Design, Synthesis, and Bioactivities of Novel Tryptophan Derivatives Containing 2,5-Diketopiperazine and Acyl Hydrazine Moieties

Based on the scaffolds widely used in drug design, a series of novel tryptophan derivatives containing 2,5-diketopiperazine and acyl hydrazine moieties have been designed, synthesized, characterized, and evaluated for their biological activities. The bioassay results showed that the target compounds possessed moderate to good antiviral activities against tobacco mosaic virus (TMV), among which compounds 4, 9, 14, 19, and 24 showed higher inactivation, curative, and protection activities in vivo than that of ribavirin (39 ± 1, 37 ± 1, 39 ± 1 at 500 mg/L) and comparable to that of ningnanmycin (58 ± 1, 55 ± 1, 57 ± 1% at 500 mg/L). Thus, these compounds are a promising candidate for anti-TMV development. Most of these compounds showed broad-spectrum fungicidal activities against 13 kinds of phytopathogenic fungi and selective fungicidal activities against Alternaria solani, Phytophthora capsica, and Sclerotinia sclerotiorum. Additionally, some of these compounds exhibited larvicidal activities against Tetranychus cinnabarinus, Plutella xylostella, Culex pipiens pallens, Mythimna separata, Helicoverpa armigera, and Pyrausta nubilalis.


Introduction
Plant viruses, which are composed of nucleic acids and proteins [1], cause global economic losses as high as USD 60 billion every year [2][3][4][5][6]. They can change the normal metabolic process of host plants, interfere with or destroy the activity of respiratory photosynthetic enzymes and the metabolism of auxin and other hormones, in addition to robbing some nutrients of infected plants. Thus far, about 1100 kinds of viruses have been found. TMV (tobacco mosaic virus) is one of the oldest known plant viruses and ranks first among the top 10 plant viruses, causing economic losses of more than USD 100 million per year. There is no antiviral agent that can completely inhibit plant viruses, and the development of novel and more practical antiviral reagents is sorely needed [7,8].
Natural products are secondary metabolites retained by natural selection after a long time of evolution. Natural products are often characterized by chemical structure and biological activity diversity, which makes them of great value in drug development and utilization [9,10]. By September 2019, among the 185 small molecule anticancer drugs approved for sale by the FDA, 120 are related to natural products, accounting for 64.9% [11].
Tryptophan is a biosynthetic precursor in notable bioactive compounds [12][13][14][15], it also has a central role in metabolism, protein structure, and signaling, and analogs are frequently used to probe enzyme function or alter enzyme properties. In our previous work, we found, for the first time, that tryptophan showed moderate anti-plant virus activity [16], which can be used as an antiviral lead for subsequent studies. The acyl hydrazone structure is a complex of hydrogen bond donors and receptors. In our previous work, it was found that the acyl hydrazone structure could enhance the anti-TMV activity of the compound, possibly because the hydrogen bond receptor or donor of the acyl hydrazone enhanced the interaction with the amino acid residues of TMV CP, thus preventing the assembly of the virus [21][22][23].
In this work, to improve the anti-virus activity of tryptophan, we designed and synthesized a series of novel tryptophan derivatives containing diketopiperazine (DKP) and acyl hydrazon moieties and first evaluated their biological activities ( Figure 2). In addition, the fungicidal and larvicidal activities of the newly synthesized tryptophan derivatives were also studied to expand their potential agricultural applications. The acyl hydrazone structure is a complex of hydrogen bond donors and receptors. In our previous work, it was found that the acyl hydrazone structure could enhance the anti-TMV activity of the compound, possibly because the hydrogen bond receptor or donor of the acyl hydrazone enhanced the interaction with the amino acid residues of TMV CP, thus preventing the assembly of the virus [21][22][23].
