Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx

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Modified quaternary ammonium salts as potential antimalarial agents Nicoletta Basilico b, , Mara Migotto a, , Denise Patoinewende Ilboudo c, Donatella Taramelli c, Riccardo Stradi a, Elena Pini a,⇑ a b c

Dipartimento di Scienze Farmaceutiche – Sezione di Chimica Generale e Organica ‘A.Marchesini’, Università di Milano, Via Venezian 21, 20133 Milano, Italy Dipartimento di Scienze Biomediche, Chirurgiche e Odontoiatriche, Università di Milano, Via Pascal 36, 20133 Milano, Italy Dipartimento di Scienze Farmacologiche e Biomolecolari, Università di Milano, Via Pascal 36, 20133 Milano, Italy

a r t i c l e

i n f o

Article history: Received 8 April 2015 Revised 28 May 2015 Accepted 29 May 2015 Available online xxxx Keywords: Malaria P. falciparum Antimalarial drugs Quaternary ammonium salts (2E,4E,6E)-Octatrien-1-ol

a b s t r a c t A series of new quaternary ammonium salts containing a polyconjugated moiety has been synthesized and characterized; their biological activity as potential antimalarial agents was investigated, as well. All compounds were screened against chloroquine resistant W-2 (CQ-R) and chloroquine sensitive, D-10 (CQ-S) strains of Plasmodium falciparum showing IC50 in the submicromolar range and low toxicity against human endothelial cells. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Malaria is the most prevalent tropical disease in the world with 207 million estimated new cases in 2012 and an annual death toll of 627,000 people. At present, there are 104 countries in which malaria is considered endemic.1 The increasing drug resistance of Plasmodium falciparum (P. falciparum) responsible of the most lethal form of malaria and the resistance of mosquitoes to various pesticides stimulates the search for new weapons to fight this disease. This is particularly urgent now that the resistance to the artemisinin derivatives, essential component of the combination treatments presently available, has been confirmed in SE Asia.2 Quaternary ammonium salts are known to play an active role inhibiting the growth of P. falciparum parasites and have gained attention as a new, effective, and relatively cheap anti-malarial drugs. Ancielin et al.3,4 reported that the in vitro lethal effect of quaternary ammonium compounds on P. falciparum are predominantly related to the inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis as a consequence of a reduced choline transport into infected erythrocytes. Choline cannot be synthesized by the parasites which relies on exogenous ⇑ Corresponding author. Tel.: +39 02 5031 4606; fax: +39 02 5031 4615.  

E-mail address: [email protected] (E. Pini). Both authors contributed equally to this work.

supply. The treatment with phospholipids polar head analogs interferes with natural phospholipids biosynthesis by competition or substitution.5,6 From these observations, a series of bisthiazolium compounds have been tested and shown to possess potent antimalarial activity.7,8 The bis-thiazolium series provided the clinical candidate albitiazolium9 (albitiazolium bromide, SAR97276), that is in clinical phase II trials to treat severe malaria by the parenteral route.10 As widely reported in literature,11,12 polyconjugated systems such as retinoids and carotenoids possess many biochemical and pharmacological properties including antioxidant and antimicrobial activities. The retinol and some retinoid-like compounds are able to inhibit the in vitro growth of P. falciparum suggesting that this parasite is retinol-sensitive and that, in patients with malaria, adjunctive retinol therapy may accelerate parasite dysfunction and death.13 Moreover, it is also reported the high antiplasmodial activity of a marine carotenoid, fucoxanthin, extracted from marine brown seaweeds, macroalgae, diatoms and microalgae which activity could be related to its antioxidant properties.14 In the last decade our interest was turned on the synthesis of polyunsaturated molecules15,16 whose structure is shown in Figure 1 which share with carotenoids and retinoids some structural features, antioxidant properties, effects on cell proliferation and antibacterial activity.17

http://dx.doi.org/10.1016/j.bmc.2015.05.055 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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N. Basilico et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

R

R = CHO, CH2OH, COOH, Ph n=2-7

n Figure 1. Polyunsaturated compounds.

