Synthesis and Antifungal Activity of Novel Tetrahydrogeranyl Quaternary Ammonium Salts

Abstract

Due to the excessive use of antifungal agents, drug resistance and ecological problems are increasing. Some antifungal agents are difficult to degrade and have high toxicity and several side effects. In this study, 15 novel tetrahydrogeranyl quaternary ammonium salts (8a–8o) were synthesized from the natural compound citral. The structures of the quaternary ammonium salts were characterized by Fourier transform infrared, proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance spectroscopy, and mass spectrometry, and the antifungal activities of these compounds at a concentration of 0.25 mg/mL against 10 plant pathogenic fungi were tested. The results showed that compound 8i had the best antifungal activity, and its inhibition rates against Rhizoctonia solani, Phytophthora parasitica var. nicotianae, Sphaeropsis sapinea, Fusarium oxysporum f. sp. niveum, and Poria vaporaria reached 100%. For Fusarium verticillioides, the inhibition rate of compound 8i was 93.28%, which was higher than that of chlorothalonil. In addition, it was found that the inhibition rates of compounds with N,N-di-n-propyl group (8l, 8m) against R solani, F oxysporum f. sp. niveum, S sapinea, P parasitica var. nicotianae, F verticillioides, Colletotrichum acutatum, and Coriolus versicolor were higher than compounds with N,N-diethyl and N,N-dimethyl groups (8a, 8b, 8j, 8k). The inhibition rates of compounds with morpholine groups (8n, 8o) were generally low.

Introduction

Plant pathogenic fungi are the main factors causing various plant diseases that result in reduced production in agriculture and forestry. ¹ Antifungal agents are prepared from compounds with certain activities that can kill or inhibit the reproduction of bacteria, fungi, and other microorganisms within a certain period of time.^2–4 However, the excessive use of antifungal agents will lead to drug resistance and ecological problems. Some highly toxic antifungal agents are difficult to degrade and have serious side effects. ⁵ It is very important to develop new plant antifungal agents with low toxicity and high efficiency. Citral is the main component of Litsea cubeba essential oil and possesses excellent biological and pharmacological properties. ⁶ It has been reported that citral derivatives have different activities against some plant pathogenic fungi.^7,8

It is generally recognized that a new generation of antifungal agents with environmental friendliness and high efficiency can hopefully be synthesized from some natural products. Rastija et al ⁹ studied a series of coumarin derivatives that were effective against Macrophomina phaseolina and Sclerotinia sclerotiorum. Quaternary ammonium salts, which are synthesized by the reaction of amines and haloalkanes, have stable chemical properties and good solubility in water. In recent years, quaternary ammonium salts have been reported to be used as fungicides because of their strong antifungal activity.^10–15 Feng et al^15–17 synthesized a series of quaternary ammonium salts with bridge ring structures using β-pinene as raw material and tested the antifungal activity of these compounds against plant pathogenic fungi. In this paper, citral was selected as the raw material to synthesize tetrahydrogeranyl quaternary ammonium salts through hydrogenation, bromination, amination,^18,19 and halogenation reactions, and the synthesized compounds contained tetrahydrogeranyl groups, N,N-diethyl, N,N-dimethyl, and N-morpholine groups. The active differences of these compounds were investigated based on the germicidal results of the tetrahydrogeranyl quaternary ammonium salts against 10 plant pathogenic fungi, and the structure–activity relationship was also examined preliminarily. These research results provide significant evidence for the processing and utilization of L cubeba essential oil.

Results

Synthesis

The synthetic routes of tertiary amines and quaternary ammonium salts are shown in Figures 1 and 2. The tertiary amines (compounds 4-7) were prepared in our laboratory, and 15 new tetrahydrogeranyl quaternary ammonium salts were synthesized by alkylation of compounds 4-7 with haloalkanes (methyl bromide, ethyl bromide, n-propyl bromide, n-butyl bromide, n-hexyl bromide, methyl iodide, ethyl iodide, n-propyl iodide, and n-butyl iodide). Quaternization was relatively easy to realize under mild conditions.

Figure 1.

Synthetic route of tetrahydrogeranyl tertiary amines and the structure of chlorothalonil.

Figure 2.

Synthetic route of tetrahydrogeranyl quaternary ammonium salts.

The molecular structures of the tetrahydrogeranyl quaternary ammonium salts were characterized by Fourier transform infrared (FTIR), proton nuclear magnetic resonance (¹H NMR), and carbon-13 nuclear magnetic resonance (¹³C NMR) spectroscopy and mass spectrometry (MS), and the results showed that the target derivatives 8a-8o were successfully synthesized.

