“Carbon Assimilation” Inspired Design and Divergent Synthesis of

Sep 26, 2017 - The accompanying introduction of versatile heterocycles and application of easily available feedstocks are beneficial for novel green a...
0 downloads 9 Views 3MB Size
Download PDF
Article pubs.acs.org/JAFC

Cite This: J. Agric. Food Chem. 2017, 65, 9013-9021

“Carbon Assimilation” Inspired Design and Divergent Synthesis of Drimane Meroterpenoid Mimics as Novel Fungicidal Leads Shasha Zhang,† Dangdang Li,† Zehua Song,† Chuanli Zang,† Lu Zhang,† Xiushi Song,† and Shengkun Li*,†,‡ †

Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, Weigang 1, Xuanwu District, Nanjing 210095, People’s Republic of China ‡ Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, People’s Republic of China S Supporting Information *

ABSTRACT: With structural diversity and versatile biological properties, drimane meroterpenoids have drawn remarkable attention in drug development. The stagnant progress made in the structure optimization and SAR study of this kind of natural product for agrochemicals was mainly a result of inefficient construction. Compared with the reported challenging coupling reaction (“1 + 1” tactic), “carbon assimilation” was conceived and used for the rapid construction of drimanyl meroterpenoid mimics, in which the newly formed covalent bond was directly from the old one of the drimanyl subunit (“2 + 0” tactic), which features atom economy, step economy, and facile preparation. The accompanying introduction of versatile heterocycles and application of easily available feedstocks are beneficial for novel green agrochemical discovery, in view of economic efficiency and improvement of physicochemical properities. Heterocyclic mimics 3a and 3c are presented as potent fungicidal leads with novel skeletons against Botrytis cinerea, >25-fold and >40-fold more promising than the commercial fungicide carbendazim, respectively. Our design was also rationalized by the 6-step synthesis and antifungal assay of the original model of natural meroterpenoids. This tactic can also be fostered or transferred directly to the design of novel natural product mimics for medicinal chemistry or other related biological exploration. KEYWORDS: drimane meroterpenoids, diverse synthesis, chiral pesticide, lead discovery, structure−activity relationship

■

INTRODUCTION A dramatic increase in efficiency of crop production is necessary for the ever-growing world population in the 21st century. Among different powerful approaches to guaranteeing an adequate production of food, chemical crop protection will remain the predominant strategy for the treatment of biotic stresses.1 The ever-increasing resistance to common pesticides presents challenges of a magnitude never seen before. With respect to the multifaceted awkward problem, novel agrochemicals with different scaffolds or action mode should be designed and investigated. Natural products have played a pivotal role in the discovery of new active ingredients in the agrochemical industry. Compared with being used directly, natural products exerted a much stronger influence on novel agrochemical discovery by serving as novel scaffolds. The proverbial example is the development of scenarios of strobilurin fungicides.2 Keeping the importance of natural products for the discovery of novel agrochemicals in mind, our research group was devoted to the design, synthesis, and SAR of natural products, hybrids, and their mimics. Meroterpenoids are broadly defined as compounds of mixed polyketide-terpenoid origin. Drimane-related meroterpenoids have drawn considerable attention, with the abundance of distribution, structural diversity, and versatile biological properties ranging from antifungal to anti-HIV, anticancer, etc.3 The typical drimane-related meroterpenoid consists of a drimane attached to an (un)substituted or oxidized phenyl moiety, © 2017 American Chemical Society

exemplified by yahazunol, chromazonarol, zonarol, pelorol, dictyvaric acid, and related natural products4 (Figure 1). Several research groups5,6 have made significant efforts to this kind of natural products mainly relying on the incorporation of a suitable arene nucleophile to a terpene unit. Light-induced radical based decarboxylative coupling was developed by the Theodorakis group7,8 to prepare rearranged drimane sesquiterpenoids. Biomimetic cyclizations of polyprenoids to prepare a small set of meroterpenoids were also developed.9 To access drimane-related meroterpenoids divergently and efficiently, Prof. Baran and co-workers prepared “borono-sclareolide” and developed it as a stable terpenoid donor for coupling with different aromatic acceptors.4 These tactics often suffered from multiple steps, tedious manipulations, poor yields, mediation by transition metals, or isomerization, which is not suitable for agrochemical exploration. Rapid and divergent construction and biological exploration of mimics are highly desirable for novel agrochemicals.

■

MOLECULAR DESIGN AND TACTIC DEVELOPMENT Fsp is the fraction of sp3 hybridized carbon atoms,10,11 which is an important index representing drug-likeness, and the mean 3

Received: Revised: Accepted: Published: 9013

July 7, 2017 September 20, 2017 September 26, 2017 September 26, 2017 DOI: 10.1021/acs.jafc.7b03126 J. Agric. Food Chem. 2017, 65, 9013−9021

Article

Journal of Agricultural and Food Chemistry

Figure 1. Drimane meroterpenoid natural products and the structure model.

Scheme 1. Synthetic Tactics of Drimane Meroquiterpenoid Mimics

value of Fsp3 of the actual drugs is reported to be about 0.47.12 According to the fungicides list in FRAC, most of the commercially launched fungicides are rich in flat aromatic rings or equivalents, with the lower metric value of Fsp3, while all the carbons in the drimane scaffold are sp3 hybridized, lacking the flat factors. We proposed that there is a latent balance between the sp3 hybridized carbons and aromatic effect for potential drug candidates, exemplified by the aforementioned bioactive drimane meroterpenoids. Considering the importance of physicochemical properties in the design of new pesticides,13 we envision that drimane is an ideal scaffold for the discovery of novel agrochemicals and may produce promising bioactivity if conjugated subunits were introduced. In another way, the introduction of the drimane skeleton will help many fungicides to “escape” from “flat”. Herein, we would like to document the design, divergent synthesis, and exploration of the agrochemical potentials of novel drimane meroterpenoid mimics. Considering cumbersome manipulations, the rapid and powerful construction of drimane meroterpenoid mimics will be highly desirable. This kind of natural product can be traced back to the model A, in which “conjugated subunits” were linked to the drimane unit. For the natural products, the “conjugated subunits” were mainly limited to substituted phenyl rings. The retrosynthetic analysis always provides the convergent aggregation of reactive drimane donor and “conjugated acceptors”. This synthetic tactic can be deemed as a “1 + 1” model (“drimane donor + conjugated acceptor”), in which the newly formed covalent bond or the linker was provided by the donor and acceptor synergistically. The

