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Synthesis of novel phytol-derived γ-butyrolactones and evaluation of their biological activity

The synthesis of phytol-derived γ-butyrolactones as well as their evaluation for deterrent activity towards peach-potato aphid Myzus persicae and antiproliferative activity against four selected cancer cell lines are reported. Products were obtained in good yields (19–96%) and their structures were fully characterized by spectroscopic data (NMR, HRMS). Four synthesized δ-halo-γ-lactones (4–7) are new and have not been previously described in the literature. In the choice test phytol (1) appeared deterrent to M. persicae, whereas modifications of its structure did not cause the avoidance of the treated leaves by the aphids. In contrast, aphids were attracted to the leaves treated with the new trans-δ-chloro-γ-lactone (6). Electrical Penetration Graph (EPG) technique applied to explore the aphid probing and feeding activity revealed that neither phytol nor lactone 6 affected aphid probing and the consumption of phloem sap, which means that both phytol and the lactone 6 might have acted as postingestive modifiers of aphid behavior. The results of in vitro antitumor assays showed that obtained phytol derivatives exhibit cytotoxic activity against studied cancer cell lines (leukemia, lung and colon carcinoma and its doxorubicin resistant subline). Halolactones 4–6 were identified as the compounds, which arrest cell cycle of leukemia cells mainly in G2/M and S phases.

www.nature.com/scientificreports/ attractant properties 24 . In some cases, like for racemic δ-halo-γ-lactone synthesized from farnesol it was shown that γ-butyrolactones are also able to inhibit the growth of cancer cell lines, A549 (human lung adenocarcinoma) and HL-60 (human promyelocytic leukemia) 16,23 . Therefore, looking among isoprenoids for a biologically active natural carbon skeleton that could be the basis structure for developing its active lactone derivatives which can be useful as a new type of insecticides and cytostatic molecules we selected phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol) (PYT) (1). Our interest in phytol (1) was inspired by its easy availability and reports on its broad spectrum of biological activity. PYT is an acyclic monounsaturated diterpene alcohol widely distributed in the plant world but can be also found in algae 25,26 , in bacteria 27,28 , in gut of the ruminant's animals and their dairy products 29 . Biosynthesis of PYT goes mainly on the mevalonic acid (MVA) pathway 30 or 2-C-methylerythritol-4-phosphate (MEP) pathway 28 . Phytol (1) plays an important role in the plant world being a structural element of chlorophyll responsible for its anchoring to the thylakoid membrane of chloroplast 31 . It is a cost-effective chemical widely used in the cosmetics industry as a fragrance component and well known as a non-mutagenic food additive. The world use of PYT reaches 0.1-1 metric ton per year and it's a non-toxic effect for the mammalian was evaluated in the studies on albino mice and rats. The calculated lethal doses (LD 50 ) of PYT for them are above 5 g and 10 g/kg, respectively 32 .
PYT is known to exhibit insecticidal activities against S. zeamais and some other insect species 33,34 . This diterpene alcohol was found as the major metabolite in the ethanolic extracts of Petiveria alliaceae, responsible for the insecticidal activity against eggs and nymphs of Bemisia tabaci 35 . Methew and Thoppil 36 reported phytol to be an important constitute of essential oil of Salvia splendens and postulated its role in insecticidal activity against Aedes albopictus larvae. Many papers indicate also that phytol (1) exhibits cytotoxic potential against certain cancer cell lines: leukemia (MV4-11 and HeLa), breast (MCF-7), prostate (PC-3) and lungs (A-549) [37][38][39] and is the substance promising for the treatment of cancer.
