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. 2023 May 12;86(5):1307–1316. doi: 10.1021/acs.jnatprod.3c00137

Synthesis of Carlina Oxide Analogues and Evaluation of Their Insecticidal Efficacy and Cytotoxicity

Eleonora Spinozzi †,*, Marta Ferrati †, Cecilia Baldassarri †, Filippo Maggi †, Roman Pavela ‡,§, Giovanni Benelli ∥, Cristina Aguzzi ⊥, Laura Zeppa ⊥, Loredana Cappellacci †, Alessandro Palmieri #, Riccardo Petrelli †
PMCID: PMC10226105  PMID: 37172063

Abstract

graphic file with name np3c00137_0007.jpg

Compounds isolated from botanical sources represent innovative and promising alternatives to conventional insecticides. Carlina oxide is a compound isolated from Carlina acaulis L. (Asteraceae) essential oil (EO) with great potential as bioinsecticide, being effective on various arthropod vectors and agricultural pests, with moderate toxicity on non-target species. Since the production from the wild source is limited, there is the need of exploring new synthetic routes for obtaining this compound and analogues with improved bioactivity and lower toxicity. Herein, the chemical synthesis of carlina oxide analogues was developed. Their insecticidal activity was assessed on the vectors Musca domestica L. and Culex quinquefasciatus Say, and their cytotoxicity was evaluated on a human keratinocyte cell line (HaCaT). The compounds’ activity was compared with that of the natural counterparts EO and carlina oxide. In housefly tests, the analogues were comparably effective to purified carlina oxide. In Cx. quinquefasciatus assays, the meta-chloro analogue provided a significantly higher efficacy (LC50 of 0.71 μg mL–1) than the EO and carlina oxide (LC50 1.21 and 1.31 μg mL–1, respectively) and a better safety profile than carlina oxide on keratinocytes. Overall, this study can open the way to an agrochemical production of carlina oxide analogues employable as nature-inspired insecticides.

The use of conventional insecticides has an enormous impact, boosting food production and contributing significantly to the improvement of human health, including the reduction of the onset of vector-borne diseases.1,2 However, the misuse and overuse of insecticides led to several negative consequences such as the accumulation in food, water, and soil, the development of pesticide resistance, and nontarget effects on human health and the ecosystem.3−6 In this context, botanical insecticides represent innovative and safe alternatives to conventional products due to their promising efficacy on a wide spectrum of vectors and agricultural pests and their moderate to low impact on the environment, as well as on human and animal health.7−9 However, the limited supply of the raw material from botanical sources may lead to exploring alternative routes for obtaining these insecticidal agents. Carlina acaulis L. is a medicinal plant belonging to the Asteraceae (Compositae) family and native to the calcareous soils of southern and central Europe10 with documented biological activities.11−14 Its root essential oil (EO) is characterized by the predominance (>95%) of 2-(3-phenylprop-1-ynyl)furan, commonly known as carlina oxide (1).15graphic file with name np3c00137_0006.jpg

This compound (1) belongs to the class of polyacetylenes, which are well recognized as phytoalexins, i.e., defense substances produced by plants in response to living microorganisms, products of microbial origin, and environmental stress such as UV light exposure and cold.16C. acaulis EO, carlina oxide, and formulations encapsulating these products have been tested against vectors (Culex quinquefasciatus Say and Musca domestica L.), agricultural pests (Lobesia botrana (Denis & Schiffermüller), Bactrocera oleae (Rossi), Ceratitis capitata (Wiedemann), and Meloidogyne incognita (Kofoid & White)), and stored-products pests (Acarus siro L., Alphitobius diaperinus (Panzer), Oryzaephilus surinamensis L., Prostephanus truncatus (Horn), Rhyzopertha dominica (F.), Sitophilus oryzae L., Tribolium confusum Jacquelin du Val, Tribolium castaneum (Herbst), Tenebrio molitor L., and Trogoderma granarium Everts.), showing noteworthy results.17−20 The above-mentioned studies also demonstrated the limited toxicity of C. acaulis EO on nontarget species, as well as its promising safety profile in terms of LD50 and IC50 values determined on rats and human cells, respectively.18,21,22 Since carlina oxide is responsible for the C. acaulis EO insecticidal potential20 and given the unlikely possibility of obtaining it in a scalable manner from the natural source, this research aimed at the production of differently functionalized carlina oxide analogues through a three-step synthetic approach and the evaluation of their insecticidal activity against the adults of M. domestica, an ubiquitous fly pest and vector of several pathogens,23 and Cx. quinquefasciatus 3rd instar larvae, a lymphatic filariasis and arbovirus vector.24,25 The cytotoxicity of these analogues was also evaluated on immortalized human keratinocytes (HaCaT) and compared with that of their natural counterparts EO and carlina oxide.

Results and Discussion

Synthesis of Carlina Oxide Analogues

Since the carlina oxide scaffold resulted to be a promising lead compound for the development of new botanical insecticides, we synthesized a new series of carlina oxide analogues differently substituted on the benzyl moiety to evaluate their insecticidal activity. The synthetic protocol was developed using a retrosynthetic approach (Scheme 1) employing commercially available benzyl bromide substrates.

Scheme 1. Retrosynthetic Analysis of the Synthetic Approach for Carlina Oxide Analogues.

Scheme 1

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The developed process involved three steps: (I) the substitution reaction of ethynyltrimethylsilane on the benzyl bromide substrates, (II) the deprotection of the terminal C–H of the alkynes, and (III) the Sonogashira coupling between the differently substituted terminal alkynes and 2-bromofuran. The model substrate used for the assessment of this synthetic approach was 4-methylbenzyl bromide (compound 9). Inspired by the work of Hameury et al.,26 the reaction of 8 with 9 (Scheme 2) was investigated using initially ethylmagnesium bromide (EtMgBr) as base and copper bromide (CuBr) as catalyst, but no reactivity was observed. After different attempts, characterized by the change of the base or of the catalyst, the use of isopropylmagnesium chloride (i-PrMgCl) and of CuBr-dimethylsulfide complex in tetrahydrofuran (THF) for 16 h led to 15 formation in 89% yield (Scheme 2).

Scheme 2. Substitution Reaction of Ethynyltrimethylsilane on 4-Methylbenzyl Bromide.

