Discovery of Hyrtinadine A and Its Derivatives as Novel Antiviral and Anti-Phytopathogenic-Fungus Agents

Plant diseases caused by viruses and fungi have a serious impact on the quality and yield of crops, endangering food security. The use of new, green, and efficient pesticides is an important strategy to increase crop output and deal with the food crisis. Ideally, the best pesticide innovation strategy is to find and use active compounds from natural products. Here, we took the marine natural product hyrtinadine A as the lead compound, and designed, synthesized, and systematically investigated a series of its derivatives for their antiviral and antifungal activities. Compound 8a was found to have excellent antiviral activity against the tobacco mosaic virus (TMV) (inactivation inhibitory effect of 55%/500 μg/mL and 19%/100 μg/mL, curative inhibitory effect of 52%/500 μg/mL and 22%/100 μg/mL, and protection inhibitory effect of 57%/500 μg/mL and 26%/100 μg/mL) and emerged as a novel antiviral candidate. These compound derivatives displayed broad-spectrum fungicidal activities against 14 kinds of phytopathogenic fungi at 50 μg/mL and the antifungal activities of compounds 5c, 5g, 6a, and 6e against Rhizoctonia cerealis are higher than that of the commercial fungicide chlorothalonil. Therefore, this study could lay a foundation for the application of hyrtinadine A derivatives in plant protection.


Introduction
Plant diseases caused by viruses, fungi, and bacteria have long been a great threat to agricultural production [1]. In general, crop losses caused by plant diseases account for 25% of the world's crop output every year [2]. As the earliest and deepest studied model virus, the tobacco mosaic virus (TMV) has been found to infect more than 400 crops including tobacco, cucumber, banana, etc. [3]. Because the TMV is parasitic to the host cells, and plants have no complete immune system, it is extremely difficult to control the plant diseases caused by the TMV [4]. In addition, the reduction in resources, climate change, and population growth all contribute to the need to maximize crop production to ensure food security [5][6][7]. Therefore, plant disease prevention is becoming increasingly important and urgent. Although scientists have created some bactericides and antiviral agents in the past decades, some of them have developed serious drug resistance during long-term use [8,9]. As a widely used antiviral agent, ribavirin showed a less than 50% anti-TMV effect at 500 µg/mL. With the enhancement of people's awareness of environmental protection, anti-TMV effect at 500 μg/mL. With the enhancement of people's awareness of environmental protection, batches of problematic pesticides have been banned [10]. Thus, the discovery of new, green, and efficient pesticides is becoming increasingly important.
In the vast ocean, there are a large number of secondary metabolites with novel chemical structures, diverse biological activities, and unique mechanisms of action beyond people's imagination. Marine natural products (MNPs) have become the main source of important drug leads. These compounds have made significant contributions to the treatment of many diseases, including cancer and infectious diseases, as well as other therapeutic areas, such as multidrug resistance (MDR), cardiovascular disease, and multiple sclerosis [11,12]. Compared with traditional synthetic molecules, MNPs have unique structural characteristics and complex skeletons, which are conducive to drug discovery. MNPs usually have a high molecular weight and a large number of carbon and oxygen atoms. In addition, marine bioactive compounds have fewer but unique nitrogen atoms and halogen atoms. To date, more than 20 MNP drugs or pesticides have been approved for use, such as Nereistoxin (insecticide for the control of rice insects), Cytarabine (anticancer drug), Ziconotide (analgesic), Trabectedin (anticancer drug), and Brentuximab (a drug for lymphoma) [13][14][15]. Hyrtinadine A is a bis-indole alkaloid isolated from an Okinawan marine sponge Hyrtios sp. (SS-1127; order Dictyoceratida; family Thorectidae; collected off Unten-Port, Okinawa, Japan) [16]. Its novel chemical structure aroused the interest of chemists, and the total synthesis of hyrtinadine A has been successfully completed with the Masuda borylation-Suzuki reaction and Kosugi-Migita-Stille reaction [17,18]. At present, the research on the activity of hyrtinadine A mainly focuses on its antitumor properties, and the research on its bioactivity spectrum and structure-activity relationship is relatively lacking. Therefore, it is of considerable significance to carry out research on the synthesis, structure optimization, and biological activity of hyrtinadine A in order to deeply understand its behavior and explore its application.
We have been committed to the discovery of novel pesticide leads based on MNPs for a long time and have found that a number of marine natural products can be used as candidates for antiviral, fungicidal, and insecticidal agents [19][20][21]. Here, alkaloid hyrtinadine A was selected as the parent structure, and a series of its derivatives were designed (Figure 1), synthesized, and evaluated for their antiviral and antifungal activities.