These compounds are chemically homologous of sorbic acid (2E,4E-hexadienoic acid), a natural preservative used as antimicrobial agent especially in food, drinks and cosmetics. From the preliminary biological evaluations18 (2E,4E,6E)-octatrien-1-ol, the corresponding acid and potassium salt showed interesting pharmacological properties. With the aim to develop a new class of compounds with potential antimalarial activity, in this work we describe the synthesis, the molecular characterization and the preliminary evaluation of the antimalarial activity in vitro of new quaternary ammonium salts derived from (2E,4E,6E)-octatrien-1-ol. 2. Results and discussion 2.1. Chemistry The compounds 4a–e and 6a–e were obtained following Scheme 1. Firstly, (2E,4E,6E)-octatrien-1-ol of industrial origin was oxidized to (2E,4E,6E)-octatrienal 1 with manganese dioxide and then reacted in anhydrous toluene with primary amines with long alkyl chain to give the corresponding Schiff bases 2a–e. The quantitative reduction of imines with sodium borohydride in refluxed anhydrous ethanol allowed to obtain the secondary amines 3a–e which were directly quaternized in a stainless steel autoclave using an excess of methyl iodide in presence of a stoichiometric amount of an hindered base to give ammonium salts 4a–e. Otherwise for the synthesis of ammonium salts 6a–e, the secondary amines 3a–e were at first reacted with benzyl chloride in the presence of a stoichiometric amount of diisopropylethylamine and finally reacted with methyl iodide. All synthesized compounds gave satisfactory analytical and spectroscopic data which were in full accordance with their depicted structures. 2.2. Biological activity Quaternary ammonium compounds with a long alkyl chain on the nitrogen atom are emerging as potentially drug alternatives to conventional antimalarial chemotherapy. The studies on antimalarial activity have suggested that choline transport from plasma to the infected erythrocyte might be the primary target of these compounds19,20 and this activity seems to be related to shape, electronegativity, and lipophilicity of these compounds. The aim of this study was to synthesize a new class of lipophilic quaternary ammonium salts bearing at the nitrogen atom a lipophilic electron rich eight carbon polyconjugated system and to evaluate their potential antimalarial effects in vitro. The lipophilicity and the electronic density of these compounds was then further modulated by introducing a C12–C18 saturated alkyl chain and dimethyl (compounds 4a–e) or methyl-benzyl (compounds 6a–e) substituents. The antimalarial activity of a fully saturated dimethylammonium salt (compound 7) and of octatrienoic potassium salt was also evaluated as control. The compounds under study were tested for their antimalarial activity against chloroquine resistant, W-2 (CQ-R) and chloroquine sensitive, D-10 (CQ-S) strains of P. falciparum. Their antimalarial activity was quantified as inhibition of parasite growth, measured as the activity of parasite lactate dehydrogenase (pLDH). The

results are summarized in Table 1 which shows the concentration of drugs inducing 50% of growth inhibition (IC50). The majority of the tested compounds exhibited moderate activity against both parasite strains, whereas the (2E,4E,6E)-octatrienoic acid potassium salt was inactive. Indeed, seven compounds (4b, 4c, 4d, 6a, 6b, 6c, 6e) showed IC50 values less than 1 lM against both strains. The remaining three compounds were considered inactive with IC50 well above 1 lM. Moreover, compound 7, analogous of 4a with a saturated alkyl chain with eight carbon units, exhibited an activity comparable with that of compounds 4c, 6a and 6c, suggesting that the impact of the polyconjugated chain is modest. In both series the effects of chain-length modifications on the antimalarial activity is maximal at 14 carbon chain. However, in the series 4a–e, a linear trend of IC50 values is observed even beyond the influence of the carbon chain length; whereas there is not such a close correlation for the compounds 6a–e, suggesting the influence of the steric hindrance of the benzyl system and of the electronic density distribution. Varying a methyl group on the nitrogen atom of compounds 4 with a benzyl residue, the activity patterns is slightly improved with the exclusion of compound 6d. What is interesting is the fact that the most potent compounds (6a and 6c) showed comparable activity against both the CQ-S and the CQ-R strains. Indeed, all the compounds of this study were equally active against both the CQ-S and the CQ-R strains. The resistance index (RI), that is the ratio between the IC50 of each compound against the two strains of P. falciparum, and thus an indication of possible cross-resistance with CQ, was significantly lower than that of CQ (Table 1); this is a characteristic shared with other quaternary ammonium salts.20 The cellular cytotoxicity on a human microvascular cell line (HMEC-1) was also assayed. All compounds exhibited low toxicity against this human cell line with a selectivity index (SI), calculated on D10 and W2 strains, ranging between 7 and 102. Interestingly, the most active compounds (4c, 6a, 6c) were also the less toxic which is indicative of selective action against parasitized red blood cells. 3. Conclusions In this paper, two new series of quaternary ammonium salts derived from all trans 2,4,6-octatrienol have been synthesized and evaluated for their in vitro activity against D-10 (CQ-S) and W-2 (CQ-R) strains of P. falciparum. The most active of the tested compounds displayed a significant inhibitory activity with IC50 in the submicromolar range with no cross resistance with CQ and very low cytotoxicity against a human endothelial cell line, suggesting a high therapeutic index. Considering that the structural requirements for antiplasmodial activity (polar head and lipophilicity around nitrogen) of these compounds are similar to quaternary ammonium derivatives previously described,8 we can suppose that they act through inhibition of choline transport. The complete saturated analog 7 was approximately equipotent to the most active compounds. The introduction of the polar head on the polyconjugated chain allowed to obtain compounds suitable of further optimization to improve in vitro activity against the parasites before considering in vivo experiments and studies for target identification. 4. Experimental 4.1. General All commercial available solvents and reagents were used after distillation or treatment with drying agents. (2E,4E,6E)-octatrienol, a gift of Giuliani s.p.a. (Milan, Italy), was purified before use by