Scheme 2

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At this point, following the study of Konno et al.,27 tetrabutylammonium fluoride (TBAF) was initially chosen as the C–H bond deprotecting agent for the synthesis of 22, and several optimization efforts were conducted for the achievement of the optimal reaction conditions. Unfortunately, all those efforts have not paid off in terms of product formation. Thus, following the work of Louvel et al.,28 this step was performed using K2CO3 in MeOH for 3 h at 0 °C, obtaining a quantitative yield (Scheme 3).

Scheme 3. Synthesis of the Alkyne 22.

Scheme 3

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quantit., quantitative yield.

Finally, for the synthesis of compound 2 a Sonogashira coupling between 22 and 2-bromofuran (21) was performed according to the procedure previously reported by Tomas-Mendivil et al.,29 using palladium(II)bis(triphenylphosphine) dichloride (PdCl2(PPh3)2), copper iodide (CuI), and diisopropylamine (i-Pr2NH) in toluene at 50 °C for 3 h. This step led to product formation in 40% yield (Scheme 4).

Scheme 4. Synthesis of Compound 2.

Scheme 4

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This synthetic protocol developed for the synthesis of compound 2 was applied to other substituted benzyl bromides to obtain compounds 3–7 in different overall yields ranging from 29% to 48% (Scheme 5).

Scheme 5. General Synthetic Protocol Developed for Carlina Oxide Analogues (a); Chemical Structures and Yield of Novel Carlina Oxide Analogues 2–7 (b).

Scheme 5

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The ortho-chloro analogue synthesis was also attempted, but the isolation of the final product was not achieved. Detailed chemical procedures for the preparation of the presented compounds and the full characterization data are reported in the Experimental Section.

Chemical modifications of the carlina oxide scaffold were previously performed by Mami et al.30 The authors developed a hemisynthesis to produce a series of carlina oxide analogues by functionalization of the polyacetylene structure on the furan ring, with yields ranging from 17% to 30%. Therefore, we developed a novel and simple synthetic approach to produce carlina oxide analogues, and six new prototypes were obtained over three steps. Even if an overall yield optimization is still needed for the above-presented study, the latter represents the starting point for the synthesis of other carlina oxide derivatives. Moreover, this preliminary study aimed at the exploration of carlina oxide derivatives, and further work is needed to better understand structure–activity relationship.

Carlina acaulis Essential Oil Chemical Analysis

In this work, C. acaulis EO was used for comparative purposes. Therefore, for the sake of completeness, its chemical composition is also reported, with carlina oxide (97.8%) representing the predominant constituent (Section S4, Supporting Information). Other minor compounds (<1%) detected in the EO were benzaldehyde (0.9%), ar-curcumene (0.7%), β-sesquiphellandrene (0.2%), and α-zingiberene (0.1%). This composition was fully consistent with those reported in the literature.17,19,31

Topical Bioassays on Musca domestica

Carlina oxide analogues displayed high efficacy in terms of M. domestica female mortality. The estimated lethal doses are presented in Table 1. As indicated by the results, all the tested analogues showed an efficacy comparable to that of purified carlina oxide (1), for which the LD50 was estimated as 5.5 μg female–1 and LD90 as 11.5 μg female–1. Only 3, 4, and 5 provided significantly poorer efficacy; for the LD50(90) of other products, the CI95 values overlapped in at least one LD parameter, and thus they cannot be considered as significantly more effective, although, for example, the LD50 for 2 was estimated as 2.9 μg female–1.

Table 1. Mortality of Musca domestica Females after 24 h from Topical Application of the Tested Products.

product tested LD50 (μg female–1) CI95 LD90 (μg female–1) CI95 χ2b df p-value
Carlina acaulis EOa 5.3 3.2–7.1 11.7 8.9.21.8 6.451 3 0.521
Carlina oxide (1) 5.5 3.3–7.5 11.5 8.6–18.7 3.562 3 0.128
2 2.9 1.3–5.7 16.3 12.7–25.8 3.502 3 0.321
3 10.1 8.1–15.6 46.5 35.8–56.2 1.835 3 0.607
4 9.7 7.2–12.9 55.1 38.7–65.9 4.218 4 0.377
5 11.6 10.3–13.1 24.5 20.4–32.1 1.861 3 0.601
6 5.1 4.1–6.2 18.4 16.2–18.5 3.814 3 0.282
7 4.7 3.8–5.8 15.8 12.3–23.7 1.262 3 0.531
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a

EO, essential oil. LD50 and LD90 values in μg female–1 and CI95 are 95% confidence intervals; products’ activity is considered significantly different when the 95% CI fail to overlap.

b

Chi-square value, not significant (ns, p > 0.05) level.

Acute toxicity on Culex quinquefasciatus larvae. The lethal concentrations estimated for Cx. quinquefasciatus are reported in Table 2. All the tested analogues showed very promising mortality on mosquito larvae. Nevertheless, based on LC50(90) comparison, 6 was found to provide a remarkably better efficacy, exhibiting a significantly lower lethal concentration (LC50(90) = 0.71(0.85) μg mL–1) compared with the EO and carlina oxide (1), which showed LC50(90) 1.21(2.28) μg mL–1 and 1.31(2.31) μg mL–1, respectively.

Table 2. Mortality of Culex quinquefasciatus Larvae (3rd Instar) after 24 h of Exposure to the Tested Products.

product tested LC50 (μg mL–1) CI95 LC90 (μg mL–1) CI95 χ2b df p-level
Carlina acaulis EOa 1.21 1.12–1.31 2.28 2.27–2.59 1.721 4 0.786
Carlina oxide (1) 1.31 1.12–1.47 2.31 1.96–3.05 7.869 5 0.163
2 2.25 1.47–2.69 3.61 2.74–6.41 4.446 3 0.188
3 1.44 1.23–1.86 2.41 2.11–3.28 3.852 3 0.421
4 1.21 1.11–1.31 2.36 2.13–2.67 3.251 5 0.128
5 1.11 0.93–1.23 2.12 1.95–2.23 2.529 4 0.896
6 0.71 0.55–0.78 0.85 0.81–1.25 2.189 3 0.334
7 0.73 0.62–1.11 1.43 1.22–2.02 3.181 3 0.364
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a

EO, essential oil. LC50 and LC90 values in μg mL–1 and CI95 are 95% confidence intervals; products’ activity is considered significantly different when the 95% CI fail to overlap.

b

Chi-square value, not significant (ns, p > 0.05) level.