The low and well-characterized toxicity of PYT and its high tolerance by mammals makes this compound a strong candidate for the development of new and environmentally friendly insecticides and cytostatic agents. Therefore, the aim of this work was to obtain more biologically active halolactone derivatives of phytol (1) and evaluate them as the antifeedant and anticancer agents. We synthesized a series of novel PYT derivatives and evaluated their feeding-deterrent activity against the peach-potato aphid Myzus persicae (Sulz.) (Hemiptera: Aphididae) and antiproliferative activity against four selected cancer cell lines. The influence of novel PYT derivatives on aphid activities was assessed by monitoring the settling behavior of freely moving aphids in choice situation. We also monitored the individual phases of aphid probing and feeding in the no-choice situation using the Electrical Penetration Graph (EPG) technique which visualizes the movements of aphid mouthparts within individual plant tissues. The antiproliferative activity of synthesized phytol derivatives was evaluated against selected cancer cell lines (leukemia, lung and colon carcinoma and its doxorubicin resistant subline) whereas their molecular mechanism of action including of cell cycle, cell death evaluation and their influence on the activity of caspase 3/7 were studied towards human biphenotypic B myelomonocytic leukemia cell line MV4-11.
Analytical Thin Layer Chromatography (TLC) was carried out on silica gel coated aluminium plates (DC-Alufolien Kieselgel 60 F254, Merck, Darmstadt, Germany) with a mixture of hexane, acetone and diethyl ether in various ratios as the developing systems. Compounds were visualized by spraying the plates with solution of 1% Ce(SO 4 ) 2 and 2% H 3 [P(Mo 3 O 10 ) 4 ] (2 g) in 10% H 2 SO 4 , followed by heating to 120-200 °C.
The products of chemical synthesis were purified by column chromatography on silica gel (Kieselgel 60, 230-400 mesh ASTM, 40-63 μm, Merck) using a mixture of hexane, acetone, and diethyl ether (in various ratios) as eluents.
General procedure for the synthesis of compounds (2)(3)(4)(5)(6)(7). The preparation of ester 2 and acid 3 has been illustrated in detail in our previous work 39 , and so the synthesis method would not be listed here.
To a solution of acid 3 (7.8 mmol) in THF (30 mL) the N-bromosuccinimide (7.8 mmol) or N-chlorosuccinimide (7.8 mmol) was added. The mixture was stirred at room temperature for 48-96 h. When the substrate reacted completely (TLC, GC) the mixture was diluted with diethyl ether and washed with saturated NaHCO 3 solution and brine. Organic layer of ether extract was separated and dired over anhydrous magnesium sulfate and evaporated on a rotary evaporator. New δ-halogeno-γ-lactones (4-7) were separated by silica gel column Deterrent activity of phytol and its derivatives. Aphids, plants and compound application. The peach potato aphids Myzus persicae (Sulzer) and the Chinese cabbage Brassica rapa subsp. pekinensis (Lour.) Hanelt were reared in laboratory at 20 °C, 65% r.h., and L16:8D photoperiod. One to seven days old apterous females of M. persicae and 3-week old plants with 4-5 fully developed leaves were used for experiments. M. persicae were obtained from the laboratory culture maintained at the Department of Botany and Ecology for many generations since 2000. All experiments were carried out under the same conditions of temperature, relative humidity, and photoperiod. The bioassays were started at 10-11.a.m. Each compound was dissolved in 70% ethanol to obtain the recommended 0.1% solution 40 . All compounds were applied on the adaxial and abaxial leaf surfaces by immersing a leaf in the ethanolic solution of a given compound for 30 s. 20 . Control leaves of similar size were immersed in 70% ethanol that was used as a solvent for the studied compounds. Experiments were performed 1 h after the compounds application to allow the evaporation of the solvent. Every plant and aphid were used only once.
Aphid settling (choice test). This bioassay allows the study of aphid host preferences under semi-natural conditions 41 . In the present study, aphids were given free choice between control and treated excised leaves that were placed in a Petri dish. Aphids were placed in the dish equidistance from treated and untreated leaves, so that aphids could choose between treated (on one half of a Petri dish) and control leaves (on the other half of the dish). Aphids that settled, i.e. they did not move, and the position of their antennae indicated feeding, on each leaf were counted at 1 h, 2 h, and 24 h intervals after access to the leaf. Each experiment was replicated 8 times (n = 8 replicates, 20 viviparous apterous females/replicate). Aphids that were moving or not on any of the leaves were not counted.