Taking compound 8i as an example to analyze the characterization of tetrahydrogeranyl quaternary ammonium salts, in its ¹H NMR spectrum the protons of C₈-H and C₁₀-H were observed as multiplets at 0.876 and 0.859 ppm, respectively, and the protons of C₁-H, C_α-H, and C₁₁-H as single peaks at 3.555, 3.511, and 3.391 ppm, respectively. In the ¹³C NMR spectrum of compound 8i, the δ values of carbon protons around N⁺ (C_-11, C_-1, and C_-α) were observed at 62.44, 63.52, 51.26, and 13.83 ppm, respectively, and that of C_-16 was observed at 13.83 ppm. The mass spectrum of 8i showed an expected molecular ion peak at m/z 270.31 (M⁺ − Br) and 428.15, 430.15, and 432.15 (M⁺ + Br) corresponding to the molecular formula.

Biological Activity

The inhibition rates of 15 tetrahydrogeranyl quaternary ammonium salts for the growth of 10 phytopathogenic fungi are shown in Table 1.

Table 1.

Inhibition Rates of Compounds for the Mycelium Growth of Plant Pathogenic Fungi.

Compounds	Inhibition rate at 0.25 mg/mL/% against the different fungi
	A		B	C	D	E	F	G	H	I	J
8a	41.04		31.05	36.78	63.68	29.31	16.60	32.32	38.95	22.31	100
8b		46.29	37.83	52.03	68.41	62.79	19.46	28.04	33.33	17.68	100
8c		50.06	32.22	55.73	85.36	29.51	23.63	29.95	40.08	16.62	100
8d		53.54	48.86	64.36	94.34	60.85	25.90	26.22	43.07	28.27	100
8e		88.17	20.10	29.08	88.17	50.87	21.18	34.38	13.52	9.37	100
8f		98.97	100	98.52	98.97	61.09	25.64	28.64	44.04	38.01	100
8g		75.21	40.97	46.94	90.59	70.43	29.12	34.94	16.78	13.82	100
8h		76.35	100	100	100	70.48	29.74	32.28	44.03	42.76	100
8i		100	100	100	100	93.28	57.64	43.72	73.67	61.79	100
8j		58.94	36.85	46.13	74.79	30.26	12.53	32.26	35.57	17.68	100
8k		55.47	54.93	66.89	78.37	36.00	15.47	30.91	30.71	16.25	100
8l		70.29	55.22	75	81.84	37.87	21.98	31.52	31.52	25.47	100
8m		74.94	60.86	60.05	71.29	45.06	20.15	26.79	38.31	35.07	100
8n		23.06	18.78	37.57	33.79	16.31	5.67	24.21	17.37	9.40	100
8o		52.92	28.93	33.82	46.00	14.22	6.96	25.52	29.95	10.66	100
Chlorothalonil		100	100	100	71.40	65.95	68.73	100	100	100	100

Notes: A. Rhizoctonia solani; B. Phytophthora parasitica var. nicotianae; C. Sphaeropsis sapinea; D. Fusarium oxysporum f. sp. niveum; E. Fusarium verticillioides; F. Colletotrichum acutatum; G. Colletotrichum gloeosporioides; H. Colletotrichum fructicola; I. Coriolus versicolor; J. Poria vaporaria.

At a concentration of 0.25 mg/mL, the inhibition rates of compound 8i against the 10 plant pathogenic fungi were the highest among compounds 8a-8o. The inhibition rates of compounds 8f, 8h, and 8i against Phytophthora parasitica var. nicotianae, Sphaeropsis sapinea, and Fusarium oxysporum f. sp. niveum were more than 95%. For Rhizoctonia solani, the inhibition rates of compounds 8f and 8i were 98.87% and 100%, respectively. In addition, the inhibition rates of compound 8i against F oxysporum f. sp. niveum and Fusarium verticillioides were higher than that of chlorothalonil, and the inhibition rates of 15 compounds (8a-8o) against Poria vaporaria reached 100%. In contrast, the inhibition rates of these compounds (except 8i) against Colletotrichum acutatum, Colletotrichum gloeosporioides, Colletotrichum fructicola, and Coriolus versicolor were below 50%.

From the structural differences of these compounds, it can be seen that compounds 8b, 8d, 8f, 8h, 8k, 8m, and 8o containing iodine anions had higher inhibitory activity than compounds 8a, 8c, 8e, 8g, 8j, 8l, and 8n with bromine ions against P parasitica var. nicotianae. For R solani, P parasitica var. nicotianae, S sapinea, and F oxysporum f. sp. niveum, the inhibition rates of compounds 8l and 8m with N,N-dipropyl groups were higher than those of compounds with N,N-diethyl (8j, 8k) and N,N-dimethyl (8a, 8b) groups. The inhibition rates of compounds with N-morpholine groups (8n, 8o) were generally lower than those of other compounds. In addition, the antifungal effect was generally better as the carbon chain length increased for some plant pathogenic fungi.