The introduction of a substituent on the benzyl ring differently modulated the biological activity of the analogues. In detail, the meta-substitution (compounds 6 and 7) led to the best LC50 values. This kind of substitution is probably crucial for the promising larvicidal activity on Cx. quinquefasciatus, and the mechanism of action should be further investigated. The best acute toxicity was observed by the meta-chloro analogue. Indeed, the chlorine substituent has been shown to be essential for several insecticidal compounds like dichlorodiphenyltrichloroethane (DDT), but also for natural products used as antibiotics and antitumor agents, such as clindamycin and vancomycin32,33 or cryptophycin and clavulone.34,35 The presence of chlorine on an aromatic moiety generally causes an increase of lipophilicity, nonbonding interactions with the binding sites, prevention of metabolic hydroxylation at that position, and increase of the electrophilicity of proximate parts of the molecule due to its electronegativity.36 However, also the meta-methoxy analogue showed a good toxicity on Cx. quinquefasciatus larvae. The presence of the methoxy group in the meta-position of compound 7 could enhance its lipophilicity and hence increase membrane permeability.37 However, this hypothesis should be further investigated.

Sublethal Effect against Culex quinquefasciatus Larvae

Exposing mosquito larvae to the LC30 estimated through acute toxicity assays achieved subsequent significant mortality on Cx. quinquefasciatus (except for compound 7) despite a relatively short exposure period (24 h). Compared with untreated larvae, a significantly lower percentage of adults emerged from insecticide-exposed larvae (Table 3). Nevertheless, differences could be observed between the individual tested products. Compound 3 provided the highest efficacy, causing mortality of 71% larvae and hatching of only 28.7% adults upon application at the concentration of 1 μg mL–1. This result was identical to that observed for the EO and even significantly better compared with carlina oxide (1).

Table 3. Being Exposed for 24 h to the LC30 of Carlina acaulis Essential Oil, Carlina Oxide, and Its Synthesized Analogues Affected Culex quinquefasciatus Larval and Pupal Mortality as Well as the Percentage of Successfully Emerged Adults.

product tested concentration (μL L–1) larval mortality (% ± SE) pupal mortality (% ± SE) emerged adults (% ± SE)
Carlina acaulis EO 0.9 79.0 ± 5.7e 0.7 ± 0.9 20.3 ± 5.2a
Carlina oxide (1) 0.8 58.3 ±7.4d 0.03 ± 0.5 41.3 ± 6.9b
2 1.5 53.0 ± 2.2d 0.0 ± 0.0 47.0 ± 2.2b
3 1.0 71.0 ± 5.0e 0.3 ± 0.5 28.7 ± 4.7a
4 0.8 32.7 ± 3.4c 0.7 ± 0.5 66.7 ± 3.9c
5 0.8 33.8 ± 3.1c 0.5 ± 0.2 69.5 ± 3.2c
6 0.5 39.0 ± 8.6c 1.3 ± 0.4 59.7 ± 8.2c
7 0.5 8.7 ± 2.4b 0.3 ± 0.5 91.0 ± 2.8d
Control   3.0 ± 2.2a 0.0 ± 0.0 97.0 ± 2.2d
ANOVA F8, 32, p-valuea   98.3; 0.000 ns 184.3; 0.000
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a

ANOVA parameters. In the same column, means followed by different letters are significantly different (ANOVA, Tukey’s HSD test, p < 0.05). EO = essential oil; ns = not significant (p > 0.05).

The results reported above pointed out a better efficacy of compound 3, bearing a bromine atom in the para-position, with respect to the meta-chloro-substituted analogue (6) that was the most active in the acute toxicity assays.

Larval Mortality Dynamics in Time, upon Application of LC90

The dynamics of Cx. quinquefasciatus larval mortality upon application of a concentration corresponding to the LC90 are presented in Table 4. No or almost no mortality was observed for the first 8 h from the application. A dynamic increase in larval mortality was observed only 12 h from exposure, and fatal mortality occurred within 24 h from application in all variants except 6, which caused 86.7% mortality, and 3, which led to 55.1% larval mortality. Compound 2 showed the highest dynamics, exhibiting the same course of the mortality increase rate as the EO, although it should be noted that a much higher applied concentration was used, i.e., 3.6 μg mL–1, while the EO was applied at the concentration of 2.3 μg mL–1.

Table 4. Culex quinquefasciatus Larval Mortality over Time When Exposed to the Estimated LC90 Values of Carlina acaulis Essential Oil, Carlina Oxide, and Its Synthesized Analogues.

  time
product concentration (μL L–1) 4 h 8 h 12 h 16 h 20 h 24 h
Carlina acaulis EO (2.3) 0.0 ± 0.0 0.0 ± 0.0a 78.6 ± 4.1f 100 ± 0.0e 100 ± 0.0d 100 ± 0.0d
Carlina oxide (1) (2.3) 0.0 ± 0.0 0.0 ± 0.0a 62.9 ± 2.8g 96.7 ± 2.4e 100 ± 0.0d 100 ± 0.0d
2 (3.6) 0.0 ± 0.0 0.0 ± 0.0a 71.9 ± 4.1f 100 ± 0.0e 100 ± 0.0d 100 ± 0.0d
3 (2.4) 0.0 ± 0.0 5.2 ± 1.2b 28.9 ± 2.5d 45.0 ± 4.1b 46.7 ± 4.1b 55.1 ± 4.1b
4 (2.4) 0.0 ± 0.0 0.0 ± 0.0a 12.8 ± 1.2b 93.3 ± 6.2e 98.3 ± 2.4d 100 ± 0.0d
5 (2.1) 0.0 ± 0.0 0.0 ± 0.0a 43.8 ± 3.5e 78.7 ± 4.7c 95.2 ± 1.5d 100 ± 0.0d
6 (1.0) 0.0 ± 0.0 0.0 ± 0.0a 18.9 ± 2.1c 75.0 ± 8.2c 81.7 ± 2.4c 86.7 ± 6.1c
7 (1.5) 0.0 ± 0.0 0.0 ± 0.0a 45.7 ± 3.2e 86.7 ± 4.7c 98.3 ± 1.2d 100 ± 0.0d
Control 0.0 ± 0.0 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a
ANOVA F8, 32, p-valuea ns 7.0; 0.005 395.6; 0.000 874.2; 0.000 1528.2; 0.000 1335.3; 0.000
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a

ANOVA parameters. In the same column, means followed by different letters are significantly different (ANOVA, Tukey’s HSD test, p < 0.05). EO = essential oil; ns = not significant (p > 0.05).