Behavioral responses of aphids Myzus persicae during probing and feeding (no-choice test). Aphid probing and the phloem sap uptake by M. persicae was monitored using the technique of electronic registration of aphid probing in plant tissues, known as EPG (= Electrical Penetration Graph), that is fre- www.nature.com/scientificreports/ quently employed in insect-plant relationship studies considering insects with sucking-piercing mouthparts [42][43][44] .
In this experimental set-up, aphid and plant are connected to electrodes and thus made parts of an electric circuit, which is completed when the aphid inserts its stylets into the plant. Weak voltage is supplied in the circuit, and all changing electric properties are recorded as EPG waveforms that can be correlated with aphid activities and stylet position in plant tissues 45,46 . The parameters describing aphid behaviour during probing and feeding, such as total time of probing, proportion of phloem patterns E1 and E2, number of probes, etc., are good indicators of plant suitability or interference of probing by chemical or physical factors in individual plant tissues [44][45][46] .
In the present study, aphids were attached to a golden wire electrode with conductive silver paint (epgsystems. eu) and starved for 1 h prior to the experiment. Probing behaviour of 12 apterous females/studied compound and control was monitored for 8 h continuously with Giga-4 and Giga-8 DC EPG with 1 GΩ of input resistance recording equipment (EPG Systems, Wageningen, The Netherlands). Each aphid was given access to a freshly prepared plant and each aphid/plant combination was used only once. Various behavioural phases were labelled manually using the Stylet + software (www.epgsy stems .eu). The following aphid behaviours were distinguished: no penetration (waveform 'np' -aphid stylets outside the plant), pathway phase-penetration of non-phloem tissues (waveforms ' ABC'), phloem phase (salivation into sieve elements, waveform 'E1' and ingestion of phloem sap, waveform 'E2'), and xylem phase (ingestion of xylem sap, waveform 'G'). Waveform 'G' occurred rarely irrespective of the treatment. Therefore, in all calculations the xylem phase was added to the pathway phase and termed as probing in non-phloem tissues. The E1/E2 transition patterns were included in E2. Waveform patterns that were not terminated before the end of the experimental period (8 h) were not excluded from the calculations. The parameters derived from EPG recordings were analyzed according to their frequency and duration in configuration related to activities in peripheral and vascular tissues.

Statistical analysis.
The data of the choice-test were analyzed using Student's t-test (STATISTICA 13.1. package). If aphids showed clear preference for the leaf treated with the tested compound (p < 0.05), the compound was described as having attractant properties. If aphids settled mainly on the control leaf (p < 0.05), the compound tested in the respective choice-test was stated a deterrent. From the data thus obtained the relative index of deterrence (DI) was calculated: DI = (C − T/C + T) where C was the number of aphids settled on control leaf, T was the number of aphids settled on the leaf treated with the tested compound. The value of DI ranged between 1 (ideal deterrent) and − 1 (ideal attractant) 41 . All statistical calculations related to data of the no-choice test were performed using StatSoft, Inc. (2014) STATISTICA (data analysis software system), version 12, www.stats oft.com. EPG parameters describing aphid probing behaviour (Table 2) were calculated manually and individually for every aphid and the mean and standard errors were subsequently calculated using the EPG analysis Excel worksheet created for this study. The parameters derived from EPGs were analyzed according to their frequency and duration in configuration related to activities in peripheral and vascular tissues. The results were statistically analyzed using Mann-Whitney U-test where the values of EPG parameters recorded from aphids on treated plants were compared to control 41 .
Antiproliferative activity of phytol and its derivatives. Cell lines. Human biphenotypic B myelomonocytic leukemia MV4-11, human colon cancer LoVo cell line and normal mouse fibroblast BALB/3T3 cells were obtained from American Type Culture Collection (Rockville, Maryland, USA), human lung carcinoma A549 cells were obtained from European Collection of Authenticated Cell Cultures (UK). All the cell lines are being maintained at the Hirszfeld Institute of Immunology and Experimental Therapy, PAS, Wroclaw, Poland and were cultured according the procedure described before 47 .
