Synthesis and Antimicrobial Activity of Calycanthaceous Derivatives

Abstract

A series of calycanthaceous alkaloid analogs have been synthesized in excellent yields. All the target compounds were evaluated in vitro for biological activity against a broad range of plant pathogen fungi, bacteria and human pathogenic fungi, and some of the designed compounds exhibited potential activity in the primary assays. Notably, Compound b7 illustrated higher degrees of activity against Aspergillu sflavu than amphotericin B, with a minimal inhibitory concentration value of 15.63 µg·mL⁻¹. Compound b7 displayed the most effective activity among the tested calycanthaceous analogs and might be a novel potential leading compound for further development of antifungal agent.

Introduction

The use of synthetic pesticides in agriculture worldwide is still the most widespread method for the control of plant diseases. However, the extensive application of agrochemicals over the years has led to the development of resistance in pest populations and environmental problems. Consequently, the discovery of new agrochemicals with high efficacy and selectivity against target species directly or indirectly from natural product has recently been crucial in the research and development of agrochemicals.

The hexahydropyrroloindole skeletons are very important moieties that are widespread in a large family of natural products with wide range of attractive bioactivities. Calycanthaceous alkaloids^1-4 (Figure 1), which contain hexahydropyrroloindole skeletons, are an important class of alkaloid that can be isolated from roots, leaves, flowers, and fruits of chimonanthus praecox. ⁵ The Calycanthaceae plants have been used as traditional Chinese medicines for the treatment of fungal, ⁶ hypertension, tumor, and inflammatory.^7,8

Figure 1.

Structures of calycanthaceous alkaloids.

Our group has recently reported the preparation and potent antimicrobial activity of calycanthaceous alkaloid derivatives.^9-15 These findings inspired us to further modify the structure of calycanthaceous alkaloids with functional motifs so as to acquire potential agrochemical leads for plant disease control.

As part of ongoing efforts to discover new natural-product-based antifungal agent, 2 novel series of N-substituted calycanthaceous alkaloid derivatives were designed and synthesized, and their structures were identified on the basis of satisfactory analytical and spectral (¹H NMR, ¹³C NMR, and ESI-MS) data.

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

Design and Synthesis of Calycanthaceous Alkaloids Analogs

The synthetic route to the target compounds is shown in Figure 2. A total of 36 calycanthaceous analogs were prepared from indole-3-acetonitrile via acylation at the N-position according to a previously reported procedure in our group and the spectral data were characterized by ¹H-NMR, ¹³C-NMR spectroscopy, and ESI-MS.^16-25

Figure 2.

Synthetic route to the title compounds a1 to a18 and b1 to b18.

Antimicrobial Acitivity

The results of the biological testing against a wide range of plant pathogen fungi, Gram-negative bacteria, Gram-positive bacteria, and human pathogenic fungi are listed in Table 1. The minimal inhibitory concentration (MIC) were evaluated with Carbendazim, Amphotericin B, Chlorothalonil, Gentamicin, Streptomycin, Penicillin, and Fluconazole as positive controls, to evaluate the activity of the synthesized calycanthaceous alkaloid analogs against Verticillium dahliae, Fusarium oxysperium sp vasinfectum, Cytospora juglandis, Aspergillu sflavu, Penicillium citrinum, Fusarium oxysporum, Colletotrichum orbiculare, Aspergillus niger, Bcinerea pers, Curvularia lunaia, Escherichia sp, Pseudomonas aeruginosa, Ralstonia solanacearum, Bacillus cereus, Staphylococcus aureus, Candida krusei, Crytococcus neofonmans, and C tropicalis.

Table 1.

MIC of Compounds Against Plant Pathogenic Fungi, Gram-Negative Bacteria, Gram-Positive Bacteria, and Human Pathogenic Fungi.