Conversely to the acute toxicity results, herein the less active analogues after 24 h were 6 (86.7% mortality) and 3 (55.1% mortality). These analogues bear a chlorine and a bromine atom in the meta- and para-positions, respectively. These results suggest that the presence of an electronegative atom on the benzyl moiety probably does not significantly affect the mosquito larval mortality over time when exposed to concentrations corresponding to LC90 values. Further experiments on the reported synthesized compounds elucidating their efficacy on different targets as well as their possible modes of action are needed.

Cell Viability Assay

The cytotoxicity assay was performed to assess the safety profile of the synthesized analogues on immortalized human keratinocytes (HaCaT). Indeed, one of the main routes of exposure to insecticides is through the skin, and this represents a significant risk causing great concern in terms of operator safety.38 Results obtained from the cytotoxicity assay showed that compounds 2 and 5 induced a significant reduction in cell viability (HaCaT cell line) compared with carlina oxide (1) and C. acaulis EO, with IC50 values of 20.26 ± 1.2 and 9.18 ± 0.3 μg mL–1, respectively, compared with 34.85 ± 2.4 μg mL–1 for carlina oxide and 54.05 ± 5.0 μg mL–1 for the EO. Moreover, compound 7 showed a similar effect to carlina oxide (1) with an IC50 of 37.39 ± 2.8 μg mL–1. On the other hand, compounds 3 and 4, with an IC50 of 60.38 ± 3.5 and 52.68 ± 3.7 μg mL–1, respectively, showed a better safety profile compared with carlina oxide (1), similar to that of EO (Table 5, Figure 1).

Table 5. Carlina acaulis Essential Oil, Carlina Oxide (1), and Carlina Oxide Analogues’ Cytotoxic Effects on HaCaT Cells.

product tested IC50 ± SD (μg mL–1)
Carlina acaulis EOa 54.05 ± 5.0a
Carlina oxide (1) 34.85 ± 2.4b
2 20.26 ± 1.2d
3 60.38 ± 3.5c
4 52.68 ± 3.7a
5 9.18 ± 0.3e
6 58.40 ± 3.1c
7 37.39 ± 2.8b
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a

EO, essential oil. Data shown are expressed as mean ± standard deviation (SD) of three separate experiments. IC50 ± SD within a column followed by the same letter do not differ significantly (ANOVA, Tukey’s HSD test, p ≤ 0.05).

Figure 1.

Figure 1

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Cell viability was determined in the HaCaT cell line by the MTT assay after treatment for 72 h with different concentrations of Carlina acaulis Essential Oil, Carlina Oxide (1), and Carlina Oxide Analogues. Data are expressed as mean ± standard deviation (SD) of three separate experiments. *p < 0.05 vs vehicle (Vhc).

Analogue 6 displayed the highest efficacy with respect to carlina oxide (1) in the acute toxicity assay on Cx. quinquefasciatus. It also showed a lower cellular toxicity (IC50 of 58.40 ± 3.1 μg mL–1) if compared with its precursor (IC50 of 34.85 ± 2.4 μg mL–1) and a similar toxicity to that of the EO (IC50 of 54.05 ± 5.0 μg mL–1). This result is highly encouraging, given the crucial importance of developing novel effective candidate insecticides with also a low cytotoxicity.

C. acaulis EO was previously tested on the primary human fibroblast cell line (NHFA12), showing a moderate toxicity (IC50 of 115.92 ± 6.1 μg mL–1). Moreover, it was also demonstrated that this toxicity became negligible when the EO was encapsulated into a microemulsion (ME) (IC50 of 5392.8 ± 315 μg mL–1).22 These results suggest that once encapsulated into nanocarriers (at 5–10% level), such as nanoemulsions, nanoparticles, or liposomes, carlina oxide analogues could be less cytotoxic according to the International Standard Organization (ISO) guidelines,39 where the reduction of cell viability should not exceed 70% at 100 ppm.22

The indiscriminate use of conventional insecticides causes an increase in arthropod pest and vector resistance along with negative effects on human and animal health and environmental pollution. Thus, the need for new, effective, and safe insecticides is growing. In this respect, botanicals represent a promising option to face these concerning issues. C. acaulis EO has already shown great potential against several insect pests and vectors. This study highlighted the possibility of synthesizing some carlina oxide analogues with better insecticidal activity than the natural polyacetylene. The meta-chloro substitution on the benzyl moiety led to improved mosquito larval toxicity and reduced cytotoxicity on human cells. On the other hand, most of the analogues, when used at their LC90, caused an almost complete mortality of mosquito larvae within 24 h and a similar efficacy in topical assays on housefly adults. In conclusion, further work is needed to better understand structure–activity relationships of carlina oxide derivatives as well as their mechanism of action in order to propose them as new insecticide leads to be exploited by agrochemical industries.

Experimental Section

General Experimental Procedures

All reagents and solvents were purchased from Merk KGaA (Darmstadt, Germany) and used without additional purification, except THF (freshly distilled over metallic sodium) and toluene (dried over 3 Å molecular sieves). 1H (400 or 500 MHz) and 13C (100 MHz) spectra were acquired on Varian Mercury 400 and 500 (Varian, Inc., Palo Alto, CA, USA). IR spectra (cm–1) were recorded with a PerkinElmer FT-IR spectrometer Spectrum Two UATR (PerkinElmer, Inc., Waltham, MA, USA). GC-MS analysis of the synthesized products was performed using a Hewlett-Packard GC/MS 6890N working with the EI technique (70 eV). Compound purity was evaluated through GC-MS and NMR analyses and was >90% for all the compounds. Elemental analyses (C, H, N, S) were conducted using a Fisons Instruments EA-1108 CHNS-O elemental analyzer (Thermo Fisher Scientific Inc., Waltham, MA, USA).

