Synthesis and Phytotoxic Evaluation of 3-Indolylglycine Derivatives as New Natural-Like Herbicides

A novel class of potential herbicides, 3-indolylglycines, was synthesized using an effective, green and rapid approach via multicomponent reaction (aza-Friedel-Crafts) between indoles, aldehydes and anilines in a water/sodium dodecyl sulfate system. The main advantages of this methodology are ease of handling, scalable to gram scale and a metal-free approach. The reaction is compatible with a variety of functional groups, obtaining the desired products in moderate to excellent yields. The biological activity of the 3-indolylglycines was evaluated, and it was found that these compounds interfere with the germination and radicle growth of Lactuca sativa, mainly the compound 4k.


Introduction
The advance of Chemistry in the discovery of new herbicides with weed control and crop selectivity is a great challenge to be explored.It is known that the presence of weeds has a great impact on crop productivity, as they compete for the same resources: soil, water, and nutrients.They can also harbor insect and disease pests. 1 The use of synthetic pesticides is the most effective way to control weeds.However, the concern with the environment, the high toxicity that pesticides bring to mammals, and the resistance acquired by weeds are some of the concerns faced by science.Therefore, the search for new pesticides that are more selective to weeds and less toxic to the environment and mammals continues to be a challenge for chemical companies. 2 strategy that has been used in a promising way is based on the synthesis of molecules containing the core of natural products as a role model for the preparation of "natural-like" herbicides.In this sense, indolylglycines derivatives are presented as a new class of "bioinspired" potential herbicides since they are analogues of 3-indolylacetic acid, a plant growth regulator of the auxins family. 3,4][11][12][13][14][15][16] Recently, many advances in the synthesis of indolylglycine derivatives have been developed. 17iang et al. 7 described a protocol that uses glyoxalate imines without the use of any acid catalyst.However, this methodology required an extended reaction time (days) to obtain the desired products and the prior preparation of the imine precursor (Scheme 1a). 6n 2015, Wu and co-workers 18 demonstrated the functionalization of amino acid derivatives by C-H bond activation using Ru and Co as photosensitizer and catalyst, where a variety of indolylglycine derivatives can be converted into the desired products (Scheme 1b).Feng and co-workers 19 reported a mild and general C-H activation using α-amino carbonyls with indoles catalyzed by Fe(ClO 4 ) 3 with TBHP (tert-butyl hydroperoxide) as the oxidant, obtaining the indolylglycines in good yields (Scheme 1c).However, these methodologies require the prior synthesis of the α-amino carbonyls precursors, the use of an oxidant agent and a metal as catalyst.
In this context, the synthesis of indolylglicines from three-component Friedel-Crafts reaction, using primary amines as substrate, is rare in the literature. 20,21For example, the catalyst-and solvent-free approach described by Zhao et al. 21Despite the success of this reaction, this protocol has their limitation regarding the scope of the reaction, since it is applied to a small variety of amines as starting materials.
Thus, in this work, we report the synthesis of indolylglicines by an effective, green, and rapid approach based on the three-component (aldehyde, primary amine and indole) Friedel-Crafts reaction (Scheme 1d).Furthermore, the phytotoxic activity of these compounds was assayed in vitro on germination and root growth of Lactuca sativa and was compared to commercial herbicides (glyphosate and atrazine), showing significant and very promising potential herbicidal activity.