Determination of antiproliferative activity. The solutions of the compounds (50 mM) were prepared by dissolving the substances in DMSO (Sigma Aldrich, Germany). Then the tested compounds were diluted in culture medium to reach the final concentrations of 625, 125, 25 and 5 μM. Before adding of the tested compounds (24 h prior), the cells were plated in 96-well plates (Sarstedt, Germany) at a density of 1 × 10 4 or 0.5 × 10 4 (A549) cells per well. The assay was performed after 72 h of exposure to 625, 125, 25 and 5 μM of the tested agents. The in vitro cytotoxic effect of all agents was examined using the MTT (MV4-11) or SRB assay, described previously 47 . The results were calculated as an IC 50 (inhibitory concentration 50%) the concentration of tested agent, which is cytotoxic for 50% of the cancer cells. IC values were calculated for each experiment separately and mean values ± SD are presented in Tables 3. Each compound in each concentration was tested in triplicate in a single experiment, which was repeated 3-5 times.
Cell cycle analysis. Cell cycle analysis was carried out according to the procedure previously described 48 .
The MV4-11 cells were seeded at the density of 1 × 10 5 cells/well of culture medium on 24-well plates (Sarstedt, Germany) to the final volume of 2 mL and were exposed to the test compounds at concentrations 75 μM for 72 h. After incubation, the cells were collected and 1 × 10 6  Caspase-3/7 activity determination. Caspase-3/7 activity determination was performed according to the protocol previously described 48 . The MV4-11 cells were seeded at the density of 1 × 10 5 cells/mL of culture medium on 24-well plates (Sarstedt, Germany) to the final volume of 2 mL. The cells were exposed to the test compounds at concentrations 75 μM or campthothecin (0.05 μg/mL) as a positive control, for 72 h. After 72 h of the incubation, the cells were collected and centrifuged (5 min., 4 °C, 250×g). Cells were suspended in 50 µL of ice-cold lysis buffer (50 mM HEPES, 10% (w/v) sucrose, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, pH 7.3, IIET, Poland) and incubated 30 min. at 4 °C. After the incubation, 40 µL of each sample was transferred to a white, 96-well plate (Corning, USA) containing 160 µL of the reaction buffer (20 mM HEPES, 10% sucrose, 100 mM NaCl, 1 mM EDTA, 10 mM DTT, 0.02% Trition X-100, pH 7.3) (IIET, Wroclaw, Poland) with 9 µM Ac-DEVD-ACC fluorogenic substrate (λex = 360 nm, λem = 460 nm). The fluorescence increase correlated with the caspase-3/7 level was continuously recorded at 37 °C for 120 min using a Biotek Synergy H4 (Biokom, Warsaw, Poland). Compounds were tested in duplicates in single experiment and each experiment was repeated at least three times independently. Results were normalized to the number of cells in each well and are reported as mean relative caspase-3/7 activity compared to untreated control sample ± SD.
Statistical analysis. Statistical analysis was performed in Statsoft Statistica 10. All datasets were analyzed using t-test. p Values lower than 0.05 were considered as statistically significant.