drimane donor (15 carbon skeleton) was always prepared from the degradation of natural diterpenoids through multistep treatments, while the homodrimanes (16 carbon skeleton) are quite more easily accessible. Direct incorporation of the homodrimane for the construction of drimane meroterpenoid mimics will be a good choice in view of atom economy, in which the linker was formally provided only by the “homodrimane part”; this is “2 + 0” model, and the excess one carbon of the homodrimane part was assimilated to the “conjugated acceptor” (Scheme 1). The inserted Michael acceptor can serve as an important intermediate for the construction of diverse heterocycles through readily available methods. Keeping in mind the importance of “Michael acceptor” fragment14,15 and chalcones16 in pharmaceutical chemistry as well as agrochemicals, an α,β-unsaturated ketone fragment was formally inserted to the drimane meroterpenoids to get the expanded “conjugated subunits”. More importantly, versatile heterocycles can be prepared easily based on the “Michael acceptor” fragments. The newly formed “conjugated acceptors” including the inserted Michael acceptor and the following synthesized heterocycles are structurally related to the related natural products. Importantly, the accompanying modification of physicochemical properties may be beneficial for the improvement of bioactivities and the affinity with potent protein targets because of innate characteristics.

■

MATERIALS AND METHODS

Instruments, Chemicals, and Related Materials. All solvents and reagents were purchased from commercial sources (Energy or Meryer Chemicals etc.); they were analytically pure and used as

9014

DOI: 10.1021/acs.jafc.7b03126 J. Agric. Food Chem. 2017, 65, 9013−9021

Article

Journal of Agricultural and Food Chemistry Scheme 2. Design and Synthesis of α,β-Unsaturated Ketone Inserted Mimics

Table 1. Antifungal Bioactivity of Different Drimanyl Merosesquiterpenoid Mimicsa antifungal bioactivity (%, 50 mg/L) compd

R.S.

S.S.

B.C.

F.G.

2a 3a 3c 3d 3e 3f DM CK1 CK2

48.46 ± 0.04 d 58.15 ± 1.14 c 57.45 ± 1.10 c 35.17 ± 0.28 e 34.00 ± 0.12 f 18.36 ± 0.39 g 62.89 ± 0.02 b − 100 ± 0.01 a

37.12 ± 0.05 e 57.05 ± 1.80 c 69.50 ± 1.29 b 54.73 ± 0.21 d 1.85 ± 0.02 h 18.82 ± 0.12 f 9.82 ± 0.01 g − 100 ± 0.01 a

34.25 ± 1.30 d 73.76 ± 0.84 b 78.85 ± 2.11 a 39.58 ± 0.11c 32.86 ± 0.15 d 5.68 ± 0.29 g 18.07 ± 0.11 f − 20.39 ± 0.38 e

34.30 ± 0.54 d 36.43 ± 0.43 c 73.81 ± 1.03 b 27.97 ± 0.10 e 17.01 ± 0.11 f 10.00 ± 0.01 g 34.98 ± 0.31 d − 100 ± 0.01 a

G.G. 38.00 14.34 32.02 35.38 42.00 38.43 55.75 − N.T.

± ± ± ± ± ± ±

0.37 0.21 0.11 0.12 0.28 0.17 0.61

ClogP d g f e b c a

5.037 4.6985 4.5810 6.7962 7.3190 8.474 4.949

a R.S.: Rhizoctonia solani. S.S.: Sclerotinia sclerotiorum. B.C.: Botrytis cinerea. F.G.: Fusarium graminearum. G.G.: Gaeumanomyces graminis. CK1: DMSO. CK2: carbendazim. Inhibitory rates are mean values of triplicate experiments. N.T.: Not tested. “”: No inhibitory effect. Different small letters in the same column showed significant difference at P < 0.05 level, through Duncan’s multiple range test in SPSS statistics 22.0. The alphabetical order is consistent with the high to low order of the antifungal activity.

mL) and ethyl acetate (15 mL). The organic layer was separated, and the aqueous phase was extracted with ethyl acetate (2 × 15 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated. The residue was purified by flash chromatography (200−300 mesh) with PE/EtOAc = 8:1 (v/v) as eluent to give α,β-unsaturated ketone inserted merosesquiterpenoid mimic 2d as a white solid (263 mg, yield 67.6%). Mp: 164.5−164.7 (°C). 1H NMR (400 MHz, CDCl3): δ 0.80 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.88 (s, 3H, CH3), 1.03 (dd, J1 = 12.24 Hz, J2 = 2.32 Hz, 1H, H in naphthane ring), 1.15 (td, J1 = 13.64 Hz, J2 = 3.92 Hz, 1H, H in naphthane ring), 1.16 (s, 3H, CH3), 1.28(td, J1 = 12.52 Hz, J2 = 3.24 Hz, 1H, H in naphthane ring), 1.34−1.42(m, 3H, H in naphthane ring), 1.49(td, J1 = 12.52 Hz, J2 = 4.04 Hz, 1H, H in naphthane ring), 1.58(m, 1H, H in naphthane ring), 1.68−1.74(m, 2H, H in naphthane ring), 1.78(br, 1H, OH), 1.96 (dt, J1 = 12.48, J2 = 3.20 Hz, 1H, H in naphthane ring), 2.08 (dd, J1 = J2 = 4.92 Hz, 1H, CH−CH2−CO), 2.72 (dd, J1 = J2 = 4.92 Hz, 2H, CH2−CO), 6.79(d, J = 16.08 Hz, 1H, CH−CO), 7.35 (d, J = 6.52 Hz, 2H, aromatic H), 7.49 (d, J = 6.52 Hz, 2H, aromatic H), 7.54(d, J = 16.08 Hz, 1H, CHCH−CO). 13C NMR (100 MHz, CDCl3): δ 15.70 (CH3), 18.41 (CH2), 20.62 (CH2), 21.43 (CH3), 23.35 (CH3), 33.24 (C), 33.34 (CH3), 37.56 (CH2), 38.62 (C), 39.54 (CH2), 41.75 (CH2), 44.63 (CH2), 55.91 (CH), 56.12 (CH), 73.16 (C), 126.56 (CH), 129.18 (2 × CH), 129.46 (2 × CH), 133.14 (C), 136.24 (C), 140.73 (CH), 201.06 (C). ESI-MS: calcd for C24H33ClNaO2 [M + Na+] 411.21 and 413.20, found 411.32 and 413.21; C24H32ClO [M − H2O + H+] 371.21 and 373.21, found 371.30 and 373.21. The α,β-unsaturated ketone inserted merosesquiterpenoid mimics in Table 1 and Table 2 were synthesized according to a procedure similar to that shown in Scheme 2. Synthesis of Drimanyl Heterocycles 3a and 3c. To a solution of α,β-unsaturated ketone mimic 2a (355 mg, 1.0 mmol, 1.0 equiv) in 10 mL of ethanol were added potassium tert-butoxide (449 mg, 4.0 mmol, 4.0 equiv) and hydroxylamine hydrochloride (139 mg, 2.0 mmol, 2.0 equiv); subsequently, the flask was immersed into a preheated oil bath at 90 °C. The mixture was stirred, and the reaction progress was monitored by TLC until the reaction was complete (∼24 h). The solvent was removed under reduced pressure, and to the

