Synthesis and Antifungal Activity of Carabrone Derivatives

Nine derivatives 6-14 of carabrone (1) were synthesized and tested in vitro against Colletotrichum lagenarium Ell et Halst using the spore germination method. Among all of the derivatives, compounds 6-8 and 12 showed more potent antifungal activity than 1. Structure-activity relationships (SAR) demonstrated that the γ-lactone was necessary for the antifungal activity of 1, and the substituents on the C-4 position of 1 could significantly affect the antifungal activity.


Introduction
Carabrone (1, Figure 1), containing cyclopropane and sesquiterpene lactone moieties, was first isolated from the fruits of Carpesium abrotanoides [1], and is widely distributed in feverfew and other plant species [2][3][4][5][6][7][8][9]. It was demonstrated that compound 1 displays cytotoxic [10], antibacterial [11,12], and antitumor activity [13]. In our course of screening for novel naturally occurring phytofungicides from the plants in northwestern China, compound 1 was obtained from Carpesium macrocephalum, and exhibited antifungal activities in vitro and in vivo against Botrytis cinerea, Colletotrichum lagenarium, and Erysiphe graminis [14]. Subsequently, we prepared four derivatives (2-5, Figure 1) from 1, and found that the 11,13-double bond and the carbonyl group on the C-4 position of 1 are two OPEN ACCESS active sites [15,16]. In order to further investigate the effect of lactone and substituents on the C-4 position of 1 on the antifungal activity, herein we synthesized nine new carabrone derivatives 6-14 as potential antifungal agents.

Results and Discussion
Nine carabrone derivatives 6-14 were synthesized as shown in Scheme 1. Compound 6 was prepared by the reaction of 2,4-dinitrophenyl hydrazine (DNPH) and 1 in the presence of hydrogen chloride (HCl). Benzhydrazide or semicarbazide reacted with 1 to give compounds 7 and 8, respectively. Compound 9 was prepared from 1 with dry HCl. Compound 10 was synthesized by the reduction of the carbonyl group of 1 in the presence of NaBH 4 , followed by chlorination of the 4-OH group of 2 with thionyl chloride (SOCl 2 ). Compound 2 reacted with acyl chlorides in the presence of pyridine to afford compounds 11-14. All compounds were characterized by 1 H-NMR, IR, and HR-MS spectra. Scheme 1. The synthetic route to carabrone derivatives 6-14. The antifungal activity was assayed in vitro against Colletotrichum lagenarium Ell et Halst by the spore germination method. Chlorothalonil was used as a positive control. As described in Table 1, compounds 6-8 exhibited the most potent antifungal activity with the EC 50 values of 2.24, 4.32 and 3.03 μg/mL, respectively, i.e., the antifungal activity of 6, 7 and 8 was 1.5-3 times more potent than that of 1. However, the antifungal activity of other compounds was 1-8 times less than that of 1. Obviously, substituents on the C-4 position of 1 could significantly affect the antifungal activity. For example, introducing the hydrazone substituents on the C-4 position of 1 lead to the most potent compounds (e.g., 6-8), while when other substituents, such as the hydroxy group, chloro atom, and ester groups (except isobutyryloxy group), were introduced on the C-4 position of 1, the corresponding compounds showed the less potent activity than 1 (e.g., 10, 11, 13 and 14). Interestingly, when the isobutyryloxy group was introduced on the C-4 position of 1 to give 12, the EC 50 value of 12 was 6.39 μg/mL, which was more potent than that of 1. Meanwhile, compound 1 was nearly eightfold more potent than 9 (EC 50 7.10 μg/mL for 1 vs. EC 50 56.30 μg/mL for 9). This demonstrated that the γlactone was necessary for the antifungal activity of 1, and opening the lactone would lead to a less potent compound (1 vs. 9).

General
All the solvents were of analytical grade and the reagents were commercially available. Thin-layer chromatography (TLC) and silica gel-column chromatography were performed with silica gel plates using silica gel 60 GF 254 , and 200-300 mesh (Qingdao Haiyang Chemical Co., Ltd., China). Melting points were determined on a digital melting-point apparatus and uncorrected. All compounds were characterized by proton nuclear magnetic resonance ( 1 H-NMR), high-resolution mass spectra (HR-MS), mass spectra (MS-ESI), and infrared spectra (IR), respectively.

Spore Germination Assay
Microorganisms and maintenance: the strain of Colletotrichum lagenarium (36199) was provided by Agricultural Culture Collection of China and maintained on potato dextrose agar (PDA). Compounds 1 and 6-14 were dissolved in acetone or DMSO and added to 2% water agar medium after sterilization to produce concentrations of 100, 75, 50, 25, 10, and 5 µg/mL or 10, 5, 2, 1, 0.5 and 0.25 µg/mL of medium. Conidial suspensions (0.2 mL) containing 1 × 10 5 condia/mL, derived from cultures grown for 12 d on PDA plates, were spread on 2% water agar. Conidia were allowed to germinate 25 ± 1 ºC for 8 h. Germination was quantified at three sites by counting 100 conidia per site. A conidium was scored as germinated if the germ tube had reached at least half the length of the conidium. Three plates for each concentration were used and the experiment was performed thrice, along with 98% chlorothalonil (Syngenta Crop Protection Co., Ltd., China) as a positive control. The EC 50 for inhibition of spore germination was calculated for each isolate. Analysis of parameters was made with the statistical analysis system (SAS institute, Inc., Cary, NC, USA) [21].

Conclusions
In summary, nine new carabrone derivatives were synthesized and evaluated in vitro against Colletotrichum lagenarium Ell et Halst. Compounds 6-8, and 12 displayed the more potent antifungal activity than 1. Meanwhile, the structure-activity relationship (SAR) demonstrated that a γ-lactone moiety was necessary for the antifungal activity of 1, and the substituents on the C-4 position of 1 could significantly affect their antifungal activity, e.g., introduction of the hydrazone substituents on the C-4 position of 1 lead to more potent compounds.