Discussion

Citral is a natural plant resource with a unique active structure, and it was found that the biological activities of citral derivatives were higher than those of citral. For example, Zhou et al ²⁰ studied the minimum inhibitory concentration and minimum fungicidal concentration of citral against Geotrichum citri-aurantii, which were 0.50 and 1.00 μL/mL, respectively. Moon et al ²¹ reported that citral had a strong inhibitory effect against Aspergillus flavus growth at 1000 μg/mL, but this effect was not observed at 10 μg/mL. On the other hand, Halli and Sumathi ²² reported the antioxidant antibacterial activities of citral and its metal complexes in vitro. The results indicated that the biological activity of citral increased after complexation with metal. ²² Saddiq and Khyyat ²³ reported that the epoxide of citral showed a higher activity for inhibiting the growth of methicillin-resistant Staphylococcus aureus and fungi than that of citral.

In this study, 15 tetrahydrogeranyl quaternary ammonium salt derivatives were synthesized from citral. The antifungal activity of the synthesized derivatives against 10 plant pathogens that cause significant damage to crops was tested in this study. R solani is a soil-borne phytopathogenic species complex as well as a necrotic fungus that causes significant crop yield losses worldwide. ²⁴ S sapinea is known as a major cause of damage to red pine seedlings in nurseries. The fungus can also be a latent pathogen of red pine seedlings, persisting in the absence of gross symptoms and later proliferating under conditions that induce host stress, ²⁵ and watermelon Fusarium wilt is one of the most severe soil-borne diseases caused by F oxysporum f. sp. Niveum. ²⁶ The results showed that 15 compounds had certain antifungal activities against the 10 tested strains at a concentration of 0.25 mg/mL. The inhibition rates of compound 8i against F oxysporum f. sp. niveum and F verticillioides were 100% and 93.28%, respectively, which were higher than the positive control chlorothalonil (71.4% and 65.95%, respectively).

In addition, some relationships between the structure of compounds and the antifungal rates were found. For F oxysporum f. sp. niveum and F. verticillioides, the longer the carbon chain was, the higher the antifungal rates of the compound (8i > 8g > 8e > 8c > 8a, 8b > 8d > 8f > 8h), which indicated that the inhibitory activity of quaternary ammonium salts with long alkyl chains against fungi was generally higher than that of salts with short alkyl chains.^27,28 For P parasitica var. nicotianae, the inhibitory activity of quaternary ammonium salts with iodine anions was obviously better than that of salts with bromine anions (8b > 8a, 8d > 8c, 8f > 8e, 8h > 8g, 8k > 8j, 8m > 8l, 8o > 8n), which may indicate that iodide ions play a key role in inhibiting the growth of P parasitica var. nicotianae. Wei et al ¹⁰ and Jin et al ²⁹ reported that the introduction of halogen groups into quaternary ammonium salt structures was beneficial to enhance antifungal activity. For R solani, P parasitica var. nicotianae, S sapinea and F oxysporum f. sp. niveum, compounds containing an N,N-di-n-propyl group showed higher antifungal activity than compounds with N,N-diethyl and N,N-dimethyl groups (8l > 8j > 8a, 8m > 8k > 8b). Compounds with N-morpholine (8n, 8o) showed poor inhibition rates against plant pathogenic fungi in this experiment (except P vaporaria).

Materials and Methods

General

Methyl bromide, ethyl bromide, n-propyl bromide, n-butyl bromide, n-hexyl bromide, methyl iodide, ethyl iodide, n-propyl iodide, n-butyl iodide, ethyl acetate, and light petroleum (purity >99%) were purchased from Aladdin Co. Ltd Citral (purity >97%, cis and trans) was purchased from Macklin Co. Ltd. Tetrahydrogeranyl dimethylamine, tetrahydrogeranyl diethylamine, tetrahydrogeranyl dipropylamine, and N-tetrahydrogeranyl morpholine (purity >95%, chiral) were prepared in our laboratory.

Plant pathogenic fungi used in the study were preserved and provided by the Administration on Forest Ecosystem Protection and Restoration of Poyang Lake Watershed: R solani, P parasitica var. nicotianae; S sapinea, F oxysporum f. sp. niveum, F verticillioides, C acutatum, C gloeosporioides, C fructicola, C versicolor, and P vaporaria.