received. Anhydrous solvents were dried and distilled by standard techniques before use. Silica gel GF254 and column chromatography silica gel for isolation (200−300 mesh) were both purchased from Qingdao Broadchem Industrial Co., Ltd. Reaction progress was monitored by thin-layer chromatography (TLC) on silica gel GF254 with ultraviolet (UV254 nm) detection and phosphomolybdic acid. Yields of all the title compounds were not optimized. Melting points (mp) were recorded on a Shenguang WRS-1B melting point apparatus and are uncorrected. 1H NMR and 13C NMR spectra were carried out utilizing a Bruker AV400 spectrometer with CDCl3 as solvent and tetramethylsilane as the internal standard. Electrospray ionization mass spectrometry (ESI-MS) data were obtained with a Waters Xevo TQ-S Micro-Spectrometer. The single crystal diffraction was carried out on a Bruker SMART APEX CCD diffractometer. The fungi were provided by the Department of Pesticide, College of Plant Protection, Nanjing Agricultural University (Nanjing, China). General Procedure for the Synthesis of α,β-Unsaturated Ketone Inserted Mimics. To a solution of (+)-sclareolide (20.0 g, 79.8 mmol) in anhydrous Et2O (250 mL) was added MeLi (1.5 M in Et2O, 100 mL, 150 mmol) dropwise at −78 °C. The reaction mixture was stirred at −78 °C, and the reaction progress was monitored by TLC until the reaction was complete (about 1 h). The mixture was quenched by slow addition of 10% H2SO4 aqueous solution and was warmed gradually to room temperature. The aqueous phase was extracted with Et2O (2 × 150 mL). The combined organics were washed sequentially with saturated NaHCO3 aqueous solution (2 × 150 mL), H2O (150 mL), and brine (100 mL), then dried over anhydrous MgSO4, filtered, and concentrated in vacuo to yield drimanyl methyl ketone 1 with the yield >90%, which was used directly for next step without further purification (Scheme 2). Sodium methoxide (65 mg, 1.2 mmol, 1.2 equiv) was added to the cooled solution of drimanyl methyl ketone 1 (267 mg, 1.0 mmol, 1.0 equiv) in 10 mL of MeOH with an ice bath (0−5 °C). After 20 min of stirring, 4-chlorobenzaldehyde (141 mg, 1.0 mmol, 1.0 equiv) was added and the mixture was moved to the preheated oil bath at 80 °C. The reaction progress was monitored by TLC until the reaction was complete (∼48 h). The solvent was removed under reduced pressure, and to the residue was added saturated aqueous solution of NH4Cl (15 9015

DOI: 10.1021/acs.jafc.7b03126 J. Agric. Food Chem. 2017, 65, 9013−9021

Article

Journal of Agricultural and Food Chemistry Table 2. Fungicidal Bioactivity of Michael Acceptor Inserted Meroterpenoidsa antifungal bioactivity (%)

a

compd

Ar

R.S. (100 mg/L)

R.S. (50 mg/L)

G.G. (100 mg/L)

1 2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k 2l 2m 2n 2o 2p 2q 2r 2s 2t 2u 2v 2w 2x 2y 2z CK1 CK2

− Ph 4-NO2-Ph 4-F-Ph 4-Cl-Ph 4-Br-Ph 4-CH3-Ph 4-CH3O-Ph 4-(CH3)2N-Ph 3-NO2-Ph 3-CF3-Ph 3-Cl-Ph 3-Br-Ph 3-CH3-Ph 3-OH-Ph 2-Br-Ph 2-CH3O-Ph 2-naphthyl 2,4-Cl-Ph 2,5-Cl-Ph 2,5-F-Ph 3,5-F-Ph 2,6-Cl-Ph 3,4-CH3-Ph 2-furyl 2-thienyl 3-pyridyl

61.54 ± 0.41 f 57.01 ± 0.31 g 40.00 ± 0.23 l 60.59 ± 0.29 f 68.50 ± 0.35 d 36.81 ± 0.17 n 23.60 ± 0.09 p 38.24 ± 0.13 mn 42.42 ± 0.19 k 49.33 ± 0.31 j 54.11 ± 0.30 h 68.55 ± 0.41 d 38.21 ± 0.16 mn 32.44 ± 0.12 o 77.43 ± 0.51 bc 23.68 ± 0.09 p 51.64 ± 0.31 i 36.43 ± 0.20 n 38.10 ± 0.12 mn 39.05 ± 0.17 lm 78.35 ± 0.45 b 61.69 ± 0.38 f 37.31 ± 0.15 mn 37.22 ± 0.12 mn 66.53 ± 0.26 e 75.82 ± 0.42 c 76.27 ± 0.56 c − 100 ± 0.01 a