General experimental procedures
All reagents used in the synthesis were purchased from commercial suppliers (Sigma-Aldrich, Steinheim, Germany) and used without further purification.Solvents used in liquid-liquid extraction and as eluents for chromatographic purification, hexane and ethyl acetate, were purchased from commercial suppliers (Quimidrol, Joinvile, Brazil) and were distilled before use.The reactions were monitored by thin-layer chromatography (TLC) using Merck Silica Gel GF254, 0.25 mm thickness and visualized with UV light, iodine tub or stained with vanillin.Flash column chromatography was performed using Merck Silica Gel 60 (230-400 mesh).All compounds were characterized by 1 H nuclear magnetic resonance (NMR) and 13 C NMR; the spectra can be found in the Supplementary Information (SI) section.The spectra were obtained using a Bruker DPX 300 ( 1 H at 300 MHz and 13 C at 75 MHz), using CDCl 3 and acetone-d 6 as solvents.All 1 H NMR shifts are reported in d units, parts per million (ppm), and were measured relative to the signal for tetramethylsilane (TMS, 0.00 ppm).Data are reported as follows: chemical shift (d), multiplicity, coupling constant (J) in hertz and integrated intensity.Abbreviations to denote the multiplicity of a particular signal are as follows: s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet).All 13 C NMR shifts are reported Scheme 1. Methodologies for synthesis of indolylglycines derivatives.
in ppm relative to deuterated-chloroform (77.23 ppm) or deuterated-acetone (206.0),unless otherwise stated, and all spectra were obtained with 1 H decoupling. High-resolution mass spectra (HRMS) were recorded on a Shimadzu ESI-TOF (Kyoto, Japan) mass spectrometer.Infrared spectra were recorded on a PerkinElmer Frontier Optica SD-10 (Vienna, Austria), with attenuated total reflection (ATR) module.

Biological assays
The bioassays of the phytotoxic activities of 3-indolylglycine derivatives were evaluated using an adaptation of the methodology described by Cunico et al. 22 The bioassays used Petri dishes (90 mm diameter) with one sheet of Whatman No. 1 filter paper as a substrate.The target plant was Lactuca sativa (cv Grand Rapids) seeds purchased from Isla Pak, RS, Brazil.It is noteworthy that seeds of the species Lactuca sativa were chosen for these bioassays because of their rapid germination rate, high sensitivity and cultivation practically worldwide.
For the germination test, 25 lettuce seeds were placed per dish with 5 mL of solution test (10, 25, 50, 100 and 150 ppm) or a control solution.All solutions of the compound synthetized were prepared dissolving the compound in a mixture of dimethyl sulfoxide (DMSO, 30 μL) and the surfactant Tween 20 (60 μL) and MES (buffered with 10 mM 2-(N-morpholino) ethanesulfonic acid) were adjusted to 6.0-6.5 with NaOH solution.The negative control was aqueous solution containing DMSO but no test compound.
The Petri dishes were covered with parafilm to reduce evaporation and incubated at 25 ± 2 ºC, 75% humidity, with light simulating day and night for 5 days.After this time, the number of germinated seeds was counted (a seed was considered to be germinated when the radicle was at least 0.2 mm long).Treatments were carried out in a completely randomized design with four replications.
The percentage of germination was calculated in relation to the germination of the control, using equation 1.
(1) where S corresponds to the average value of seed germination and C corresponds to the average germination of the control.
To plant growth bioassay, 10 seeds of Lactuca sativa previously sprouted (2 mm radicle) in distilled water, were placed in the Petri dishes with 5 mL of the test solution (10, 25, 50, 100 and 150 ppm).After the exposition period, the root and aerial part lengths of each radicle were measured manually to the nearest millimeter, using a ruler.The test was performed in quadruplicate.The percentage of growth inhibition was calculated in relation radicle length of the control, according to equation 2. ( where AGs corresponds to the average value of the radicle (root and shoot) length and AGc corresponds to the average growth of the control.The test data were subjected to statistical analysis using Origin ® software (version 8.0) 23 and Microsoft Excel ® (version 2016). 24neral procedure for the synthesis of 4a-4o In a 10 mL tube, sodium dodecyl sulfate (SDS) (0.2 mmol), aniline 3 (1.2mmol), aldehyde 2 (1.2 mmol) and 1.0 mL of water were added and stirred at 50 °C.After 5 min, indole 1 (1.0 mmol) and more 1.0 mL of water were added.The reaction was monitored by TLC to determine reaction time and it was indicated in Scheme 2. The organic compounds were then directly extracted with ethyl acetate (3 × 5 mL), dried over MgSO 4 and concentrated under vacuum.The crude product was purified by flash column chromatography on silica gel using an appropriate mixture of hexane and ethyl acetate as the eluent.The identity and purity of the products were confirmed by 1 H NMR, 13 C NMR and melting points, all spectral data were in perfect agreement with those reported in the literature (see SI section).