Results and discussion
Chemistry. The synthesis of phytol-derived γ-butyrolactones is outlined in Fig. 1. The starting compound was commercially available mixture of cis/trans (35% : 65%) isomers of phytol (1) (PYT) (97% purity). This natural allylic alcohol (1) was subjected in the first step of the synthesis to the Johanson-Claisen rearrangement using triethylorthoacetate in the presence of a catalytic amount of propionic acid. The reaction afforded known ethyl 3,7,11,15-tetramethyl-3-vinylhexadecanoate (2) in 86% yield, which was subsequently hydrolyzed in ethanolic KOH solution to the corresponding γ,δ-unsaturated acid (3) in 96% yield 39 . The acid 3 was next the sub- www.nature.com/scientificreports/ strate in the halolactonization reactions, which were carried out in tetrahydrofuran with N-bromosuccinimide or N-chlorosuccinimide, respectively and afforded four novel δ-halo-γ-lactones (4-7). The bromolactonization afforded a mixture of two new products (4, 5) in a ratio of 28% : 72% according to GC (after 2 days). We separated them using column chromatography and their structures were established based on spectroscopic methods. The products of cyclization were trans-δ-bromo-γ-lactone (4) (minor, 28%) and cisδ-bromo-γ-lactone (5) (major, 72%). The lactones were obtained in 25 and 46% yield, respectively. The reaction of γ,δ-unsaturated acid (3) with N-chlorosuccinimide (NCS) was carried out to obtain chlorolactones (6, 7). Using chloride as an electrophilic agent, we observed a similar situation as in the process of bromolactonization, but this time the reaction proceeded longer, 4 days. Two new δ-chloro-γ-lactones (6) and (7) were formed as products of cyclization. According to GC, the reaction mixture consisted 31% of trans-δ-chloro-γ-lactone (6) and 69% of cis-δ-chloro-γ-lactone (7), which were obtained in 21 and 39% yield, respectively. The structures of halolactones 4-7 were confirmed by 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy. Correlation spectroscopy (COSY), heteronuclear single quantum correlation spectroscopy (HSQC), nuclear overhauser effect spectroscopy (NOESY) and mass spectra (HRMS) were also applied for this purpose (all data are presented in the Experimental Section and all spectra are in Supplementary Materials). 1  The spatial orientation of the halogenomethyl groups of synthesized bromo-and chlorolactones relative to the isoprenoid chain at carbon atom C-4 has been confirmed by spin-spin coupling through the space on the spectrum made in NOESY technique. Observed in the spectra of lactone 5 and 7 correlation signals confirmed the coupling of protons of halogenomethyl group with protons methyl group at C-4, indicating their close location in space (cis to each other). These correlations were not observed in the spectra of 4 and 5 made in NOESY technique. Formation of higher amounts of trans-lactones 5 and 7 where in their structure is trans location of halogenomethyl substituent and isoprenoid chain at C-4 can be explained by the preferential formation of bromonium/chloronium ion from the less shadowed side of double bound it means from the side of the methyl group, not a isoprenoid substituent. Carboxylate ion, according to the mechanism of halolactonization, formed a lactone ring, reacting from the side opposite to the bromine/chlorine atom.
Biological studies. Behavioral response of Myzus persicae during settling: choice test. The effect of phytol and its analogues was assessed by monitoring settling behavior of freely moving aphids. Aphids settle on a plant only when they accept it as a food source 49 . Therefore, the number of aphids that settle and feed on a given substrate is a good indicator of its suitability. Exogenously applied substances may alter plant suitability to aphids 18,20,50 . In the present work, we determined the settling success of the peach potato aphid after the application of phytol (1) and its newly obtained derivatives (2-7). The settling preferences of aphids depended on the compound and the time after the exposure of plants to the compound applied. The application of phytol (1) caused an initial acceptance of the treated leaves by M. persicae but from the second hour after application onwards, aphids tended to prefer the untreated to the treated leaves (Fig. 2, Table 1).
This indicates the deterrent properties of phytol (1) to M. persicae. Such switches in aphid responses in the course of time have previously been reported for other substances, such as piperitone-derived halolactones and hydroxylactones 24,51 . In contrast, aphids preferred the leaves treated with the new trans-δ-chloro-γ-lactone 6, which indicates the attractant properties of this lactone to M. persicae. The aphid responses to phytol and lactone 6 were the strongest and statistically significant 24 h after exposure. The phytol derivatives 2-5 and 7 did not affect aphid settling behavior significantly. However, aphids showed a detectable tendency to avoid the leaves treated with the new γ,δ-unsaturated acid 3 and two new δ-bromo-γ-lactones 4 and 5 and prefer the leaves treated with the new cis-δ-chloro-γ-lactone 7 (Fig. 2, Table 1). Considering M. persicae response to phytol and phytol-derived lactones in the present study, it may be concluded that the freely moving aphids are less likely to settle on plants treated with phytol and more likely to settle on trans-δ-chloro-γ-lactone 6-treated leaves than on untreated plants.
