Novel Insect Antifeedant and Ixodicidal Nootkatone Derivatives

Naturally occurring nootkatone, with reported insecticidal and acaricidal properties, has been used as a lead to generate molecular diversity and, consequently, new insect antifeedant and ixodicidal compounds. A total of 22 derivatives were generated by subjecting this molecule to several reactions including dehydrogenation with the iodine/DMSO system, oxidation with SeO2, epoxidation with mCPBA, oxidation or carbon homologations of the α-carbonyl position with TMSOTf (trimethylsilyl trifluoromethanesulfonate) followed by Rubottom and Dess Martin periodane oxidations, condensation with formaldehyde using Yb(OTf)3 as catalyst and dehydroxilation using the Grieco protocol. The insect antifeedant (against Myzus persicae and Ropaloshysum padi) and ixodicidal (against the tick Hyalomma lusitanicum) activities of these compounds were tested. Compound 20 was the most active substance against M. persicae and R. padi, and twice more efficient than nootkatone in the antitick test.


Introduction
The well-known advantages of biopesticides compared to conventional synthetic pesticides account for the increasing number of research papers published in this field in recent years [1,2]. Conventional pesticides and biopesticides can be categorized according to the origin of their active ingredients, with one of these categories encompassing synthetic natural derived (SND) substances [2]. In this context, we focused our attention on nootkatone (1), a sesquiterpene which was isolated for the first time from the essential oil of the heartwood of Alaska yellow cedar (Cupressus nootkatensis) [3,4], and is also present in plants of Citrus genus [5], Vetiveria species [6] and others natural sources [7]. Our interest in this molecule was based on its very interesting insecticidal and acaricidal properties [4,[8][9][10][11][12][13][14]. In this sense, formulations acting as pesticides against mosquitoes and ticks are currently being developed [15]. However, the mode of action of this product remain uncertain [16].
Considering the repellent effects and insecticidal activity of nootkatone, we planned to use this renewable and chiral molecule as natural lead to generate molecular diversity with the ultimate goal of obtaining new SND insect antifeedants (against Rhopalosiphum padi, Myzus persicae, Spodoptera littoralis) and ixodicidal (against Hyalomma lusitanicum) compounds. The moth S. littoralis, also known as Egyptian cotton leaf worm, is reported to be a pest of this plant and of a high variety of vegetable crops [17]. The aphid M. persicae is known to infect many species from 40 plant families, with special prevalence in Brassica crops [18]. R. padi is also an aphid and is considered one of the most important Apart from its widely known detrimental effects on several species of cattle, this pest is an efficient vector transmitter of the Crimean-Congo hemorrhagic fever disease [20].
Nootkatone features an appropriate structure with functionalizations that may allow an easy and efficient synthesis of a series of derivatives following a Diversity Oriented Synthesis (DOS) approach [21]. Thus, the presence of an unsaturated ketone would enable the production of further insaturations at C3-C4 or C8-C9. Also, the carbonyl group at C2 should permit the generation of new derivatives functionalized at C3. Additionally, the direct and allylic oxidations of the double bonds present in the molecule will help increase structural diversity (Figure 1). To improve persistence of biological activity in open areas, new less volatile oxygenated nootkatone derivatives have been designed.

Chemicals and Instrument
Silica gel 60 (35-70 µm) was used for flash column chromatography. NMR spectra (see Supplementary Materials) were obtained in a Varian Direct-Drive 600 ( 1 H 600 MHz/ 13 C 150 MHz), Varian Direct-Drive 500 ( 1 H 500 MHz/ 13 C 125 MHz) and Varian Direct-Drive 400 ( 1 H 400 MHz/ 13 C 100 MHz). Accurate mass determinations were performed on a SYNAPT G2-Si Q-TOF mass spectrometer (Waters, Milford, MA, USA) equipped with high-efficiency T-Wave ion mobility and an orthogonal Z-spray™ electrospray ionization (ESI) source. MassLynx v.4.1 software was used for HRMS instrument control, peak detection, and integration. Reactions were monitored by TLC on 0.25-mm E. Merck silica gel plates (60F-254) visualized under UV light and by applying a phosphomolybdic acid solution in EtOH followed by heat. High quality reagents were purchased at the highest quality that was commercially available and were used without further purification. Table 1 To a solution of nootkatone 1 (655 mg, 3 mmol) in 20 mL of benzene, under an argon atmosphere, was added DDQ (749 mg, 3.3 mmol) and 0.22 mL of acid acetic. The reaction mixture was stirred for 2 days in reflux. Then, the solution was filtered at room temperature and the solvent was evaporated under low pressure. The crude reaction mixture was then re-dissolved in EtOAc, and washed with saturated NaHCO 3 and brine, dried with anhydrous Na 2 SO 4 , and concentrated in vacuo. Purification by flash chromatography (H:MTBE, 1:1) provided 328 mg of 3 [23] (51%) and 103 mg of 1 (16%).