FTIR spectra of the compounds were recorded on a Nicolet IS10 FTIR spectrometer, and ¹H NMR and ¹³C NMR spectra on a Bruker AVANCE 400 NMR spectrometer (Bruker) using deuterium oxide (D₂O) as a solvent and trimethylsilane as the internal standard. Electrospray ionization MS was conducted on a Bruker Amazon SL mass spectrometer, and electron impact (EI)-MS on a Bruker Amazon SL mass spectrometer (Bruker). The purity of the compounds was detected by liquid chromatography (Agilent Technologies Singapore International Pte., Ltd). Melting points were determined in a WRS-2 melting point apparatus (Shanghai Precision & Scientific Instrument Co., Ltd) and were uncorrected, and a GHP-250 Intelligent incubator (Shanghai Sanfa Scientific Instrument Co., Ltd) was selected to culture strains during the experiment.

Quaternary Ammonium Salt Synthesis

Tertiary amine 4 was reacted with methyl bromide, methyl iodide, ethyl bromide, ethyl iodide, n-propyl bromide, n-propyl iodide, n-butyl bromide, n-butyl iodide, and n-hexyl bromide. Tertiary amines 5-7 were reacted with methyl bromide and methyl iodide. Tertiary amines (0.2 mol), ethyl acetate (20 mL), and haloalkane (0.22 mol) were added to a flask equipped with a stirrer, condenser, and thermometer, and the reaction was carried out at 75 °C for 6 h. The reaction mixture was evaporated to remove ethyl acetate, and the residue was washed with light petroleum 3 to 5 times, recrystallized with a mixture of ethanol and ethyl acetate, and freeze-dried to obtain the final compounds.

The characterization data of the compounds are shown in Table 2.

Table 2.

Structural Analysis of 15 Quaternary Ammonium Salts.

