Design, Semisynthesis, Insecticidal and Antibacterial Activities of a Series of Marine-Derived Geodin Derivatives and Their Preliminary Structure–Activity Relationships

To enhance the biological activity of the natural product geodin (1), isolated from the marine-derived fungus Aspergillus sp., a series of new ether derivatives (2–37) was designed and semisynthesized using a high-yielding one-step reaction. In addition, the insecticidal and antibacterial activities of all geodin congeners were evaluated systematically. Most of these derivatives showed better insecticidal activities against Helicoverpa armigera Hübner than 1. In particular, 15 showed potent insecticidal activity with an IC50 value of 89 μM, comparable to the positive control azadirachtin (IC50 = 70 μM). Additionally, 5, 12, 13, 16, 30 and 33 showed strong antibacterial activity against Staphylococcus aureus and Aeromonas salmonicida with MIC values in the range of 1.15–4.93 μM. The preliminary structure–activity relationships indicated that the introduction of halogenated benzyl especially fluorobenzyl, into 1 and substitution of 4-OH could be key factors in increasing the insecticidal and antibacterial activities of geodin.


Introduction
Cotton bollworm (Helicoverpa armigera Hübner) is one of the most destructive agricultural pests known. This organism causes severe damage to a wide range of crops, such as cotton, sunflower, okra, tomato, chickpea, maize, potato and cabbage [1,2]. H. armigera is native to Africa, Asia, Europe, and Australasia and has become the most globally widespread species of Helicoverpa [3]. Consequently, H. armigera Hübner is responsible for about two billion dollars in direct economic losses each year [4]. Moreover, it has developed resistance against newer chemistries and conventional insecticides because of the extensive use of these chemical insecticides [5]. With the emergence of resistance in H. armigera Hübner to commercial insecticides, research on new insecticides has become particularly important. Marine natural products (MNPs) continue to play highly significant roles in the pesticide development process and are potential sources of new drugs for treating plant diseases [6,7]. Introducing structural modifications to MNPs is one important strategy for treating plant diseases [6,7]. Introducing structural modifications to MNPs is one important strategy for drug development initiatives to improve bioavailability, enhance biological activity and reduce toxicity [8].
During the course of our ongoing research into bioactive compounds from marinederived fungi [9][10][11][12][13][14][15], a secondary metabolite belonging to the griseofulvin family, geodin (1), was isolated from the soft coral-derived fungus Aspergillus sp. (CHNSCLM-0151), collected from the South China Sea in 2015. Griseofulvin is a classic antifungal agent used clinically for the treatment of dermatomycoses and is an inhibitor of centrosomal clustering of cancer cell lines [16]. Griseofulvin and its analogues have been rigorously studied and some structure-activity relationships (SARs) clearly identified [16,17]. A library of 53 griseofulvin analogues with modifications on the 4, 5, 6, 2′, 3′ and 4′positions have been found to be less bioactive than the parent compound. Notably, a 2′-benzyloxy derivative with low activity against Trichophyton rubrum and Trichophyton metagrophytes was found to be among the most potent compounds identified against MDA-MB-231 cancer cells [16]. Geodin (1) shares the same grisan backbone as griseofulvin ( Figure 1) and displays various interesting biological activities such as antimicrobial, glucose uptake for modulatory (rat adipocytes), fibrinolytic enhancement, antiviral and cytotoxic activities [18][19][20]. Most noteworthy from the current semisynthetic perspective, we have previously noted that geodin (1) exerts weak insecticidal activity [19]. Moreover, the previous study SAR study of 1 demonstrated that the phenolic hydroxyl group is not required for insecticidal activity [19]. In order to enhance the biological activity of 1, 36 new derivatives of geodin (1) were semisynthesized to evaluate insecticidal and antimicrobial activities; using this new panel of analogs, we also have extended SAR knowledge of 1. Figure 1. The structures of griseofulvin and geodin (1) which share the same grisan backbone (rings A, B and C).