Synthesis of Compounds 16 and 17
To a solution of the compounds 14 and 15 (40 mg, 0.17 mmol) dry DCM (1 mL), DMP (89 mg, 0.21 mmol) was added under argon atmosphere. The mixture was stirred for 90 min. Then, a saturated solution of Na 2 S 2 O 3 -NaHCO 3 (3 mL) was added and the resulting mixture was stirred for 30 additional min. Upon consumption of the starting material, the reaction mixture was diluted in MTBE (10 mL), washed with NaHCO 3 (3 × 7 mL) and brine (3 × 7 mL), dried with anhydrous Na 2 SO 4 , and concentrated in vacuo. Purification by flash chromatography (H:TMBE, 3:1) gave 10 mg of 16 (23%) and 6 mg of 17 (14%). To a solution of nootkatone 1 (0.23 mL, 1 mmol) in dry toluene (4 mL) cooled at −78 • C, 1.25 mL of collidine were added and the resulting mixture was stirred for 15 min, then TMSOTf (0.45 mL, 2.5 mmol) was added and stirred for 10 additional min. Upon consumption of the starting material, the reaction mixture was diluted with hexane (15 mL), washed with brine (3 × 10 mL) and dried over anhydrous Na2SO4 and concentrated in vacuo. The crude mixture was dissolved in THF (10 mL), then 4 mL of formaldehyde (37%, water) and Yb(OTf) 3 (550 mg, 0.9 mmol) were added under argon atmosphere and stirred for 24 h. The reaction mixture was then diluted with a solution of saturated NaHCO 3 (60 mL) and extracted with EtOAc (3 × 15 mL). The organic layer was washed with 1N HCl (3 × 15 mL), brine (3 × 15 mL), dried with anhydrous Na 2 SO 4 and concentrated in vacuo. The crude reaction mixture was purified by column chromatography using mixtures of solvents with increasing polarity to obtain 126 mg of 1 (54.  To a solution of 18 (75 mg, 0.3 mmol) in dry TFH (2.5 mL), 0.13 mL of n-Bu 3 P (0.46 mmol) and 1.3 mg of o-NO 2 PhSeCN (0.46 mmol) were added under argon atmosphere and the resulting solution was stirred for 80 min at room temperature. Upon consumption of starting material, the reaction mixture was diluted with MTBE (10 mL), was washed with saturated NH 4 Cl (3 × 5 mL), brine (3 × 5 mL), dried with anhydrous Na 2 SO 4 , and concentrated in vacuo. The crude reaction mixture was dissolved in THF (30 ml) and 30% H 2 O 2 (0.06 mL, 0.48 mmol) was added under argon atmosphere. To solution of 8 (290 mg, 1.05 mmol) in dry toluene (5 mL) cooled at −78 • C, 1.5 mL of collidine was added under argon atmosphere and the resulting mixture was stirred for 15 min at room temperature, then 0.47 mL of TMSOTf (2.5 mmol) were added and stirred for another 10 min. The reaction mixture was diluted in hexane (20 mL) and was washed with brine (3 × 15 mL), dried with anhydrous Na 2 SO 4 , and concentrated in vacuo. The crude mixture was dissolved in dry THF (10 mL), and then 5 mL of formaldehyde (37% water solution) and 1 g of Yb(OTf) 3 (1.6 mmol) were added under argon atmosphere and stirred for 24 h at room temperature. Upon consumption of the starting material, the reaction mixture was diluted with 60 mL of saturated NaHCO 3 solution and then was extracted with EtOAc (3 × 35 mL). The combined organic layers were washed with 1N HCl (3 × 20 mL), and brine (3 × 20 mL), dried with anhydrous Na 2 SO 4 , and concentrated in vacuo. The