Compounds	Characterization data
8a	White solid; m.p. 85 °C-89 °C; yield 85%; purity 92%; IR (cm−1): 2955, 2928, 2870, 1468, 1384, 1366, 1169; 1H NMR (D2O, 400 MHz) δH: 3.185 (m, 2H, 1−CH2), 2.934 (s, 9H, 11−CH3, 2 α−CH3), 1.604 (m, 1H, 3−CH), 1.461-1.347 (m, 3H, 7−CH, 2−CH2), 1.161-0.986 (m, 6H, 4−CH2, 5−CH2, 6−CH2), 0.755 (d, J = 6 Hz, 3H, 9−CH3), 0.687 (d, J = 7.2 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 65.52 (C−1), 52.80 (2C−α, C−11), 38.53 (C−2), 36.09 (C−6), 30.17 (C−5), 28.90 (C−4), 27.29 (C−7), 23.81 (C−3), 22.02 (C−8, C−10), 18.56 (C−9); EI-MS m/z: 200.24 (M+ − Br); 358.07, 360.07, 362.07 (M+ + Br).
8b	Light yellow solids; m.p. 89 °C-92 °C; yield 85%; purity 92%; IR (cm−1): 2955, 2929, 2870, 1470, 1384, 1366; 1H NMR (D2O, 400 MHz) δH: 3.420 (m, 2H, 1−CH2), 3.093 (s, 9H, 11−CH3, 2 α−CH3), 1.692 (m, 1H, 3−CH), 1.436 (m, 3H, 7−CH, 2−CH2), 1.280-1.015 (m, 6H, 4−CH2, 5−CH2, 6−CH2), 0.861 (d, J = 5.6 Hz, 3H, 9−CH3), 0.762 (d, J = 6.4 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 65.27 (C−1), 53.45 (2C−α, C−11), 39.04 (C−2), 36.95 (C−6), 30.66 (C−5), 29.67 (C−4), 27.82 (C−7), 24.58 (C−3), 22.79 (C−8), 22.70 (C−10), 19.33 (C−9); EI-MS m/z: 200.24 (M+ − I); 454.05 (M+ + I), 126.90 (I−).
8c	White solid; m.p. 78 °C-82 °C; yield 80%; purity 90%; IR·Vmax (cm−1): 2955, 2928, 2870, 1467, 1384, 1366, 1024; 1H NMR (D2O, 400 MHz) δH: 3.244 (8, J = 7.2 Hz, 2H, 11−CH2), 3.148 (m, 2H, 1−CH2), 2.910 (s, 6H, 2 α−CH3), 1.603 (m, 1H, 3−CH), 1.413 (m, 3H, 7−CH, 2−CH2), 1.231-0.902 (m, 9H, 4−CH2, 12−CH3, 5−CH2, 6−CH2), 0.799 (d, J = 6 Hz, 3H, 9−CH3), 0.726 (d, J = 6.4 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 64.09 (C−1), 61.35 (C−11), 52.25 (2C−α), 40.88 (C−2), 38.63 (C−6), 32.58 (C−3), 30.78 (C−4), 29.67 (C−7), 26.32 (C−5), 24.56 (C−8), 24.46 (C−10), 20.93 (C−9), 9.78 (C−12); EI-MS m/z: 214.25 (M+ − Br); 372.09, 374.09, 376.09 (M+ + Br).
8d	Light yellow solids; m.p. 88 °C-91 °C; yield 80%; purity 91%; IR (cm−1): 2954, 2927, 2869, 1467, 1384, 1022; 1H NMR (D2O, 400 MHz) δH: 3.41 (8, J = 7.2 Hz, 2H, 11−CH2), 3.35 (m, 2H, 1−CH2), 3.07 (s, 6H, 2 α−CH3), 1.73 (m, 1H, 3−CH), 1.55 (m, 3H, 7−CH, 2−CH2), 1.30∼1.14 (m, 9H, 4−CH2, 12−CH3, 5−CH2, 6−CH2), 0.931 (d, J = 6 Hz, 3H, 9−CH3), 0.831 (d, J = 6.4 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 61.75 (C−1), 59.07 (C−11), 50.15 (2 C−α), 38.52 (C−2), 36.37 (C−6), 30.22 (C−3), 28.68 (C−4), 27.29 (C−7), 24.04 (C−5), 22.26 (C−8), 22.16 (C−10), 18.83 (C−9), 7.79 (C−12); EI-MS m/z: 214.25 (M+ − I); 468.06 (M+ + I), 126.90 (I−).
8e	Light yellow solids; m.p. 72 °C-75 °C; yield 75%; purity 90%; IR (cm−1): 2954, 2929, 2870, 1467, 1384, 1365, 1167; 1H NMR (D2O, 400 MHz) δH: 3.267 (m, 2H, 1−CH2), 3.192 (m, 2H, 11−CH2), 2.993 (s, 6H, 2α−CH3), 1.656 (m, 3H, 3−CH, 2−CH2), 1.454 (m, 3H, 7−CH, 4−CH2), 1.253 (m, 4H, 12−CH2, 5−CH2), 1.098 (m, 2H, 6−CH2), 0.891 (t, J = 7.2 Hz, 3H, 9−CH3), 0.870 (t, J = 6.8 Hz, 3H, 13−CH3), 0.791 (d, J = 6.4 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 65.00 (C−11), 62.52 (C−1), 50.89 (2C−α), 38.81 (C−2), 36.51 (C−6), 30.48 (C−3), 28.72 (C−4), 27.59 (C−7), 24.19 (C−5), 22.45 (C−8), 22.37 (C−10), 18.86 (C−9), 15.78 (C−12) , 10.07 (C−13); EI-MS m/z: 228.27 (M+ − Br); 386.11, 388.10, 390.10 (M+ + Br).