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CH2OH

a

CHO

3 1 c

b

+ NH2 (CH2)nCH3

3

CH=N(CH2)nCH3 3

n = 11, 12, 13, 15, 17 CH3

NH(CH2 )nCH3

d

2a-e

I

N(CH2)nCH3

3

3 4a-e

H

3a-e

CH3

e

CH3

I N(CH2)nCH3

f N(CH2 )nCH3 3

3 6a-e

5a-e

Scheme 1. General method of the preparation of compounds 4a–4e and 6a–e. Reagents and conditions: (a) MnO2, CH2Cl2, 35–40 °C, (75%); (b) toluene, rt, 94–0%); (c) NaBH4, EtOH, rt (90–99%); (d) CH3I, diisopropylethylamine, 80 °C (61–67%); (e) benzyl chloride, diisopropylethylamine, 120 °C (54–65%); (f) CH3I, acetone, 58 °C (75–90%).

Table 1 In vitro antiplasmodial activity and cellular cytotoxicity of the compounds under study Compounds

D10 IC50a (lM)

W2 IC50a (lM)

RIb

HMECc IC50a (lM)

SId HMEC/D10

SIe HMEC/W2

4a 4b 4c 4d 4e 6a 6b 6c 6d 6e 7 Octatrienoic potassium salt CQ

4.28 ± 1.45 0.36 ± 0.03 0.21 ± 0.13 0.62 ± 0.04 1.15 ± 0.23 0.25 ± 0.97 0.87 ± 0.1 0.22 ± 0.04 >8.635 0.45 ± 0.06 0.19 ± 0.08 >28.37 0.011 ± 0.004

4.71 ± 0.22 0.63 ± 0.05 0.53 ± 0.17 0.95 ± 0.06 1.53 ± 0.48 0.33 ± 0.043 0.52 ± 0.36 0.31 ± 0.07 >8.635 0.61 ± 0.14 0.48 ± 0.18 >28.37 0.32 ± 0.08

1.1 1.7 2.5 1.5 1.3 1.2 0.6 1.4 — 1.3 2.5 — 29

>20 5.63 ± 0.04 7.89 ± 2.96 7.074 ± 1.78 16.72 ± 5.61 5.90 ± 1.43 21.24 ± 1.67 22.99 ± 10.86 5.99 ± 0.93 7.02 ± 1.18 8.69 ± 3.73

ND 15 36 11 14 23 24 102 ND 15 44 ND ND

ND 8 14 7 10 18 41 75 ND 11 18 ND ND

>20

ND = not determined. a The results are expressed as IC50 ± SD of three different experiments each performed in duplicate. b Ratios between the IC50 values of each compound against W-2(CQ-R) or D-10(CQ-S) strains of P. falciparum. c HMEC: Human Microvascular Endothelial Cells. d Selectivity index = IC50 on HMEC-1/IC50 on P. falciparum, D10 strain. e Selectivity index = IC50 on HMEC-1/IC50 on P. falciparum, W2 strain.

