	G.	B. c.	C. l. B.	Sporegermination
Carbendazim	98.9	98.9	97.5	99.8	98.3	99.4	98.7	-
a1	9.8	18.5	22.4	10.7	31.0	23.0	20.1	0.6
a2	21.2	14.1	34.6	7.6	16.2	23.1	31.4	0.8
a3	26.3	15.8	17.3	30.9	10.0	14.5	25.4	0.4
a4	37.7	9.2	15.2	27.5	30.4	12.1	22.7	0.6
a5	37.2	57.1	61.1	38.5	47.4	50.5	29.7	0.5
a6	26.9	29.9	18.7	15.7	17.4	26.9	29.4	0.2
a7	18.9	28.5	10.9	9.8	29.3	17.8	21.2	0.7
a8	35.1	26.6	27.8	20.5	26.3	32.3	13.9	0.3
a9	28.5	46.6	57.3	40.2	31.2	26.6	18.3	0.4
a10	21.1	12.7	9.2	21.0	38.0	18.1	10.2	0.4
a11	10.6	14.1	9.7	25.4	9.2	20.5	17.7	0.2
b1	33.5	37.2	15.9	28.4	37.4	29.2	17.2	0.4
b2	43.6	28.1	38.5	16.0	27.8	34.1	26.6	0.6
b3	25.8	19.5	38.2	25.7	25.9	38.1	17.5	0.6
b4	19.7	11.9	37.9	52.4	9.2	19.1	24.4	0.4
b5	17.3	37.9	10.7	21.6	60.2	10.7	17.1	0.7
c1	18.6	24.4	34.8	25.6	34.0	13.2	36.3	0.4
c2	16.7	11.3	33.9	24.4	18.5	28.4	25.8	0.6
c3	47.6	29.6	18.3	22.7	23.2	45.9	26.9	0.6
c4	26.1	16.3	34.8	33.5	25.1	44.5	27.9	0.4
c5	42.8	28.6	37.6	18.2	20.3	22.5	25.5	0.3

Abbreviations: Abbreviations: A.a., Alternaria alternata; B. c., Botrytis cinerea; C. g, Colletotrichum gloesporioides; C. l. B., Curvularia lunata (Walk) Boedijn; EC₅₀, half maximal effective concentration; FON, Fusarium oxysporum f. sp. niveum; G, Gibberella zeae; S. s., Sclerotinia sclerotiorum.

Note. The carbendazim was used as the positive control.

Although it is difficult to extract apparent structure-activity relationships from the bioassay results, some conclusions can still be obtained. First, N-substituents (a5-a10, b2, and b5) with a halogen-substituted aryl displayed more antibacterial activities than the compounds (a1-a4, b1, b3, b4, and c1-c5) without a halogen atom. Second, compounds (a1-a11 and b1-b5) with aryl-substituted groups at the N-position showed greater antibacterial activities than the compounds (c1-c5) with alkyl groups. Third, compounds with the halogen atom on the aryl positions of the N-substituents (a5, a6, a8-a10, b2, and b5) showed better antibacterial activities than compounds with the halogen atom attached to the aliphatic chain on the aryl substituent (a7).

Conclusions

Twenty-one luotonin derivatives were synthesized using 2-aminobenzamide as the starting material via either alkylation or acylation at the N-position, and their antimicrobial activities have been evaluated against 8 strains of bacteria and many plant pathogen fungi. The majority of the synthesized compounds showed potent activities. Notably, compounds a5, a9, b4, and b5 demonstrated remarkably activities and might be novel potential lead compounds for further development of antifungal agents.

Supplemental Material

online supplementary file 1 - Supplemental material for Synthesis and Antimicrobial Characterization of Luotonin Derivatives

Supplemental material, online supplementary file 1., for Synthesis and Antimicrobial Characterization of Luotonin Derivatives by Rui Zhu, Rui Guo, Ke Han, Wenying Xi, Shaojun Zheng and Jiwen Zhang in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was supported by the National Key Research and Development Program of China (No. 2017YFD0201402), the National Natural Science Foundation of China (21502073), the Natural Science Foundation of Jiangsu Province (Grants No BK20150465 and BK20180978), and the Key Research and Development Program (Modern Agriculture) of Zhenjiang City (NY2018002), and Zhoushan Public Welfare Science and Technology Project (2019C31066).

ORCID iD

Shaojun Zheng

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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