Chemistry
The fungal strain Aspergillus sp. (CHNSCLM-0151) was cultivated in 50 L PDB medium at 28 °C with shaking for one week. Then the fermentation broth was extracted three times with an equal volume of EtOAc. The organic extracts were combined and concentrated under vacuum to afford a dry crude extract (35.7 g). The extract was subjected to vacuum liquid chromatography (VLC) on silica gel and eluted with a stepwise gradient of petroleum ether (PE)-EtOAc to afford six fractions (Fr. 1-Fr. 6). Fr. 5 was applied to reverse phase silica gel column and eluted with MeOH-H2O to obtain seven sub-fractions (Fr. . Fr. 5-B was then purified with 65% MeOH-H2O to yield compound 1 (2.2 g).
The chemical structure of 1 was elucidated by analysis of NMR data and comparisons with literature [17]. Compound 1 contains an exchangeable proton, two singlet protons, two oxygenated methyl groups and a methyl. The substituents at the 4-OH were semisynthetically modified (Scheme 1) using benzyl bromide at a temperature of 40 °C for 6-12 h in the presence of K2CO3 in dry acetone to produce compounds 2-37 ( Figure 1 and Table   Figure 1. The structures of griseofulvin and geodin (1) which share the same grisan backbone (rings A, B and C).

Chemistry
The fungal strain Aspergillus sp. (CHNSCLM-0151) was cultivated in 50 L PDB medium at 28 • C with shaking for one week. Then the fermentation broth was extracted three times with an equal volume of EtOAc. The organic extracts were combined and concentrated under vacuum to afford a dry crude extract (35.7 g). The extract was subjected to vacuum liquid chromatography (VLC) on silica gel and eluted with a stepwise gradient of petroleum ether (PE)-EtOAc to afford six fractions (Fr. 1-Fr. 6). Fr. 5 was applied to reverse phase silica gel column and eluted with MeOH-H 2 O to obtain seven sub-fractions (Fr. 5-A and Fr. 5-G). Fr. 5-B was then purified with 65% MeOH-H 2 O to yield compound 1 (2.2 g).
The chemical structure of 1 was elucidated by analysis of NMR data and comparisons with literature [17]. Compound 1 contains an exchangeable proton, two singlet protons, two oxygenated methyl groups and a methyl. The substituents at the 4-OH were semisynthetically modified (Scheme 1) using benzyl bromide at a temperature of 40 • C for 6-12 h in the presence of K 2 CO 3 in dry acetone to produce compounds 2-37 ( Figure 1 and Table 1). The structures of these derivatives  were fully characterized by extensive spectroscopic analyses (Figures S1-S111). 1). The structures of these derivatives (2−37) were fully characterized by extensive spectroscopic analyses (Figure S1−S111).
Scheme 1. General semisynthetic strategy employed to make ether derivatives of 1.

Insecticidal Activity against Helicoverpa armigera Hübner
In this study, the insecticidal activities against Helicoverpa armigera Hübner of geodin (1) and 36 new derivatives modified at the 4-OH position were assessed. Most derivatives showed better insecticidal activity than marine natural product 1 ( Table 2). Most of the derivatives were functionalized with benzyl analogues except for 37 which was modified with only an ethyl group and had no insecticidal activity. Compounds 2-4 and 35 were

Insecticidal Activity against Helicoverpa armigera Hübner
In this study, the insecticidal activities against Helicoverpa armigera Hübner of geodin (1) and 36 new derivatives modified at the 4-OH position were assessed. Most derivatives showed better insecticidal activity than marine natural product 1 ( Table 2). Most of the derivatives were functionalized with benzyl analogues except for 37 which was modified with only an ethyl group and had no insecticidal activity. Compounds 2-4 and 35 were

Insecticidal Activity against Helicoverpa armigera Hübner
In this study, the insecticidal activities against Helicoverpa armigera Hübner of geodin (1) and 36 new derivatives modified at the 4-OH position were assessed. Most derivatives showed better insecticidal activity than marine natural product 1 ( Table 2). Most of the derivatives were functionalized with benzyl analogues except for 37 which was modified with only an ethyl group and had no insecticidal activity. Compounds 2-4 and 35 were

