# Synthesis and insecticidal activities of novel solanidine derivatives

Remi Beaulieu, Eric Grand, Imane Stasik, Jacques Attoumbre, Quentin Chesnais, Virginie Gobert, Arnaud Ameline, Philippe Giordanengo, José Kovensky

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## To cite this version:

Remi Beaulieu, Eric Grand, Imane Stasik, Jacques Attoumbre, Quentin Chesnais, et al., Synthesis and insecticidal activities of novel solanidine derivatives. Pest Management Science, 2019, 75 (3), pp.793-800. 10.1002/ps.5180. hal-02617912

HAL Id: hal-02617912

https://hal.inrae.fr/hal-02617912

Submitted on 9 Oct 2023

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# Synthesis and Insecticidal Activities of Novel Solanidine Derivatives

Rémi Beaulieu<sup>a,b</sup>, Eric Grand<sup>a</sup>, Imane Stasik<sup>a</sup>, Jacques Attoumbré<sup>b,c</sup>, Quentin Chesnais<sup>c</sup>, Virginie Gobert<sup>b</sup>, Arnaud Ameline<sup>c</sup>, Philippe Giordanengo<sup>d</sup>, José Kovensky<sup>a*</sup>

<sup>a</sup> Laboratoire de Glycochimie, des Antimicrobiens et des Agroressources (LG2A) CNRS UMR 7378, and Institut de Chimie de Picardie FR 3085, Université de Picardie - Jules Verne, 80039 Amiens, France.

<sup>b</sup> Semences, Innovation, Protection, Recherche et Environnement (SIPRE), rue des Champs Potez, 62217 Achicourt, France.

<sup>c</sup> Ecologie et Dynamique des Systèmes Anthropisés (EDYSAN) CNRS UMR 7058, Université de Picardie Jules Verne, 1 rue des Louvels 80037 Amiens, France.

<sup>d</sup> Université de Picardie Jules Verne, Amiens, France – Institut Sophia Agrobiotech, CNRS 7254 – INRA 1355 – Université de Nice Sophia Antipolis, Sophia Antipolis, France.
1 Abstract

2

3 BACKGROUND: Potato (Solanum tuberosum) is the fourth culture in the world and is
4 widely used in the agri-food industries. They generate by-products where α-chaconine and α-
5 solanine, the two major solanidine based glycoalkaloids of potato, are present. As secondary
6 metabolites, they play an important role in the protecting system of potato and are involved in
7 plant protection against insects. To add value to these by-products, we described herein new
8 glycoalkaloids that could have phytosanitary properties.

9 RESULTS: Solanidine, as a renewable source, was modified with an azido linker and coupled
10 by Copper catalyzed alkyne azide cycloaddition (CuAAC) to alkynyl derivatives of the
11 monosaccharides found in the natural potato glycoalkakoids: D-glucose, D-galactose and L-
12 rhamnose. The efficacy of our compounds was evaluated on the potato aphid *Macrosiphum
13 euphorbiae*. The synthetic compounds have stronger aphicidal properties against nymphs than
14 unmodified solanidine. They also showed strong aphicidal activities on adults and a negative
15 impact on fecundity.

16 CONCLUSION: Our synthetic neoglycoalkaloids affected *Macrosiphum euphorbiae* survival
17 at the nymphal stage as well as at the adult stage. Furthermore, they induced a decrease of
18 fecundity. Our results show that chemical modifications of by-products may afford new
19 sustainable compounds for crop and plant protection.

20

21 **Keywords**

22 glycoalkaloid, insecticide, solanidine, synthesis, solanum, potato.

23

24 Running Title: Solanidine Derivatives
25

26

# 1 Introduction

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Plants constitute an important source of bioactive compounds, and one of the best alternatives to fossil resources in the development of sustainable chemistry. Potato is the fourth world agricultural production with about 380 million metric tons (FAOSTAT, http://www.fao.org/faostat/en/#data/QC). The consumption per capita increases each year and goes along with the consumption of processed products, to the detriment of fresh potato. In the last decades, the development of industrial potato-based food products led to a large amount of by-products (mainly skin and tuber). Their management is an environmental and economic challenge. Nowadays, these by-products are partially used as farm animal feed, in the production of fuel-grade ethanol and in anaerobic digesters. However, they may be a source of bioactive compounds such as phenolic compounds and glycoalkaloids¹ which may be considered as high added-value residues for industrial purposes.

Glycoalkaloids are secondary metabolites found in solanaceae species. α-Chaconine and α-solanine are the main glycoalkaloids found in potato. They have the same aglycone, solanidine, but they differ in their saccharide moiety, chacotriose and solatriose respectively (Figure S1). The glycoalkaloids content is highly dependent on the varieties of potatoes. They are located all over the plant: tubers, sprouts or aerial parts as reported by Friedman² and analyzed by mass spectrometry.³ Their amounts vary from a few milligrams per kilogram of fresh matter in the inner part of the tuber to several grams per kilogram of fresh matter in the sprouts.

