LOW TOXICITY COMPOUNDS FOR USE AS INSECTICIDES AND METHOD OF PRODUCING SAID COMPOUNDS

Abstract

The present application relates to a compound of Formula (I) and its analogues, which are obtained by chemical synthesis. The disclosed compounds are suitable to be used as insecticides by killing insect cells in a selective fashion, namely by not being toxic to human cells. These compounds are different from existing insecticide solutions given their potency, selectivity and non-toxicity for human cells, and mechanism of action via caspase activation.

Claims

1. A compound of Formula (I) and its analogues for use as insecticide, wherein Formula (I) is: ##STR00008## wherein R and R1 are selected from hydrogen or carboxyl group; when R is hydrogen, R1 is a carboxyl group; and when R is a carboxyl group, R1 is hydrogen; and R2 is an alkyl group substituted or unsubstituted.

2. The compound of Formula (I) and its analogues for use as insecticide according to claim 1, wherein the alkyl group comprises 12 to 20 carbon atoms.

3. The compound of Formula (I) and its analogues for use as insecticide according to claim 2, wherein the alkyl group comprises 12 to 15 carbon atoms.

4. The compound of Formula (I) and its analogues for use as insecticide according to claim 1, wherein the alkyl group is selected from the group consisting of dodecyl, tridecyl and pentadecyl groups, substituted or unsubstituted.

5. The compound Compound of Formula (I) and its analogues for use as insecticide according to claim 1, wherein the compound is a 2-(dodecylamino)benzoic acid of Formula (II): ##STR00009##

6. The compound of Formula (I) and its analogues for use as insecticide according to claim 1, wherein the compound is a 3-(dodecylamino)benzoic acid of Formula (III): ##STR00010##

7. A formulation for use as insecticide, comprising the compound of Formula (I) or its analogues described in claim 1.

8. A method of producing the compound of Formula (I) and its analogues described in claim 1, comprising the following steps: adding a solution of 1-chloroalkene or 1-bromoalkene to a solution of 2-aminobenzoic acid or of 3-aminobenzoic acid to obtain a reaction mixture; heating the reaction mixture at a temperature between 50? C. and 70? C., using an alcohol as solvent, and with a final concentration of 0.05 to 0.2 M of the reaction mixture, for a time period between 24 to 64 hours; evaporating the solvent under reduced pressure.

9. The method according to claim 8, wherein the solution of 1-chloroalkene or of 1-bromoalkene has a concentration between 0.1 to 0.4 M.

10. The method according to claim 8, wherein the solution of 2-aminobenzoic acid or of 3-aminobenzoic acid has a concentration between 0.05 to 0.2 M.

11. The method according to claim 8, wherein the alcohol solvent is selected from methanol or ethanol in a concentration from 80 to 97% (w/w).

12. The method according to claim 8, wherein the evaporation step is performed at a temperature between 20 and 70? C.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0044] For easier understanding of this application, figures are attached in the annex that represent the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein.

[0045] FIG. 1 shows the structure of 2-(heptadec-10-en-1-yl)-6-hydroxybenzoic acid (1), the synthesis pathway to obtain 2-(heptadec-10-en-1-yl)-6-methoxybenzoic acid (2), 2-(pentadecylamino) benzoic acid (4), 2-(tridecylamino) benzoic acid (5), 2-(dodecylamino) benzoic acid (6a) and 3-(dodecylamino) benzoic acid (6b), from 2-aminobenzoic acid (3a) and 3-aminobenzoic acid (3b), in case of compound (6b).

[0046] FIG. 2 shows the viability of insect cells (Sf9 cells) in control conditions (control), with the compound (6a), other related compounds, namely 2-(heptadec-10-en-1-yl) -6-hydroxybenzoic acid (1), 2-(heptadec-10-en-1-yl) -6-methoxybenzoic acid (2), 2-aminobenzoic acid (3a), 3-aminobenzoic acid (3b), 2-(pentadecylamino) benzoic acid (4), 2-(tridecylamino) benzoic acid (5), 3-(dodecylamino) benzoic acid (6b), and in the presence of the commercial insecticide chlorpyrifos (CHPY). Results represent the mean+SEM of at least three independent experiments, each of them performed in triplicate: *p<0.05, **p<0.01, ***p<0.001.

