Natural products as sources of new fungicides ( I ) : synthesis and antifungal activity of Kakuol derivatives against phytopathogenic fungi

In order to establish an advanced structural-activity relationship (SAR) and to explore the feasibility of kakuol analogues with better anti-fungi activity, a series of 2-hydroxy-4,5-methylenedioxyaryl ketones were conveniently synthesized by the Friedel-Crafts acyl reaction, etherification reaction, reduction reaction and oximation reaction. Their structures characterized by H and C NMR and HRMS methods. Their in vitro antifungal activities were assayed. Most of the derivatives showed a remarkable in vitro activity, and some of them appeared significantly more effective than a commercial fungicide hymexazol as positive control. In particular compounds 2h and 2i, were found active with a IC50 value of 3.1 mg/ml and 2.9 mg/ml against Glomerella cingulate, which suggested that 2-hydroxy-4,5-methylenedioxyaryl ketones might be a promising candidates in the development of new antifungal compounds. Compounds 2e, 5 and 6 also exhibited high antifungal activities on a wide range of organisms, which might be considered as leading compounds in the development of new antifungal compounds.


INTRODUCTION
Crops are facing a tremendous damage from pests, diseases and weed damages which results in direct economic losses including reduction in grain yield and quality [1].Although these losses may be attenuated by the use of disease-tolerant cultivars, crop rotation, or sanification practices, the use of fungicides will remain the predominant method of crop protection for the foreseeable future.Despite the availability of effective fungicides [2], new fungicides with novel chemical structures which have higher potency and broader spectrum of activity against resistant fungal strains have attracted great attentions recently.Recent studies have demonstrated that kakuol and its derivatives can be seen as important intermediates for the synthesis of some agrochemicals and pharmaceuticals.For example, kakuol (1-[2-hydroxy-4, 5 -(methylenedioxy)phenyl]propan -1 -one, Fig. 1) isolated from the rhizomes of Asarum sieboldii (Miq.)Maek, exhibited antifungal activity against some important plant pathogens such as Colletotrichum orbiculare, Rhizoctonia solani, Botrytis cinerea, Phytophthora capsici and Cladosporium cucumerinum [3], Asarum sieboldii isolated from flower buds of Magnolia fargesii had been reported to have antifungal and insecticidal activity [4,5].Several analogous propiophenones, such as 2-methoxy-3,4-(methylenedioxy)propiophenone [6] methylkakuol and 2,4,5-trimethoxypropiophenone [7,8], isolated from Asarum, Anethum and Piper species, respectively, yet their antifungal activity have not been reported.In 2010, Loana Musso et al. reported of a structure-activity relationship (SAR) kakuol derivatives, and found that the compounds bearing a C=C bond conjugated to the C=O group was beneficial to their antifungal activity [9].Some data related to structure-activity relationship (SAR) kakuol and methylkakuol have been reported in a recent patent and studies [10][11][12].These facts justify for urther studies on this class of molecules to identify the essential structural requirements for their antifungal activity.The urther study about the synthesis and the determination of a structure-activity relationship (SAR) for a series of kakuol analogues still needed.In Loana's study, they prepared 2-hydroxy-4,5methylenedioxyaryl ketones by two steps which involving Grignard reaction, but with a low yields (from 5% -40%).Based on their study, in our work the synthesis method has been improved, the reaction time has been shortened, and the yield has been raised.Several new compounds have been prepared by one step using Friedel-Crafts acyl reaction.In this study, we prepared a series of alkyl and benzyl kakuol derivatives 2a-2n, 3,4, 5 and 6, moreover evaluated in vitro antifungal activities against several phytopathogens.Herein, we report the interesting findings of these kakuol derivatives.