dissolution in dichloromethane; the precipitate was filtered and the solution evaporated under reduced pressure. The residue was then recrystallized from petroleum ether. All of the reactions that involved the use of reagents sensitive to oxygen or hydrolysis were carried out under an inert atmosphere and the glassware was previously dry in oven at 110 °C. The reactions were monitored by thin layer chromatography (TLC) on Merck precoated silica GF254 plates or Alumina using when necessary iodine vapor for spot visualization. Mps: Optolab (Italy) instrument are uncorrected. FTIR spectra: Spectrum One (Perkin Elmer) (MA, USA) in a spectral region between 4000 and 450 cm 1 and analyzed by transmittance technique with 32 scansions and 4 cm 1 resolution. Solid samples were mixed in a mortar with KBr (1:100) and pressed in a hydraulic press (14 tons) to small tablets, while for liquid samples one drop was placed between two plates of sodium chloride. The 1H and 13C NMR and Bi-dimensional analyses were taken on a Bruker Avance 500 (Billerica, MA, USA) operating at 500 MHz for 1H and 125.75 MHz for 13C or with a Varian Mercury Plus 200, operating at 200 MHz for 1H and 50.3 MHz for 13C. Chemical shifts were expressed as

ppm (d) using the central peak of deuterated chloroform as the internal reference (dH = 7.23 ppm; dC = 77.3 ppm), J in Hertz. The APT sequence was used to distinguish methine and methyl carbon signals from those arising from methylene and quaternary carbon atoms. MS analyses: Thermo Finnigan (MA, USA) LCQ Advantage system equipped with a quaternary pump, Diode Array Detector (working wavelength 254 nm) and MS spectrometer with an Electrospray ionization source and an Ion Trap mass analyser; ionization: ESI positive or ESI negative; capillary temperature: 250 °C; source voltage: 5.50 kV; source current: 4.00 lA; multipole 1 and 2 offset, 5.50 V and 7.50 V, respectively; intermultipole lens voltage: 16.00 V; trap DC offset voltage: 10.00 V. 4.2. Synthesis of (2E,4E,6E)-octatrienal (1) Under nitrogen atmosphere, (2E,4E,6E)-octatrienol (8 mmol) purified as above reported, was dissolved in anhydrous dichloromethane (15 mL) and MnO2 powder (57 mmol) was added in small portions. The mixture was stirred for 5 h at 40 °C and then at room temperature for 48 h monitoring by TLC using as eluent

