Chinese Chemical Letters  2018, Vol. 29 Issue (6): 977-980   PDF    
Synthesis and nematicidal activity of piperazinedione derivatives based on the natural product Barettin
Haiyang Sun, Hui Li, Jiayi Wang, Gonghua Song    
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
Abstract: Nematodes are serious constraints of crop production worldwide. However, the traditional nematicides suffer from the side-effects, including environmental and human toxicity. Herein, more than 70 novel piperazinedione derivatives based on the natural product Barettin were synthesized and evaluated against the root-knot nematode Meloidogyne incognita (M. incognita). While most of synthesized compounds exhibited certain nematicidal activity at high concentration, the best one showed a nematicidal activity of 75% at 2.4 μmol/L.
Key words: 5-HT     Barettin     Nature product     Nematicidal activity     Piperazinedione derivatives    

Phytonematodes are one kind of the major crop pests which can cause significant loss in crop yields worldwide [1]. The most common host plants of phytonematodes include corn, cotton, soybean, peanut, wheat, rice, sugarcane, sorghum, tobacco, numerous vegetable crops, fruit and nut crops, and golf greens [2]. While chemical nematicides such as methyl bromide, carbamates and organophosphates have largely eliminated the damages of nematodes on agriculture, they also exerted detrimental effects on both environment and human health such as groundwater pollution and human poisoning [3-5]. The repeat applications of traditional nematecides have also resulted in serious resistance [6, 7]. Hence, the development of potent nematicides with novel mechanism of action, low off-target toxicity and high nematicidal efficiency is extremely urgent.

Natural products can offer powerful leads for the development of valuable pesticide candidates [8-10]. For example, insecticide Cartap was successfully developed based on nereistoxin [11], Resorcinols, the products from Lithraea molleoides have shown strong activity against nematodes [12], and the essential oils of Lantana camara also revealed certain nematicidal activity [13].

More recently, marine natural products Barettin and 8, 9-dihydrobarettin (Fig. 1) produced by the cold water marine sponge Geodia barretti were shown to be able to bind specifically to a mammalian serotonin (5-hydroxytryptamine, 5-HT) receptor (5- HT2) [14]. 5-HT is a significant monoamine neurotransmitter which plays vital roles in mammalian endocrine function, as well as in the central and peripheral nervous system [15, 16]. 5-HT was also found to be an important regulator of various physiological activities in nematodes including feeding, movement and reproduction [17-20].

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Fig. 1. Structures of barettin, 8, 9-dihydrobarettin and target compounds

On the other hand, some mammalian 5-HT receptor ligands have shown definite nematicidal activity. For example, the selective 5-HT3 receptor antagonist MDL 72222 can cause dosedependent mortality to C. elegans [21]. With our continuous effort in searching for novel pesticides potentially acting on the 5-HT receptors of pests [22-24], a series of novel 2, 5-diketopiperazine derivatives (Fig. 1) which were analogous in structure to Barettin and 8, 9-dihydrobarettin were synthesized and screened against the root-knot nematode M. incognita herein.

The synthesis route for title compounds is shown in Scheme 1. Intermediates 5a, 5b, and 5c were prepared according to the literature [25].

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Scheme 1. General synthetic procedure for target compounds

The (S)-2, 5-diaminopentanoic acid hydrochloride (1a) (337 mg, 2 mmol), di(tert-butyl) carbonate ((Boc)2O) (1310 mg, 6 mmol), and triethylamine (Et3N) (404 mg, 4 mmol) were added in 20 mL of acetone and 10 mL of water. The mixture was stirred at room temperature for 12 h, and the solvent and triethylamine were distilled using a rotary evaporator. The residue was extracted with dichloromethane (20 mL × 3). The resulting organic phase was concentrated to afford (S)-2, 5-bis((tert-butoxycarbonyl)amino) pentanoic acid (2a) as a colorless liquid in 96% yield.