[0047] FIG. 3 shows the viability of human keratinocytes in control conditions (control), in the presence of compound (6a) (no toxicity), and in the presence of compound (1) and the commercial insecticide chlorpyrifos, all at 50 ?g/mL. Results represent the mean?SEM of at least three independent experiments, each of them performed in triplicate: **<0.01, ***p<0.001.

[0048] FIG. 4 shows the caspase-like activity in insect cells in control conditions (basal level, (1), in the presence of compound (6a) (50 ?g/mL, 400% increase), in the presence of compound (1) (50 ?g/mL, no increase) and in the presence of the commercial insecticide chlorpyrifos (50 ?g/mL, no statistically significant changes). Results represent the mean+SEM of at least three independent experiments, each of them performed in triplicate: ***p<0.001.

[0049] FIG. 5 discloses the compound of formula (I).

DESCRIPTION OF EMBODIMENTS

[0050] Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings. However, they are not intended to limit the scope of this application.

[0051] Prior studies showed that natural molecules such as ginkgolic acids (compound (1) in FIG. 1), extracted from Ginkgo biloba leaves, have high toxicity towards insect cells (toxicity towards Sf9 insect cells, causing a loss of ca. 75% of viability at 100 ?g/mL). Derivate and improved compound (2) in FIG. 1 had a low yield and had lower activity in Sf9 insect cells.

[0052] However, compound (1) exists in very low percentages in its natural source, and a low yield of compound (2) was obtained, therefore obtaining these compounds is extremely time-consuming and expensive. Their low availability makes them inviable to be used as an alternative to currently available insecticides.

[0053] The present invention attempts to provide compounds with high toxicity towards insect cells, with high potency, low toxicity to human cells, which can be readily synthesized to be used as insecticides.

[0054] Thus, some derivatives of 2-aminobenzoic and 3-aminobenzoic acids possessing side chains with variable number of atoms, derived from compound of Formula (I) were obtained as shown in FIG. 5. Compound (4), compound (5), and compounds (6a, 6b), in FIG. 1, were obtained as examples.

[0055] The compound of the present application and its analogues for use as insecticide have the Formula (I):

##STR00007##

wherein R and R1 are selected from hydrogen or carboxyl group,
when R is hydrogen, R1 is a carboxyl group; and when R is a carboxyl group, R1 is hydrogen,
and R2 is an alkyl group substituted or unsubstituted.

[0056] In the present application, the term alkyl relates to a linear hydrocarbon group comprising from 12 to 20 carbon atoms, preferably 12 and 15 carbon atoms. The alkyl group can be selected from the dodecyl, tridecyl and pentadecyl groups, substituted or unsubstituted.

[0057] FIG. 1 shows a schematic representation of the synthesis pathway to obtain compound 2-(heptadec-10-en-1-yl) -6-methoxybenzoic acid (2) from 2-(heptadec-10-en-1-yl) -6-hydroxybenzoic acid (1) as comparison to the presently disclosed compounds of Formula (I) and its analogues. This same schematic shows the synthesis pathway of the present application in order to obtain compounds such as compounds (6a) and (6b) related to Formula (I).

[0058] The method of producing the compound of Formula (I) and its analogues comprises the following steps: [0059] Adding a solution of 1-chloroalkane or of 1-bromoalkane to a solution of 2-aminobenzoic acid or of 3-aminobenzoic acid; [0060] Heating the reaction mixture at a temperature between 50? C. and 70? C., using an alcohol as solvent, and with a final concentration of 0.05 to 0.2 M of the reaction mixture, for a time period between 24 to 64 hours; [0061] Evaporating the solvent under reduced pressure.

[0062] The evaporation step can be performed at a temperature between 20 and 70? C. The evaporation step can also be performed at a reduced pressure between 100 and 600 mmHg.