Behavioral responses of Myzus persicae during probing and feeding: no-choice test. The electronic monitoring of probing avtivities of M. persicae was applied to reveal the behavioral and physiological background of the activity of the studied compounds that was exposed in the observation of freely moving aphids in the aphid settling choice-test. The values of parameters derived from EPG recordings are reliable and accurate indicators of aphid behavioral responses to alteration in plant suitability due to exogenous application of xenobiotics 50,52 .
The typical behavior of M. persicae on control untreated plants consisted of non-probing (10% time of the 8-h experiment), probing in non-phloem tissues (35%), and probing in phloem tissues (55%). Sap ingestion occupied 98% of the phloem phase. Aphid probing activities were divided into 24.2 (± 4.8) probes on average and these probes were approximately 0.7 (± 0.3) hours long. M. persicae needed approximately 2.1 (± 0.4) hours and 14.1 (± 3.7) probes to reach phloem vessels and commence sap ingestion. 8.8 (± 2.5) of the probes before the www.nature.com/scientificreports/ first phloem phase were short eipdermal probes. The first contact with sieve elements (the first phloem phase) was 1.8 (± 0.7) hours long ( Table 2). Stylet penetration in plant tissues was the main aphid activity throughout the duration of the experiment and phloem phase predominated among all probing activities (Fig. 2). Generally, the behavior of M. persicae on plants treated with phytol was similar to the aphid behavior on control untreated plants. Aphids spent similar time on probing activities and the values of most EPG-derived parameters were similar to control (Table 2, Fig. 2). However, among the stylet probing activities, pathway activity predominated, which caused a slight decrease in the duration of the phloem phase. Aphids needed 2.7 (± 5.3) hours to reach phloem vessels. During that time, aphids inserted the stylets 27.0 (± 5.3) times, which was significantly more than in aphids on control plants. At the same time, the short epidermal probes were twice more numerous than on the control plants. The proportion of salivation during the phloem phase was four times higher than on control plants.

Figure 2.
Settling success of Myzus persicae on Brassica rapa subsp. pekinensis exposed to phytol (1) and phytolderived lactones (2-7). The same plant species was used to maintain the aphid stock culture. Relative index of deterrence (DI) after 1, 2, and 24 h. '*' Asterisks indicate statistically significant differences between the numbers of aphids on control and treated leaves at p < 0.05 (Student t-test).  Fig. 4).
The values of parameters derived from EPG recordings are reliable and accurate indicators of aphid behavioral responses to alteration in plant suitability due to exogenous application of xenobiotics 20,24,41 .
Insect feeding can be constrained at three levels: preingestive (immediate effect associated with host finding and host selection processes involving gustatory receptors), ingestive (related to food transport and production, release, and digestion by salivary enzymes), and postingestive (long-term effects involving various aspects of digestion and absorption of food) 53 . In the aphid settling choice test, we established that only phytol and the new trans-δ-chloro-γ-lactone 6 showed deterrent or attractant properties against M. persicae, respectively. The effects of these compounds on aphid behavior were revealed with a considerable delay in relation to the time when the aphids had access to the treated plants (Fig. 3). At the same time, we demonstrated that neither phytol nor any of the studied phytol-derivatives constrained aphid probing. The duration of the phloem sap ingestion activity was not altered on the treated plants, either, which means that aphids consumed similar amount of sap as on control plants during the experimental period. The rate of ingestion of the phloem sap is constant, therefore the amount of the consumed sap depends on the duration of ingestion 54 . Nevertheless, we may conclude that phytol had negative effect on M. persicae by changing the aphid behavior. Aphids walk away from the phytol-treated plants apparently after the sap has been consumed. We also found that aphids egested considerable amount of saliva during the phloem phase on phytol-treated plants, which was several times more than on control. The high proportion of salivation during the phloem phase suggests that phytol is not easily accepted by M. persicae. Watery saliva contains