General Synthesis of Compounds 15–20

Compounds 15–20 were synthesized according to the procedure of Hameury et al.26 with some modifications. i-PrMgCl (2 M in THF, 20.0 mmol, 4.0 equiv) was slowly added at 0 °C to a solution of ethynyltrimethylsilane (20.0 mmol, 4.0 equiv) in THF (10 mL). The reaction mixture was stirred for 30 min at 0 °C and for an additional 30 min at rt. Then, CuBr-dimethylsulfide complex (3 mmol, 0.6 equiv) was added all at once, and the mixture was stirred at rt for 30 min before the addition of substrates 9–14 (5.0 mmol). The reaction was refluxed for 16 h. After that time, the solution was cooled to rt and poured into a saturated aqueous solution of NH4Cl (200 mL). The aqueous phase was extracted with Et2O (2 × 250 mL), and the organic layers were washed with H2O (200 mL), dried over MgSO4, filtered, and concentrated. The crude products were purified by silica gel chromatography (100% n-hexane) to afford the products in different yields.

(3-(p-Tolyl)-prop-1-yn-1-yl)-trimethylsilane (15):

colorless oil (174 mg, 86% yield); IR (neat) 2959, 2176, 1514, 1418, 1249, 1030, 1020, 838, 793, 758, 638, 476 cm–1; NMR spectra were in accordance with data reported in the literature:281H NMR (CDCl3, 400 MHz) δ 7.26–7.18 (m, 2H), 7.17–7.08 (m, 2H), 3.61 (s, 2H), 2.33 (s, 3H), 0.18 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 136.3, 133.5, 129.3, 127.9, 104.8, 86.7, 25.9, 21.2, 0.2; MS (EI) m/z = 202 (M+), 187 (100%), 172, 157, 128, 73; anal. C 77.14, H 9.01%, calcd for C13H18Si, C 77.16, H 8.97%.

(3-(4-Bromophenyl)-prop-1-yn-1-yl)-trimethylsilane (16):

colorless oil (216 mg, 81% yield); IR (neat) 2959, 2178, 1515, 1405, 1248, 1071, 1028, 1012, 841, 791, 759, 635, 474 cm–1; 1H NMR (CDCl3, 500 MHz) δ 7.46–7.42 (m, 2H), 7.24–7.19 (m, 2H,), 3.60 (s, 2H), 0.19 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 131.7, 129.8, 129.2, 128.4, 103.6, 87.6, 25.9, 21.2, 0.2; MS (EI) m/z = 267 (M+), 252 (100%), 222, 194, 172, 128, 73; anal. C 53.90, H 5.62%, calcd for C12H15BrSi, C 53.93, H 5.66%.

(3-(4-(Methylthio)phenyl)-prop-1-yn-1-yl)-trimethylsilane (17):

colorless oil (169 mg, 72% yield); IR (neat) 2958, 2175, 1492, 1404, 1248, 1091, 967, 908, 839, 794, 732, 642, 484 cm–1; 1H NMR (CDCl3, 500 MHz) δ 7.31–7.27 (m, 2H), 7.27–7.24 (m, 2H), 3.64 (s, 2H), 2.50 (s, 3H), 0.21 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 136.6, 133.6, 128.5, 127.3, 104.3, 87.1, 25.8, 16.4, 0.2; MS (EI) m/z = 234 (M+, 100%), 187, 159, 109, 73; anal. C 66.62, H 7.72, S 13.66%, calcd for C13H18SSi, C 66.60, H 7.74, S 13.68%.

(3-(4-Chlorophenyl)-prop-1-yn-1-yl)-trimethylsilane (18):

colorless oil (174 mg, 78% yield); IR (neat) 2963, 2165, 1498, 1400, 1204, 1037, 1018, 1009, 829, 801, 773, 622, 482 cm–1; NMR spectra were in accordance with data reported in the literature:401H NMR (CDCl3, 500 MHz) δ 7.29–7.27 (m, 4H), 3.61 (s, 2H), 0.19 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 135.0, 130.5, 129.4, 128.7, 103.7, 87.5, 25.8, 0.2; MS (EI) m/z = 222 (M+), 207 (100%), 179, 128, 73; anal. C 64.68, H 6.80%, calcd for C12H15ClSi, C 64.69, H 6.79%.

(3-(3-Chlorophenyl)-prop-1-yn-1-yl)-trimethylsilane (19):

colorless oil (138 mg, 62% yield); IR (neat) 2959, 2178, 1431, 1250, 1075, 1029, 1010, 840, 759, 649, 474 cm–1; 1H NMR (CDCl3, 400 MHz) δ 7.38–7.32 (m, 1H), 7.25–7.14 (m, 3H), 3.63 (s, 2H), 0.20 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 138.5, 134.4, 129.8, 128.3, 127.0, 126.2, 103.3, 87.8, 26.0, 0.2; MS (EI) m/z = 222 (M+), 207 (100%), 179, 128, 73; anal. C 64.65, H 6.74%, calcd for C12H15ClSi, C 64.69, H 6.79%.

(3-(3-Methoxyphenyl)-prop-1-yn-1-yl)trimethylsilane (20):

yellow oil (181 mg, 83% yield); IR (neat) 2958, 2176, 1435, 1249, 1026, 1015, 840, 759, 650, 441 cm–1; 1H NMR (CDCl3, 400 MHz) δ 7.26–7.18 (m, 1H), 6.95–6.86 (m, 2H), 6.79–6.73 (m, 1H), 3.79 (s, 3H), 3.62 (s, 2H), 0.17 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 159.9, 138.0, 129.6, 120.4, 113.6, 112.3, 104.3, 87.2, 55.3, 26.3, 0.2; MS (EI) m/z = 218 (M+), 189 (100%), 173, 73; anal. C 71.53, H 8.29%, calcd for C13H18OSi, C 71.50, H 8.31%.

Synthesis of Compounds 22–27 and 21

The synthesis of compounds 22–27 was performed following the work of Louvel et al.28 Compound 21 was prepared according to the procedure previously reported by Gilman and Wright,41 and its IR, NMR, and MS spectra were in line with those reported.41

1-Methyl-4-(prop-2-yn-1-yl)benzene (22):

colorless oil (130 mg, quantitative yield); IR (neat) 2256, 1489, 1157, 1125, 990, 915, 732, 660, 597, 493 cm–1; NMR spectra were in accordance with data reported in the literature:421H NMR (CDCl3, 500 MHz) δ 7.44–7.38 (m, 2H), 7.33–7.28 (m, 2H), 3.74 (d, J = 2.7 Hz, 2H), 3.03 (s, 1H), 2.49 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 136.4, 135.4, 129.2, 128.4, 82.4, 70.3, 24.5, 21.2; MS (EI) m/z = 130 (M+), 105 (100%), 91; anal. C 92.25, H 7.71%, calcd for C10H10, C 92.26, H 7.74%.