As secondary metabolites, α-chaconine and α-solanine play an important role in the immune system of potato and are involved in plant protection against insects.⁴ The potato leafhopper (*Empoasca fabae*) is sensitive to glycoalkaloids. α-Chaconine and α-solanine at 0.09% (about
1 mM) have a toxic effect with a mortality of 59% and 8%, respectively. When the concentration is increased to 0.27%, mortality was higher than 80% for both compounds.⁵ A toxic effect was also observed against beetle (*Trogoderma granarium*). This pest of stored grains and cereal products is drastically affected after 96 h, by a topical application of α-chaconine (LD₅₀ = 18.1 µg/mg insect) and α-solanine (LD₅₀ = 22.5 µg/mg insect) as shown by Nenaah.⁶ α-Chaconine or α-solanine containing phyto extracts also affect the development and reproduction of *Drosophila melanogaster*.⁷ On the contrary, colorado potato beetle (*Leptinotarsa decemlineata*) survival is not affected by the two glycoalkaloids.⁸

Aphids can be harmful to a lot of cultures because of their phytophagous activity, but also as carriers of pathogens leading to plant diseases. The survival of *Schizaphis graminum*, an aphid enfeoffed to *Poaceae* plants, is affected by either α-chaconine or α-solanine by ingestion. After 24 h on an artificial diet, 48% of aphids died with α-chaconine at 250 µM and 61% with α-solanine at 250 µM.⁹ Similarly, an extract of potato glycoalkaloids at 160 mg.L⁻¹ increased mortality of the green peach aphid, *Myzus persicae*.¹⁰ The authors also demonstrated that glycoalkaloids alter life history traits of this aphid by reducing diet uptake and fecundity. These results have been partially confirmed for another aphid species, the potato aphid *Macrosiphum euphorbiae*.¹¹ α-Chaconine at 250 ppm allowed the reproduction to decrease while α-solanine did not. None of these two glycoalkaloids induced mortality, even at high concentration. Nevertheless, the authors demonstrated a toxic effect of solanidine (the aglycone of α-chaconine and α-solanine) at 250 ppm.

The toxicity of potato glycoalkaloids is mainly due to three mechanisms of action. They are cytotoxic (affecting the cell membranes containing sterols) with a synergistic action,¹² they disturb the ionic flux,¹³ and they inhibit cholinesterases.¹⁴
Considering the literature, it is possible to evaluate more precisely the influence of the saccharide moiety. Inhibition of cholinesterases by solanidine glycoalkaloids is slightly dependent on the saccharide moiety. α-Chaconine and α-solanine inhibited acetyl or butyrylcholinesterase to the same level, few more than β₂-chaconine.¹⁵ The cytotoxicity of solanidine glycoalkaloids is more dependent on the saccharide moiety, as it has been shown on liposome models.¹⁶ α-Chaconine showed significant lytic activity on liposomes while α-solanine or β₂-chaconine did not. As a consequence, the toxicity of the solanidine based glycoalkaloids can be affected by the sugar composition. α-Chaconine and β₁-chaconine were more toxic on frog embryos than α-solanine, β₂-chaconine or γ-chaconine.¹⁷ The same trend was observed for teratogenicity. α-Chaconine and β₁-chaconine were more teratogenic for frog embryos than β₂-chaconine or γ-chaconine.

Synthetically modified solanidine derivatives could be the base of a new sustainable crop and plant protection strategy. Herein, we describe the synthesis of new glycoalkaloids starting from solanidine as a renewable source of hemisynthetic bioactive molecules. We combined solanidine and the monosaccharides found in the natural potato glycoalkaloids (chacotriose and solatriose): D-glucose, D-galactose and L-rhamnose. A spacer arm was incorporated between the aglycon and the saccharide moiety. The efficiency of our compounds was evaluated on the aphid *Macrosiphum euphorbiae*. As this insect is polyphagous, found worldwide and a main pest of many cultivated plants such as potato, tomato, peas, etc., it is a good model for testing new sustainable pesticides. The insecticidal activity of our compounds was compared to that of the solanidine in order to evaluate the effects of structural modifications.

## 2 Materials and Methods

### 2.1 Chemicals and Instruments
Solanidine was extracted from potato as described previously.<sup>18</sup> Skin and sprouts from *S. tuberosum* cv. Pompadour was supplied by the Comité Nord Plants de Pomme de Terre and was used as starting material. All purchased materials were used without further purification.

Dichloromethane was distilled from calcium hydride and tetrahydrofuran over sodium and benzophenone. Analytical thin-layer chromatography (TLC) was carried out using Merck D.C.-Alufolien Kieselgel 60 F<sub>254</sub>. Flash chromatography (FC) was performed on a Reveleris iES System supplied by Grace (USA) using pre-packed silica cartridges and ELSD/UV detection.

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## 2.2 Synthetic procedures

### 2.2.1 Procedure A - 1,3-dipolar cycloaddition (Meldal conditions).
The azido solanidine derivative (1 mmol) and the peracetylated propargyl sugar derivative (1.2 mmol) were dissolved in toluene (25 mL). N,N-Diisopropylethylamine (DIPEA) (1.2 mmol) and CuI (0.2 mmol) were added and the reaction mixture was stirred for 8 h at 110 °C. After filtration through celite, the solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography (1:0 to 1:1 cyclohexane-EtOAc containing 0.1% Et<sub>3</sub>N). The purified product was finally dried in a desiccator overnight.

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### 2.2.2 Procedure B – Deacetylation with sodium methanolate.
Sodium (1 mmol) was added to MeOH (45 mL). The sodium methanolate solution obtained was then added to a solution of the acetylated compound (1 mmol) in MeOH (45 mL). The reaction mixture was stirred at room temperature for 4 h. Acid resin Amberlite® IR120 [H<sup>+</sup>] was added until a pH value of 5-6 was reached. The reaction mixture was stirred at room temperature for 30 min and then filtered to remove the resin. The solvent was removed under reduced pressure.