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hexane/AcOEt 7:3. After filtration through a celite cake, the yellow solution was evaporated under reduced pressure and directly used for the next step. Yield 75%. Mp 57 °C; FTIR (KBr) m = 3021, 2969, 2932, 2875, 2746, 1720, 1678, 1638, 1610, 1446, 1376, 1163, 1118, 1016, 995 cm 1. 1H NMR (200 MHz, CDCl3) d: 9.54 (d, 1H, J = 8.1 Hz, CHO), 7.11 (dd, 1H, J = 11.0, J = 15.4 Hz, H3), 6.65 (dd, 1H, J = 10.3, J = 14.7 Hz, H5), 6.39–5.95 (m, 4H, H2, H4, H6, H7), 1.85 (d, 3H, J = 6.4 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 193.9, 152.8, 143.5, 137.5, 131.6, 131.2, 128.1, 19.1. 4.3. Synthesis of Schiff bases 2a–e General method: Under nitrogen atmosphere, (2E,4E,6E)-octatrienal 1 (2.5 mmol) was dissolved in anhydrous toluene (10 mL); a solution of the appropriate amine (2.5 mmol) diluted in anhydrous toluene (15 mL) was slowly added dropwise and the mixture was magnetically stirred at room temperature for 24 h. The solvent was then evaporated under reduced pressure and the residue directly used for the next step. 4.3.1. (E)-N-((2E,4E,6E)-Octatrien-1-ylidene)-dodecan-1-amine (2a) Yield: 94%. Mp 68–69 °C. FTIR (KBR): m = 3014, 2956, 2917, 2851, 1622, 1610, 1471, 1439, 999, 716 cm 1. 1H NMR (200 MHz, CDCl3) d: 7.86 (d, 1H, J = 9.2 Hz, H8); 6.58 (dd, 1H, J = 15.0, J = 10.3 Hz, H6); 6.40–6.24 (m, 4H, @CH); 5.87 (dq, 1H, J = 13.60, J = 7.0 Hz, H2); 3.43 (t, 2H, J = 7.0 Hz, NCH2); 1.80 (d, 3H, J = 7.0 Hz, CH3CH@); 1.61 (q, 2H, NCH2CH2); 1.25 (m, 18H, CH2); 0.87 (t, 3H, J = 6.2 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 162.4, 141.4, 137.2, 132.8, 131.8, 131.1, 129.3, 61.8, 32.1, 31.2, 29.8, 29.7, 29.6, 29.5, 27.5, 22.8, 18.5, 14.2. 4.3.2. (E)-N-((2E,4E,6E)-Octatrien-1-ylidene)-tridecan-1-amine (2b) Yield: 99%. Mp 71–72 °C. FTIR (KBR): m = 3014, 2953, 2916, 2850, 1620, 1610, 1471, 1439, 1001, 716 cm 1. 1H NMR (200 MHz, CDCl3) d: 7.85 (d, 1H, J = 8.8 Hz, H8); 6.57 (dd, 1H, J = 15.4, J = 10.3 Hz, H6); 6.25–6.06 (m, 4H, @CH); 5.85 (dq, 1H, J = 7.0, J = 13.5 Hz, H2); 3.43 (t, 2H, J = 7.0 Hz, NCH2); 1.80 (d, 3H, J = 7.0 Hz, CH3CH@); 1.60 (q, 2H, NCH2CH2); 1.24 (m, 20H, CH2); 0.87 (t, 3H, J = 5.9 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 162.4, 141.4, 137.2, 132.8, 131.8, 131.1, 129.3, 61.8, 32.1, 31.1, 29.8, 29.7, 29.6, 29.5, 27.5, 22.8, 18.5, 14.2. 4.3.3. (E)-N-((2E,4E,6E)-Octatrien-1-ylidene)-tetradecan-1-amine (2c) Yield: 97%. Mp 72–73 °C. FTIR (KBR): m = 3014, 2956, 2916, 2851, 1622, 1610, 1471, 1439, 998, 716 cm 1. 1H NMR (200 MHz, CDCl3) d: 7.86 (d, 1H, J = 8.8 Hz, H8); 6.59 (dd, 1H, J = 15.4, J = 10.3 Hz, H6); 6.44–6.26 (m, 4H, @CH); 5.86 (dq, 1H, J = 6.6, J = 14.6 Hz, H2); 3.44 (t, 2H, J = 7.0 Hz, NCH2); 1.81 (d, 3H, J = 6.9 Hz, CH3CH@); 1.61 (q, 2H, NCH2CH2); 1.25 (m, 22H, CH2); 0.89 (t, 3H, J = 6.2 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 162.4, 141.4, 137.3, 132.8, 131.8, 131.1, 129.3, 61.9, 32.1, 31.2, 29.9, 29.8, 29.7, 29.6, 29.5, 27.5, 22.8, 18.5, 14.2. 4.3.4. (E)-N-((2E,4E,6E)-Octatrien-1-ylidene)-hexadecan-1-amine (2d) Yield: 80%. Mp 77–78 °C. FTIR (KBR): m = 3014, 2957, 2916, 2851, 1622, 1610, 1472, 1000, 716 cm 1. 1H NMR (200 MHz, CDCl3) d: 7.85 (d, 1H, J = 9.1 Hz, H8); 6.57 (dd, 1H, J = 15.4, J = 10.3 Hz, H6); 6.24–6.08 (m, 4H, @CH); 5.83 (dq, 1H, J = 6.6, J = 14.6 Hz, H2); 3.43 (t, 2H, J = 7.0 Hz, NCH2); 1.80 (d, 3H, J = 6.9 Hz, CH3CH@); 1.61 (q, 2H, NCH2CH2); 1.24 (m, 26H, CH2); 0.87 (t, 3H, J = 5.9 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 162.5,