49.65 ± 0.29 g 48.46 ± 0.04 g 25.00 ± 0.11 no 56.77 ± 0.23 f 61.42 ± 0.28 e 35.52 ± 0.12 j 9.92 ± 0.07 r 33.33 ± 0.14 k 30.74 ± 0.11 l 45.71 ± 0.30 h 48.05 ± 0.26 g 64.98 ± 0.39 d 32.38 ± 0.18 kl 23.37 ± 0.14 op 67.44 ± 0.48 c 22.63 ± 0.11 p 39.10 ± 0.19 i 29.00 ± 0.17 m 26.41 ± 0.07 n 14.92 ± 0.06 q 44.59 ± 0.29 h 61.59 ± 0.49 e 24.77 ± 0.09 no 31.20 ± 0.16 l 64.96 ± 0.39 d 61.94 ± 0.30 e 73.58 ± 0.39 b − 100 ± 0.01 a

19.00 49.16 22.00 39.92 47.00 41.05 31.09 49.21 42.80 40.00 53.78 62.00 55.00 36.00 33.00 41.18 48.10 52.11 50.00 55.26 59.46 54.83 45.00 51.35 50.00 64.63 33.19 − N.T.

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.11 0.21 0.12 0.13 0.23 0.19 0.13 0.21 0.18 0.19 0.17 0.34 0.23 0.18 0.21 0.21 0.21 0.36 0.19 0.23 0.24 0.23 0.16 0.33 0.18 0.28 0.16

r gh q m i m p gh k m d b d n o lm hi e fg d c d j ef fg a o

G.G. (50 mg/L) 6.00 ± 0.12 q 38.00 ± 0.37 e 17.00 ± 0.09 o 28.00 ± 0.15 k 35.00 ± 0.16 fg 30.00 ± 0.13 ij 12.00 ± 0.11 p 27.34 ± 0.15 kl 30.00 ± 0.12 ij 33.00 ± 0.10 h 43.00 ± 0.15 cd 49.00 ± 0.32 b 42.00 ± 0.20 d 26.00 ± 0.05 lm 25.00 ± 0.13 m 31.00 ± 0.20 i 22.63 ± 0.13 n 28.95 ± 0.20 jk 42.00 ± 0.12 d 44.21 ± 0.18 c 36.00 ± 0.19 f 28.00 ± 0.11 k 34.00 ± 0.14 gh 21.00 ± 0.15 n 43.00 ± 0.21 cd 52.83 ± 0.24 a 29.00 ± 0.11 jk − N.T.

See footnote a in Table 1.

Figure 2. Crystal structures of drimanyl isoxazoline 3a and drimanyl pyrimidine 3c. 2.47 (m, 2H, CH2-CO), 3.02 (dd, J1 = 16.92 Hz, J2 = 8.60 Hz, 1H, Ph−CH−CH2), 3.41 (dd, J1 = 16.92 Hz, J2 = 10.64 Hz, 1H, Ph−CH− CH2), 5.52 (dd, J1 = 10.64 Hz, J2 = 8.60 Hz, 1H, Ph−CH−CH2), 7.29−7.36 (m, 5H, aromatic H). 13C NMR (100 MHz, CDCl3): δ 15.14 (CH3), 18.42 (CH2), 20.41 (CH2), 21.48 (CH3), 23.63 (CH3), 23.73 (CH2), 33.26 (C), 33.40 (CH3), 38.90 (C), 39.65 (CH2), 41.71 (CH2), 44.32 (CH2), 46.08 (CH2), 55.93 (CH), 57.87 (CH), 73.67 (C), 81.51 (CH), 125.92 (2 × CH), 127.96 (CH), 128.61 (2 × CH), 141.21 (C), 161.30 (C). ESI-MS: calcd for C24H35NNaO2 [M + Na+] 392.26, found 392.30; C24H34NO [M − H2O + H+] 352.26, found 352.31. The white solid was dissolved in ethyl acetate to afford colorless square crystal easily. The molecular structure was further confirmed by X-ray single crystal diffraction (CCDC: 1555836), as shown in Figure 2; the isoxazoline is enantioselectively formed, and the chiral carbon was assigned as S configuration.

residue was added a saturated aqueous solution of NH4Cl (15 mL) and ethyl acetate (15 mL). The organic layer was separated, and the aqueous phase was extracted with ethyl acetate (2 × 15 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated. The residue was purified by flash chromatography (200−300 mesh) with PE/EtOAc = 5:1 (v/v) as eluent to give drimanyl isoxazoline 3a as a white solid (171 mg, yield 46.3%). Mp: 122.9−124.5 (°C). 1H NMR (400 MHz, CDCl3): δ 0.80 (s, 3H, CH3), 0.83 (s, 3H, CH3), 0.88 (s, 3H, CH3), 1.15 (dd, J1 = 14.20 Hz, J2 = 4.24 Hz, 1H, H in naphthane ring), 1.19 (td, J1 = 9.02 Hz, J2 = 4.08 Hz, 1H, H in naphthane ring), 1.21 (s, 3H, CH3), 1.29 (td, J1 = 12.08 Hz, J2 = 3.24 Hz, 1H, H in naphthane ring), 1.40−1.46 (m, 3H, H in naphthane ring), 1.57(dt, J1 = 12.72 Hz, J2 = 3.60 Hz, 1H, H in naphthane ring), 1.58−1.64 (m, 3H, H in naphthane ring), 1.66 (br, 1H, OH), 1.74 (dd, J1 = J2 = 4.80 Hz, 1H, H in naphthane ring), 1.91 (dt, J1 = 12.40 Hz, J2 = 3.32 Hz, 1H, H in naphthane ring), 9016

DOI: 10.1021/acs.jafc.7b03126 J. Agric. Food Chem. 2017, 65, 9013−9021

Article

Journal of Agricultural and Food Chemistry Scheme 3. Diverse Synthesis of Drimanyl Heterocycle Mimics