Chemistry
To date, the total synthesis of hyrtinadine A has been reported by Sarandeses [22], Müller [17], and Söderberg [18]. Müller's route is more suitable for the large-scale preparation and derivation of hyrtinadine A and can provide intermediates with rich structures. Therefore, hyrtinadine A was prepared in five steps from 5-methoxyindole with a 42% overall yield (Scheme 1) by Müller's method with modification [17].

Chemistry
To date, the total synthesis of hyrtinadine A has been reported by Sarandeses [22], Müller [17], and Söderberg [18]. Müller's route is more suitable for the large-scale preparation and derivation of hyrtinadine A and can provide intermediates with rich structures. Therefore, hyrtinadine A was prepared in five steps from 5-methoxyindole with a 42% overall yield (Scheme 1) by Müller's method with modification [17]. products 7a−7f. Then, the protective group of the mono-acylated products 7a and 7d could be removed under the condition of CF3COOH to create products 8a−8b. Acylhydrazone is a good pharmacophore, and we found that the introduction of this group can improve the antiviral activity of the molecule [24]. As depicted in Scheme 4, compound 5g went through hydrolysis and esterification hydrazinolysis to yield 8c. Then, 8c reacted with the corresponding aldehyde in ethanol under the refluxed condition to yield acylhydrazones 9a−9g. Scheme 1. The synthesis of alkaloid hyrtinadine A and compounds 5a−5d. Scheme 1. The synthesis of alkaloid hyrtinadine A and compounds 5a-5d.
In order to investigate the structure-activity relationship (SAR), a series of hyrtinadine A derivatives (5a-5d, 6a-6e, 7a-7f, 8a-8b, and 9a-9g) were designed and synthesized. As depicted in Scheme 1, compounds 5a-5d with different groups at the indole ring were prepared using the Masuda borylation-Suzuki reaction as a key step. Scaffold hopping is a common and efficient strategy for drug lead optimization [23]. In order to investigate the importance of the bis-indole ring on the compound's activity and further simplify the molecular structure, we designed and synthesized compounds 6a-6e by replacing the indole ring with a substituted benzene ring (Scheme 2). As shown in Scheme 3, the NH 2 group in 6e can be replaced with a corresponding acyl chloride to create mono-acylated products 7a-7f. Then, the protective group of the mono-acylated products 7a and 7d could be removed under the condition of CF 3 COOH to create products 8a-8b. Acylhydrazone is a good pharmacophore, and we found that the introduction of this group can improve the antiviral activity of the molecule [24]. As depicted in Scheme 4, compound 5g went through hydrolysis and esterification hydrazinolysis to yield 8c. Then, 8c reacted with the corresponding aldehyde in ethanol under the refluxed condition to yield acylhydrazones 9a-9g.

Phytotoxic Activity
The phytotoxic activity tests demonstrated that compounds 5a−5d, 6a−6e, 7a−7f, 8a−8b, and 9a−9g were safe for testing on plants at 500 μg/mL. The detailed test procedures can be seen in the Supplementary Materials.

Phytotoxic Activity
The phytotoxic activity tests demonstrated that compounds 5a-5d, 6a-6e, 7a-7f, 8a-8b, and 9a-9g were safe for testing on plants at 500 µg/mL. The detailed test procedures can be seen in the Supplementary Materials.

Structure-Activity Relationship (SAR)
On the whole, the structural optimization of the natural product hyrtinadine A is very successful, and the biological activities of most derivatives are better than that of hyrtinadine A. The substituent groups at the 5-position of the indole had a significant influence on biological activity, and methoxy and chlorine are the most prominent (inhibitory effect: 5a ≈ 5c > 5d > 5b > 5e). The electron effect does not show obvious regularity. The substitution of Br or CN for an indole ring leads to a decrease in activity (inhibitory effect: 5a > 5f and 5g), which indicates that an aromatic ring in this area is necessary. A substituted benzene ring instead of an indole ring on hyrtinadine A is also unfavorable to its activity (inhibitory effect: 5a > 6a-6c and 6e). The introduction of an acyl group into the benzene ring amino group has little effect on the activity (inhibitory effect: 6e > 7a-7f). In contrast, increasing the electron cloud density on the benzene ring is beneficial to its biological activity (inhibitory effect: 6e > 6a-6c and 6e). The activity of compound 7a was significantly increased after further deprotection (inhibitory effect: 8a > 7a), but the activity of compound 7d decreased after deprotection (inhibitory effect: 7d > 8b). Acylhydrazone is a good pharmacophore; most of the compounds containing acylhydrazone functional groups show good antiviral activity (inhibitory effect: 9e, 9f ≈ 5a > 9a, 9b, 9d ≈ 5g > 9c, 9g). The successful discovery of highly active molecules 8a, 9e, and 9f provides guidance for us to further simplify the molecular structure.