141.5, 137.4, 133.0, 131.7, 131.1, 129.3, 61.9, 32.1, 31.2, 29.9, 29.8, 29.6, 29.5, 27.6, 22.9, 18.6, 14.3. 4.3.5. (E)-N-((2E,4E,6E)-Octatrien-1-ylidene)-octadecan-1-amine (2e) Yield: 95%. Mp 70–72 °C. FTIR (KBR): m = 3014, 2953, 2918, 2849, 1472, 1462, 993, 719 cm 1. 1H NMR (200 MHz, CDCl3) d: 7.85 (d, 1H, J = 9.1 Hz, H8); 6.57 (dd, 1H, J = 15.4, J = 10.6 Hz, H6); 6.24–6.06 (m, 4H, @CH); 5.85 (dq, 1H, J = 6.6, J = 14.7 Hz, H2); 3.43 (t, 2H, J = 6.9 Hz, NCH2); 1.8 (d, 3H, J = 6.9 Hz, CH3CH@); 1.60 (q, 2H, NCH2CH2); 1.24 (m, 30H, CH2); 0.87 (t, 3H, J = 5.9 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 162.5, 141.5, 137.4, 133.0, 131.7, 131.0, 129.3, 61.9, 32.1, 31.2, 29.9, 29.8, 29.6, 29.5, 27.6, 22.9, 18.6, 14.3. 4.4. Synthesis of amines 3a–e General method: Under nitrogen atmosphere to a solution of Schiff base 2a–e (1.73 mmol) in dry ethanol (25 mL), sodium borohydride was added in small portion ensuring that the temperature did not rise. After 3 h of magnetic stirring at room temperature, water (20 mL) was added and the mixture was extracted with diethyl ether. The organic phase was dried over anhydrous Na2SO4, filtered and then evaporated to dryness. 4.4.1. N-((2E,4E,6E)-Octatrien-1-yl)-dodecan-1-amine (3a) Yield: 99%. Mp 51–52 °C. FTIR (KBR): m = 3285, 3016, 2956, 2921, 2850, 2814, 2760, 1637, 1472, 1461, 1447, 1373, 1117, 989, 787, 719 cm 1. 1H NMR (200 MHz, CDCl3) d: 6.24–5.99 (m, 4H, @CH); 5.78–5.65 (m, 2H, H2, H7); 3.28 (d, 2H, J = 7.3 Hz, H8); 2.61 (t, 2H, J = 7.0 Hz, NCH2); 1.77 (d, 3H, J = 7.3 Hz, CH3CH@); 1.48 (q, 2H, NCH2CH2); 1.25 (m, 19H, CH2, NH); 0.87 (t, 3H, J = 6.2 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 132.5, 131.9, 131.8, 131.7, 130.1, 129.7, 51.8, 49.6, 32.1, 30.3, 29.8, 29.7, 29.5, 27.6, 22.8, 18.3, 14.2. 4.4.2. N-((2E,4E,6E)-Octatrien-1-yl)-tridecan-1-amine (3b) Yield: 99%. Mp 54–55 °C. FTIR (KBR): m = 3285, 3015, 2922, 2850, 2815, 2758, 1594, 1472, 1462, 1371, 1117, 998, 789, 719 cm 1. 1H NMR (200 MHz, CDCl3) d: 6.25–6.09 (m, 4H, @CH); 5.78–5.65 (m, 2H, H2, H7); 3.26 (d, 2H, J = 6.6 Hz, H8); 2.58 (t, 2H, J = 7.0 Hz, NCH2); 1.76 (d, 3H, J = 7.0 Hz, CH3CH@); 1.46 (q, 2H, NCH2CH2); 1.24 m, 21H, CH2, NH); 0.87 (t, 3H, J = 7.2 Hz, CH3). 13 C NMR (50.3 MHz, CDCl3) d: 132.4, 131.9, 131.8, 131.7, 130.1, 129.7, 51.9, 49.7, 32.1, 30.3, 29.8 (3C), 29.7 (2C), 29.5, 27.6, 22.8, 18.3, 14.2. 4.4.3. N-((2E,4E,6E)-Octatrien-1-yl)-tetradecan-1-amine (3c) Yield: 92%. Mp 57–58 °C. FTIR (KBR): m = 3286, 3016, 2955, 2920, 2850, 2814, 2761, 1637, 1472, 1462, 1374, 1118, 989, 787, 719 cm 1. 1H NMR (200 MHz, CDCl3) d: 6.24–6.09 (m, 4H, @CH); 5.69–5.56 (m, 2H, H2, H7); 3.27 (d, 2H, J = 6.6 Hz, H8); 2.58 (t, 2H, J = 7.0 Hz, NCH2); 1.77 (d, 3H, J = 7.0 Hz, CH3CH@); 1.46 (q, 2H, NCH2CH2); 1.25 (m, 23H, CH2, NH); 0.87 (t, 3H, J = 7.0 Hz, CH3). 13 C NMR (50.3 MHz, CDCl3) d: 132.4, 131.9, 131.8, 130.1, 129.7, 51.9, 49.7, 32.1, 30.4, 29.8, 29.5, 27.6, 22.8, 18.3, 14.2. 4.4.4. N-((2E,4E,6E)-Octatrien-1-yl)-hexadecan-1-amine (3d) Yield: 94%. Mp 60–62 °C. FTIR (KBR): m = 3286, 3016, 2955, 2920, 2850, 2814, 2759, 1636, 1472, 1462, 1373, 1118, 988, 787, 719 cm 1. 1 H NMR (200 MHz, CDCl3) d: 6.18–6.02 (m, 4H, @CH); 5.73–5.65 (m, 2H, H2, H7); 3.27 (d, 2H, J = 6.6 Hz, H8); 2.58 (t, 2H, J = 7.0 Hz, NCH2); 1.77 (d, 3H, J = 7.0 Hz, CH3CH@); 1.41 (q, 2H, NCH2CH2); 1.25 (m, 27H, CH2, NH); 0.87 (t, 3H, J = 6.5 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 132.5, 131.9, 131.8, 131.7, 130.1, 129.8, 51.9, 49.7, 32.1, 30.4, 29.9, 29.8, 29.7, 29.6, 27.6, 22.9, 18.5, 14.3.