The 2a (664 mg, 2 mmol), (S)-methyl 2-amino-3-(1H-indol-3-yl)propanoate hydrochloride (610 mg, 2.4 mmol), Et3N (505 mg, 5 mmol), N-hydroxybenzotriazole (HOBt) (324 mg, 2.4 mmol) and 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDCI) (460 mg, 2.4 mmol) were combined in 30 mL of dichloromethane, and the mixture was cooled to 0 ℃ with stirring for 2 h and then stirred at room temperature overnight. The reaction liquid was washed with water (20 mL × 3). The organic phase was dried over anhydrous magnesium sulfate, and the solvent was evaporated to afford a brown liquid that was purified by silica gel flash column chromatography to afford (S)-methyl 2-((S)-2, 5-bis((tert-butoxycarbonyl)amino)pentanamido)-3-(1H-indol-3-yl)propanoate (3a) as a pale yellow liquid in 77% yield.

The 3a (1064 mg, 2 mmol) and trifluoroacetic acid (TFA) (1140 mg, 10 mmol) were added to 20 mL of dichloromethane and stirred at room temperature overnight. The solvent and TFA were distilled using a rotary evaporator. The residue was dissolved in 20 mL of dichloromethane and then washed with water (10 mL × 3). The organic phase was dried over anhydrous magnesium sulfate and concentrated to afford (S)-methyl 2-((S)-2, 5-diaminopentanamido)-3-(1H-indol-3-yl)propanoate (4a) as a brown liquid in 93% yield.

The 4a (664 mg, 2 mmol) was dissolved in 10 mL of n-butyl alcohol and added dropwise to 10 mL of n-butyl alcohol under reflux. The mixture was then maintained refluxing for 6 h. The n-butyl alcohol was removed by rotary evaporation, and the residue was washed with dichloromethane and methanol (95:5, v/v). The precipitate was filtered and dried to afford (3S, 6S)-3-((1H-indol-3-yl)methyl)-6-(3-aminopropyl)piperazine-2, 5-dione (5a) as a pale powder in 79% yield. Intermediates 5b and 5c were prepared similarly to 5a.

Aniline (140 mg, 1.5 mmol) and Et3N (202 mg, 2 mmol) were dissolved in 5 mL of dichloromethane and slowly added dropwise to a solution of triphosgene (BTC) (223 mg, 0.75 mmol) in 10 mL of dichloromethane at 0 ℃. The mixture was then stirred at 0 ℃ for 1 h. Then, 5a (300 mg, 1 mmol) in 1 mL of DMF was added at 0 ℃, and the mixture was then stirred at room temperature for 30 min, and the solvent was distilled with a rotary evaporator. The residue was washed with water (10 mL × 3) and purified via silica gel flash column chromatography to afford 1-(3-((2S, 5S)-5-((1H-indol-3-yl) methyl)-3, 6-dioxopiperazin-2-yl)propyl)-3-phenylurea (6a) as a pale powder in 74% yield. Compounds 6b-v, 7a-k and 8a-k were prepared similar to 6a.

CS2 (152 mg, 1.5 mmol) was dissolved in 5 mL of THF and then dropped into a solution of aniline (140 mg, 1.5 mmol) and 1, 4- diazabicyclo[2.2.2]octane (DABCO) (336 mg, 3 mmol) in 5 mL of THF. The mixture was stirred at room temperature for 24 h. The reaction liquid was then filtered, and the residue was dissolved in 5 mL of dichloromethane. The BTC (223 mg, 0.75 mmol) was dissolved in 3 mL of dichloromethane and then dropped into a liquid suspension of the above residue with stirring at room temperature for 4 h. The reaction liquid was filtered, and the filtrate was added to 5a (300 mg, 1 mmol) and Et3N (121 mg, 1.2 mmol) dissolved in 1 mL of DMF. The reaction liquid was stirred at room temperature for 1 h. The solvent was then distilled with a rotary evaporator, and the residue was washed with water and purified via silica gel flash column chromatography to afford 1-(3- ((2S, 5S)-5-((1H-indol-3-yl)methyl)-3, 6-dioxopiperazin-2-yl)propyl)-3-phenylthiourea (9a) as a pale powder in 68% yield. Compounds 9b-i were prepared similar to 9a.