Drimanyl pyrimidines 3c was prepared through similar manipulation, replacing hydroxylamine hydrochloride by guanidine hydrochloride. Data for the drimanyl pyrimidine 3c was as follows. Yield: 30.2%. Mp: 205.5−206.3 (°C). 1H NMR (400 MHz, CDCl3): δ 0.81 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.95 (dd, J1 = 12.20 Hz, J2 = 2.28 Hz, 1H, H in naphthane ring), 1.09 (td, J1 = 13.12 Hz, J2 = 3.88 Hz, 1H, H in naphthane ring), 1.25 (s, 3H, CH3), 1.29 (m, 1H, H in naphthane ring), 1.32−1.42 (m, 3H, H in naphthane ring), 1.52(td, J1 = 12.76 Hz, J2 = 3.88 Hz, 1H, H in naphthane ring), 1.61 (dt, J1 = 13.32 Hz, J2 = 3.52 Hz, 1H, H in naphthane ring), 1.66− 1.76 (m, 3H, H in naphthane ring), 2.01 (dt, J1 = 12.60 Hz, J2 = 3.28 Hz, 1H, H in naphthane ring), 2.66 (dd, J1 = 15.20 Hz, J2 = 3.16 Hz, 1H, pyrimidyl-CH2), 2.82 (dd, J1 = 15.20 Hz, J2 = 4.96 Hz, 1H, pyrimidyl-CH2), 5.13(s, br, 2H, NH2), 6.93 (s, 1H, H in pyrimidyl ring), 7.46−7.49 (m, 3H, aromatic H), 7.96−7.99 (m, 2H, aromatic H). 13C NMR (100 MHz, CDCl3): δ 15.64 (CH3), 18.40 (CH2), 20.51 (CH2), 21.43 (CH3), 24.68 (CH3), 33.29 (C), 33.35 (CH3), 33.50 (CH2), 39.55 (CH2), 39.61 (C), 41.81 (CH2), 44.17 (CH2), 56.17 (CH), 60.47 (CH), 72.40 (C), 106.90 (CH), 127.17 (2 × CH), 128.74 (2 × CH), 130.57 (CH), 137.35 (C), 162.47 (C), 165.99 (C), 173.72 (C). ESI-MS: calcd for C25H34N3 [M − H2O + H+] 376.28, found 376.32; C25H36N3O [M + H+] 394.29, found 394.38. The structure of 3c was also confirmed by X-ray single crystal diffraction as shown in Figure 2 (CCDC: 1555837); the naphthane scaffold is trans fused, and adopts chair conformations, keeping the original stereocenters untouched. Synthetic details and compound list of the other drimanyl heterocycles and derivatives in Scheme 3 are provided in the Supporting Information. General Procedure for Biological Assay. The fungicidal activity of the target compounds was tested in vitro against the aforementioned plant pathogenic fungi using the mycelium growth rate test according to our previous report.17 All the tested compounds were dissolved in DMSO at a concentration of 20 mg/mL. The media containing compounds at a concentration of 100 μg·mL−1 were then poured into Petri dishes for initial screening. In the precision antifungal test of a specific compound, the 20 mg/mL solutions were diluted to 50, 25, 12.5, 6.25, 3.125 μg/mL and the above experiments were repeated three times; the inhibition rates were calculated separately. The solvent DMSO and the broad-spectrum fungicide carbendazim18 were chosen as a negative control and positive control, respectively. The statistical analyses were performed by SPSS software version 20.0.

as agrochemical discovery, while only stagnant progress was made in the structure optimization and SAR study (especially for agrochemicals). In our continuing efforts to the exploration of drimane natural products as novel fungicide candidates,17 we envisage that more efficient synthesis of meroterpenoid mimics will be a good choice for the discovery of potentially bioactive molecules, especially if some subunits can be incorporated to improve the innate properties. The subunit of α,β-unsaturated ketone was perceived as a specific Michael acceptor, involved in many important chemical translations and chemical biology. The drimanyl methyl ketone 1 was prepared in good yield with the commercially available (+)-sclareolide as starting material. A tiny modification was made for the ring-opening of lactone by nucleophilic attack of MeLi based on a previous report,19 and the nucleophile was dissolved in anhydrous ethyl ether and added slowly under −78 °C. The workup was also finished under low temperature by the slow addition of aqueous H2SO4 to quench the excrescent MeLi. MeONa mediated aldol condensation was performed for the conversion of drimanyl methyl ketone 1 to drimane meroterpenoid mimics 2 in good to excellent yields. Formally, the α,β-unsaturated ketone fragment was inserted to the meroterpenoids as a linker for drimane and aromatics. The meroterpenoid mimics with expanded conjugated effect were constructed efficiently and confirmed by ESI-mass and NMR. The newly formed C−C double bond geometries were confirmed exclusively as trans configuration, with the coupling constants greater than 12 Hz (J = 16.12 Hz for 2a), which are thermodynamically more stable. The purity was determined by 1H NMR analysis. Design and Synthesis of Heterocycle Mimics. Approximately 70% of all the 2400 pharmaceuticals listed in the online version of “pharmaceutical substances” bear at least one heterocyclic ring; and a similar percentage was also found for the pesticides among its about 900 entries in the latest edition of the “pesticide manual”.20,21 The intrinsic superiority of heterocycles in fine-tuning the physicochemical properties led us to carry out diversity-oriented optimization with the readily available Michael acceptors 2. As can be seen in Scheme 3, a great variation of popular heterocycles in modern crop protection20 were established successfully with commercially available nucleophiles. The heterocycle formation will not only increase the rigidity of the original Michael acceptors but also

■

RESULTS AND DISCUSSION Design and Synthesis of α,β-Unsaturated Ketone Inserted Mimics. Drimane meroterpenoids make up a promising library of metabolites for drug development as well 9017

DOI: 10.1021/acs.jafc.7b03126 J. Agric. Food Chem. 2017, 65, 9013−9021

Article

Journal of Agricultural and Food Chemistry Scheme 4. Derivative Synthesis of Drimanyl Pyrimidine Mimics