Plant pathogen fungi											Gram-negative bacteria			Gram-positive bacteria		Human pathogenic fungi
Comp.	V.d	F.v	C.j	A.s	P.c	F.o	C.o	A.n	B.p	C.l	E.s	P.a	R.s	B.c	S.a	C.k	C.n	C.t
MIC (µg mL)
a1	–	–	–	31.25	–	–	–	–		–	–		–	–	–	–	–	–
a2	–	–	–	250	–	–	–	–		–	–		–	–	–	–	–	250
a3	–	–	–	125	–	–	–	–		–	–		–	–	–	–	–	250
a4	–	–	–	125	–	–	–	–		–	–		–	–	–	–	–	–
a5	–	–	–	250	–	–	–	–		–	–		–	–	250	–	–	–
a6	250	–	250	62.5	–	250	–	–		125	250		–	–	250	–	–	–
a7	–	–	250	62.5	–	250	250	250		125	250		–	250	–	–	–	–
a8	62.5	–	250	–	–	125	250	250		–	250		–	–	250	250	–	–
a9	–	–	–	–	–	–	–	–		–	–		–	–	–	–	–	–
a10	–	–	–	–	–	–	–	–		250	–		–	–	–	–	–	–
a11	–	–	–	250	–	–	–	–		–	–		–	–	–	–	–	–
a12	–	–	–	62.5	–	–	–	–		–	–		–	–	–	–	–	–
a13	–	–	–	125	–	–	–	–		–	–		–	–	–	–	–	–
a14	–	–	–	–	–	–	–	–		–	–		–	–	–	–	–	–
a15	–	–	–	125	–	–	–	–		–	–		–	–	–	–	–	–
a16	–	–	–	62.5	–	–	–	–		–	–		–	–	–	–	–	–
a17	–	–	–	–	–	–	–	–		–	–		–	–	–	–	–	–
a18	–	–	–	125	–	–	–	–		–	–		–	–	–	–	–	–
b1	–	–	–	31.25	–	–	–	–		–	–		–	–	–	–	–	–
b2	–	–	–	125	–	–	–	–		–	–		–	–	–	–	–	250
b3	–	–	–	250	–	–	–	–		–	–		–	–	–	–	–	250
b4	–	–	–	125	–	–	–	–		–	–		–	–	–	–	–	–
b5	–	–	–	125	–	–	–	–		–	–		–	–	250	–	–	–
b6	250	–	250	62.5	–	250	250	250		125	250		–	–	250	–	–	250
b7	250	–	250	15.63	–	250	250	250		125	250		–	250	–	–	–	–
b8	125	–	250	125	–	125	250	250		–	250		–	250	250	250	–	–
b9	–	–	–	250	–	–	–	–		–	–		–	–	–	–	–	–
b10	–	–	–	–	–	–	–	–		–	–		–	–	–	–	–	–
b11	–	–	–	62.5	–	–	–	–		–	–		–	–	–	–	–	–
b12	–	–	–	–	–	–	–	–		–	–		–	–	–	–	–	–
b16	–	–	–	62.5	–	–	–	–		–	–		–	–	–	–	–	–
b17	–	–	–	250	–	–	–	–		–	–		–	–	–	–	–	–
b18	–	–	–	125	–	–	–	–		250	250		–	–	–	–	–	–
Ca	7.8	62.5	31.3	7.8	1.9	125	125	–	1.9	250						250	–	250
A	1.9	–	250	31.3	31.3	62.5	250	3.9	–	7.8						250	1.9	–
Ch	31.3	250	62.5	7.8	15.6	62.5	250	15.6	31.3	125						7.8	–	1.9
G											1.9	1.9	62.5
S											31.3	–	250
P														7.8	15.6
F																62.5	–	250

Note: The Carbendazim and chlorothalonil were used as the positive controls; “─” means no inhibition effect.

Abbreviations: MIC, minimal inhibitory concentration; V.d, Verticillium dahliae; F.v, Fusarium oxysperium sp. vasinfectum; C.j, Cytospora juglandis; A.s, Aspergillu sflavu; P.c, Penicillium citrinum; F.o, F oxysporum; C.o, Colletotrichum orbiculare; A.n, A niger; B.p, Bcinerea pers; C.l, Curvularia lunaia; E.s,Escherichia sp; P.a, Pseudomonas aeruginosa; R.s, Ralstonia solanacearum; B.c, Bacillus cereus; S.a, Staphylococcus aureus; C.k, C krolimus; C.n, Crytococcus neofonmans; C.t, Candida tropicalis; Ca, Carbendazim; A, amphotericin B; Ch, chlorothalonil; G, gentamicin; S, streptomycin; P, penicillin; F, fluconazole.