8f	White solid; m.p. 75 °C-78 °C; yield 75%; purity 90%; IR (cm−1): 2955, 2926, 2867, 1467, 1384; 1H NMR (D2O, 400 MHz) δH: 3.27 (m, 2H, 1−CH2), 3.17 (m, 2H, 11−CH2), 2.99 (s, 6H, 2 α−CH3), 1.68 (m, 3H, 3−CH, 2−CH2), 1.47 (m, 3H, 7−CH, 4−CH2), 1.26 (m, 4H, 12−CH2, 5−CH2), 1.10 (m, 2H, 6−CH2), 0.89 (t, J = 7.2 Hz, 3H, 9−CH3), 0.87 (t, J = 6.8 Hz, 3H, 13−CH3), 0.78 (d, J = 6.4 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 64.74 (C−11), 62.38 (C−1), 50.31 (2 C−α), 38.13 (C−2), 35.76 (C−6), 29.85 (C−3), 28.14 (C−4), 26.93 (C−7), 23.48 (C−5), 21.69 (C−8), 21.69 (C−10), 18.38 (C−9), 15.31 (C−12), 9.53 (C−13); EI-MS m/z: 228.27 (M+ − I); 482.08 (M+ + I)), 126.90 (I−).
8g	Light yellow solids; m.p. 71 °C-74 °C; yield 72%; purity 92%; IR (cm−1): 2956, 2929, 2871, 1467, 1384, 1366, 1176; 1H NMR (D2O, 400 MHz) δH: 3.336 (m, 2H, 1−CH2), 3.243 (m, 2H, 11−CH2), 3.043 (s, 6H, 2 α−CH3), 1.650 (m, 3H, 3−CH, 2−CH2), 1.483 (m, 3H, 7−CH, 4−CH2), 1.361-1.223 (m, 6H, 5−CH2, 12−CH2, 13−CH2), 1.116 (m, 2 H, 6−CH2), 0.902 (t, J = 7.2, 4.8 Hz, 6H, 14−CH3, 9−CH3), 0.814 (d, J = 6.4 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 62.96 (C−1), 61.89 (C−11), 51.36 (2C−α), 38.98 (C−2), 36.73 (C−6), 30.62 (C−3), 28.78 (C−4), 27.76 (C−7), 24.39 (C−5), 24.17 (C−12), 22.61 (C−8), 22.53 (C−10), 19.25 (C−13) , 19.03 (C−9), 13.29 (C−14); EI-MS m/z: 242.28 (M+ − Br); 400.12, 402.12, 404.12 (M+ + Br)
8h	Brown yellow liquid; yield 72%; purity 93%; IR (cm−1): 2957, 2929, 2871, 1466, 1366, 1384, 1169; 1H NMR (D2O, 400 MHz) δH: 3.288 (m, 4H, 1−CH2, 11−CH2), 3.016 (s, 6H, 2 α−CH3), 1.650 (m, 3H, 3−CH, 2−CH2), 1.626 (m, 3H, 3−CH, 2−CH2), 1.473 (m, 3H, 7−CH, 2−CH2), 1.292 (m, 6H, 5−CH2, 12−CH2, 13−CH2), 1.140 (m, 2 H, 6−CH2), 0.930 (t, J = 7.2 Hz, 3H, 14−CH3), 0.891 (d, J = 6.0 Hz, 3H, 9−CH3), 0.855 (d, J = 6.8 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 62.63 (C−1), 61.78 (C−11), 49.99 (2C−α), 38.59 (C−2), 36.21 (C−6), 30.33 (C−3), 28.31 (C−4), 27.34 (C−7), 23.92 (C−5), 23.75 C−12), 22.54 (C−8), 22.43 (C−10), 19.25 (C−13), 19.18 (C−9), 13.45 (C−14); EI-MS m/z: 242.28 (M+ − I); 496.09 (M+ + I), 126.90 (I−).
8i	Light yellow liquid; yield 72%; purity 95%; IR (cm−1): 2956, 2929, 2871, 1468, 1384, 1367, 1169; 1H NMR (D2O, 400 MHz) δH: 3.555 (m, 2H, 11−CH2), 3.511 (m, 2H, 1−CH2), 3.391 (s, 6H, 2 α−CH3), 1.720 (m, 3H, 3−CH, 2−CH2). 1.538 (m, 3H, 7−CH, 2−CH2), 1.336 (m, 8H, 5−CH2, 12−CH2, 13−CH2, 14−CH2), 1.149 (m, 4H, 6−CH2, 15−CH2), 0.977 (d, J = 6.4 Hz, 3H, 9−CH3), 0.893 (t, J = 6.8 Hz, 16−CH3), 0.867 (d, J = 6.8 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 Hz) δC: 63.52 (C−1), 62.44 (C−11), 51.26 (2C−α), 38.88 (C−2), 36.94 (C−6), 31.17 (C−12), 30.72 (C−3), 29.33 (C−4), 27.79 (C−7), 25.75 (C−13), 24.76 (C−15), 24.46 (C−5), 22.59 (C−14), 22.46 (C−10), 22.30 (C−8), 19.35 (C−9), 13.83 (C−16); EI-MS m/z: 270.31 (M+ − Br); 428.15, 430.15, 432.15 (M+ + Br).
8j	Light yellow solids; m.p. 85 °C-89 °C; yield 75%; purity 93%; IR (cm−1): 2954, 2928, 2869, 1464, 1366, 1384, 1177; 1H NMR (D2O, 400 MHz) δH: 3.201 (8, J = 7.2 Hz, 4H, 2 α−CH2), 3.119 (m, 2H, 1−CH2), 2.822 (s, 3H, 11−CH3), 1.550 (m, 1H, 3−CH), 1.390 (m, 3H, 7−CH, 2−CH2), 1.153 (m, 9H, 4−CH2, 2 β−CH2, 5−CH), 1.027 (m, 3H, 5−CH, 6−CH2), 0.804 (s, 3H, 9−CH3), 0.720 (d, J = 6.4 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 60.79 (C−1), 58.34 (2 C−α), 49.07 (C−11), 40.78 (C−2), 38.51 (C−6), 32.55 (C−3), 30.23 (C−4), 29.56 (C−7), 26.14 (C−5), 24.45 (C−8), 24.35 (C−10), 20.86 (C−9), 9.30 (2 C−β); EI-MS m/z: 228.27 (M+ − Br); 386.11, 388.10, 390.10 (M+ + Br).