Antifeedant Activity
Insect colonies maintained at ICA-CSIC were used for conducting the bioassays. M. persicae and R. padi were reared on bell pepper (Capsicum annuum) and Hordeum vulgare plants, respectively, and maintained at 22 ± 1 • C and >70% relative humidity, with a photoperiod of 16:8 h (L:D) in a growth chamber.
Antifeedant bioassays: The upper surface of C. anuum and H. vulgare leaf disks or fragments (1.0 cm 2 ) were treated with 10 µl of the test substance. The products were tested at an initial dose of 5 mg/ml (50 µg/cm 2 ) respectively. Twenty ventilated plastic boxes (2 × 2 cm) with 10 apterous aphid adults (24-48 h old) each were allowed to feed in a growth chamber (24 h, environmental conditions as above). Each experiment was repeated 2 (SE < 10%) and terminated after 24 h. Aphid settling was measured by counting the number of aphids on each leaf fragment. Settling inhibition (%FI or %SI) was calculated as % FI / %SI = [1 − (T/C) × 100], where T and C represent settling on treated and control leaf disks, respectively. The antifeedant effects (% SI) were analyzed for significance by the nonparametric Wilcoxon signed-rank test. Extracts and compounds with an SI > 70% were further tested in a dose-response experiment (3-4 serial dilutions) to calculate their relative potency (EC50, the effective dose to give a 50% settling reduction) from linear regression analysis (% FI/SI on Log-dose) [27].

Ixodicidal Activity
H. lusitanicum engorged female ticks were collected in central Spain (Finca La Garganta, Ciudad Real) from their host (deer) and maintained at 22-24 • C and 70% RH until oviposition and egg hatching. Resulting larvae (4-6 weeks old) were used for the bioassays [12]. Briefly, 50 µL of test solution were added to 25 mg of powdered cellulose at different concentrations (initial concentration of 10 mg/ml) and the solvent was evaporated. For each test, three replicates with 20 larvae each were used. Dead ticks were counted after 24 h of contact with the treated cellulose at the environmental conditions described, using a binocular magnifying glass. The larvicidal activity data are presented as percent mortality corrected according to Schneider-Orelli's formula. Effective lethal doses (LC 50 and LC 90 ) were calculated by Probit Analysis

Synthesis of Nootkatone Derivatives
The generation of chemical diversity from nootkatone (1) started by subjecting this molecule to the iodine/DMSO dehydrogenating system [28,29]. The reaction of 1 with 0.1 equiv of I 2 and 5 equiv of DMSO in refluxing toluene led to the production of three dehydrogenated products 2-4 ( Table 1, entry 1). Under these conditions, dienone 3 was obtained as the major compound [23]. Compound 2 is a natural component of grapefruit juice [22]. Compound 4 is described for the first time here. Its stereochemistry at C3 was assigned based on the proton J value (J = 12.5 Hz) of the proton at this position. stereochemistry at C3 was assigned based on the proton J value (J = 12.5 Hz) of the proton at this position. When 0.2 mmol of I2 were used (Table 1, entry 2), the same mixture of compounds was obtained, with compound 2 being the main reaction product (50% yield). When the quantity of I2 increased to 1 mmol (Table 1, entry 3), a different proportion of the same mixture 2-4 was obtained, with compounds 2 (28%) and 4 (25%) being then the main reaction products. A mechanistic proposal for the formation of these substances is shown in Scheme 1. Thus, when 1 reacts via Ia, compounds 2 and 4 are obtained, whereas if Ib is the reacting species, the process leads to the generation of 3. Scheme 1. Proposed mechanism for the generation of 2-4.
The regioselective dehydrogenation of nootkatone also afforded dienone 3 (62%) after its When 0.2 mmol of I 2 were used ( Table 1, entry 2), the same mixture of compounds was obtained, with compound 2 being the main reaction product (50% yield). When the quantity of I 2 increased to 1 mmol (Table 1, entry 3), a different proportion of the same mixture 2-4 was obtained, with compounds 2 (28%) and 4 (25%) being then the main reaction products. A mechanistic proposal for the formation of these substances is shown in Scheme 1. Thus, when 1 reacts via Ia, compounds 2 and 4 are obtained, whereas if Ib is the reacting species, the process leads to the generation of 3.  When 0.2 mmol of I2 were used (Table 1, entry 2), the same mixture of compounds was obtained, with compound 2 being the main reaction product (50% yield). When the quantity of I2 increased to 1 mmol (Table 1, entry 3), a different proportion of the same mixture 2-4 was obtained, with compounds 2 (28%) and 4 (25%) being then the main reaction products. A mechanistic proposal for the formation of these substances is shown in Scheme 1. Thus, when 1 reacts via Ia, compounds 2 and 4 are obtained, whereas if Ib is the reacting species, the process leads to the generation of 3. The regioselective dehydrogenation of nootkatone also afforded dienone 3 (62%) after its treatment with DDQ/AcOH in benzene.
The oxidation of 1 with SeO2 in refluxing EtOH for 3 h led to compounds 5-7 (Scheme 2), with alcohol 5 being the major product. Compound 7 was formed after allylic oxidation at C7 and subsequent Michael addition of this hydroxyl group to C10. Alcohol 5 is a natural product isolated The regioselective dehydrogenation of nootkatone also afforded dienone 3 (62%) after its treatment with DDQ/AcOH in benzene.
The oxidation of 1 with SeO 2 in refluxing EtOH for 3 h led to compounds 5-7 (Scheme 2), with alcohol 5 being the major product. Compound 7 was formed after allylic oxidation at C7 and subsequent Michael addition of this hydroxyl group to C10. Alcohol 5 is a natural product isolated from Alpinia oxyphylla [30], and it was also found in P450-catalyzed transformations of nootkatone [31]. Compound 6 was previously synthesized from nootkatone by a photochemical process [24]. from Alpinia oxyphylla [30], and it was also found in P450-catalyzed transformations of nootkatone [31]. Compound 6 was previously synthesized from nootkatone by a photochemical process [24].