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**2.2.3 Propargyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (2).** 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose (1, 25.62 mmol, 10.0 g) was dissolved in anhydrous CH₂Cl₂ (200 mL) under argon. Propargyl alcohol (30.72 mmol, 1.82 mL) was added followed by BF₃.Et₂O (102.48 mmol, 12.64 mL). The mixture was stirred for 2 h at room temperature. Then, potassium carbonate (38.43 mmol, 5.31 g) was added and the reaction stirred for 30 min at room temperature. After filtration, the mixture was washed with distilled water (2 x 200 mL). The aqueous layers were combined and extracted with CH₂Cl₂ (2 x 100 mL). The organic layers were combined, dried over MgSO₄, filtered and the solvent was removed under reduced pressure. The crude product was dissolved in a minimum volume of CH₂Cl₂, then cyclohexane was added until the precipitation started. The mixture was stirred for 20 min at room temperature. Compound 2 was obtained after filtration through sintered glass, with 84% yield (8.3 g) as a white solid.

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**2.2.4 Propargyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (4).** 1,2,3,4,6-penta-O-acetyl-β-D-galactopyranose (3, 12.81 mmol, 5.0 g) and silver trifluoroacetate (19.21 mmol, 4.28 g) were dissolved in anhydrous CH₂Cl₂ (150 mL). Propargyl alcohol was added (19.21 mmol, 1.13 mL) followed by SnCl₄ (38.43 mmol, 4.5 mL). The reaction mixture was stirred for 1.5 h at room temperature. CH₂Cl₂ (400 mL) was added and the mixture was washed with, successively, saturated solution of NaHCO₃ (300 mL), distilled water (3 x 300 mL) and brine (300 mL). The organic layer was dried over MgSO₄, filtered and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (1:0 to 1:1 cyclohexane-EtOAc). Compound 4 was obtained in 87% yield (4.3 g) as a white solid.

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**2.2.5 1,2,3,4-tetra-O-acetyl-L-rhamnopyranose (6).** L-rhamnose (5, 109.0 mmol, 20.0 g)

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was dissolved in pyridine (80 mL). Acetic anhydride (80 mL) was then added and the reaction

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mixture was stirred for 15 h at room temperature. After evaporation of the solvent, the residue was

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dissolved in EtOAc (250 mL) and washed with distilled water (3 x 100 mL). The organic layer

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was dried over MgSO₄, filtered, and the solvent was removed under reduced pressure.

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Compound 6 was obtained in 98% yield (35.1 g) as a colorless syrup (α/β: 86/14).

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**2.2.6 Propargyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranoside (7).** Compound 6 (11.14 mmol,

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3.8 g) was dissolved in anhydrous CH₂Cl₂ (50 mL) under argon. Propargylic alcohol

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(13.72 mmol, 0.81 mL) was added followed by BF₃.Et₂O (45.74 mmol, 5.65 mL). The

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mixture was stirred for 2 h at room temperature. Then, potassium carbonate (17.15 mmol,

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2.37 g) was added and the reaction stirred for 30 min at room temperature. After filtration,

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the solution was diluted with CH₂Cl₂ (100 mL) and washed with distilled water (2 x 100

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mL). The aqueous layers were combined and extracted with CH₂Cl₂ (2 x 50 mL). The

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organic layers were combined, dried over MgSO₄, filtered and the solvent was removed

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under reduced pressure. The crude product was purified by flash chromatography (1:0 to 3:2

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cyclohexane-EtOAc). Compound 7 was obtained in 79% yield (2.9 g) as a white solid.

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**2.2.7 3-azidopropan-1-ol (9).** 3-chloropropan-1-ol (8, 0.11 mol, 10.0 g) was dissolved in

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distilled water (45 mL). Sodium azide (0.21 mmol, 13.78 g) was added and the reaction

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mixture was stirred for 15 h at 80°C. After cooling to room temperature, the mixture was

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extracted with diethyl ether (3 x 50 mL). The organic layers were combined and dried over

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MgSO₄. After filtration, the solvent was removed under reduced pressure. Compound 9 was

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obtained in 99% yield (11.0 g) as a colorless liquid.

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2.2.8 **3-azidopropyl p-toluenesulfonate (10).** Compound **9** (0.11 mol, 11.5 g) was dissolved in **CH<sub>2</sub>Cl<sub>2</sub>** (110 mL). 4-Dimethylaminopyridine (DMAP) (23.0 mmol, 2.78 g) and **Et<sub>3</sub>N** (0.17 mol, 23.8 mL) were added and the reaction mixture was placed at 0°C. A solution of *p*-Toluenesulfonyl chloride (0.17 mol, 32.41 g), in **CH<sub>2</sub>Cl<sub>2</sub>** (60 mL) was added and the mixture was stirred at room temperature for 15 h. Then, the mixture was diluted with **CH<sub>2</sub>Cl<sub>2</sub>** (200 mL) and washed with, successively, a saturated solution of **NaHCO<sub>3</sub>** (200 mL), a 10% solution of **HCl** (200 mL) and distilled water (200 mL). The organic layer was dried over **MgSO<sub>4</sub>** and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (1:0 to 3:2 cyclohexane-Et<sub>2</sub>O). Compound **10** was obtained in 76% yield (21.4 g) as a colorless liquid.

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2.2.9 **3-O-(3-azidopropyl)solanidine (12).** Solanidine (11, 0.25 mmol, 100 mg) was dissolved in anhydrous THF (4 mL) and the solution was placed under argon. NaH 95% (1.26 mmol, 33 mg) was added and the mixture stirred for 30 min at room temperature. Compound **10** (1.26 mmol, 0.32 g) was then added and the reaction mixture stirred for 48 h at 60 °C. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was dissolved in chloroform and filtered through celite. After concentration under reduced pressure, the crude product was purified by flash chromatography (1:0 to 9:1 CHCl<sub>3</sub>-MeOH). The product was finally recrystallized in acetonitrile. Compound **12** was obtained in 71% yield (85 mg) as a white solid.

