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 in reflux 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 °C. The mixture was then stirred at 0 °C for 1 h.
Then, 5a (300 mg, 1 mmol) in 1 mL of DMF was added at 0 °C, 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 °C 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 °C 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 °C 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 °C. 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