Scheme 5. Synthesis of Merosesquiterpenoid DM

respectively. The degradation, transition metal catalysis, and regioselective borylation are key transformations. Compared with the multistep manipulation and isomerization, the superiority of our current research is becoming more evident for biological research, especially for agrochemical exploration. Structure and Activity Relationships (SAR). To verify the reasonability of our initial design, the antifungal activities against a number of agriculturally important plant pathogens were measured through the mycelium growth rate test. The limited exploration of the agrochemical potential of drimanyl meroterpenoids promotes us to screen a wide variety of plant pathogens (up to 10 kinds, see Supporting Information for details; typical results are listed in Table 1). The merosesquiterpenoid mimic 2a possessed moderate antifungal bioactivity against all the test fungi and demonstrated promising inhibition against Rhizoctonia solani and Gaeumanomyces graminis. Fine-tuning of α,β-unsaturated ketone inserted drimanyl meroterpenoids was implemented to investigate the electronic and steric effects of the substituents on fungicidal activity, and up to 26 analogues were synthesized specifically starting from versatile and easily available benzaldehydes. As can be seen in Table 2, the antifungal bioactivity was affected by both the inherent electron properties and the substituted positions. The electron-withdrawing groups on the phenyl ring are more beneficial for the improvement in antifungal activity. The antifungal activity of monosubstituted phenyl counterparts confirmed the benefits of electron withdrawal, regardless of the substituent position (Table 2, 2c−2g, 2j−2m). The inhibitory rate of mono-para-substituted molecules against Rhizoctonia solani increased following 4-(CH3)2N ≈ 4-CH3O < 4-F ≈ 4-Cl (Table 2, 2c, 2d, 2g, and 2h). The synergistic improvement was not evident when more electron-withdrawing groups were introduced (2k vs 2r, 2s, 2v). It was noteworthy that the decorated position on the phenyl ring also had a significant impact on the antifungal bioactivity, especially for nitrated (Table 2, 2i > 2b) and chlorinated candidates (2k > 2d). Heterocycles have a proven track record as ideal bioisosteric replacements of a benzene ring; replacement by either furyl, thienyl, or pyridyl can deliver much better biological efficacy against both Rhizoctonia solani and Gaeumanomyces graminis; interestingly, the inhibition against Rhizoctonia solani followed thienyl < furyl < pyridyl, while the tendency changed reversely for Gaeumanomyces graminis (Table 2, 2x−2z).

modify the innate hydrophobicity while keeping the stereocenters and rigid conformation untouched. The isoxazoline was synthesized from the condensation of 2a and hydroxylamine hydrochloride under refluxed basic ethanol. Gratifyingly, this translation leads selectively to the Risoxazoline diastereomer, as confirmed by X-ray crystallography (Figure 2, 3a). The dehydrogenative transformation of isoxazoline will occur with improved temperature under air atmosphere when the media is changed to DMF. Variation of the Michael donor from NH2OH to the readily available guanidine will produce the aminopyrimidine mimic 3c with a slight decrease in yield (30.2%), and the structure was confirmed by mass, NMR, and X-ray crystallography (Figure 2, 3c). Exposure of 2a to 2-aminobenzimidazole in refluxing DMF results in the ring fused benzoimidazolopyrimidine mimic 3d in up to 53.5% yield, in which 2-aminobenzimidazole can be deemed as masked guanidine. Pyrazoline mimic 3e will be accessed successfully when phenylhydrazine hydrochloride is submitted under similar conditions. The iodine mediated condensation of 2a and phenylhydrazine hydrochloride under refluxing ethanol was conducted to form the drimanyl-pyrazole successfully and efficiently. It is worth mentioning that the dehydration will happen with poor regioselectivity favoring for the internal alkene products (Δ7,8 and Δ8,9). Exocyclic alkene product (Δ8,12 isomer) will become relatively obvious when phenylhydrazines with electron-donating substituents are employed. A number of pyrazole mimics were purified through careful gradient elution on silica gel column chromatography (22 entries, including the modification of both phenylhydrazines and analogues of 2). Modification of the pyrimidine mimic 3c was conducted as shown in Scheme 3 and Scheme 4 (12 entries). All three isomeric alkenes (3ca−3cc) were successfully obtained from trifluoroacetic acid mediated dehydration of 3c (Scheme 3). NH2-protected product 3cd was produced in good yield through copper catalyzed Chan−Lam reaction. Variation of α,β-unsaturated ketone mimics will lead correspondingly to different drimanyl pyrimidine mimics 3ce−3ck (Scheme 4). The simplified natural drimane meroterpenoid DM was also synthesized through Baran’s method (Scheme 5) and recently by our own developed coupling method (will be reported elsewhere). The preparation of DM was finished in 6 steps or 7 steps starting from commercial available sclareolide or sclareol, 9018

DOI: 10.1021/acs.jafc.7b03126 J. Agric. Food Chem. 2017, 65, 9013−9021

Article

Journal of Agricultural and Food Chemistry Table 3. Fungicidal Bioactivity of Drimanyl Pyrimidine Mimicsa antifungal bioactivity (%, 50 mg/L)

a

compd

S. sclerotiorum

R. solani

B. cinerea

F. graminearum

3c 3ca 3cb 3cc 3cd 3ce 3cf 3cg 3ch 3ci 3cj 3ck CK1 CK2

69.50 ± 2.29 d 33.39 ± 0.22 g 27.64 ± 0.08 h 20.73 ± 0.10 i 36.40 ± 0.24 f 82.40 ± 0.41 b 17.23 ± 0.06 j 81.27 ± 0.41 b 10.86 ± 0.05 k 41.98 ± 0.18 e 71.91 ± 0.50 c 68.54 ± 0.31 d − 100 ± 0.02 a

57.45 ± 1.10 c 35.20 ± 0.29 gh 30.10 ± 0.11 i 36.22 ± 0.14 g 23.21 ± 0.11 j 48.18 ± 0.23 e 33.58 ± 0.11 h 47.81 ± 0.21 e 40.15 ± 0.21 f 51.82 ± 0.20 d 7.04 ± 0.04 k 74.45 ± 0.38 b − 100 ± 0.15 a

78.85 ± 2.11 a 24.60 ± 0.19 e 20.31 ± 0.10 g 27.73 ± 0.12 d 10.63 ± 0.06 i 15.60 ± 0.13 h 28.00 ± 0.13 d 38.07 ± 0.12 c 5.00 ± 0.02 j 22.48 ± 0.11 f 5.00 ± 0.06 j 56.42 ± 0.29 b − 20.39 ± 0.38 g

73.81 ± 1.03 b 14.50 ± 0.09 f 18.00 ± 0.10 e 19.50 ± 0.11 e 50.00 ± 0.24 c 8.10 ± 0.09 ij 9.70 ± 0.10 hi 19.50 ± 0.06 e 6.80 ± 0.04 j 11.86 ± 0.05 g 10.17 ± 0.09 h 41.53 ± 0.39 d − 100 ± 0.01 a

See footnote a for Table 1.