In this work, to improve the anti-virus activity of tryptophan, we designed and synthesized a series of novel tryptophan derivatives containing diketopiperazine (DKP) and acyl hydrazon moieties and first evaluated their biological activities ( Figure 2). In addition, the fungicidal and larvicidal activities of the newly synthesized tryptophan derivatives were also studied to expand their potential agricultural applications.
For acylhydrazone derivatives, 4-25, the types, position, and number of substituents on the benzene ring had an important influence on the anti-TMV activity. The introduction of strong electron-withdrawing groups on the benzene ring, such as nitro (5,17), and trifluoromethyl (10), was detrimental to the activity. For the substituents at the para position of the benzene ring, electron-donating groups (6,9) and weak electron-withdrawing group (8) were favorable for maintaining the activity. The position of the substituents on the benzene ring had a significant effect on the activity and showed a significant ortho-position effect; that is, the activities of the ortho-substituted derivatives were significantly better than that of the derivatives substituted at other positions (14 versus 8, 9, and 19 versus 9, 18). For example, when the benzene ring has a methoxy substituted on the benzene ring, the order of bioactivity levels is 19 (2-OMe) > 9 (4-OMe) > 18 (3-OMe); different from this, when the substituent was chlorine, the order changed to 14 (2-Cl) > 13 (3-Cl) > 8 (4-Cl). The anti-TMV activities of 14 (inhibition rate for inactivation, curative, and protection activities in vivo: 54 ± 3, 50 ± 3, 45 ± 2% at 500 mg/) and 19 * When the inactivation effect of a compound was less than 40%, its protection and curative effects were not determined.
For acylhydrazone derivatives, 4-25, the types, position, and number of substituents on the benzene ring had an important influence on the anti-TMV activity. The introduction of strong electron-withdrawing groups on the benzene ring, such as nitro (5,17), and trifluoromethyl (10), was detrimental to the activity. For the substituents at the para position of the benzene ring, electron-donating groups (6,9) and weak electron-withdrawing group (8) were favorable for maintaining the activity. The position of the substituents on the benzene ring had a significant effect on the activity and showed a significant ortho-position effect; that is, the activities of the ortho-substituted derivatives were significantly better than that of the derivatives substituted at other positions (14 versus 8, 9, and 19 versus 9, 18). For example, when the benzene ring has a methoxy substituted on the benzene ring, the order of bioactivity levels is 19 (2-OMe) > 9 (4-OMe) > 18 (3-OMe); different from this, when the substituent was chlorine, the order changed to 14 (2-Cl) > 13 (3-Cl) > 8 (4-Cl). The anti-TMV activities of 14 (inhibition rate for inactivation, curative, and protection activities in vivo: 54 ± 3, 50 ± 3, 45 ± 2% at 500 mg/) and 19 * When the inactivation effect of a compound was less than 40%, its protection and curative effects were not determined.
For acylhydrazone derivatives, 4-25, the types, position, and number of substituents on the benzene ring had an important influence on the anti-TMV activity. The introduction of strong electron-withdrawing groups on the benzene ring, such as nitro (5,17), and trifluoromethyl (10), was detrimental to the activity. For the substituents at the para position of the benzene ring, electron-donating groups (6,9) and weak electron-withdrawing group (8) were favorable for maintaining the activity. The position of the substituents on the benzene ring had a significant effect on the activity and showed a significant ortho-position effect; that is, the activities of the ortho-substituted derivatives were significantly better than that of the derivatives substituted at other positions (14 versus 8, 9, and 19 versus 9, 18). For example, when the benzene ring has a methoxy substituted on the benzene ring, the order of bioactivity levels is 19 (2-OMe) > 9 (4-OMe) > 18 (3-OMe); different from this, when the substituent was chlorine, the order changed to 14 (2-Cl) > 13 (3-Cl) > 8 (4-Cl). The anti-TMV activities of 14 (inhibition rate for inactivation, curative, and protection activities in vivo: 54 ± 3, 50 ± 3, 45 ± 2% at 500 mg/) and 19 * When the inactivation effect of a compound was less than 40%, its protection and curative effects were not determined.