[0063] The evaporation step can be followed by a purification step performed, for example, through chromatography, to obtain a pure compound of Formula (I) and its analogues.

[0064] In one embodiment the solution of 1-chloroalkane or of 1-bromoalkane is used in a concentration from 0.1 to 0.4 M. In one embodiment the 2-aminobenzoic acid or 3-aminobenzoic acid is used in a concentration from 0.05 to 0.2 M.

[0065] In one embodiment methanol or ethanol are used as solvent in a concentration from 80 to 97% (w/w).

[0066] The compound (6a) of the present application is more potent than the commercial insecticide chlorpyrifos (FIG. 2), as assessed by using cultivating Spodoptera frugiperda cells in the same cell density, exposing them to either compound (6a) or chlorpyrifos at the same concentration (100 ?g/mL) and assessing cell viability after 24 hours using resazurin. The loss of cell viability caused by compound (6a) was more than the triple of that of chlorpyrifos.

[0067] The compound (6a) of the present application has no toxicity in human cells, contrarily to the ginkgolic acid (1), or the synthetic commercial insecticide evaluated as benchmark (chlorpyrifos) (FIG. 3), as assessed by cultivating human keratinocytes in the same cell density, exposing them to either compound (6a) or chlorpyrifos at the same concentration (50 ?g/mL) and assessing cell viability after 24 hours using resazurin. Compound (6a) did not cause any loss of cell viability, while chlorpyrifos resulted in ca. 20% of viability loss.

[0068] The compound (6a) of the present application presents a mechanism of action distinct of the commercial insecticide chlorpyrifos, which may be of interest as a strategy to overcome pesticide resistances. Specifically, the compound (6a) is capable of activating insect effector caspase DRICE (involved in the process of cell death), while the commercial insecticide is unable to trigger this effect and has no impact in this target (FIG. 4). This was evaluated by incubating cells with the same cellular density with the proluminescent substrate DEVD-aminoluciferin, which becomes fluorescent after cleavage by effector caspases, being detected subsequently in a luminescence detector.

[0069] Similarly, to compound (6a) specifically, it is expected that all compounds derived from compound of Formula (I), such as compounds (4), (5) and (6b), present similar potency, no toxicity to human cells, and the same mechanism of action in insect cells. Therefore, the compounds derived from Formula (I) are suitable to be used as insecticide.

[0070] In one embodiment, the compound of Formula (I) and its analogues can be incorporated into a formulation for use as insecticide.

EXAMPLES

[0071] Method of producing 2-(dodecylamino) benzoic acid (6a): 1-Bromododecane (0.7 mL, 2.9 mmol) was added to a solution of 2-aminobenzoic acid (3a) (0.2 g; 1.5 mmol). The reaction mixture was refluxed in ethanol (14 mL) for 64 hours and monitored by TLC (DCM/MeOH 9:1). The solvent was evaporated, and the residue obtained was subjected to purification by column chromatography, with DCM/MeOH, as eluent, to afford compound (6a) as a grey solid (0.276 g, 9.0 mmol, 60%). R.sub.f=0.33 (DCM/MeOH 9:1).

[0072] UV (EtOH)=?max: 222 (?616 M.sup.?1 cm.sup.?1), 249 1664 M.sup.?1 cm.sup.?1)and 355 (?3224 M.sup.?1 cm.sup.?1) nm.

[0073] FTIR (DCM) ?.sub.max=2918, 2848, 1669, 1579, 1640, 1574, 1550, 1415, 1255, 1160 cm.sup.?1.

[0074] 1H RMN ?.sub.H (Dimethyl Sulfoxide (DMSO), 400 MHz): 7.76 (dd, J =6.4 and 1.6 Hz, 1H, H-6), 7.33 (dt, J=6.8 and 1.6 Hz, 1H, H-4), 6.69 (d, J=8.4 Hz, 1H, H-3), 6.52 (t, J=6.8 Hz, 1H, H-5), 3.13 (t, J=7.2 Hz, 2H, NHCH.sub.2), 1.60-1.53 (m, 2H, NHCH.sub.2CH.sub.2), 1.25-1.35 (m, 18H, 9?CH.sub.2), 0,84 (t, J=6.8 Hz, 3H, CH.sub.3) ppm.