various enzymes associated with detoxification of plant allelochemicals, such as UPDglucose transferases, polyphenol oxidases, and peroxidases 55 . Therefore, the long duration or frequent periods of watery salivation that interrupt sustained ingestion may indicate problems in sap acceptance 45 . It is very likely that phytol has postingestive deterrent effect on M. persicae behavior. In contrast to phytol, the lactone 6 did not cause excessive salivation during the phloem phase. At the same time, significantly more aphids settled on the new trans-δ-chloro-γ-lactone 6-treated leaves 24 h after exposure. I our opinion, lactone 6 may be considered postingestive attractant. However, the hypotheses that refer to deterrent or attractant properties of phytol and lactone 6, respectively need further testing before the practical application of these compounds is proposed. Table 2. Probing activities (EPG parameters) of M. persicae on B. pekinensis treated with phytol (1) and its derivatives (2-7) during 8-h EPG monitoring. Values represent means ± SE; n = number of replicates. *Asterisks denote statistically significant differences in relation to control at p < 0.05; Mann-Whitney U-test. a Probing phase: pathway phase + xylem phase + phloem phase (C + G + E). b Pathway (probing in non-vascular tissues) with cell punctures (C). c Phloem phase (E). d Index calculated as: duration of phloem phase E/duration of probing phase C + G + E. e First phloem phase: first salivation into sieve elements or first salivation and phloem sap ingestion (E1 or E1 + E2); all individuals were included in analysis; when an aphid did not show phloem phase, the time/number of probes to the first E1 or the first E1 + E2 was the time/number of probes until the end of the experiment. f (duration of phloem salivation E1/duration of phloem phase E1 + E2)*100.

EPG parameters/compounds
Control phytol (1) (2) (3) (4) (5) (6) General aspects of aphid probing behavior n = 12 n = 12 n = 12 n = 12 n = 12 n = 12 n = 12 n = 12 Activities in non-phloem tissues n = 11 n = 12 n = 10 n = 12 n = 12 n = 12 n = 10 n = 10   Table 3. www.nature.com/scientificreports/ Cell viability analysis indicates that synthesized derivatives show more pronounced cytotoxic activity than phytol (1). According to the values summarized in Table 3, it is shown that structural modification of PYT induces increase of the cytotoxic activity of obtained products and makes them more selective towards evaluated cell lines (Table 4). Among the tested cancer lines leukemia cells were the most sensitive to the inhibitory effect of all studied compounds (1-7). The phytol derivatives 2-7 were active towards leukemia at concentration range of 39.28-110.5 μM. In this group the highest activity was reported for 3,7,11,15-tetramethyl-3-vinylhexadecanoic acid (3) and for trans-δ-bromo-γ-lactone (4) with IC 50 = 39.28 and 46.9 μM, respectively. Considering the effect of phytol derivatives on the normal cells the potential application exhibit mainly δ-halo-γ-lactones (4-6) for which strong selectivity action towards cancer cells was observed. The obtained results of the antiproliferative effect of lactones 4-7 proved some correlation between their structures and cytotoxicity. While the differences in the activity of individual lactones containing bromine or chlorine atoms are almost not noticeable, in the case of orientation of the halogenomethyl group at carbon atom C-5 are clearly visible. trans-δ-Bromo-γ-lactone (4) and trans-δ-chloro-γ-lactone (6) exhibit higher activity than their cis analogues 5 and 7.
In contrast to the cytotoxicity of phytol-derived products against MV4-11 cells the proliferation of human lung adenocarcinoma (A549) was inhibited only by acid 3 at the concentration 49.5 μM which was slightly lower than this appointed for phytol (1). The proliferation of colon cancer LoVo cells was also inhibited only by acid 3 and the established IC 50 value 59.5 μM was 4 times lower than this observed for phytol (1) (IC 50 255.2 μM).
To evaluate agents activity against the MDR (multidrug resistance) phenotype cancer cell line LoVo together with its drug resistant subline LoVo/DX were tested and indexes of resistance (IR) were calculated. The IR value indicates how many times more a resistant subline is chemoresistant relative to its parental cell line. The data presented in Table 1 show that all compounds were more active against drug resistant LoVo/DX cells than against drug sensitive LoVo cells. Phytol (1) and compounds 3 and 6 were able to overcome drug resistance (IR < 2).