1-Bromo-4-(prop-2-yn-1-yl)benzene (23):

yellow oil (168 mg, 86% yield); IR (neat) 2256, 1488, 1220, 1157, 989, 910, 729, 652, 595, 490 cm–1; NMR spectra were in accordance with data reported in the literature:431H NMR (CDCl3, 500 MHz) δ 7.47–7.43 (m, 2H), 7.25–7.22 (m, 2H), 3.56 (d, J = 2.7 Hz, 2H), 2.20 (t, J = 2.7 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ 135.3, 131.8, 129.8, 120.8, 81.4, 71.0, 24.5; MS (EI) m/z = 195 (M+), 115 (100%), 89, 63; anal. C 55.44, H 3.64%, calcd for C9H7Br, C 55.42, H 3.62%.

Methyl-(4-(prop-2-yn-1-yl)phenyl)sulfane (24):

yellow solid (153 mg, 94% yield); IR (neat) 2253, 1492, 1210, 1153, 985, 904, 727, 649, 592, 488 cm–1; NMR spectra were in accordance with data reported in the literature:421H NMR (CDCl3, 500 MHz) δ 7.32–7.28 (m, 2H), 7.28–7.23 (m, 2H), 3.59 (d, J = 2.7 Hz, 2H), 2.50 (s, 3H), 2.21 (t, J = 2.7 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ 136.8, 133.3, 128.5, 127.3, 82.0, 70.6, 24.4, 16.3; MS (EI) m/z = 162 (M+), 147, 115 (100%), 89, 63; anal. C 73.04, H 6.18, S 19.73%, calcd for C10H10S, C 74.03, H 6.21, S 19.76%.

1-Chloro-4-(prop-2-yn-1-yl)benzene (25):

yellow oil (122 mg, 81% yield); IR (neat) 2263, 1501, 1203, 1174, 995, 923, 756, 674, 640, 470 cm–1; NMR spectra were in accordance with data reported in the literature:441H NMR (CDCl3, 500 MHz) δ 7.24–7.18 (m, 2H), 7.04–6.97 (m, 2H), 3.55 (d, J = 2.6 Hz, 2H), 2.17 (t, J = 2.8 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ 139.9, 130.1, 129.5, 128.9, 81.6, 71.1, 24.5; MS (EI) m/z = 150 (M+), 115 (100%), 89, 63; anal. C 71.76, H 4.71%, calcd for C9H7Cl, C 71.78, H 4.69%.

1-Chloro-3-(prop-2-yn-1-yl)benzene (26):

yellow oil (151 mg, quantitative yield); IR (neat) 2154, 1491, 1212, 1160, 990, 906, 730, 652, 593, 489 cm–1; 1H NMR (CDCl3, 400 MHz) δ 7.38–7.36 (m, 1H), 7.25–7.22 (m, 3H), 3.61–3.58 (m, 2H), 2.22 (t, J = 2.7 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ 138.2, 134.5, 129.9, 128.7, 127.1, 126.4, 81.1, 71.2, 24.6; MS (EI) m/z = 150 (M+), 115 (100%), 89, 63; anal. C 71.80, H 4.72%, calcd for C9H7Cl, C 71.78, H 4.69%.

1-Methoxy-3-(prop-2-yn-1-yl)benzene (27):

yellow oil (135 mg, 92% yield); IR (neat) 3292, 2958, 2835, 1600, 1585, 1488, 1454, 1212, 1159, 996, 737, 638, 489 cm–1; NMR spectra were in accordance with data reported in the literature:271H NMR (CDCl3, 400 MHz) δ 7.30–7.22 (m, 1H), 6.98–6.90 (m, 2H), 6.83–6.76 (m, 1H), 3.82 (s, 3H), 3.62–3.58 (m, 2H), 2.20 (t, J = 2.8 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ 159.9, 137.8, 129.7, 120.3, 113.7, 112.3, 81.9, 70.7, 55.3, 24.9; MS (EI) m/z = 146 (M+, 100%), 131, 115, 103, 89, 63; anal. C 82.13, H. 6.94%, calcd for C10H10O, C 82.16, H 6.90%.

Synthesis of Compounds 2–7

Compounds 2–7 were synthesized according to the methodology developed by Tomas-Mendivil et al.,29 except for the products’ purification, which was performed through silica gel column chromatography (100% n-hexane).

2-(3-(p-Tolyl)-prop-1-yn-1-yl)furan (2):

yellow oil (66 mg, 40% yield); IR (neat) 3032, 2921, 2216, 1513, 1486, 983, 900, 793, 738, 592, 475 cm–1; 1H NMR (400 MHz, CDCl3) δ 7.35 (m, 1H), 7.27 (d, J = 7.9 Hz, 2H), 7.15 (d, J = 7.7 Hz, 2H), 6.52 (d, J = 2.6 Hz, 1H), 6.39–6.35 (m, 1H), 3.81 (s, 2H), 2.34 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 142.7, 137.2, 136.2, 132.7, 129.1, 127.7, 114.0, 110.5, 92.1, 72.5, 25.2, 20.8; MS (EI) m/z = 196 (M+, 100%), 181, 167, 152, 128, 115, 91, 51; anal. C 85.65, H 6.19%, calcd for C14H12O, C 85.68, H 6.16%.

2-(3-(4-Bromophenyl)-prop-1-yn-1-yl)furan (3):

yellow oil (107 mg, 49% yield); IR (neat) 3033, 2925, 2218, 1515, 1488, 986, 906, 796, 740, 596, 477 cm–1; 1H NMR (500 MHz, CDCl3) δ 7.49 (t, J = 2.0 Hz, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.39–7.38 (m, 1H), 7.29–7.27 (m, 2H), 6.56 (d, J = 3.2 Hz, 1H), 6.40 (dd, J = 3.3, 1.9 Hz, 1H), 3.83 (s, 2H); 13C NMR (CDCl3, 100 MHz) δ 143.1, 138.2, 137.1, 135.0, 130.5, 129.7, 129.7, 123.2, 114.5, 110.7, 91.2, 73.4, 25.4; MS (EI) m/z = 261.5 (M+), 181 (100%), 168, 152, 115 91, 51; anal. C 59.84, H 3.49%, calcd for C13H9BrO, C 59.80, H 3.47%.