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2.2.10 **3-O-{3-[4-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyloxymethyl)-1,2,3-triazol-1-yl]propyl}solanidine (13).** Compound **13** was prepared from compound **12** (0.34 mmol, 165 mg) and compound **2** (0.72 mmol, 170 mg) according to the procedure A and obtained in 73% yield (217 mg) as a white solid.
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**2.2.11** **3-O-{3-[4-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxymethyl)-1,2,3-triazol-1-yl]propyl}solanidine** (14). Compound 14 was prepared from compound 12 (0.21 mmol, 100 mg) and compound 4 (0.25 mmol, 96 mg) according to the procedure A, and obtained in 72% yield (129 mg) as a white solid.

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**2.2.12** **3-O-{3-[4-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyloxymethyl)-1,2,3-triazol-1-yl]propyl}solanidine** (15). Compound 15 was obtained from compound 12 (0.66 mmol, 320 mg) and compound 7 (0.80 mmol, 262 mg) according to the procedure A and obtained in 71% yield (379 mg) as a white solid.

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**2.2.13** **3-O-{3-[4-(β-D-glucopyranosyloxymethyl)-1,2,3-triazol-1-yl]propyl}solanidine** (16). Compound 16 was prepared from compound 13 (0.14 mmol, 125 mg) according to the procedure C and purified by flash chromatography (1:0 to 4:1 CHCl₃-MeOH containing 0.1% Et₃N). Compound 16 was obtained in 68% yield (68 mg) as a white solid.

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**2.2.14** **3-O-{3-[4-(β-D-galactopyranosyloxymethyl)-1,2,3-triazol-1-yl]propyl}solanidine** (17). Compound 17 was synthesized from compound 14 (0.15 mmol, 129 mg) according to the procedure C purified by flash chromatography (1:0 to 4:1 CHCl₃-MeOH containing 0.1% Et₃N). Compound 17 was obtained in 72% yield (76 mg) as a white solid.

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**2.2.15** **3-O-{3-[4-(α-L-rhamnopyranosyloxymethyl)-1,2,3-triazol-1-yl]propyl}solanidine** (18). Compound 18 was prepared from compound 15 (0.53 mmol, 430 mg) according to the procedure C and purified by flash chromatography (1:0 to 9:2 CHCl₃-MeOH containing 0.1% Et₃N). Compound 18 was obtained in 77% yield (279 mg) as a white solid.

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## 2.3 Insects and feeding assays

Macrosiphum euphorbiae was mass-reared on potato plants (Solanum tuberosum cv Désirée) in environmental chambers maintained at 20 ± 1° C, 60 ± 5% relative humidity, and a photoperiod of 16:8 h (L:D) to induce parthenogenesis. The colony was initiated from a single virginiparous female supplied by INRA/INSA Villeurbanne (France) in 2004 and initially collected in 1995 from an eggplant field in the Rhône Alpes region (southern France). A standard artificial diet, diet pouches and feeding chambers were prepared as described on the literature.<sup>19,20</sup> It used as a carrier for natural glycoalkaloids (α-chaconine, α-solanine, solanidine) and synthesis derivatives (<b>16, 17, 18</b>) dilution and as a control. The concentrations of the above compounds incorporated into the artificial diet were 0 (control), 2, 20 and 200 µM. For nymphal survival, pools of synchronized first instar nymphs (less than 24 h old) were obtained from parthenogenetic females placed on pouches of control diet. Groups of five first instar nymphs were then transferred on pouches of each diet with a sample of 50 nymphs per conditions. Survival was recorded every 2 days for 10 days (until nymphs become adults). For adult experiments, the development of synchronized first instar nymphs until adult state was done in control diet for 10 days. Then, groups of five young adults were transferred on pouches of each diet with a sample of 40 nymphs per conditions. Survival and fecundity was recorded every 2 days for 16 days. Pouches of artificial diet were changed every 2 days to avoid bacterial or fungal contamination.<sup>21,22</sup>

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## 2.4 Statistical analyses

Mean values are given with their standard error of the mean (SEM). The effect of compounds and different concentrations on aphid survival (after 10 for nymphs and 16 days for adults) that were not normally distributed was analyzed using a Kruskal-Wallis one-way analysis of
247 variance (H), followed by multiple comparison tests using the R package "nparcomp" (type: 248 Dunnet for comparison to control). Aphid survival was analyzed using a Cox-proportional 249 hazards model. The effect of compounds and different concentrations on aphid fecundity that 250 was not normally distributed was analyzed using a Kruskal-Wallis one-way analysis of 251 variance (H), followed by multiple comparison tests using the R package "PMCMR" 252 (pairwise comparison Dunn test). All statistical analyses were carried out using the statistical 253 program "R" (R 3.2.2—R Development Core Team, 2015). 254

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## 3 Results and Discussion

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256

### 3.1 Synthetic route of new glycoalkaloids

257 The hemisynthesis of our new glycoalkaloids was designed with a convergent strategy. 258 Solanidine (isolated by extraction of potato sprouts) was first functionalized with an azido- 259 propyl chain by S<sub>N</sub>2 substitution on the 3-OH position. Nucleophilic substitution has been 260 previously used to prepare 3-O-steroid derivatives.<sup>23,24</sup> Among the synthetic options, a leaving 261 group have been placed on the linker, the 3-OH acting as the nucleophile, was chosen in order 262 to prevent inversion/epimerization of the chiral carbon 3 atom. 263