Many chemotherapeutics are toxic not only against malignant cells but also normal ones. Therefore, a very serious problem is the degree of toxicity of drugs depending on the doses required to achieve response in clinical Table 3. Antiproliferative activity of phytol (1) and its synthesized derivatives against selected cancer cell lines and normal mice fibroblasts. n.a. no activity in concentration of 5, 25, 125, 625 μM. IC 50 -compound concentration leading to 50% inhibition of cell proliferation. Data are presented as mean ± standard deviation (SD) calculated using Prolab-3 system based on Cheburator 0.4 software 56 . IR was calculated according to the formula IR = (IC 50 estimated against resistant cell line)/(IC 50 estimated against non-resistant cell line); values range: 0 < IR < 2-indicate that the tested compound is able to overcome drug resistance; 2 < IR < 10-defines the moderate ability of the compound to overcome drug resistance; IR > 10-defines no influence on the drug resistance phenomenon. (2) n.a n.a n.a n.a -n.a  www.nature.com/scientificreports/ trials. Selectivity of the cytotoxic activity of the phytol derivatives was determined by comparing the cytotoxic activity (IC 50 ) of each compound against each cancerous cell line with that of the normal fibroblasts BALB/3T3 (Table 3). Results were expressed as the values of the Selectivity Index (SI). The selectivity indexes for new δ-haloγ-lactones (4-6) against MV4-11 cells were much higher than for cisplatin. Phytol (1) also demonstrated lower selectivity towards cancer cells than its synthesized derivatives 3-7. The 3,7,11,15-tetramethyl-3-vinylhexadecanoic acid (3) had efficacy against all tested tumor cells greater than toxicity against normal cells.
Effect of phytol derivatives on cell cycle distribution of human leukemia cells MV4-11. In the next step of the study the cell cycle of leukemia MV4-11 cells was analyzed after 72 h treatment of compounds in concentration 75 µM (Fig. 4). Compounds 3 and 6 induced death of 50% cells, which resulted lowering of cells number in G0/G1, S and G2/M phases (statistically significance in comparison to control cells, p < 0.05). This result is with agreement with result of analysis of caspase 3 activity (Fig. 5). Compounds 4, 5 and 7 also induced death of 20-30% of cells.

Conclusion
In summary, a series of phytol-derived compounds were synthesized in good yields and their aphid behavior modifying activity and cytotoxic potency were evaluated. The observation of freely moving aphids and the electronic monitoring of aphid probing in response to phytol and phytol-derived lactones demonstrated the deterrent potential of phytol and the attractant potential of the new trans-δ-chloro-γ-lactone 6. Both phytol and the chlorolactone 6 acted as postingestive modifiers of aphid behavior. The phytol derivatives 2-5, and 7 did not affect the feeding activity or settling behavior of M. persicae.
Phytol derivatives were also evaluated towards four human cancer cell lines (MV4-11, A549, LoVo, LoVo/ DX) and normal mice fibroblasts (BALB/3T3). Obtained results confirmed that introduction of carboxy group and lactone ring into the structure of phytol increase its anticancer potential. Halolactones 4-6 were identified as the compounds, which arrest cell cycle of leukemia cells mainly in G2/M and S phases. We have found that the antiproliferative activity of individual lactones depends mainly on the orientation of halogenomethyl group at carbon atom C-5, whereas we did not observe bigger differences in the activity of bromo-and chlorolactones. However, further laboratory research in the area of the synthesis of more derivatives of phytol and broaden studies on their molecular mechanism of action are needed to be able to analyze the structure activity relationships. In the aspect of obtained results so far, the important fact is that calculated selectivity factors of almost all phytol derivatives were higher than those of cisplatin, what makes those products promising for further biological investigations. Intermediate acid 3 derived from phytol effectively inhibits the growth of all four types of tested cancer cells and is pro-apoptotic agent of leukemia cells that acts by activation of caspase-3/7 on the level comparable with camptothecin.