Table 4. Bioactivity and Structural Analysis of Fungicidal Drimanyl Meroterpenoid Mimicsa antifungal bioactivity (EC50, mg/L)

Properties

S.S.

R.S.

F.G.

Fsp

ClogP

TPSA

LE

3a 3c

27.01 ± 0.25 B 14.06 ± 0.19 C

32.31 ± 1.11 D 22.81 ± 2.91 C

3.84 ± 0.41 A 5.41 ± 0.15 B

>25 9.51 ± 0.37

0.7083 0.60

4.6985 4.581

41.82 70.97

2z 2a CK1 CK2

>25 >50 − 0.18 ± 0.01 A

4.47 ± 0.42 B >50 − 0.48 ± 0.04 A

>25 >50 − 158.65 ± 10.15 C

>25 >50 − 0.59 ± 0.12

0.6522 0.625 − −

3.54 5.037 − −

49.66 37.3 − −

−0.0258b −0.0364b −0.0462c −0.0343d 25-fold and >40-fold more promising than the commercial fungicide carbendazim, respectively (Table 4). The bioactivity against Botrytis cinerea of both 3a and 3c seems to be less sensitive to the concentration than that of carbendazim. (Figure 3). Further structural optimizations are in progress aimed at the discovery of more active and selective chiral drimane meroterpenoid mimics.

mimics. As can be seen in Table 1, the fungicidal potential of the pre-engineered mimics decreased with the increment of ClogP values. The more potent fungicidal candidate 3c possessed the ClogP value of 4.581 and correlated well with the statistical analysis. It is known that the polar regions of a molecule’s surface were closely associated with various bioavailability related properties, and topological PSAs calculated in ChemBioOffice showed that all the TPSA values were lower than 90. Some studies have shown the benefits of a higher Fsp3, and this value for the actual drugs is 0.47,12 while an analysis of medicinal compounds synthesized over the past 50 years demonstrates that the average Fsp3 is declining.24,25 We envision that optimization of the Fsp3 enriched drimane is a good choice for the acquisition of appropriate complexity with a wide range of Fsp3 and untouched chirality, which is otherwise not so easy to achieve. Analysis of the synthesized compounds with prominent fungicidal activity showed that all the Fsp3 values are over 0.6. The concept of ligand efficiency (LE) was also introduced and widely accepted as a reliable index of drug-like qualities;26 all the EC50 values of synthesized compounds with conspicuous activity were tested and further used for the calculation of LEs according to the former report27 (Table 4). The target plant pathogen changed from Rhizoctonia solani to Botrytis cinerea or Fusarium graminearum, with the structural alteration from unsaturated ketone inserted meroterpenoids to the heterocycle mimics. The drimane meroterpenoid mimics 3a, 3c, and 2z can be chosen as fungicidal leads for the inhibition against Botrytis cinerea, Rhizoctonia solani, or Fusarium graminearum, among which 3c may be a good choice in the view of both bioactivities and physicochemical characteristics. In summary, “carbon assimilation” was conceived and used for the rapid construction of drimanyl meroterpenoid mimics, in which the newly formed covalent bond was directly from the old one of the drimanyl subunit (“2 + 0” tactic). This tactic can formally link the drimanyl part and aromatic unit diversely and efficiently, instead of solving the challenging coupling of one atom from each part (“1 + 1” tactic). With the easy availability of the homodrimane skeleton, the redundant one carbon was “assimilated” or “donated” to the aromatic part for the construction of drimane meroterpenoid mimics. This “assimilation” or “2 + 0” tactic is a powerful tool in the exploration of potential of drimane meroterpenoid mimics as novel agrochemicals, which facilitated rapid, practical, efficient, and diverse construction of molecular diversity. This tactic can be fostered or transferred directly to medicinal chemistry or other related biological exploration. Specifically, a series of α,βunsaturated ketone inserted drimane meroterpenoid mimics

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b03126. Synthetic details, list of synthesized compounds, antifungal data, and NMR spectra (PDF) Crystallographic data for 3a (CIF) Crystallographic data for 3c (CIF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Shengkun Li: 0000-0001-5458-0811 Author Contributions

S.L. conceived and designed this work, S.Z. performed synthetic and biological experiments, Z.S. and D.L. carried out NMR and MASS detection, C.Z. and L.Z. helped to conduct antifungal experiments, and S.L. and S.Z. analyzed the data. X.S. helped to prepare the regression curves and histogram, and S.L. wrote the paper. Funding

This work was financially supported by National Natural Science Foundation of China (No. 31401777), Natural Science Foundation of Jiangsu Province (No. BK20140684), the opening foundation of the Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University (2014GDGP0102), and Hong Kong Scholars Program for S.L. (XJ2016011). 9020