[0075] .sup.13C RMN ?c (DMSO, 100.6 MHz): 170.06 (CO.sub.2H), 150.96 (C-2), 134.45 (C-4), 131.67 (C-6), 113.90 (C-5), 111.08 (C-3) , 109.71 (C-1), 41.95 (NHCH.sub.2), 31.28 (CH.sub.2), 29.02 (CH.sub.2), 28.99 (CH.sub.2), 28.95 (CH.sub.2), 28.72 (CH.sub.2), 28.70 (CH.sub.2), 28.56 (CH.sub.2), 26.52 (CH.sub.2), 22.08 (CH.sub.2), 13.93 (CH.sub.3) ppm. HRMS: m/z (ESI-TOF): Found [M+1].sup.+: 306.2429; C.sub.19H.sub.32NO.sub.2 requires [M+1]+: 306.2428.

[0076] Method of producing 3-(dodecylamino) benzoic acid 6b: 1-Bromododecane (0.7 mL, 2.9 mmol, 0.5 eq.) was added to a solution of 3-aminobenzoic acid (3b) (0.2 g; 1.5 mmol). The reaction mixture was refluxed in ethanol (14 mL) for 50 hours and monitored by TLC (DCM/MeOH 9:1). The solvent was evaporated, and the residue obtained was subjected to purification by column chromatography, with DCM/MeOH 97:3, as eluent, to afford compound (6b) as a grey solid (0.21 g, 0.61 mmol, 41%).

[0077] Rf=0.23 (DCM/MeOH 9:1).

[0078] UV (EtOH)=?max: 227 (?526 M.sup.1 cm.sup.?1), 257 (?1579 M?1 cm.sup.?1) and 340 (?6380 M.sup.?1 cm.sup.?1) nm;

[0079] FTIR (DCM) ?.sub.max=2955, 2922, 1679, 1606, 1587 cm.sup.?1.

[0080] .sup.1H RMN ?.sub.H (DMSO, 400 MHz): 7.15 (d, J=7.6 Hz, 3H, H-6) , 7.12-7.08 (m, 2H, H-2 and H-5), 6.6 (d, J=6.8 Hz, 1H, H-4), 5.81 (br s, 1H, NH), 2.96 (t, J=7.2 Hz, 2H, NHCH.sub.2), 1.45-1.50 (m, 2H, NHCH.sub.2CH.sub.2), 1.17-1.29 (m, 18H, 9?CH.sub.2), 0.79 (t, J=7.2 Hz, 3H, CH.sub.3) ppm.

[0081] .sup.13C RMN ?.sub.c (DMSO, 100.6 MHz): 166.31 (CO2H), 149.15 (C-3), 130.47 (C-5), 129.05 (C-1), 117.3 (C-6), 116.05 (C-4), 115.90 (C-2), 60.32 (CH.sub.2), 42.69 (NHCH.sub.2), 31.28 (CH.sub.2), 29.03 (CH.sub.2), 29.01 (CH.sub.2), 28.98 (CH.sub.2), 28.84 (CH.sub.2), 28.69 (CH.sub.2), 28.47 (CH.sub.2), 26.60 (CH.sub.2), 22.07 (CH.sub.2), 14.17 (CH.sub.3) ppm. HRMS: m/z (ESI-TOF): Found [M+1].sup.+: 306.2428; C.sub.19H.sub.32NO.sub.2 requires [M+1]+: 306.2430.

REFERENCES

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[0083] 2Europe Insecticides By Type, By Crop Type, By Mode of Application, By Form, And By RegionIndustry Analysis, Share, Size, Growth, Trends, and Forecasts (2020-2025), Market Data Forecast, February 2020;3Eur. JOC, 2007, 24, 4111-4115;

[0084] 4Adv. Synth. Catal. 2009, 351, 1671-1676;

[0085] 5Pharmaceutical Chemistry Journal, 1971, vol. 5, 1, 7-10.