Scheme 2. Synthesis of nootkatone derivatives 5-8.
Due to the overlapping signals observed for compound 7 in its 1 H-NMR spectrum, the configurations at C17 and C10 could only be only assigned after analysis of the corresponding stepNOESY experiments (selective TOCSY edited preparation NOESY experiences) [32] (Figure 2).  Epoxidation of 1 with mCPBA at 0 °C for 3 h afforded mainly monoepoxide 11 [33]. With longer reaction times reaction times, excess of mCPBA and at room temperature, the mixture of epoxides 11 and 12 [26] was obtained along with diepoxide 13, generated via the Baeyer-Villiger oxidation of the C1-C2 bond of diepoxide 12 (Scheme 4).

Scheme 2. Synthesis of nootkatone derivatives 5-8.
Due to the overlapping signals observed for compound 7 in its 1 H-NMR spectrum, the configurations at C17 and C10 could only be only assigned after analysis of the corresponding stepNOESY experiments (selective TOCSY edited preparation NOESY experiences) [32] (Figure 2). from Alpinia oxyphylla [30], and it was also found in P450-catalyzed transformations of nootkatone [31]. Compound 6 was previously synthesized from nootkatone by a photochemical process [24].

Scheme 2. Synthesis of nootkatone derivatives 5-8.
Due to the overlapping signals observed for compound 7 in its 1 H-NMR spectrum, the configurations at C17 and C10 could only be only assigned after analysis of the corresponding stepNOESY experiments (selective TOCSY edited preparation NOESY experiences) [32] (Figure 2).  Epoxidation of 1 with mCPBA at 0 °C for 3 h afforded mainly monoepoxide 11 [33]. With longer reaction times reaction times, excess of mCPBA and at room temperature, the mixture of epoxides 11 and 12 [26] was obtained along with diepoxide 13, generated via the Baeyer-Villiger oxidation of the C1-C2 bond of diepoxide 12 (Scheme 4). from Alpinia oxyphylla [30], and it was also found in P450-catalyzed transformations of nootkatone [31]. Compound 6 was previously synthesized from nootkatone by a photochemical process [24].