Compound 5a (300 mg, 1 mmol), benzoic acid (146 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol), Et3N (121 mg, 1.2 mmol) and EDCI (230 mg, 1.2 mmol) were added to 5 mL of DMF and 20 mL of dichloromethane, and the mixture was cooled to 0 ℃ under stirring for 1 h and then stirred at room temperature overnight. The reaction liquid was washed by water (10 mL × 3). The organic phase was dried over anhydrous magnesium sulfate and purified with silica gel flash column chromatography to afford N-(3- ((2S, 5S)-5-((1H-indol-3-yl)methyl)-3, 6-dioxopiperazin-2-yl)propyl) benzamide (10a) as pale yellow liquid in 71% yield. Compounds 10b-j were prepared similar to 10a.

Compound 5a (300 mg, 1 mmol), 4-methoxybenzaldehyde (163 mg, 1.2 mmol), and sodium triacetoxyborohydride (STAB) (297 mg, 1.4 mmol) were added to 2 mL of DMF and stirred at room temperature overnight. The reaction liquid was washed by water and purified by silica gel flash column chromatography to afford (3S, 6S)-3-((1H-indol-3-yl)methyl)-6-(3-((4-methoxybenzyl)amino)propyl)piperazine-2, 5-dione (11a) as a yellow powder in 55% yield. Compounds 11b-h were prepared similar to 11a.

In order to reduce the influence of different stereoisomers on bioactivity, the key intermediates (5a-c) were synthesized by using chiral amino acids as the starting materials (Scheme 1). The N-Boc amino acids (2a-c) were easily obtained in high yields by reacting amino acids (1a-c) with Boc anhydride. Subsequently, 2a-c reacted with (S)-methyl 2-amino-3-(1H-indol-3-yl)propanoate hydrochloride to afford the N-Boc dipeptides (3a-c). Following the method given in literature [25], HOBt was used in the preparation of 3a-c and the reaction mixture was kept at 0 ℃ to inhibit the possible racemization of 2a-c. The dipeptides (4a-c) were prepared by deprotection of 3a-c using TFA. Then 4a-c were cyclized by heating to afford the key intermediates 5a-c.

The urea compounds (6a-v, 7a-k and 8a-k) and thiourea compounds (9a-i) were prepared by reaction of intermediate 5a-c with isocyanate and isothiocyanate, respectively. The amide compounds (10a-j) were prepared by the acylation reaction of 5a. The secondary amine compounds (11a-h) were prepared by reductive amination of aromatic aldehyde with 5a.

During melting point studies, decomposition was observed at about 230 ℃ for all target compounds before melting. Finally, the three-dimensional structure of 11b in its salt form with TFA was determined via X-ray crystallography (Fig. S1 in Supporting information). This demonstrated that the chirality introduced by the amino acids did not change during the synthetic process, which was consistent with the result reported in literatures [26, 27].

The characterization data of key intermediates and all target compounds can be found in the Supporting information.

All target compounds were evaluated against the root-knot nematode M. incognita by using the following method. Cucumber seeds were sprouted for 3 days in moist paper towels. Acceptable sprouts should be 3 cm to 4 cm long with several lateral roots just emerging. All compounds were dissolved in DMSO and diluted to required concentration with H2O containing Triton X-100 (0.1 mg/L). The cucumber seedlings were planted in test tubes, and the sample solution was added. The test tubes were inoculated two days after planting by adding 2000 root-knot nematodes M. incognita second-instar larvae to each tube. The treated seedlings were cultivated with a sun exposure of 10 h each day at 20-25 ℃. After 20 days, the number of root knots was counted, graded and scored. Distilled water containing Triton X-100 (0.1 mg/L) and DMSO was used as blank control. The negative control was run with M. incognita and distilled water containing Triton X-100 (0.1 mg/L) and DMSO, and the positive control was run with a solution of Fenamiphos or Avermectin [28]. For each sample, the bioassay was repeated three times with four replicates in each trial. The nematicidal activity is indicated by the average inhibition ratio on root knots of three trials.