8k	Brown solid; m.p. 78 °C-80 °C; yield 75%; purity 92%; IR (cm−1): 2954, 2927, 2869, 1462, 1384, 1366, 1176; 1H NMR (D2O, 400 MHz) δH: 3.397 (8, J = 7.2 Hz, 4H, 2 α−CH2), 3.298 (m, 2H, 1−CH2), 3.003 (s, 3H, 11−CH3), 1.694 (m, 1H, 3−CH), 1.537 (m, 3H, 7−CH, 2−CH2), 1.392 (m, 4H, 4−CH2, 5−CH2), 1.273 (t, J = 7.2, 6.8 Hz, 6H, 2 β−CH3), 1.134 (m,2H, 6−CH2), 0.935 (d, J = 5.2 Hz, 9−CH3), 0.826 (d, J = 6.4 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 59.15 (C−1), 56.71 (2C−α), 47.75 (C−11), 39.09 (C−2), 36.92 (C−6), 30.85 (C−3), 28.87 (C−4), 27.85 (C−7), 24.61 (C−5), 22.83 (C−8), 22.73 (C−10), 19.44 (C−9), 8.05 (2 C−β); EI-MS m/z: 228.27 (M+ − I); 482.08 (M+ + I), 126.90 (I−).
8l	Light yellow solids; m.p. 85 °C-87 °C; yield 75%; purity 94%; IR (cm−1): 2955, 2929, 2873, 1467, 1366, 1384, 1171; 1H NMR (D2O, 400 MHz) δH: 3.284 (m, 2H, 1−CH2), 3.181 (t, J = 6.8 Hz, 4H, 2 α−CH2), 2.976 (s, 3H, 11−CH3), 1.676 (m, 5H, 3−CH, 2 β−CH2), 1.486 (m, 3H, 7−CH2, 4−CH2), 1.282 (m, 4H, 4−CH2, 5−CH2), 1.119 (m, 2H, 6−CH2), 0.913 (t, J = 7.2, 6.4 Hz, 9H, 9−CH3,2 γ−CH3), 0.820 (d, J = 6.4 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 63.05 (2 C−α), 59.83 (C−1), 48.45 (C−11), 39.01 (C−2), 36.73 (C−6), 30.71 (C−3), 28.50 (C−4), 27.78 (C−7), 24.38 (C−5), 22.66 (C−8), 22.59 (C−10), 19.70 (C−9), 15.55 (2 C−β), 10.26 (2 C−γ); EI-MS m/z: 256.30 (M+ − Br); 414.14, 416.13, 418.13 (M+ + Br).
8m	Light yellow solids; m.p. 85 °C-89 °C; yield 75%; purity 92%; IR (cm−1): 2955, 2928, 2870, 1468, 1384, 1169; 1H NMR (D2O, 400 MHz) δH: 3.320 (m, 6H, 1−CH2, 2 α−CH2), 2.871 (s, 3H, 11−CH3), 1.634 (m, 5H, 3−CH, 2 β−CH2), 1.443 (m, 3H, 7−CH, 2−CH2), 1.204 (m, 4H, 4−CH2, 5−CH2), 1.080 (m, 2H, 6−CH2), 0.831 (t, J = 7.2, 6.4 Hz, 9H, 9−CH3, 2 γ−CH3), 0.752 (d, J = 6.4 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 63.03 (2 C−α), 60.27 (C−1), 48.05 (C−11), 38.52 (C−2), 36.02 (C−6), 30.31 (C−3), 28.01 (C−4), 27.31 (C−7), 24.38 (C−5), 24.56 (C−5), 22.08 (C−8), 22.02 (C−10), 18.77 (C−9), 15.35 (2 C−β), 9.91 (2 C−γ); EI-MS m/z: 256.30 (M+ − I); 510.08 (M+ + I), 126.90 (I−).
8n	Light yellow solids; m.p. 105 °C-108 °C; yield 75%; purity 92%; IR (cm−1): 2955, 2928, 2870, 1468, 1384, 1161; 1H NMR (D2O, 400 MHz) δH: 3.890 (m, 4H, 2 β−CH2), 3.340 (s, 6H, 1−CH2, 2 α−CH2), 3.024 (s, 3H, 11−CH3), 1.644 (m, 1H, 3−CH), 1.476∼1.361 (m, 3H, 7−CH, 2−CH2), 1.201 0.991 (m, 6H, 4−CH2, 5−CH2, 6−CH2), 0.784 (d, J = 5.2 Hz, 3H, 9−CH3), 0.701 (d, J = 6 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 64.21 (C−1), 60.42 (2 C−β), 59.51 (2C−α), 46.55 (C−11), 38.48 (C−2), 36.03 (C−6), 30.19 (C−3), 27.42 (C−4), 27.27 (C−7), 23.77 (C−5), 21.99 (C−8,C−10), 18.62 (C−9); EI-MS m/z: 242.25 (M+ − Br); 400.08, 402.08, 404.08 (M+ + Br).
8o	Light yellow solids; m.p. 113 °C-115 °C; yield 75%; purity 93%; IR (cm−1): 2953, 2926, 2870, 1464, 1384, 1117; 1H NMR (D2O, 400 MHz) δH: 3.871 (s, 4H, 2β−CH2), 3.322 (s, 6H, 1−CH2, 2α−CH2), 3.005 (s, 3H, 11−CH3), 1.627 (m, 1H, 3−CH), 1.616∼1.325 (m, 3H, 7−CH, 2−CH2), 1.203∼0.894 (m, 6H, 4−CH2, 5−CH2, 6−CH2), 0.771 (d, J = 5.2 Hz, 3H, 9−CH3), 0.680 (d, J = 6 Hz, 6H, 8−CH3, 10−CH3); 13C NMR (D2O, 100 MHz) δC: 66.18 (C−1), 62.40 (2 C−β), 61.53 (2 C−α), 48.63 (C−11), 40.43 (C−2), 37.98 (C−6), 32.15 (C−3), 29.38 (C−4), 29.21 (C−7), 25.73 (C−5), 24.00 (C−8, C−10), 20.58 (C−9); EI-MS m/z: 242.25 (M+ − I); 496.05 (M+ + I), 126.90 (I−).