Results and Discussion
Prompted by our recent work 25 on the synthesis of bis(indolyl)methane derivatives under ytterbium triflate catalysis in relation to expanding our interest in the Friedel-Crafts alkylation reaction as well as in the design of eco-friendly processes, we decided to investigate this methodology to obtain 3-indolylglycine derivatives.In this regard, we initially evaluated the use of Yb(OTf) 3 /SDS in H 2 O to obtain 3-indolylglycine derivatives using indole (1a), ethyl glyoxylate (2a) and p-anisidine (3a) as model substrates.
Thus, when the reaction was performed with 5 mol% Yb(OTf) 3 and 15 mol% SDS at room temperature the product 4a was obtained in 80% yield (Table 1, entry 1).In order to increase the yield, the catalyst and surfactant loading was increased two-fold, obtaining 90% yield (Table 1, entry 2).To investigate the importance of SDS, the reaction was conducted in the absence of SDS, but the yield decreased to 72% (Table 1, entry 3).
Next, we investigated the influence of other ytterbium sources, such as YbCl 3 .6H 2 O and Yb(OAc) 3 .4H 2 O.However, the product was obtained in only moderate yield (Table 1, entries 4 and 5).Moreover, the influence of triflate source in the catalysts was also studied, such as Sc(OTf) 3 , In(OTf) 3 and Cu(OTf) 2 .However, the yields were lower than those obtained with Yb(OTf) 3 (Table 1, entries 6-8).Other catalysts were tested (CuCl and FeCl 3 .6H 2 O), but with no increases in yield (Table 1, entries 9 and 10).
Next, the reaction was carried out in the absence of Yb(OTf) 3 , and the product 4a was obtained but in a lower yield even after 24 h (Table 1, entry 11).Thus, we decided to test the effect of temperature on the reaction, based on the article described by Kumar et al. 26 for the synthesis of amino alkylated indoles, and to our surprise the product was obtained in a high yield (Table 1, entry 12), using lower temperature and shorter reaction time when compared to the methodology described.However, when the amount of SDS was decreased, the yield also decreased.(Table 1, entry 13).
With the best reaction conditions obtained (Table 1, entry 12), we evaluated the generality and scope of the reaction in order to study reactivity and electronic effects, as shown in Scheme 2. Thus, we explored the reaction of different indoles 1a-1h.However, no significant differences in reactivity were observed when indoles with electronwithdrawing or electron-donating groups were employed, obtaining the products 4a-4h in good to excellent yields (58-98%), except when an -OH group was at position 5 of the indole, which resulted in 4g at a lower yield than the other substrates.
In addition, different substituted anilines were tested (3a-3e) and afforded the respective 3-indolylglycines 4i-4k in 77-96% yields.When p-nitroaniline was employed, the reaction did not occur 4l, whereas the nitro group is strongly electron-withdrawing and influences imine formation.We have also investigated the behavior of different aldehydes on the reaction 2a-2d, affording the desired 3-indolylmethanamines 4m and 4n in satisfactory yields.It is worth to note that product 4n can act as part of new therapy to reduce prostate cancer cell growth and enables investigations toward the function of vitamin D receptor-coactivator interactions during gene regulation. 27It is notable that the use of an alkyl aldehyde did not provide the desired product 4o even after a long reaction time.
Moreover, to demonstrate the synthetic utility of this methodology, a scale-up reaction at 5 mmol was performed between indole 1a, ethyl glyoxylate 2a and p-anisidine 3a under optimized conditions (Table 1, entry 12), affording 4a with no decrease in yield (Scheme 3), indicating that this protocol could be used as a robust method for the larger scale synthesis of 3-indolylglycine derivative.