For acylhydrazone derivatives, 4-25, the types, position, and number of substituents on the benzene ring had an important influence on the anti-TMV activity. The introduction of strong electron-withdrawing groups on the benzene ring, such as nitro (5,17), and trifluoromethyl (10), was detrimental to the activity. For the substituents at the para position of the benzene ring, electron-donating groups (6,9) and weak electron-withdrawing group (8) were favorable for maintaining the activity. The position of the substituents on the benzene ring had a significant effect on the activity and showed a significant ortho-position effect; that is, the activities of the ortho-substituted derivatives were significantly better than that of the derivatives substituted at other positions (14 versus 8, 9, and 19 versus 9, 18). For example, when the benzene ring has a methoxy substituted on the benzene ring, the order of bioactivity levels is 19 (2-OMe) > 9 (4-OMe) > 18 (3-OMe); different from this, when the substituent was chlorine, the order changed to 14 (2-Cl) > 13 (3-Cl) > 8 (4-Cl). The anti-TMV activities of 14 (inhibition rate for inactivation, curative, and protection activities in vivo: 54 ± 3, 50 ± 3, 45 ± 2% at 500 mg/) and 19 * When the inactivation effect of a compound was less than 40%, its protection and curative effects were not determined.
For acylhydrazone derivatives, 4-25, the types, position, and number of substituents on the benzene ring had an important influence on the anti-TMV activity. The introduction of strong electron-withdrawing groups on the benzene ring, such as nitro (5,17), and trifluoromethyl (10), was detrimental to the activity. For the substituents at the para position of the benzene ring, electron-donating groups (6,9) and weak electron-withdrawing group (8) were favorable for maintaining the activity. The position of the substituents on the benzene ring had a significant effect on the activity and showed a significant ortho-position effect; that is, the activities of the ortho-substituted derivatives were significantly better than that of the derivatives substituted at other positions (14 versus 8, 9, and 19 versus 9, 18). For example, when the benzene ring has a methoxy substituted on the benzene ring, the order of bioactivity levels is 19 (2-OMe) > 9 (4-OMe) > 18 (3-OMe); different from this, when the substituent was chlorine, the order changed to 14 (2-Cl) > 13 (3-Cl) > 8 (4-Cl). The anti-TMV activities of 14 (inhibition rate for inactivation, curative, and protection activities in vivo: 54 ± 3, 50 ± 3, 45 ± 2% at 500 mg/) and 19 * When the inactivation effect of a compound was less than 40%, its protection and curative effects were not determined.
For acylhydrazone derivatives, 4-25, the types, position, and number of substituents on the benzene ring had an important influence on the anti-TMV activity. The introduction of strong electron-withdrawing groups on the benzene ring, such as nitro (5,17), and trifluoromethyl (10), was detrimental to the activity. For the substituents at the para position of the benzene ring, electron-donating groups (6,9) and weak electron-withdrawing group (8) were favorable for maintaining the activity. The position of the substituents on the benzene ring had a significant effect on the activity and showed a significant ortho-position effect; that is, the activities of the ortho-substituted derivatives were significantly better than that of the derivatives substituted at other positions (14 versus 8, 9, and 19 versus 9,  18). For example, when the benzene ring has a methoxy substituted on the benzene ring, the order of bioactivity levels is 19 (2-OMe) > 9 (4-OMe) > 18 (3-OMe); different from this, when the substituent was chlorine, the order changed to 14 (2-Cl) > 13 (3-Cl) > 8 (4-Cl). The anti-TMV activities of 14 (inhibition rate for inactivation, curative, and protection activities in vivo: 54 ± 3, 50 ± 3, 45 ± 2% at 500 mg/) and 19 (53 ± 2, 48 ± 4, 45 ± 2% at 500 mg/L) were better than that of ribavirin (39 ± 1, 37 ± 1, 39 ± 1 at 500 mg/L) and comparable to that of ningnanmycin (58 ± 1, 55 ± 1, 57 ± 1% at 500 mg/L). These two compounds could be further developed as antiviral drug candidates.