Abbreviations: m.p., melting point; IR, infrared. EI-MS, electron impact-mass spectrometry.

Biological Activity Evaluation

The inhibitory activities of 15 new compounds against 10 plant pathogenic fungi were determined by the mycelium rate method. The compound was quantitatively dissolved in sterile water and added to potato dextrose agar (PDA) medium to a final quaternary ammonium salts concentration of 0.25 mg/mL. PDA medium with no compound was used as a negative control, and chlorothalonil was used as a positive control. All prepared PDA medium was poured into culture dishes (9 cm in diameter). After cooling and solidification, the PDA medium was inoculated with fungal strains (0.5 cm). Each strain was cultured in 3 repeated dishes. After inoculation, mycelium was cultured in a constant temperature incubator (27 °C) for several days. When the mycelium of the negative control group grew to ∼7 cm, the diameter of the fungus cake was measured by the crossing method. The antifungal rate was calculated by equations (1) and (2):

$$\begin{array}{rl}& \mathrm{Corrected}\mathrm{diameter}(\mathrm{cm})=\mathrm{average}\mathrm{diameter}\mathrm{of}\mathrm{colonies}(\mathrm{cm})\\ & -\mathrm{diameter}\mathrm{of}\mathrm{fungal}\mathrm{cake}(0.5\mathrm{cm})\end{array}$$

(1)

$$\begin{array}{rl}& \mathrm{Inhibition}\mathrm{rate}(\text{\%})=[\mathrm{control}\mathrm{corrected}\mathrm{diameter}(\mathrm{cm})\\ & -\mathrm{treatment}\mathrm{corrected}\mathrm{diameter}(\mathrm{cm})]/\\ & \mathrm{control}\mathrm{corrected}\mathrm{diameter}(\mathrm{cm})\times 100\text{\%}\end{array}$$

(2)

5. Conclusion

Fifteen tetrahydrogeranyl quaternary ammonium salt derivatives were synthesized from citral in this study, and their structures were characterized by FTIR, MS, ¹H NMR, and ¹³C NMR. The antifungal activities of tetrahydrogeranyl quaternary ammonium salt derivatives against 10 plant pathogenic fungi were determined by the mycelium growth rate method, and the test results showed that all compounds had certain inhibitory activities against the tested strains. The structure–activity relationship analysis showed that the n-hexyl groups on tetrahydrogeranyl quaternary ammonium salt had a significant influence on the improvement of the antifungal activity against R solani, F oxysporum f. sp. niveum, S sapinea, P vaporaria, and P parasitica var. nicotianae. It was also found that the introduction of the iodide anion could improve the antifungal rate of quaternary ammonium salts. The antifungal rates of quaternary ammonium salts containing N,N-di-n-propyl were generally higher than those of quaternary ammonium salts containing N,N-diethyl and N,N-dimethyl. These results were conducive to the design and synthesis of new and more effective antifungal agents and to improve the economic value of L cubeba utilization.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221078452 - Supplemental material for Synthesis and Antifungal Activity of Novel Tetrahydrogeranyl Quaternary Ammonium Salts

Supplemental material, sj-docx-1-npx-10.1177_1934578X221078452 for Synthesis and Antifungal Activity of Novel Tetrahydrogeranyl Quaternary Ammonium Salts by Yun Peng, Jiayu Chang, Zhuangquan Xiao, Jiazong Huang, Ting Xu, Shangxing Chen, Guorong Fan, Shengliao Liao, Zongde Wang and Hai Luo in Natural Product Communications