Scheme 2. Synthesis of nootkatone derivatives 5-8.
Due to the overlapping signals observed for compound 7 in its 1 H-NMR spectrum, the configurations at C17 and C10 could only be only assigned after analysis of the corresponding stepNOESY experiments (selective TOCSY edited preparation NOESY experiences) [32] (Figure 2).  Epoxidation of 1 with mCPBA at 0 °C for 3 h afforded mainly monoepoxide 11 [33]. With longer reaction times reaction times, excess of mCPBA and at room temperature, the mixture of epoxides 11 and 12 [26] was obtained along with diepoxide 13, generated via the Baeyer-Villiger oxidation of the C1-C2 bond of diepoxide 12 (Scheme 4). Epoxidation of 1 with mCPBA at 0 • C for 3 h afforded mainly monoepoxide 11 [33]. With longer reaction times reaction times, excess of mCPBA and at room temperature, the mixture of epoxides 11 and 12 [26] was obtained along with diepoxide 13, generated via the Baeyer-Villiger oxidation of the C1-C2 bond of diepoxide 12 (Scheme 4). Then the reactivity of the α-carbonyl methylene of nootkatone to trigger either oxidations or carbon homologations was addressed. The incorporation of a hydroxyl group at C3 was achieved via treatment of 1 with TMSOTf (trimethylsilyl trifluoromethanesulfonate) and collidine to produce the TMS-dienol intermediate I, which after a Rubottom oxidation [34] evolved towards the corresponding α-hydroxy carbonyl stereoisomers 14-15. These compounds were easily oxidized with the Dess Martin periodane to afford the dicarbonyl derivatives 16-17 (Scheme 5).  Additionally, the same TMS-dienol I was condensated with formaldehyde (37% in water) using Yb(OTf)3 as catalyst [35]. Under these conditions aldol 18 was obtained in a 34% yield together with minor proportions of compound 19 (11%), generated a result of a double condensation process. Starting from 18, the elimination of the hydroxyl group was achieved in two steps following the Grieco protocol [36] to obtain a good yield of dienone 20 in good (71%) (Scheme 6). Then the reactivity of the α-carbonyl methylene of nootkatone to trigger either oxidations or carbon homologations was addressed. The incorporation of a hydroxyl group at C3 was achieved via treatment of 1 with TMSOTf (trimethylsilyl trifluoromethanesulfonate) and collidine to produce the TMS-dienol intermediate I, which after a Rubottom oxidation [34] evolved towards the corresponding α-hydroxy carbonyl stereoisomers 14-15. These compounds were easily oxidized with the Dess Martin periodane to afford the dicarbonyl derivatives 16-17 (Scheme 5). Then the reactivity of the α-carbonyl methylene of nootkatone to trigger either oxidations or carbon homologations was addressed. The incorporation of a hydroxyl group at C3 was achieved via treatment of 1 with TMSOTf (trimethylsilyl trifluoromethanesulfonate) and collidine to produce the TMS-dienol intermediate I, which after a Rubottom oxidation [34] evolved towards the corresponding α-hydroxy carbonyl stereoisomers 14-15. These compounds were easily oxidized with the Dess Martin periodane to afford the dicarbonyl derivatives 16-17 (Scheme 5).  Additionally, the same TMS-dienol I was condensated with formaldehyde (37% in water) using Yb(OTf)3 as catalyst [35]. Under these conditions aldol 18 was obtained in a 34% yield together with minor proportions of compound 19 (11%), generated a result of a double condensation process. Starting from 18, the elimination of the hydroxyl group was achieved in two steps following the Grieco protocol [36] to obtain a good yield of dienone 20 in good (71%) (Scheme 6). Then the reactivity of the α-carbonyl methylene of nootkatone to trigger either oxidations or carbon homologations was addressed. The incorporation of a hydroxyl group at C3 was achieved via treatment of 1 with TMSOTf (trimethylsilyl trifluoromethanesulfonate) and collidine to produce the TMS-dienol intermediate I, which after a Rubottom oxidation [34] evolved towards the corresponding α-hydroxy carbonyl stereoisomers 14-15. These compounds were easily oxidized with the Dess Martin periodane to afford the dicarbonyl derivatives 16-17 (Scheme 5).  Additionally, the same TMS-dienol I was condensated with formaldehyde (37% in water) using Yb(OTf)3 as catalyst [35]. Under these conditions aldol 18 was obtained in a 34% yield together with minor proportions of compound 19 (11%), generated a result of a double condensation process. Starting from 18, the elimination of the hydroxyl group was achieved in two steps following the Grieco protocol [36] to obtain a good yield of dienone 20 in good (71%) (Scheme 6). Additionally, the same TMS-dienol I was condensated with formaldehyde (37% in water) using Yb(OTf) 3 as catalyst [35]. Under these conditions aldol 18 was obtained in a 34% yield together with minor proportions of compound 19 (11%), generated a result of a double condensation process.
Starting from 18, the elimination of the hydroxyl group was achieved in two steps following the Grieco protocol [36] to obtain a good yield of dienone 20 in good (71%) (Scheme 6). The C3 stereochemistry of alcohol 18 was determined following analysis of the NOEs observed in the selective 1D-NOESY spectra of this compound (Figure 4). Following a similar sequence, the acetoxy derivative 23 was synthesized from 8 in three steps (Scheme 7). The enhancements observed in the selective 1D NOESY of 21 allowed us to assign the stereochemistry of this compound at C3 ( Figure 5). The C3 stereochemistry of alcohol 18 was determined following analysis of the NOEs observed in the selective 1D-NOESY spectra of this compound (Figure 4). The C3 stereochemistry of alcohol 18 was determined following analysis of the NOEs observed in the selective 1D-NOESY spectra of this compound (Figure 4). Following a similar sequence, the acetoxy derivative 23 was synthesized from 8 in three steps (Scheme 7). The enhancements observed in the selective 1D NOESY of 21 allowed us to assign the stereochemistry of this compound at C3 ( Figure 5). Following a similar sequence, the acetoxy derivative 23 was synthesized from 8 in three steps (Scheme 7). The C3 stereochemistry of alcohol 18 was determined following analysis of the NOEs observed in the selective 1D-NOESY spectra of this compound (Figure 4). Following a similar sequence, the acetoxy derivative 23 was synthesized from 8 in three steps (Scheme 7). The enhancements observed in the selective 1D NOESY of 21 allowed us to assign the stereochemistry of this compound at C3 ( Figure 5).  Table 2 shows the results of the aphid antifeedant effects of nootkatone (1) and derivatives. These compounds were also tested against the lepidopteran S. littoralis without significant effects (all EC50 > 50). The most active compounds were 20 (both species), 15 and 2 (R. padi) followed by 7, 10 (M. persicae), 3 (R. padi) and 14 (both species). All these derivatives were more active than nootkatone (1), with 20, 15 and 2 being over 50% more potent, followed by 10, 7, and 11 (∼50%) ( Figure 6).   Table 2 shows the results of the aphid antifeedant effects of nootkatone (1) and derivatives. These compounds were also tested against the lepidopteran S. littoralis without significant effects (all EC 50 > 50). The most active compounds were 20 (both species), 15 and 2 (R. padi) followed by 7, 10 (M. persicae), 3 (R. padi) and 14 (both species). All these derivatives were more active than nootkatone (1), with 20, 15 and 2 being over 50% more potent, followed by 10, 7, and 11 (~50%) ( Figure 6).