2-(3-(4-(Methylthio)phenyl)-prop-1-yn-1-yl)furan (4):

yellow solid (96 mg, 48% yield); IR (neat) 3035, 2253, 1492, 1093, 985, 904, 727, 649, 592 cm–1; 1H NMR (500 MHz, CDCl3) δ 7.38 (dd, J = 1.8, 0.7 Hz, 1H), 7.35–7.31 (m, 2H), 7.28–7.27 (m, 1H), 7.27–7.25 (m, 1H), 6.55 (d, J = 3.4 Hz, 1H), 6.39 (dd, J = 3.4, 1.9 Hz, 1H), 3.83 (s, 2H), 2.50 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 143.0, 137.3, 136.8, 132.9, 128.5, 128.4, 127.2, 127.1, 114.3, 110.7, 91.8, 73.0, 25.3, 16.2; MS (EI) m/z = 228 (M+), 181 (100%), 152, 126, 91, 51; anal. C 73.66, H 5.33, S 14.06%, calcd for C14H12OS, C 73.65, H 5.30, S 14.04%.

2-(3-(4-Chlorophenyl)-prop-1-yn-1-yl)furan (5):

yellow oil (82 mg, 44% yield); IR (neat) 3034, 2925, 2218, 1596, 1574, 1431, 1077, 986, 904, 775, 741, 592, 432 cm–1; 1H NMR (400 MHz, CDCl3) δ 7.39–7.37 (m, 1H), 7.37 (dd, J = 1.9, 0.7 Hz, 1H), 7.28 (s, 1H), 7.27–7.25 (m, 2H), 6.55 (d, J = 3.4 Hz, 1H), 6.38 (dd, J = 3.4, 1.9 Hz, 1H), 3.82 (s, 2H); 13C NMR (CDCl3, 100 MHz) δ 143.4, 138.1, 137.3, 134.7, 130.1, 128.4, 127.3, 126.4, 114.9, 110.9, 91.2, 73.7, 25.8; MS (EI) m/z = 216 (M+), 181 (100%), 152, 127, 92, 51; anal. C 72.10, H 4.21%, calcd for C13H9ClO, C 72.07, H 4.19%.

2-(3-(3-Chlorophenyl)-prop-1-yn-1-yl)furan (6):

yellow oil (48 mg, 37% yield); IR (neat) 3004, 1598, 1577, 1474, 1432, 1264, 1078, 895, 855, 777, 703, 643, 436 cm–1; 1H NMR (400 MHz, CDCl3) δ 7.39–7.38 (m, 1H), 7.37 (dd, J = 1.9, 0.7 Hz, 1H), 7.27 (t, J = 1.4 Hz, 1H), 7.27–7.25 (m, 2H), 6.55 (d, J = 3.4 Hz, 1H), 6.38 (dd, J = 3.4, 1.9 Hz, 1H), 3.83 (s, 2H); 13C NMR (CDCl3, 100 MHz) δ 143.4, 138.1, 137.3, 134.7, 130.1, 128.4, 127.3, 126.4, 114.9, 111.1, 91.2, 73.7, 25.8; MS (EI) m/z = 216 (M+, 100%), 152, 127, 92, 51; anal. C 72.10, H 4.21%, calcd for C13H9ClO, C 72.07, H 4.19%.

2-(3-(3-Methoxyphenyl)-prop-1-yn-1-yl)furan (7):

yellow oil (134 mg, 63% yield); IR (neat) 2938, 2835, 1600, 1585, 1487, 1464, 1453, 1257, 1152, 1048, 984, 900, 775, 737, 592, 432 cm–1; 1H NMR (400 MHz, CDCl3) δ 7.35 (dd, J = 1.9, 0.7 Hz, 1H), 7.25 (s, 1H), 6.99–6.97 (m, 1H), 6.97–6.94 (m, 1H), 6.80 (dd, J = 8.0, 2.3 Hz, 1H), 6.53 (d, J = 3.4 Hz, 1H), 6.37 (dd, J = 3.4, 1.9 Hz, 1H), 3.83 (s, 2H), 3.82 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 160.1, 143.2, 137.7, 137.5, 129.8, 120.6, 114.5, 114.0, 112.5, 110.9, 92.0, 73.2, 55.5, 26.1; MS (EI) m/z = 212 (M+, 100%), 181, 169, 152, 127, 115, 51; anal. C 79.20, H 5.75%, calcd for C14H12O2, C 79.23, H 5.70%.

Carlina acaulis Essential Oil Isolation and Chemical Characterization

Dry roots of C. acaulis were purchased from A. Minardi & Figli (Bagnacavallo, Ravenna, Italy). The EO was obtained through hydrodistillation (HD) from the roots preventively reduced to a 1.5 mm size and following the procedure reported by Benelli et al.45 A 1 kg amount of roots was soaked for 16 h with 7 L of distilled water in a 10 L round-bottom flask. The HD process was conducted accordingly employing the distillation system previously reported,45 and the EO was obtained in 0.97% yield (w/w) (yellowish color, density of 1.063 g mL–1, and refractive index of 1.584). The EO was chemically characterized by GC-MS analysis and instrumental and analytical conditions, together with a chromatogram study following those previously published.45

Carlina Oxide Isolation

Part of the obtained EO (1.403 g) was subjected to silica gel (70 g) column chromatography (70–230 mesh, 60 Å, Merck) using n-hexane (Merck, Italy) as eluent. Carlina oxide (1) (1.306 g) was isolated and then characterized by NMR and mass spectrometry analyses. The chemical identification obtained was in accordance with that already reported.31

Insecticidal Assays

Houseflies

The used houseflies, M. domestica (females, 3–5 days old), were obtained from an established laboratory colony (Crop Research Institute, Czech Republic, >20 generations). Houseflies were reared as detailed by Pavela46 and were maintained at 25 ± 1 °C, 50–70% RH, and 16:8 (L:D). Larvae were reared in a mixture of sterilized bran, milk powder, and water; adults were provided with ad libitum access to water and to milk powder.