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263 Then, the azido-solanidine derivative was linked to a propargyl glycoside by copper(I)- 264 catalyzed alkyne-azide cycloaddition (CuAAC) affording a triazole type spacer arm. CuAAC 265 has several advantages such as regioselectivity and the lack of secondary reactions. The 1,2,3- 266 triazole is stable in biological conditions and, as far as we know, is biocompatible. The use of 267 a triazole as a spacer between an aglycon (diosgenin) and chacotriose has been already 268 described to study the effect on the cytotoxic activity.<sup>25</sup> This modification was conducted by 269 click reaction of an 1-azidochacotriosyl with a propargyl-diosgenin or by the reaction of 270 propargyl chacotrioside with an azido/azidoalkyl diosgenin derivative. 271

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The synthesis had to be simple and efficient. Consequently we chose, as saccharide moiety, the three different monosaccharide units present in the natural potato glycoalkaloids (β-D-glucose, β-D-galactose and α-L-rhamnose). By addition of only one monosaccharide, we could evaluate their individual impact on the activity.

For cytotoxicity activity, the mechanism of lytic action is based on the interaction of steroids alkaloids with sterol containing in membranes.²⁶ This interaction is followed by the formation of intermolecular hydrogen bonds between saccharide moieties which induces the destruction of membranes. In our strategy, we wanted to balance the lack of some saccharide units by adding a flexible spacer arm which could enhance intermolecular hydrogen bond formation.

The presence of the triazole ring, resulting from the click reaction, could result in additional H-bond or CH/π interactions, and the flexible propyl spacer arm could facilitate the interactions between solanidine and the binding site on cholinesterases.

## 3.2 Synthesis

First, alkynyl glycosides were synthesized. The reaction of 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose 1 with propargyl alcohol, in the presence of BF₃.Et₂O at room temperature, following a reported procedure,²⁷ afforded the glycoside 2 (84% yield). For the galactoside derivative 4, only Praly's conditions²⁸ with SnCl₄ and CF₃CO₂Ag allowed to obtain β-anomer exclusively, in 87% yield. The propargyl α-L-rhamnoside 7 was obtained in 71% overall yield via the peracetate 6 in the conditions used for the glucose derivative.

On the other hand, to obtain the azido-solanidine derivative by nucleophilic substitution, we had to synthesize an appropriate linker with an azido group and a tosylate as leaving group. 3-chloropropanol was azidated with NaN₃ in water, and the resulting 3-azidopropanol was tosylated²⁹ with 75% overall yield for 10 from 8 (Figure 1).
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### 3.3 Effect of α-solanine, α-chaconine and solanidine on nymphal survival

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The effect of the natural compounds α-solanine, α-chaconine and solanidine on *Macrosiphum euphorbiae* was studied using a standard artificial diet as a carrier containing the compound at three different concentrations: 2, 20 and 200 µM. At 10 days, nymphal survival was significantly affected by treatments (Kruskal-Wallis test, *H* = 41.751, df = 9, *P* < 0.001) (Figure 3a). Compared to the control, nymphal survival was significantly reduced at the 200 µM threshold for α-chaconine (54% survival), α-solanine (64%) and solanidine (52%) (multiple nonparametric comparisons, type: Dunnet; *P* < 0.001, *P* < 0.01 and *P* < 0.001, respectively). At 20 µM, the reduction was significant only for solanidine with 64% of survival (*P* < 0.01).

When comparing the different treatments during all the experiment length (supporting information, figure S2), nymphal survival was significantly affected by the added compound (ANOVA Cox model, $\chi^2$ = 6.923, df = 2, *P* < 0.05) and by the concentration (ANOVA Cox model, $\chi^2$ = 27.955, df = 2, *P* < 0.001) indicating a dose dependent effect.
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321 Solanidine at a concentration of 200 µM has been thus used as a positive control in the
322 evaluation of synthetic compounds.

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324 **3.4 Effect of synthetic compounds 16, 17 and 18 on nymphal survival.**

325 Synthetic derivatives **16-18** were also evaluated at three concentrations: 2, 20 and 200 µM in
326 comparison of solanidine at 200 µM selected from the above experiment. After 10 days,
327 nymphal survival was significantly affected by treatments (Kruskal-Wallis test, *H* = 125.55,
328 df = 9, *P* < 0.001) (Figure 3b). Compared to solanidine at 200 µM (nymphal survival 52%),
329 the glucose-containing molecule **16** displayed higher aphicidal properties at 20 µM and at 200
330 µM, with 25% of nymphal survival for both concentration (multiple nonparametric
331 comparisons, type: Dunnet; *P* < 0.05). The galactosyl derivative **17** at 2 and 20 µM showed
332 similar effects than solanidine at 200 µM (64% and 34% survival respectively). However, at
333 200 µM, a significantly higher aphicidal effect (24% survival) was observed (multiple
334 nonparametric comparisons, type: Dunnet; *P* < 0.05). Finally, compared to solanidine at 200
335 µM, compound **18** did not show any significant difference at 20 µM but showed stronger
336 aphicidal activity at 200 µM with 6% of nymphal survival (multiple nonparametric
337 comparisons, type: Dunnet; *P* < 0.001).

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339 In conclusion, at 20 µM only the glucose-containing molecule **16** exhibited higher activity
340 than solanidine at 200 µM. At equal concentration (200 µM), the three synthetic molecules
341 were significantly more active than solanidine. The rhamnose-containing molecule **18** at 200
342 µM shows the most significant activity on nymphal survival.