Antifeedant Activity
It should be noticed that the α,β-unsaturated ketone 20, with an extra methylene, 15 with an α-hydroxy at C-3 and 2, with an additional endocyclic C8-C9 unsaturation, exhibited strong antifeedant effects followed by the exocyclic epoxide 7, and the aldehyde derivative 10. The presence of an additional Michael acceptor in compound 20 may account for the observed bioactivity of this product. In this regard, previous results showed that the presence of an exocyclic epoxide in eremophilanes resulted in strong antifeedant effects against M. persicae and S. littoralis [12].  It should be noticed that the α,β-unsaturated ketone 20, with an extra methylene, 15 with an αhydroxy at C-3 and 2, with an additional endocyclic C8-C9 unsaturation, exhibited strong antifeedant effects followed by the exocyclic epoxide 7, and the aldehyde derivative 10. The presence of an additional Michael acceptor in compound 20 may account for the observed bioactivity of this product. In this regard, previous results showed that the presence of an exocyclic epoxide in eremophilanes resulted in strong antifeedant effects against M. persicae and S. littoralis [12]. Table 3 shows the ixodicidal effects of nootkatone (1) and derivatives against H. lusitanicum larvae.   Table 3 shows the ixodicidal effects of nootkatone (1) and derivatives against H. lusitanicum larvae. Using the activity of nootkatone as reference, the tested compounds can be classified in four groups according to their relative activity (Figure 7). Compounds 5, 20 (>50%) and 3 (20 and 68%) showed an increase in acaricidal activity with respect to nootkatone (1) for both lethal doses (Table 3, Figure 5); followed by 2, 7 and 11 with an increase for LD 90 >50%. Compounds 15 and 18 showed reduced acaricidal potency at LD 50 and higher at LD 90 than 1. Lastly, the remaining compounds either displayed a significant loss of activity or were not active at all. These results are relevant considering that nootkatone was reported to be more active against the tick species Ixodes scapularis and Amblyomma americanum than Permanone, a permethrin-based commercial clothing repellent [37].