Mosquitoes

Cx. quinquefasciatus 3rd instar larvae were obtained from an established laboratory colony (Crop Research Institute, Czech Republic, >20 generations) as well. The larvae were fed on dog biscuits and yeast powder in a 3:1 ratio. Rearing conditions were 25 ± 2 °C, 70 ± 5% RH, and 16:8 (L:D) h.

Topical Bioassays on Houseflies

Acute topical toxicity of C. acaulis EO, carlina oxide, and its analogues on M. domestica adult females was evaluated according to Pavela.46 Fly females were anaesthetized using CO2. The products were diluted in acetone (p.a. purity, Sigma-Aldrich, Czech Republic), employing a concentration series to obtain the following doses upon application of 1 μL onto the housefly pronotum: 1, 3, 5, 8, 12, 15, 18, 20, 25, 30, 35, and 40 μg fly–1. To calculate the lethal doses alone, 5 or 6 doses were selected, which caused mortality in the range of 20–90%. The doses were applied using a microelectric applicator; the control flies were treated only with the solvent used for dilutions. Groups of 20 adults, replicated 4 times, were tested for each dose. After evaporation of acetone (approximately after 3–5 min), the flies were transferred to air-permeable plastic boxes (10 × 15 × 8 cm) containing food in the form of a 20% sugar solution (w:v). The experiment was performed in an air-conditioned room at 25 ± 1 °C, 70 ± 3% RH, and 16:8 h (L:D). The entire experiment was repeated 4 times. Adult mortality was evaluated 24 h after treatment.

Acute Toxicity on Mosquito Larvae

Acute toxicity of C. acaulis EO, carlina oxide, and its analogues diluted in DMSO (Sigma-Aldrich, Czech Republic) for Cx. quinquefasciatus larvae was evaluated following the World Health Organization (WHO)47 procedure with minor modifications by Benelli et al.31 Experimental treatment was prepared as follows: 1 mL of serial dilution was dissolved using DMSO in 224 mL of distilled water using a 500 mL glass bowl and shaken to produce a homogeneous test solution. The tested concentrations were 0.5, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, and 2.6 μg mL–1, while each concentration was replicated 4 times on groups of 25 larvae/beaker each. Distilled water containing the same amount of DMSO as that used to dissolve the compounds was used as the negative control. Cx. quinquefasciatus larvae were transferred into water in the bowl containing the prepared test solution (25 larvae/beaker). Four duplicate trials (100 larvae per single replication) were performed for each sample concentration, while each trial included a negative control composed of distilled water with the same amount of DMSO as the test sample. The assays were put in a growth chamber [25 ± 1 °C; 16:8 (L:D)]; mortality was recorded after 24 h.

Sublethal Effects on Mosquito Larvae

To assess the effect of low concentrations (i.e., LC30) of C. acaulis EO, carlina oxide, and its analogues on Cx. quinquefasciatus, 3rd instar larvae were exposed to each product for 24 h (the used concentrations are presented in Table 3). Subsequently, the surviving larvae were moved to clean water and nourished with a standard diet. The application methods are detailed in the paragraph dedicated to larvicidal tests. Larval and pupal mortality and emerged adults were evaluated, and 4 replicates were completed for each product tested. Again, the treated insects were positioned in a growth chamber (25 ± 1 °C; 16:9 (L:D)).

Mosquito Larval Mortality over Time When Exposed to LC90

The increase of Cx. quinquefasciatus mortality rate in time upon application of LC90 concentrations was evaluated as follows. The EO, carlina oxide, or the analogues were mixed in water using the same method as reported for acute toxicity tests. The tested concentrations are given in Table 4. Mortality was assessed at different time intervals, i.e., 4, 8, 12, 16, 20, and 24 h, from introducing the larvae in water contaminated with respective substances. Larvae not reacting to mechanical stimuli were considered dead. At the time of each mortality check, dead larvae were removed using a brush; 4 replicates (25 larvae/each) were performed for each product tested. Mosquitoes were placed in a growth chamber (25 ± 1 °C; 16:8 (L:D)).

Cytotoxicity Assays

Cell Lines

Immortalized human keratinocytes cell line (HaCaT), provided by IFO (Istituti Fisioterapici Ospitalieri, Rome, Italy), was cultured in DMEM enriched with 10% fetal bovine serum (FBS), 100 IU mL–1 penicillin/streptomycin, and 2 mM l-glutamine and kept at 37 °C with 5% CO2 and 95% humidity.

Cell Viability Assay

Cells were seeded at a density of 2 × 103/well in a 96-well plate with a final volume of 100 μL. After overnight incubation, cells were treated with different concentrations of carlina oxide analogues, C. acaulis EO, and carlina oxide (up to 100 μg mL–1) for 3 days, and then cytotoxicity was evaluated by adding MTT. After 3 h, the salt crystals were solubilized in 100 μL/well of DMSO. An ELISA reader microliter plate (BioTek Instruments, Winooski, VT, USA) was employed for the measurement of the absorbance of samples at 570 nm against a control.

Statistical Analysis

In insecticidal tests, mortality was corrected through the Abbott’s formula.48 LD50(90) and LC50(90) were estimated by probit analysis.49 Data in % were transformed using the arcsine square root transformation before being analyzed by ANOVA followed by Tukey’s HSD test (p ≤ 0.05).

Cell cytotoxicity data represent the mean with standard deviation (SD) of at least three independent experiments. Significant differences were assessed by one-way ANOVA, followed by Tukey’s HSD multiple comparisons test (p < 0.05). IC50 was calculated using GraphPad Prism software.

Acknowledgments

Roman Pavela would like to thank the Ministry of Agriculture of the Czech Republic for financial support of the botanical pesticide and basic substances research. Financial support for this work was provided by the Ministry of Agriculture of the Czech Republic (institutional support MZE-RO0423). The study was also supported by a PRIN grant from the Italian Ministry of Health to Riccardo Petrelli (PRIN 2017CBNCYT_005).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.3c00137.

  • NMR spectra of compounds 2–7, 15–20, and 22–27 and GC-MS chromatogram of Carlina acaulis EO (PDF)

Author Contributions

The manuscript was written with contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

np3c00137_si_001.pdf (3MB, pdf)

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