Inhibition ratio (%) = (1-Treat score/Negative control score) × 100%

Preliminary nematicidal activity of the target compounds against M. incognita is shown in Table 1. First, the inhibition of compounds 6a-6v was evaluated. Most of the target compounds exhibited good nematicidal activities against M. incognita at a concentration of 25 mg/L (50 × 60 μmol/L). Among these derivatives, 6e, 6g, 6i, 6o, 6p, 6r and 6v exhibited excellent nematicidal activities with the inhibition rates ranging from 90.9% to 100%. It is worth mentioning that the compound bearing an electronwithdrawing group on the phenyl generally exhibited better nematicidal activity than that with an electron-donating group. For example, the compounds with electron-donating groups at the 4-position of the phenyl (e.g., 6b, 6c) shown relatively lower nematicidal activity than their counterparts with electron withdrawing groups at the same position (6d-6i, except for 6h). This result could be further verified by comparing the biological activity of 6l and 6m. The data also suggested that the compounds having a heterocycle (6s-6v) exhibited good nematicidal activity (>85%).

Table 1
Nematicidal activities of target compounds against M. incognita

The compounds with high nematicidal activity (>90%) were then screened again at 10 mg/L (20 × 25 μmol/L) (Table 1, right column). While only 6r still had high nematicidal activity of 89.3%, the nematicidal activity of the other compounds dropped down sharply as compound concentration decreased.

For further optimization, compounds 7a-k (n = 2 in Scheme 1) and 8a-k (n = 1 in Scheme 1) were synthesized by reducing the length of the carbon chain. Unfortunately, the bioactivity was not improved when the carbon chain was shortened (Table 1). For all of those compounds, the inhibition rates against M. incognita were below 90% at 25 mg/L (50 × 60 μmol/L). The inhibition rate of the most active compound (7f) was only 81.8%.

Therefore, we synthetized more analogues such as the thiourea derivatives 9a-i, the amide derivatives 10a-j and the secondary amine derivatives 11a-h, and their bioassay results are summarized in Table 1. The thiourea derivatives 9a-i and the amide derivatives 10a-j displayed normal nematicidal activity. But the structure-activity relationship of the target compounds was unclear either based on the electronic effect or on the position effect of the substituent on biological activity. The secondary amine compounds 11a-h were more promising since all of synthesized secondary amine compounds showed nematicidal activities higher than 80% at 25 mg/L (50 × 60 μmol/L). Compounds 10c, 11b and 11h had high nematicidal activity (>95%) and were studied again at lower concentration of 10 mg/L (20 × 25 μmol/L). The nematicidal activity of 10c and 11h decreased sharply (- 50%), but 11b still had 97.2% activity at 10 mg/L (24 μmol/L).

Finally, we tested 11b at 48, 24, 12 and 2.4 μmol/L (Table 2). Even at 2.4 μmol/L, 11b showed 75% nematicidal activity against M. incognita.

Table 2
Nematicidal activity of compound 11b against M. incognita

In conclution, more than 70 diketopiperazines derived from the marine natural products Barettin and 8, 9-dihydrobarettin were synthesized. Most target compounds showed certain nematicidal activities against root-knot nematode M. incognita. After lead optimization, a potent compound 11b with a nematicidal activity of 75% at 2.4 μmol/L was found. Further studies are still in progress and will be reported in due course.

Acknowledgment

This work was financially supported by the National Natural Science Foundation of China (No. 21572060).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.cclet.2017.10.015.

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