Materials and methods:
The melting points of the products were determined on an XT-4 apparatus (Beijing Tech Instrument Co., Beijing, China) and are uncorrected.The IR spectra were recorded on a Bruker VECTOR22 spectrometer in film or KBr discs. 1 H and 13 C NMR spectra were taken on a Varian Mercury 400 MHz (400 MHz for 1 H and 100 MHz for 13 C, respectively) or a Bruker Avance 500 MHz (500 MHz for 1 H and 125 MHz for 13 C, respectively) spectrometer in CDCl 3 solution using TMS as an internal standard (d given in ppm, J in Hz).MS spectra were taken on a Bruker Daltonics esquire 6000 (ESI-ION TRAP) or on a Waters ESI-Q-TOF Ultima Global mass spectrometer.Thin-layer chromatography (TLC) was performed with silica gel plates using silica gel 60 GF254 and column chromatography (CC) was carried out on silica gel (200-300 mesh) (Qingdao Haiyang Chemical Co. Ltd.Qingdao, Shandong province, China).Synthetic procedures: All the solvents were of analytical grade, alkyl or benzyl acyl chlorides and sesamol were commercial reagents.Target compounds (2a-2n) were synthesized by the Friedel-Crafts acyl reactions of sesamol with the corresponding benzyl acyl chloride or alkyl acyl chloride (Scheme 1).General Synthetic Procedure for Compounds 2a-2n.
[13] To a three-necked flask (50 ml) equipped with a condenser, an addition funnel, an HCl trap and a stopper with nitrogen flow, AlCl 3 (1.5 mmol) and dry CH 2 Cl 2 (5 ml) were added.The flask was then cooled with ice bath, and a solution of acyl chloride (1.2 mmol) in CH 2 Cl 2 (5 ml) slowly added via the addition funnel, until all the solid AlCl 3 had dissolved.A solution of phenol (1 mmol) in CH 2 Cl 2 was then added dropwise through the addition funnel, to avoid excessive evolution of gaseous HCl.The ice bath was then removed, and the mixture was refluxed for 2 hours, then cooled to room temperature.The reaction was poured into a beaker containing ice (5 g) and concentrated HCl (5 ml), and stirred 30 min.The organic layer was extracted with ethyl acetate (3 x 10 ml), washed with saturated NaHCO 3 , brine and dried over Na 2 SO 4 and concentrated under vacuum.The concentrated mixture was subjected to silica gel column chromatography and eluted by the mixture of petroleum ether and ethyl acetate (10:1 ~ 15:1, v/v) gradient elution to give title compounds.Synthetic Procedure for Compound 4. [14] To a solution of 2b (195 mg, 1 mmol) in acetone (5 ml), K 2 CO 3 (165 mg, 1.2 mmol) and MeI (0.3 ml, 5 mmol) were added, and the mixture was refluxed for 15 h.The mixture was filtered, then the solution was removed in vacuo, the residue was extracted by ethyl acetate.The organic layer was washed with H 2 O and brine, the solution was dried (Na 2 SO 4 ), and solvent was removed in vacuo.The crude material was purified by FCC (petroleum ether and ethyl acetate 15 :1) to give 4 (108 mg, 56 %).Synthetic Procedure for Compound 5. [15] To a solution of 2b (195 mg, 1 mmol) in MeOH (5 ml), This solution was chilled to 0°C, and sodium borotetrahydride (0.05 g, 1.2 mmol) was added portion-wise over 30 mins.Upon completion by TLC the reaction was quenched by the slow addition of ice.The resulting mixture was slowly evaporated to remove the methanol.The resulting solution was taken up in 10 ml of ethyl acetate and washed with 1M HCl.The organic layer was then washed three times with water and once with brine.The organic layer was dried over sodium sulfate and concentrated in vacuo to give a brown viscous oil.The crude material was purified by FCC (petroleum ether and ethyl acetate 5 :1) to give 5 (176 mg, 90 %).Synthetic Procedure for Compound 6 [16].To a The in vitro antifungal activity of the test compounds was evaluated by using mycelial growth inhibitory rate method [17].Briefly, the media (100 ml) incorporating test samples were inoculated at the center of agar discs of the test fungi (4 mm diameter).Three replicate plates for each fungus were incubated at 26 (±2)°C for all test fungi.Control plates containing media mixed with acetone (1 ml) were included.After incubation for 72 h, the mycelial growth of fungi (mm) in both treated (T) and control (C) Petri dishes was measured diametrically in three different directions (decussation method) till the fungal growth in the control dishes was almost complete.The percentage of growth inhibition (I) was calculated using the following formula.

RESULTS AND DISCUSSION
Chemistry: As shown in Scheme 1, kakuol and the series of 2-hydroxyaryl ketones could be readily obtained in a single step by the Friedel-Crafts acylation reaction of sesamol with the corresponding benzyl acyl chloride or alkyl acyl chloride in the presence of dry AlCl 3 in good yields.Commercially unavailable acyl chlorides were prepared according to reported methods.Specifically, as stated in experimental section, benzylated products and some alkylated products with large steric hindrance group such as 2f, 2j, 2k and 2m, were obtained by increasing reaction temperature and time in order to get a reasonable yield, the best results were obtained for 2b at 65 °C for 5 h in yield of 73 % and for 2a at 60 °C for 4 h in yield of 71 %.The progress of the reactions was monitored by TLC.In some cases, for the compounds 2a-o besides the desired ortho-acyl phenols, we observed the formation of the very small amount of corresponding esters 3, as analyzed by TLC.The presence of these compounds in the reaction mixture made the purification of desired ortho-acyl phenols very difficult and in most cases, a further purification by crystallization after flash chromatography was needed.Compound 4 was obtained from kakuol(2b)by methylation with MeI and K 2 CO 3 in acetone [18] (Scheme 2).The synthesis of compound 5 was preparing in 90 % yield by refluxing a solution of 2b in methanol with NaBH 4 [19] for 1 h and 6 was performed by reacting kakuol (2b) with NH 2 OH•HCl [15] and AcONa in MeOH.