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When comparing the different treatments during all the experiment length (supporting
information, figure S3), nymphal survival was significantly affected by the compound
(ANOVA Cox model, $\chi^2 = 24.579$, df = 3, P < 0.001) and by the concentration (ANOVA Cox
model, $\chi^2 = 132.978$, df = 2, P < 0.001) indicating a dose dependent effect. As observed for α-
chaconine, α-solanine and solanidine, a severe reduction in nymphal survival was observed
within the first 4 days of treatment.

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**3.5 Effect of synthetic compounds 16, 17 and 18 on adult survival.**

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Synthetic derivatives **16-18** (at 2, 20 and 200 µM) were evaluated on *Macrosiphum euphorbiae* adults (Figure 4a). Compared to the control, adult survival was significantly affected by treatments (Kruskal-Wallis test, *H* = 130.95, df = 9, *P* < 0.001) after 16 days. The glucose-containing molecule **16** and the galactosyl derivative **17** exhibited significant aphicidal activity at the 200 µM threshold, 46% and 54% adult survival respectively (multiple nonparametric comparisons, type: Dunnet; *P*< 0.001 for both compounds). The rhamnosyl derivative **18** showed strong aphicidal properties at 20 µM (ca. 67% survival, multiple nonparametric comparisons, type: Dunnet; *P*< 0.05) and at 200 µM (ca. 7% survival, multiple nonparametric comparisons, type: Dunnet; *P*< 0.001).

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When comparing the different synthetic glycoalkaloids treatments (Figure 4b), adult survival was significantly affected by the added compound (ANOVA Cox model, $\chi^2 = 12.367$, df = 2, *P* < 0.01) and by the concentration (ANOVA Cox model, $\chi^2 = 101.299$, df = 2, *P* < 0.001) indicating a dose dependent effect. Globally, adult survival was significantly lower for the strongest concentrations. The rhamnose-based compound **18** at 200 µM showed a significant decreased of aphid survival compared to all the other treatments (pairwise comparisons using least-squares means, *P* < 0.05).

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**3.6 Effect of synthetic compounds 16, 17 and 18 on reproduction.**

The presence of synthetic glycoalkaloids in the artificial diet affected reproduction (Kruskal-Wallis test, *H* = 130.95, df = 9, *P* < 0.001) (Figure 5). Overall, reproduction was more affected by the three synthetic compounds at the highest concentrations. At low concentration (2 µM), only the glucosyl derivative **16** decreased significantly the fecundity (pairwise comparison Dunn test, *P* < 0.05), whereas compounds **17** and **18** had no effect (*P* > 0.05). At 20 µM, all three compounds decreased significantly the fecundity to a similar extent, compared to the control. At 200 µM, the three molecules **16**, **17** and **18** had a strong effect. The rhamnosyl derivative **18** showed the strongest activity, significantly higher than **16** and **17** (*P* < 0.05), and with a dose-dependent response.

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**4 Conclusion**

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All these results show that our approach to obtain strong aphicidal activity with modified glycoalkaloids is promising. Starting from solanidine, a simple and efficient synthetic strategy allowed to click different sugars in a few steps. The analogs have been designed to include a triazole-containing spacer between the sugar and the alkaloid moieties. This modification
386 increases the flexibility of the molecule and might facilitate interactions with biological
387 targets. The aphicidal activity of our synthetic neoglycoalkaloids containing only one
388 monosaccharide unit was proven. They affected *Macrosiphum euphorbiae* survival at the
389 nymphal stage as well as at the adult stage. Furthermore, they induced a decrease of
390 fecundity. In addition, our results show the influence of the monosaccharide structure on the
391 activity, as the rhamnosyl derivative **18** is clearly more active than the glucosyl and the
392 galactosyl conjugates **16** and **17** on adult survival and on reproduction. Further studies could
393 shed light on the specific mechanism of these new glycoalkaloid derivatives and to study their
394 effects on other pests. In perspective, it could also be interesting to investigate the feeding
395 behavior of aphids in the presence of our compounds using the electrical penetration graph
396 (EPG) technique.³¹ Anyway, our results show that the effect of natural glycoalkaloids can be
397 amplified through structural modifications performed by chemical synthesis, leading to new
398 sustainable compounds for crop and plant protection.
399

400 **5 Acknowledgments**

401 We thank SIPRE, the Comité Nord Plant de Pommes de Terre, the Conseil Régional de
402 Picardie, the Ministère de l'Enseignement Supérieur et de la Recherche, and the Centre
403 National de la Recherche Scientifique for financial support.

405 **6 Supporting Information**

406 Characterization of compounds **2** to **18**. ¹H and ¹³C NMR spectra of compounds **12-18**.
407 Names and chemical structures of natural hydrolysis products of α-chaconine and α-solanine
408 (Figure S1). Cox graphic representations of the nymph survival of *Macrosiphum euphorbiae*
409 reared on diets (Figures S2 and S3). Tables corresponding to graphics (Tables S1, S2, S3).
410
411 **7 References**

412 ¹ Wu ZG, Xu HY, Ma Q, Cao Y, Ma JN and Ma CM, Isolation, identification and quantification of unsaturated fatty acids, amides, phenolic compounds and glycoalkaloids from potato peel. *Food Chem* **135**:2425-2429 (2012).

415 ² Friedman M, Potato Glycoalkaloids and Metabolites: Roles in the Plant and in the Diet. *J Agr Food Chem* **54**:8655-8681 (2006).

417 ³ Ha M, Kwak JH, Kim Y and Zee OP, Direct analysis for the distribution of toxic glycoalkaloids in potato tuber tissue using matrix-assisted laser desorption/ionization mass spectrometric imaging. *Food Chem* **133**:1155-1162 (2012).