Ixodicidal Activity
Using the activity of nootkatone as reference, the tested compounds can be classified in four groups according to their relative activity (Figure 7). Compounds 5, 20 (>50%) and 3 (20 and 68%) showed an increase in acaricidal activity with respect to nootkatone (1) for both lethal doses (Table 3, Figure 5); followed by 2, 7 and 11 with an increase for LD90 >50%. Compounds 15 and 18 showed reduced acaricidal potency at LD50 and higher at LD90 than 1. Lastly, the remaining compounds either displayed a significant loss of activity or were not active at all. These results are relevant considering that nootkatone was reported to be more active against the tick species Ixodes scapularis and Amblyomma americanum than Permanone, a permethrin-based commercial clothing repellent [37]. From these data, the following structure-activity relationships were inferred for the acaricidal effects. Hydroxylation at C13 (compound 5) increased acaricidal activity, possibly due to a decrease in the volatility of the molecule. The presence of a methylene group at C3 (compound 20) also increased activity with respect to nootkatone, most likely due to the presence of a more accessible Michael acceptor. Also, an additional endocyclic unsaturation increased the ixodicidal effects (C3-C4 in 3 and C8-C9 in 2). Finally, it can be concluded that the unsaturated ketone present in the structure of 1 plays a key role in the antitick activity of the tested substances whereas the C11-C12 double bond enhances the antitick efficiency as previously shown [12].

Conclusions
Starting from the natural product nootkatone (1), a known biopesticide compound, we have synthesized 22 derivatives in a reduced number of steps. Two are known natural products (2 and 5) and 15 have been synthesized for the first time. With these derivatives, we performed antifeedant test against S. littoralis, M. persicae and R. padi, and ixodicidal tests against H. lusitanicum. Compounds 20, 14 and 2 were more antifeedant (>50% more potent) than 1, followed by 7, 10 and 11 (∼50%). Compounds 5, 20, and 3 were stronger acaricidal agents than 1 for both lethal doses, followed by 2, 7 and 11 (LD90 >50%). Compound 20 was the only compound with stronger effects than 1 on both targets (insects and ticks). In this regard, the introduction of an additional methylene moiety at C3 enhanced antifeedant and ixodicidal activities. The significance of these results is supported by the fact that nootkatone is subject to research and development to support US Environmental Protection Agency registration for commercial marketing as a repellent and biopesticide against mosquitoes and ticks [38].
Supplementary Materials: The supplementary material related to this article includes NMR spectra of new compounds and is available online. From these data, the following structure-activity relationships were inferred for the acaricidal effects. Hydroxylation at C13 (compound 5) increased acaricidal activity, possibly due to a decrease in the volatility of the molecule. The presence of a methylene group at C3 (compound 20) also increased activity with respect to nootkatone, most likely due to the presence of a more accessible Michael acceptor. Also, an additional endocyclic unsaturation increased the ixodicidal effects (C3-C4 in 3 and C8-C9 in 2). Finally, it can be concluded that the unsaturated ketone present in the structure of 1 plays a key role in the antitick activity of the tested substances whereas the C11-C12 double bond enhances the antitick efficiency as previously shown [12].

Conclusions
Starting from the natural product nootkatone (1), a known biopesticide compound, we have synthesized 22 derivatives in a reduced number of steps. Two are known natural products (2 and 5) and 15 have been synthesized for the first time. With these derivatives, we performed antifeedant test against S. littoralis, M. persicae and R. padi, and ixodicidal tests against H. lusitanicum. Compounds 20, 14 and 2 were more antifeedant (>50% more potent) than 1, followed by 7, 10 and 11 (~50%). Compounds 5, 20, and 3 were stronger acaricidal agents than 1 for both lethal doses, followed by 2, 7 and 11 (LD 90 >50%). Compound 20 was the only compound with stronger effects than 1 on both targets (insects and ticks). In this regard, the introduction of an additional methylene moiety at C3 enhanced antifeedant and ixodicidal activities. The significance of these results is supported by the fact that nootkatone is subject to research and development to support US Environmental Protection Agency registration for commercial marketing as a repellent and biopesticide against mosquitoes and ticks [38].