Evaluation of antifungal activity:
The in vitro antifungal activities of all synthetic kakuol derivatives against five phytopathogenic fungi: Glomerella cingulate, Botrytis cinerea, FusaHum graminearum Sehw., Curvularia lunata and Fusarium oxysporum f. sp.Vasinfectum, were evaluated by mycelial rate growth assay, and the results are listed in Table 1.As reported in the Table 1, the natural product compound 2b showed a broad spectrum of activity and led to a good reduction of mycelial growth on Botrytis cinerea (ca.70.82 % at 100 mg/ml).While it was less efficient on Curvularia lunata (28.03 % at 100 mg/ml) and FusaHum graminearum (25.0 % at 100 mg/ml).Among ketones derivatives 2a-2o, with aliphatic chains, compounds 2a -2g showed moderate to strong antifungal activities against the test phytopathogenic fungi, while 2e exerted strong activities (Curvularia lunata 86.03 %; Botrytis cinerea 75.02 % at 100 mg/ml).Compound 2o with cyclopropyl as side chain had strong antifungal activities against all the test phytopathogenic fungi.The same result was observed as we introduced Cl in the side chain (2n).The results revealed that the antifungal activity increased as the alkyl side chains getting longer cyclopropyl group is detrimental to the activity.On the other hand, the introduction of unsaturated aliphatic chain, gave compound 2h and 2i with increased activity.In fact, they almost completely inhibited the mycelial growth of the tested pathogens at 100 mg/ml.The results listed in the Table 1 showed that the introduction of a Me group at the α-position of the C=O group (2h) generally did not affect the activity compared to compound 2i.This finding is consistent with Loana Musso's research results.Thus, we can assume that α,β-unsaturated ketone, plays an important role.So we increased the unsaturated carbon chain by introducing benzene ring as side chain hopefully to find more active compounds.However, compound 2l led to a substantial decrease of antifungal activity against the tested organisms, 2k and 2m with electron -donating group at the benzene ring, we saw an increase of antifungal activities.While those with electron-withdrawing group (2j), even had no obvious inhibitory action on Fusarium oxysporum f. sp.Vasinfectum.Thus, we suggested that aromatic ring is not crucial.Introducting the methoxy group to the free phenol group, ether 4 presented values of mycelial growth inhibition comparable to those of kakuol (2b) with the exception for Fusarium oxysporum f. sp.vasinfectum (36 % at 100 mg/ml; Table 1).This result suggested that the presence of the OH group may not crucial for the activity.Transforming the C=O group of kakuol (2b) into an alcohol, 5, led to an increase of activity against the tested organisms, except for Fusarium oxysporum f. sp.vasinfectum (36.07 % at 100 mg/ml; Table 1).Moreover, transforming the C=O into an oxime, 6, led to a significant increase of antifungal activity against the tested organisms.This results suggested that the C=O group is important, but in some situations, is replaceable.In order to test and verify Loana Musso's finding, we also using ChemDraw Ultra Vers.5.0 calculating log P (logarithm of partition coefficient in octan-1-ol/H2O) to reveal the relevance of lipophilicity, the lipophilicity values of the synthetic compounds, [20] and [21].As showed in Table 2, compounds 2h (log P 1.77), and 2i (log P 1.42) showed remarkable higher antifungal activities than the more liphophilic 2c (log P 1.81), 2d (log P 2.66), 2f (log P 2.66), and 2g (log P 2.64), while 2e (log P 2.22) showed a relative high antifungal activities, compounds 2o (log P 1.46), 5 (log P 1.65) and 6 (log P 1.78) exhibited higher antifungal activities than the natural occurred product 2b on a wide range of organisms.The introduction of a benzene rings (i.e., 2l, log P 2.63; 2m, log P 3.19) led to a decrease of antifungal activity.This result showed that the most active compounds have a log P value in the range of 1.42 -1.80 (compounds 2h, 2i, 2o, and 5), the only exception being compound 2e that maintained a moderate antifungal activity, despite its quite high log P value (2.22).Among the tested compounds, 2h and 2i appeared to be two most promising candidates.For this reason, they were tested at lower concentrations in order to evaluate their median inhibitory concentration (IC 50 ) for the most sensitive organisms (Glomerella cingulate, FusaHum graminearum Sehw., and Fusarium oxysporum f. sp.vasinfectum).The IC 50 values for compound 2h and 2i against Glomerella cingulate were 3.1 mg/ml and 2.9 mg/ml; A slightly higher IC 50 value (2h, 6.9 mg/ml; 2i, 6.2 mg/ml) was found against the Fusarium oxysporum f. sp.vasinfectum, whereas FusaHum graminearum Sehw.were found less sensitive (IC 50 values of 15.2 mg/ml and 14.9 mg/ml resp).Taken together, a pharmacophore figure of this class of kakuol derivates using SAR data is given in Figure 2.

CONCLUSIONS
A series of kakuol derivatives were prepared and their in vitro antifungal activities against phytopathogens were evaluated.Among the synthetic derivatives, some compounds such as 2h and 2i were found to exert broad-spectrum antifungal activities, which might be promising leads for agricultural fungicides, compounds 2e, 5 and 6 also exhibited higher antifungal activities than kakuol on a wide range of organisms, which might be considered as leading compounds in the development of new antifungal compounds and there are more works to be in progress.

Table 2 .
The lipophilicity values of the compounds 1-6