Insecticidal Activity against Helicoverpa armigera Hübner
In this study, the insecticidal activities against Helicoverpa armigera Hübner of geodin (1) and 36 new derivatives modified at the 4-OH position were assessed. Most derivatives showed better insecticidal activity than marine natural product 1 ( Table 2). Most of the derivatives were functionalized with benzyl analogues except for 37 which was modified with only an ethyl group and had no insecticidal activity. Compounds 2-4 and 35 were modified with bromobenzyl moieties and exhibited moderate insecticidal activity with IC 50 values of 176 µM (although 4 was inactive). In addition, the introduction of chlorobenzyl groups at the 4-OH position of 1 afforded the analogues 5-9 and 31-34 most of which showed higher insecticidal activity. However, the 2-chlorobenzyl derivative 5, 2-chloro-4-fluorobenzyl derivative 31 and 4-chloro-2-fluorobenzyl derivative 33 displayed lower activity against H. armigera Hübner compared to parent 1. Besides, the iodobenzyl analogues 10 and 11 had no insecticidal activity. Multiple studies have demonstrated that the introduction of a fluorine atom was useful for increasing insecticidal activity because of its unique properties such as electronegativity, size, and electrostatic interactions [21][22][23][24][25]. Herein, 12-21 were modified by fluorinated benzyl and most exhibited stronger activity (16 and 19 were inactive). Especially, 2,3,4,5-tetrafluorobenzyl derivative 15 exhibited potent insecticidal activity comparable with the positive control azadirachtin with an IC 50 value of 89 µM ( Figure 2). Moreover, the result of the antifouling activity of 15 against Navicula exigua and the settlement of the Mytilus edulis showed inactive and indicated 15 was non-toxic. In addition, non-halogenated benzyl derivatives 22-30 which were modified with nitro, methyl, phenyl and nitrile groups had good insecticidal activity except for 3-methylbenzyl compound 24 and 2-cyanobenzyl compound 30. The above results indicated that the introduction of benzyl especially halogenated benzyl could enhance the insecticidal activity of 1. Strikingly, further research on 15 was worth developing the low-toxicity and high-efficiency insecticide drug.

Insecticidal Activity against Helicoverpa armigera Hübner
In this study, the insecticidal activities against Helicoverpa armigera Hübner of geodin (1) and 36 new derivatives modified at the 4-OH position were assessed. Most derivatives showed better insecticidal activity than marine natural product 1 ( Table 2). Most of the derivatives were functionalized with benzyl analogues except for 37 which was modified with only an ethyl group and had no insecticidal activity. Compounds 2-4 and 35 were modified with bromobenzyl moieties and exhibited moderate insecticidal activity with IC50 values of 176 μM (although 4 was inactive). In addition, the introduction of chlorobenzyl groups at the 4-OH position of 1 afforded the analogues 5-9 and 31-34 most of which showed higher insecticidal activity. However, the 2-chlorobenzyl derivative 5, 2chloro-4-fluorobenzyl derivative 31 and 4-chloro-2-fluorobenzyl derivative 33 displayed lower activity against H. armigera Hübner compared to parent 1. Besides, the iodobenzyl analogues 10 and 11 had no insecticidal activity. Multiple studies have demonstrated that the introduction of a fluorine atom was useful for increasing insecticidal activity because of its unique properties such as electronegativity, size, and electrostatic interactions [21][22][23][24][25]. Herein, 12-21 were modified by fluorinated benzyl and most exhibited stronger activity (16 and 19 were inactive). Especially, 2,3,4,5-tetrafluorobenzyl derivative 15 exhibited potent insecticidal activity comparable with the positive control azadirachtin with an IC50 value of 89 μM ( Figure 2). Moreover, the result of the antifouling activity of 15 against Navicula exigua and the settlement of the Mytilus edulis showed inactive and indicated 15 was non-toxic. In addition, non-halogenated benzyl derivatives 22-30 which were modified with nitro, methyl, phenyl and nitrile groups had good insecticidal activity except for 3-methylbenzyl compound 24 and 2-cyanobenzyl compound 30. The above results indicated that the introduction of benzyl especially halogenated benzyl could enhance the insecticidal activity of 1. Strikingly, further research on 15 was worth developing the lowtoxicity and high-efficiency insecticide drug.
For A. salmonicida, similarly, the introduction of halogenated benzyl strengthened the activity of geodin (1). Almost all of the compounds modified by benzyl groups substituted with fluorine, chlorine, bromine and iodine atoms showed better antibacterial activity than the parent 1. The derivatives 2, 5,

General Experimental Procedures
1 H and 13 C NMR spectra were measured on an Agilent DD2 NMR spectrometer at 500 MHz and 125 MHz frequencies, respectively. For vacuum column chromatography silica gel (200-300 mesh, Qing Dao Hai Yang Chemical Group Co, Qingdao, China) and silica gel plates for thin layer chromatography (G60, F-254, and Yan Tai Zi Fu Chemical Group Co, Yan Tai, China) were used. HR-ESI-MS spectra were recorded on a Micro-mass Q-TOF spectrometer while UPLCMS spectra were measured on Waters UPLC ® system using a C18 column [ACQUITY UPLC ® BEH C18, 2.1 × 50 mm, 1.7 μm; 0.5 mL/min] and ACQUITY QDA ESIMS scan from 150 to 1000 Da. For reverse phase Octadecylsilyl silica gel column was used. All the derivatives of compound 1 were semisynthesized by one step reaction. The products formation and reaction completion were checked by TLC at various intervals of time.