To investigate the role of R in bioactivity, we designed and synthesized compound 25, which has a methyl at the imine moiety. To our delight, it showed lower antiviral activities (43 ± 3, 38 ± 2, 40 ± 4%, 500 mg/L) than compound 4 (R = H, 51 ± 1, 46 ± 2, 48 ± 3% at 500 µg/mL). The above experimental results prove the rationality of our choice of aldimine. When the benzene ring was changed to alkyl groups (31 and 32), the activity decreased obviously.

Larvicidal Activities
We then studied the larvicidal activities of the synthesized derivatives, and different orders of pests were selected for the research, such as T. cinnabarinus, P. xylostella (lepidoptera), and C. pipiens pallens (diptera) ( Table 4). In general, some derivatives showed larvicidal activities against these pests, and at the same time, these derivatives showed obvious selectivity. The derivatives containing the structure of benzyl imines 18 (3-OMe) and 21 (1,3-dioxol) showed good larvicidal activity against T. cinnabarinus. Hydrazide derivative 3 showed no activity against T. cinnabarinus. For the lepidopteran pest P. xylostella, the overall activity was better than that against T. cinnabarinus, and most of the derivatives showed larvicidal activities. Likewise, hydrazide derivative 3 did not exhibit larvicidal activity against P. xylostella. Derivatives containing the structure of benzyl imines 4 (no substituent), 23 (4-bromo-2,6-difluoro), and heteroarylmethyl imines 29 (imidazolyl) showed >50% larvicidal activities against P. xylostella at 200 mg/L. Different from the activity rules of the former two pests, hydrazide derivative 3 has larvicidal activity against C. pipiens pallens, and its activity against C. pipiens pallens larvae was 50 ± 0% at the concentration of 2 mg/L. Derivatives containing the structure of benzyl imines 9 (4-OMe), 21 (1,3-dioxol), 23 (4-bromo-2,6-difluoro), and heteroarylmethyl imines 28 (furyl) showed >60% larvicidal activities at 5 mg/L.  To further study the larvicidal activities of these derivatives against other lepidopteran pests, the larvicidal activities against M. separate, H. armigera, and P. nubilalis were also studied (Table 5). In general, most derivatives showed larvicidal activities against these three lepidopteran pests. The structure-activity relationship was different from that of larvicidal activities against P. xylostella, where derivative 3 showed no larvicidal activity, but this derivative exhibited larvicidal activity against these three lepidopteran pests. Derivatives containing the structure of benzyl imines 12 (4-Ph), heteroarylmethyl imines 28 (furyl), alkyl imines 31 (t-Bu), and 32 (cyclohexyl) showed >60% larvicidal activities at 600 mg/L, the larvicidal activities of derivatives 31 and 32 against these three pests were 100% at 600 mg/L. This means that a good fat-soluble alkyl substituent was beneficial to larvicidal activities. Derivatives 31 and 32 can be used as insecticidal leads for further study.

Materials
The hydrazinolysis reaction was carried out in a microwave synthesis system (100 • C, 100 W, Discover S-Class, CEM). 1 H, 13 C nuclear magnetic resonance (NMR) spectra were obtained at 400 MHz using a Bruker AC-P 400. Chemical shift values (δ) were given in parts per million (ppm) and were downfield from internal tetramethylsilane. High-resolution mass spectra (HRMS) data were obtained on an FTICR-MS instrument (Ionspec 7.0 T). The melting points were determined on an X-4 binocular microscope melting point apparatus and were uncorrected. Reaction progress was monitored by thin-layer chromatography on silica gel GF-254 with detection by UV.

General Synthesis
The synthetic routes of target compounds 3-32 are depicted in Scheme 1. The spectra of target compounds 3-32 are depicted in the Supplementary Materials.