420 ⁴ Chowański S, Adamski Z, Marciniak P, Rosiński G, Büyükgüzel E, Büyükgüzel K, Falabella P, Scrano L, Ventrella E, Lelario F and Bufo SA, A Review of Bioinsecticidal Activity of *Solanaceae* Alkaloids. *Toxins* **8**:60 (2016).

423 ⁵ Sanford L, Domek J, Cantelo W, Kobayashi R and Sinden S, Mortality of potato leafhopper adults on synthetic diets containing seven glycoalkaloids synthesized in the foliage of various *Solanum* species. *Am J Potato Res* **73**:79-88 (1996).

426 ⁶ Nenaah GE, Toxic and antifeedant activities of potato glycoalkaloids against *Trogoderma granarium* (*Coleoptera: Dermestidae*). *J Stored Prod Res* **47**:185-190 (2011).

428 ⁷ Ventrella E, Adamski Z, Chudzińska E, Miądowicz-Kobielska M, Marciniak P, Büyükgüzel E, Büyükgüzel K, Erdem M, Falabella P, Scrano L and Bufo SA, *Solanum tuberosum* and *Lycopersicon esculentum* leaf extracts and single metabolites affect development and reproduction of *Drosophila melanogaster*. *PLoS ONE* **11**:e0155958 (2006).

432 ⁸ Lyytinen A, Lindström L, Mappes J, Julkunen-Tiitto R, Fasulati S and Tiilikkala K, Variability in host plant chemistry: Behavioural responses and life-history parameters of the Colorado potato beetle (*Leptinotarsa decemlineata*). *Chemoecology* **17**:51-56 (2007).
435

⁹ Soule S, Güntner C, Vázque A, Argandoña VH, Ferreira F and Moyna P, Effect of *Solanum* glycosides on the aphid *Schizaphis graminum*. *J Chem Ecol* **25**:369-374 (1999).

436

437

¹⁰ Fragoyiannis DA, McKinlay RG and D'Mello JPF, Studies of the growth, development and reproductive performance of the aphid *Myzus persicae* on artificial diets containing potato glycoalkaloids. *Entomol Exp Appl* **88**:59-66 (1998).

438

439

440

¹¹ Güntner C, González A, Reis RD, González G, Vázquez A, Ferreira F and Moyna P, Effect of *Solanum* glycoalkaloids on potato aphid, *Macrosiphum euphorbiae*. *J Chem Ecol* **23**:1651-1659 (1997).

441

442

443

¹² Yamashoji S and Matsuda T, Synergistic cytotoxicity induced by α-solanine and α-chaconine. *Food Chem* **141**:669-674 (2013).

444

445

¹³ Blankemeyer JT, Stringer BK, Rayburn JR, Bantle JA and Friedman M, Effect of potato glycoalkaloids, α-chaconine and α-solanine on membrane potential of frog embryos. *J Agr Food Chem* **40**:2022-2025 (1992).

446

447

448

¹⁴ McGehee DS, Krasowski MD, Fung DL, Wilson B, Gronert GA and Moss J, Cholinesterase inhibition by potato glycoalkaloids slows mivacurium metabolism. *Anesthesiology* **93**:510-519 (2000).

449

450

451

¹⁵ Bushway R, Savage S and Ferguson B, Inhibition of acetyl cholinesterase by solanaceous glycoalkaloids and alkaloids. *Am J Potato Res* **64**:409-413 (1987).

452

453

¹⁶ Roddick JG and Rijnenberg AL, Synergistic interaction between the potato glycoalkaloids α-solanine and α-chaconine in relation to lysis of phospholipid/sterol liposomes. *Phytochemistry* **26**:1325-1328 (1987).

454

455

456

¹⁷ Rayburn JR, Bantle JA and Friedman M, Role of carbohydrate side chains of potato glycoalkaloids in developmental toxicity. *J Agric Food Chem* **42**:1511-1515 (1994).

457

458

¹⁸ Attoumbré J, Giordanengo P and Baltora-Rosset S, Solanidine isolation from *Solanum* *Tuberosum* by centrifugal partition chromatography. *J Sep Sci* **36**:2379-2385 (2013).

459
19 Febvay G, Delobel B and Rahbé Y, Influence of the amino acid balance on the improvement of an artificial diet for a biotype of *Acyrthosiphon pisum* (Homoptera: Aphididae). *Can J Zool* **66**:2449-2453 (1988).

20 Down RE, Gatehouse AMR, Hamilton WDO and Gatehouse JA, Snowdrop Lectin Inhibits Development and Decreases Fecundity of the Glasshouse Potato Aphid (*Aulacorthum solani*) When Administered *In Vitro* and Via Transgenic Plants Both in Laboratory and Glasshouse Trials. *J Insect Physiol* **42**:1035-1045 (1996).

21 Le Roux V, Saguez J, Vincent C and Giordanengo P, Rapid Method to Screen Resistance of Potato Plants Against *Myzus persicae* (Homoptera: Aphididae) in the Laboratory. *J Econ Entomol* **97**:2079-2082 (2004).

22 Dussouy C, Bultel L, Saguez J, Cherqui A, Khelifa M, Grand E, Giordanengo P and Kovensky J, Strong Aphicidal Activity of GlcNAc(β1→4)Glc Disaccharides: Synthesis, Physiological Effects, and Chitinase Inhibition. *Chem Eur J* **18**:10021-10028 (2012).

23 Zhang X, Yang X and Zhang S, Synthesis of Triazole-Linked Glycoconjugates by Copper(I)-Catalyzed Regiospecific Cycloaddition of Alkynes and Azides. *Synth Commun* **39**:830-844 (2009).