It is manifested that compounds a1, a6, a7, a8, generally exhibit more effective antimicrobial activity than the positive control. Compounds a6 and a7 illustrated improved activity against C lunaia compared with the positive control carbendazol, both with the same MIC value of 125 µgmL⁻¹. Compound a1 manifested better activity against V dahliae than Chlorothalonil, with a MIC value of 31.25 µg mL⁻¹. The activity of Compound a17 is more potent than carbendazol, amphotericin B, and Chlorothalonil against F oxysporum, all with the same MIC value of 31.25 µg mL⁻¹.Compound a1 manifested much more activity against A sflavu than amphotericin B, with a MIC value of 31.25 µg mL⁻¹. Compound a8 showed comparable control efficacy against F oxysporum to carbendazol, with a MIC value of 125 µg mL⁻¹. Compounds a6 and a7 revealed comparable control efficacy against C lunaia to carbendazol, both with the same MIC value of 125 µg mL⁻¹. Compound b1 showed comparable control efficacy against A sflavu to amphotericin B, with a MIC value of 31.25 µg mL⁻¹. The activity of compound b7 is more potent than amphotericin B against A sflavu, with a MIC value of 15.63 µg mL⁻¹.Compounds b6 and b7 revealed improved activity against C juglandis compared with the positive control carbendazol and Chlorothalonil, both with the same MIC value of 125 µg mL⁻¹.Compounds b6, b7, and b8 illustrated greater activity against C juglandis than amphotericin B, all with the same MIC value of 250 µg mL⁻¹.Compounds b6, b7, and b8 manifested much more activity against C orbiculare than amphotericin B and Chlorothalonil, all with the same MIC value of 250 µg mL⁻¹.Compounds b2, b3, and b6 showed more effective activity against C tropicalis than carbendazol and Fluconazole, all with the same MIC value of 250 µg mL⁻¹.

Materials and Methods

Instruments and Chemicals

All reagents and solvents were reagent grade or purified according to standard methods before use. Analytical thin-layer chromatography was performed with silica gel plates using silica gel 60 GF₂₅₄ (Qingdao Haiyang Chemical Co., Ltd). Melting points were measured on an Electrothermal digital apparatus and were uncorrected. The ¹H - NMR (500 MHz), and ¹³C-NMR (125 MHz) were obtained on an AM-500 FT-NMR spectrometer (Bruker Corporation) with CDCl₃ as the solvent and tetramethylsilane as the internal standard. Mass spectrum (MS) were recorded under electrospray ionization (ESI) conditions using a LCQ Fleet instrument (Thermo Fisher). Yields were not optimized. The title compounds were synthesized under a nitrogen atmosphere.

Synthesis

Intermediates 1 to 3 Were Synthesized According to Our Previously Reported Procedure ¹²

Synthesis of Compounds a1 to a18 and b1 to b18

Compound 3 was dissolved in pyridine (10 mL), then, the corresponding desired reagent was added at 0 °C. After refluxing for 2 h. The mixture was warmed to room temperature. Then, the resulting mixture were reacted for 1.5 h. At last, the resulting mixture was quenched with methanol (2 mL), and extracted with ethyl acetate. The organic extracts were combined, washed with brine, dried over Na₂SO₄, and concentrated. Purification by flash chromatography on silica gel afforded the compounds a1 to a18 and b1 to b18 in yields from 80% to 96% (for characterization data see Supplemental Materials).

Biological Activity

The antimicrobial activity of calycanthaceous alkaloids analogs was measured according to the previously reported method.^26,27

The tested compounds dissolved in 5% dimethyl sulfoxide, to a concentration of 1.02 mg/mL, 100 μL of the solutions were added to the first well and serially diluted from first well by taking 100 μL into second. This 2-fold dilution was continued down the plate and 100 μL from the eighth column of the plated discarded. The ninth column of the plate was reserved for negative control wells (without inocula) and the tenth column, for the positive growth control wells (without antibacterial agent). The antibacterial concentrations were 256, 128, 64, 32, 16, 8, 4, and 2 µg mL, respectively. The antibacterial test plates were incubated aerobically at 37 °C for 24 h, the antifungal test plates were incubated aerobically at 28 °C for 48 h. The MICs were examined. All tests were performed in triplicate and repeated if the results differed.

Conclusions

A total of 36 novel tetrahydropyrroloindole-based calycanthaceous alkaloid analogs were prepared using indole-3-acetonitrile as the starting material via acylation at the N-position and the activity against a wide range of plant pathogen fungi were screened. Notably, compound b7 illustrated potent activity against A sflavu than that of amphotericin B, with MIC value of 15.63 µg mL⁻¹. Compound b7 displayed a remarkably activity among the tested calycanthaceous analogs and might be a novel potential leading compound for further development of antifungal agents. Further structural optimization of calycanthaceous alkaloid is well under way, alongside more detailed biological testing of the most active compounds, aiming to improve their levels of antifungal activity.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X211032611 - Supplemental material for Synthesis and Antimicrobial Activity of Calycanthaceous Derivatives

Supplemental material, sj-docx-1-npx-10.1177_1934578X211032611 for Synthesis and Antimicrobial Activity of Calycanthaceous Derivatives by Rui Zhu, Cheng Yang, Ke Han, Yongdong Gu, Jiwen Zhang, Shaojun Zheng and Hongjin Bai in Natural Product Communications