Chemicals
Reagents were purchased from commercial sources and were used as received. All anhydrous solvents were dried and purified by standard techniques prior to use.

Instruments
The melting points of the synthesized compounds were determined on an X-4 binocular microscope (Beijing Tech Instruments Co., Beijing, China) with the thermometer uncorrected. NMR spectra were obtained on a Bruker AV 400 spectrometer (Bruker Corp., Switzerland) in CDCl 3 or DMSO-d 6 solution with tetramethylsilane as the internal standard. High-resolution mass spectra were obtained with an FT-ICR MS spectrometer (Ionspec, 7.0 T, IonSpec Co., Ltd., Lake Forest, CA, USA).

General Procedures for the Preparation of 3a-3d
KOH (1.9 g, 34 mmol) was added to a solution of substituted indoles (13.6 mmol) in DMF (20 mL). Then, a solution of I 2 (3.8 g, 15 mmol) in DMF (20 mL) was added dropwise into the previous solution and stirred at room temperature for 2 h. The reaction was then quenched with ice water (300 mL, containing 4% NH 3 ·H 2 O, 1% Na 2 S 2 O 5 ) and the mixture was placed in a refrigerator to ensure complete precipitation. The precipitate was filtered, washed with 200 mL ice water, and dried in vacuo to obtain 2a-2d, which were used without further purification in the next step. tert-Butyl 5-chloro-3-iodo-1H-indole-1-carboxylate (3c). White solid; mp 105-107 • C; yield 84%. Other data are consistent with those in the reference [17].

Procedures for the Preparation 5f and 5g
Compounds 5f and 5g were obtained by following similar procedures for the preparation of compounds 5a-5d.

General Procedures for the Preparation of 6a-6e
Et 3 N (19 mL,134 mmol) and substituted phenylborate ester (0.74 mmol) were added to a solution of 5f (0.2 g, 0.5 mmol), Pd(PPh 3 ) 4 (0.06 g, 0. 05 mmol), and Cs 2 CO 3 (0.4 g, 1.25 mmol) in 1,4-dioxane (10 mL) under argon and the reaction mixture was stirred at 100 • C for 3 h. After the completion of the reaction, the solution was cooled to room temperature and the solvents were removed under reduced pressure. The residue was purified by column chromatography on silica gel with petroleum ether/ethyl acetate to yield compounds 6a-6e.
tert    13  at least three times. The activity results were evaluated according to a percentage of mortalities from 0 to 100 (0 means no activity and 100 means total kill or inhibition).

Antiviral Biological Assay
The antiviral activities against TMV were carried out using previously reported methods [20,21]. The detailed procedures can be seen in the Supplementary Materials.

Antifungal Biological Assay
The procedures of antifungal activity tests were described in the literature [20,21]. The detailed procedures can also be seen in the Supplementary Materials.

Conclusions
In summary, the marine natural product hyrtinadine A and its derivatives were synthesized, and their antiviral and fungicidal activities were evaluated systematically for the first time. Compounds 5a, 5c, 8a, 9e, and 9f exhibited excellent in vivo anti-TMV activities at 500 µg/mL which were similar to that of ningnanmycin. The research on the structure-activity relationship showed that the substituent group in the 5-position of the indole of hyrtinadine A influences the anti-TMV activity, and that methoxy and chlorine are the most prominent. In addition, the bis-indole skeleton is important to the activity, but it is not unchangeable. Moreover, the structurally simplified compounds 8a, 9e, and 9f with high antiviral activities were successfully discovered and all the synthesized compounds were found to display broad-spectrum fungicidal activities. The current research results thus provide reliable support to develop hyrtinadine A and its analogs as novel antiviral and fungicidal agents.

Conflicts of Interest:
The authors declare no conflict of interest.