24 Beaulieu R, Gottis S, Meyer C, Grand E, Deveaux V, Kovensky J and Stasik I, Cholesteryl and diosgenyl glycosteroids: Synthesis and characterization of new smectic liquid crystals. *Carbohydr Res* **404**:70-78 (2015).

25 Pérez-Labrada K, Brouard I, Morera C, Estévez F, Bermejo J and Rivera DG, ‘Click’synthesis of triazole-based spirostan saponin analogs. *Tetrahedron* **67**:7713-7727 (2011).

26 Keukens EAJ, De Vrije T, Van den Boom C, De Waard P, Plasman HH, Thiel F, Chupin V, Jongen WMF and De Kruijff B, Molecular basis of glycoalkaloid induced membrane disruption. *Biochim Biophys Acta* **1240**:216-228 (1995).
485

27 Mereyala HB and Gurrala SR, A highly diastereoselective, practical synthesis of allyl, propargyl 2,3,4,6-tetra-O-acetyl-β-D-gluco, β-D-galactopyranosides and allyl, propargyl heptaacetyl-β-D-lactosides. *Carbohydr res* **307**:351-354 (1998).

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487

488

28 Xue JL, Cecioni S, He L, Vidal S and Praly JP, Variations on the SnCl₄ and CF₃CO₂Ag-promoted glycosidation of sugar acetates: a direct, versatile and apparently simple method with either α or β stereocontrol. *Carbohydr res* **344**:1646-1653 (2009).

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490

491

29 Pak JK and Hesse M, Synthesis of Penta-N-Protected Homocaldopentamine and Its Selective Acylation. *J Org Chem* **63**:8200-8204 (1998).

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493

30 Beaulieu R, Attoumbré J, Gobert-Deveaux V, Grand E, Stasik I, Kovensky J and Giordanengo P, Novel solanidine-derived compounds. WO Patent 2015008007A1 (2015).

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31 Mondédji AD, Ketoh GK, Amévoin K, Ameline A, Giordanengo P and Glitho IA, Evaluation of neem leaves-based preparations as insecticidal agents against the green peach aphid, *Myzus persicae* (Sternorrhyncha: Aphididae). Afr J Agric Res **9**:1344-1352 (2014).

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Figure captions

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Fig. 1. Reagents: (a) NaN₃, H₂O, 80 °C, 15 h, 99%; (b) TsCl, Et₃N, DMAP, CH₂Cl₂, 15 h, 0 °C → rt, 76%; (c) NaH, THF, 60 °C, 24 h, 71%.

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Fig. 2. Reagents: (a), **12**, CuI, DIPEA, toluene, 8 h, 110 °C; (b) NaMeO, MeOH, 4 h, rt.

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Fig. 3. (a) Nymphal survival of *Macrosiphum euphorbiae* reared on diets containing 2, 20 or 200 µM of α-chaconine (light gray), α-solanine (gray) and solanidine (black) after 10 days of treatment. (b) Nymphal survival of *Macrosiphum euphorbiae* reared on diets containing 2, 20 or 200 µM of **16** (light gray), **17** (gray) and **18** (black) after 10 days of treatment. Asterisks

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509
510 indicate statistically significant differences between the control (solanidine at 200 µM) and
511 the treatment (multiple nonparametric comparisons, type: Dunnet; *P < 0.05; ** P < 0.01;
512 *** P < 0.001).

513

514 Fig. 4. (a) Adult survival of *Macrosiphum euphorbiae* reared on diets containing 2, 20 or 200
515 µM of compounds **16** (light gray), **17** (gray) and **18** (black) after 16 days of treatment.

516 Asterisks indicate statistically significant differences between the control and the treatment
517 (multiple nonparametric comparisons, type: Dunnet; *P* < 0.05; **P** < 0.01; ***P* < 0.001).

518 (b) Adult survival of *Macrosiphum euphorbiae* reared on diets containing 2 µM (full line), 20
519 µM (dashed line) or 200 µM (dotted line) of compounds **16** (light gray), **17** (gray) and **18**
520 (black).

521

522 Fig. 5. Total fecundity (± SEM) of *Macrosiphum euphorbiae* reared on diets containing 2 µM,
523 20 µM or 200 µM of compounds **16** (light gray), **17** (gray) and **18** (black) and control (white).

524 Letters indicate significant differences between treatments associated with Dunn test.

525

526 **Figure graphics**

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<figure><img src="image_3.png" /></figure>

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<figure><img src="image_4.png" /><figcaption>Fig. 1.</figcaption></figure>

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<caption>Fig. 2.</caption>

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a

<figure><img src="image_5.png" /></figure>

b

<figure><img src="image_6.png" /><figcaption>Fig. 3.</figcaption></figure>

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a

<figure><img src="image_7.png" /></figure>

b

<figure><img src="image_8.png" /><figcaption>Fig. 4.</figcaption></figure>

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<figure><img src="image_9.png" /><figcaption>Fig. 5.</figcaption></figure>

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549 **Graphical abstract**

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<figure><img src="image_10.png" /></figure>

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**Synthesis and Insecticidal Activities of Novel Solanidine Derivatives**

Rémi Beaulieu, Eric Grand, Imane Stasik, Jacques Attoumbré, Quentin Chesnais, Virginie Gobert, Arnaud Ameline, Philippe Giordanengo, José Kovensky*

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This article describes the synthesis of new glycoalkaloids starting from potato solanidine. The synthetic neoglycoalkaloids showed aphicidal activities against *Macrosiphum euphorbiae* nymphs and adults.

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