General Experimental Procedures
1 H and 13 C NMR spectra were measured on an Agilent DD2 NMR spectrometer at 500 MHz and 125 MHz frequencies, respectively. For vacuum column chromatography silica gel (200-300 mesh, Qing Dao Hai Yang Chemical Group Co., Qingdao, China) and silica gel plates for thin layer chromatography (G60, F-254, and Yan Tai Zi Fu Chemical Group Co., Yan Tai, China) were used. HR-ESI-MS spectra were recorded on a Micro-mass Q-TOF spectrometer while UPLCMS spectra were measured on Waters UPLC ® system using a C18 column [ACQUITY UPLC ® BEH C18, 2.1 × 50 mm, 1.7 µm; 0.5 mL/min] and ACQUITY QDA ESIMS scan from 150 to 1000 Da. For reverse phase Octadecylsilyl silica gel column was used. All the derivatives of compound 1 were semisynthesized by one step reaction. The products formation and reaction completion were checked by TLC at various intervals of time.

Biological Material
Aspergillus sp. (CHNSCLM-0151) was isolated from the inner soft fresh tissue of a soft coral Sinularia sp., collected in 2015 from the coastal depth of the South China Sea. It was identified as Aspergillus sp., on the basis of its morphological and RNA base sequences. The fungus 512 base pair had 98% ITS sequence similarity with Aspergillus sp., NRRL58570 (GenBank No. HQ288052.1). The sequence data have been submitted to GenBank with accession number KY235298. The strain has been stored at the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.

Extraction and Isolation
The pure spores of fungal strain were streaked onto PDA plates and incubated at 28 • C for one week. After the growth of fungus, small plugs of PDA containing the spores were aseptically put into 1000 mL Erlenmeyer flasks having about 60 g sterilized rice medium and 2% sodium chloride salt. The flasks were fermented at room temperature for 3 weeks, extracted three times with EtOAc, and evaporated under vacuum. The crude extract was subjected to normal phase silica gel column chromatography (CC) (200-300 mesh), eluting with a linear gradient of PE-EtOAc (v/v, gradient) to afford five fractions (Fr. 1-Fr. 5). Fraction 4 eluted with 40% EtOAc was further subjected to reverse phase silica gel CC, using MeOH and H 2 O as mobile phase. Compound 1 was eluted with 80% MeOH/H 2 O and further purified by recrystallization in MeOH/CH 2 Cl 2 .

General Synthetic Methods for Compounds 2-37
A corresponding benzyl bromide reagent (3-5 eq.) and anhydrous K 2 CO 3 (15 mg) were added to a stirred solution of 1 (40 mg, 0.10 mmol) in dry acetone (20 mL). The reaction mixture was stirred at 40 • C for 6-12 h. After the completion of the reaction, water was added to the reaction mixture, then the solution was extracted two times with EtOAc (40 mL). The organic layer was combined and dried under vacuum to give a crude residue which was purified through normal phase silica gel CC (200-300 mesh) eluting with a linear gradient of PE-EtOAc.

Antibacterial Activity
The methods described by Fromtling et al. were used for the evaluation of antibacterial activities [27]. Ciprofloxacin and sea-nine 211 were used as positive controls. Bacterial species were cultured at 37 • C for 8 h in LB medium and diluted to 10 6 cfu/mL, using 96-well microplates, having 2 µL test sample and 198 µL of bacterial solutions. The plates were incubated at 37 • C for 24 h while DMSO was used as the negative control.

Conclusions
In summary, 36 new derivatives of marine-derived geodin (1) were semisynthesized successfully via one mild step reaction with high yields. Among them, 15 showed significant insecticidal activity equivalent to the positive drug, azadirachtin. Meanwhile, 37 showed selective antibacterial activity against P. aeruginosa. The results revealed that modification of the 4-OH and introduction of halogen atoms, especially fluorine and chlorine atoms, could enhance the insecticidal and antibacterial activities of 1. These findings bring further evidence that geodin derivatives are active and provide new information supporting the importance of continued studies into the structure-activity relationships of griseofulvin analogs.