DOI: 10.1021/acs.jafc.7b03126 J. Agric. Food Chem. 2017, 65, 9013−9021

Article

Journal of Agricultural and Food Chemistry Notes

(21) Lamberth, C., Dinges, J., Eds. Bioactive Heterocyclic Compound Classes: Agrochemicals; Wiley-VCH Verlag & Co. KGaA: Weinheim, Germany, 2012. (22) Hao, G.; Dong, Q.; Yang, G. A Comparative Study on the Constitutive Properties of Marketed Pesticides. Mol. Inf. 2011, 30, 614−622. (23) Rao, H.; Huangfu, C.; Wang, Y.; Wang, X.; Tang, T.; Zeng, X.; Li, Z.; Chen, Y. Physicochemical Profiles of the Marketed Agrochemicals and Clues for Agrochemical Lead Discovery and Screening Library Development. Mol. Inf. 2015, 34, 331−338. (24) Feher, M.; Schmidt, J. M. Property Distributions: Differences between Drugs, Natural Products, and Molecules from Combinatorial Chemistry. J. Chem. Inf. Comput. Sci. 2003, 43, 218−227. (25) Leeson, P. D.; St-Gallay, S. A. The influence of the ’organizational factor’ on compound quality in drug discovery. Nat. Rev. Drug Discovery 2011, 10, 749−765. (26) Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A. The maximal affinity of ligands. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 9997−10002. (27) Hirata, K.; Kotoku, M.; Seki, N.; Maeba, T.; Maeda, K.; Hirashima, S.; Sakai, T.; Obika, S.; Hori, A.; Hase, Y.; Yamaguchi, T.; Katsuda, Y.; Hata, T.; Miyagawa, N.; Arita, K.; Nomura, Y.; Asahina, K.; Aratsu, Y.; Kamada, M.; Adachi, T.; Noguchi, M.; Doi, S.; Crowe, P.; Bradley, E.; Steensma, R.; Tao, H.; Fenn, M.; Babine, R.; Li, X.; Thacher, S.; Hashimoto, H.; Shiozaki, M. SAR Exploration Guided by LE and Fsp(3): Discovery of a Selective and Orally Efficacious RORgamma Inhibitor. ACS Med. Chem. Lett. 2016, 7, 23−27.

The authors declare no competing financial interest.

■

REFERENCES

(1) Scherkenbeck, J. Modern trends in agrochemistry. Bioorg. Med. Chem. 2009, 17, 4019. (2) Sauter, H.; Steglich, W.; Anke, T. Strobilurins: Evolution of a New Class of Active Substances. Angew. Chem., Int. Ed. 1999, 38, 1328−1349. (3) Shan, W.-G.; Ying, Y.-M.; Ma, L.-F.; Zhan, Z.-J. Drimane-Related Merosesquiterpenoids, a Promising Library of Metabolites for Drug Development. Stud. Nat. Prod. Chem. 2015, 45 (Chapter 6), 147−215. (4) Dixon, D. D.; Lockner, J. W.; Zhou, Q.; Baran, P. S. Scalable, Divergent Synthesis of Meroterpenoids via “Boronosclareolide. J. Am. Chem. Soc. 2012, 134, 8432−8435. (5) Akita, H.; Nozawa, M.; Shimizu, H. Synthesis of decalin type chiral synthons based on enzymic functionalization and their application to the synthesis of (−)-Ambrox and (+)-zonarol. Tetrahedron: Asymmetry 1998, 9, 1789−1799. (6) Alvarez-Manzaneda, E. J.; Chahboun, R.; Pérez, I. B.; Cabrera, E.; Alvarez, E.; Alvarez-Manzaneda, R. First Enantiospecific Synthesis of the Antitumor Marine Sponge Metabolite (−)-15-Oxopuupehenol from (−)-Sclareol. Org. Lett. 2005, 7, 1477−1480. (7) Ling, T.; Xiang, A. X.; Theodorakis, E. A. Enantioselective Total Synthesis of Avarol and Avarone. Angew. Chem., Int. Ed. 1999, 38, 3089−3091. (8) Ling, T.; Poupon, E.; Rueden, E. J.; Kim, S. H.; Theodorakis, E. A. Unified Synthesis of Quinone Sesquiterpenes Based on a Radical Decarboxylation and Quinone Addition Reaction. J. Am. Chem. Soc. 2002, 124, 12261−12267. (9) Ishibashi, H.; Ishihara, K.; Yamamoto, H. A New Artificial Cyclase for Polyprenoids: Enantioselective Total Synthesis of (−)-Chromazonarol, (+)-8-epi-Puupehedione, and (−)-11‘-Deoxytaondiol Methyl Ether. J. Am. Chem. Soc. 2004, 126, 11122−11123. (10) Yan, A.; Gasteiger, J. Prediction of Aqueous Solubility of Organic Compounds by Topological Descriptors. QSAR Comb. Sci. 2003, 22, 821−829. (11) Yan, A.; Gasteiger, J. Prediction of Aqueous Solubility of Organic Compounds Based on a 3D Structure Representation. J. Chem. Inf. Comput. Sci. 2003, 43, 429−434. (12) Lovering, F.; Bikker, J.; Humblet, C. Escape from Flatland: Increasing Saturation as an Approach to Improving Clinical Success. J. Med. Chem. 2009, 52, 6752−6756. (13) Akamatsu, M. Importance of physicochemical properties for the design of new pesticides. J. Agric. Food Chem. 2011, 59, 2909−17. (14) Wang, F.; Chen, C.; Yang, K.; Xu, Y.; Liu, X.; Gao, F.; Liu, H.; Chen, X.; Zhao, Q.; Liu, X.; Cai, Y.; Yang, H. Michael Acceptor-Based Peptidomimetic Inhibitor of Main Protease from Porcine Epidemic Diarrhea Virus. J. Med. Chem. 2017, 60, 3212−3216. (15) Heller, L.; Schwarz, S.; Perl, V.; Kowitsch, A.; Siewert, B.; Csuk, R. Incorporation of a Michael acceptor enhances the antitumor activity of triterpenoic acids. Eur. J. Med. Chem. 2015, 101, 391−399. (16) Zhuang, C.; Zhang, W.; Sheng, C.; Zhang, W.; Xing, C.; Miao, Z. Chalcone: A Privileged Structure in Medicinal Chemistry. Chem. Rev. 2017, 117, 7762−7810. (17) Li, D.; Zhang, S.; Song, Z.; Wang, G.; Li, S. Bioactivity-guided mixed synthesis accelerate the serendipity in lead optimization: Discovery of fungicidal homodrimanyl amides. Eur. J. Med. Chem. 2017, 136, 114−121. (18) Marei, G. I. K.; Rasoul, M. A. A.; Abdelgaleil, S. A. M. Comparative antifungal activities and biochemical effects of monoterpenes on plant pathogenic fungi. Pestic. Biochem. Physiol. 2012, 103, 56−61. (19) Vadapalli, S.; Kane, C. T., Jr. Improved synthesis of 11-acetoxy8α-drimanol. Org. Prep. Proced. Int. 2008, 40, 201−204. (20) Lamberth, C. Heterocyclic chemistry in crop protection. Pest Manage. Sci. 2013, 69, 1106−1114. 9021

DOI: 10.1021/acs.jafc.7b03126 J. Agric. Food Chem. 2017, 65, 9013−9021