The products 4a, 4b, 4c, 4e, 4g, 4h, 4j, 4k and 4n were evaluated for their ability to inhibit the seed germination and radicle growth of the dicotyledonous species Lactuca sativa.They were evaluated at five different concentrations (10, 25, 50, 100 and 150 ppm) and the results are shown in Figure 1.As positive control, it was used two herbicides, atrazine (Atra) and glyphosate (Gly).In Figure 1, it can be observed that most of the 3-indolylglycine derivatives had a significant influence on the germination of lettuce seeds.It is interesting to note that the phenyl substituent in C2-indole influences biological activity, since the 4h derivative (at all concentrations tested) inhibited about 100% seed germination while 4c did not significantly inhibit even at the highest concentration (3.5 ± 1.5%).Furthermore, we observed the importance of halogen in the amine precursor, once that, in the compounds 4j, 4k, 4n the inhibition was almost 100% in the highest concentration.In order to prove this effect, the precursors 1a, 3a and 3d were tested and it was noted that the halogen substituted amine (3d) at 100 and 150 ppm concentrations, as well as the indole nucleus (1a) at 150 ppm concentration had an inhibitory effect, while p-anisidine did not show a significant result (3a) (Figure 1).
The compounds 4b, 4h and 4k inhibited germination relative to control by almost 100% in the highest concentration, especially compound 4k (Figure 2).It is noteworthy that the compounds 4h and 4k at the lowest concentration showed a significant reduction in germination (73.3 ± 4.64% and 79.1 ± 3.10%, respectively).With these results, to verify which target site of compounds that may influence inhibition, the synthetic precursors 1a, 3a and 3d were tested.These precursors inhibited the germination, but only at the highest concentration.However, the 3a showed no significant inhibition.When we compare the results to compounds 4b, 4h and 4k, especially 4h, with those obtained from glyphosate and atrazine, we can observe a greater inhibitory effect on the germination of lettuce seeds for synthesized compounds, especially the compound 4h that was the most significant in all concentrations tested.This is an important result because of the resistance to weed control generated by excessive rotation of herbicides.Thus, it is of fundamental importance the search for new herbicidal chemical agents that act as specific inhibitors for use in pest control. 28e phytotoxicity of a compound may interfere with other germinal process variables, such as inhibition of root growth.Considering the results obtained in seed germination, for the growth test, only the 6 compounds that presented the best results (4a, 4b, 4h, 4j, 4k and 4n) were used.Thus, we study phytotoxic activity in relation to inhibition of root growth, as shown the Figure 3.
Analyzing the results obtained, we observed that the compounds 4b and 4h had a growth inhibition of approximately 30% in relation to the control, only in the highest concentrations.The compound 4n showed growth inhibition at higher concentrations, with 33.5 ± 1.19%.
The compounds 4a, 4j and 4k inhibited the radicle growth in all concentrations tested.It is worth noting that the best result was observed for 4k (83.8 ± 0.48%) mainly when compared to atrazine (54.9 ± 0.68%) and glyphosate (60.9 ± 0.64%) at 150 ppm.The compounds 4a and 4j showed inhibition of approximately 33% at the lowest concentration 82.7 ± 0.41 and 60.9 ± 0.8%, respectively, for the highest concentration.The compound 4k had the highest inhibition both at the lowest (41.7 ± 0.89%) and highest concentrations (83.8 ± 0.48%).This is easily seen   in Figures 3 and 4. In summary, the compounds 4a, 4j and 4k indicated greater inhibition of shoot length in the lettuce species compared to the control, glyphosate and atrazine herbicides.

Conclusions
The results showed that multicomponent reaction in an SDS/H 2 O system was a versatile strategy to obtain 3-indolylglycine derivates, this methodology showed advantages that include: no use of a metallic catalyst, ease of scale-up to gram scale, atom-economy, short reaction time, water as solvent.The phytotoxicity of 3-indolylglycine derivates was investigated against dicotyledonous specie, Lactuca sativa.The products exhibited predominantly inhibitory activity on germination and plant growth in the target species, in particular compound 4k.Many of them were more phytotoxic than the positive control (atrazine and glyphosate).These results suggested that indolylglycines derivatives, especially 4k could be promising as new natural-like herbicides.

Figure 2 .
Figure 2. Anomalous germination of Lactuca sativa rootlets after 5 days of exposure to 150 ppm solution of compound 4k (right) in comparison with the control (left).