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4.4.5. N-((2E,4E,6E)-Octatrien-1-yl)-octadecan-1-amine (3e) Yield: 93%. Mp 61–63 °C. FTIR (KBR): m = 3284, 3016, 2955, 2919, 2849, 2815, 2870, 1624, 1472, 1462, 1377, 1118, 989, 788, 719 cm 1. 1H NMR (200 MHz, CDCl3) d: 6.17–6.04 (m, 4H, @CH); 5.77–5.65 (m, 2H, H2, H7); 3.26 (d, 2H, J = 6.3 Hz, H8); 2.58 (t, 2H, J = 7.0 Hz, NCH2); 1.76 (d, 3H, J = 6.9 Hz, CH3CH@); 1.41 (q, 2H, NCH2CH2); 1.25 (m, 31H, CH2, NH); 0.87 (t, 3H, J = 6.0 Hz, CH3). 13 C NMR (50.3 MHz, CDCl3) d: 132.4, 131.9, 131.7, 130.1, 129.8, 51.9, 49.7, 32.1, 30.4, 29.9, 29.8, 29.7, 29.6, 27.6, 22.9, 18.4, 14.3. 4.5. Synthesis of N,N-dimethyl quaternary amines 4a–e General method: In a stainless steel autoclave (200 mL), amines 3a–e (5.75 mmol) in anhydrous acetone (20 mL), diisopropylethylamine (5.75 mmol, 1 mL) and methyl iodide (34.52 mmol, 2.15 mL) were stirred for 24 h at 80 °C. After cooling at room temperature, the mixture was diluted with diethyl ether (150 mL) and washed with water. The organic layer was dried under Na2SO4, filtered, evaporated under reduced pressure and the residue was purified by column chromatography (silica gel) using a mixture of AcOEt/MeOH (8:2) as eluent. 4.5.1. N,N-Dimethyl-N-((2E,4E,6E)-octatrien-1-yl)-dodecan-1ammonium iodide (4a) Yield: 68%. Mp 73 °C. FTIR (KBR): m = 3013, 2924, 2853, 1635, 1571, 1415, 1004, 721 cm 1. 1H NMR (200 MHz, CDCl3) d: 6.75 (dd, 1H, J = 10.6, J = 15.0 Hz, H6); 6.40 (dd, 1H, J = 10.3, J = 15.0 Hz, H4); 6.13–6.05 (m, 2H, H5, H3); 5.84 (dq, 1H, J = 6.3, J = 15.0 Hz, H2); 5.63 (dt, 1H, J = 8.0, J = 15 Hz, H7); 4.40 (d, 2H, J = 7.7 Hz, H8); 3.43 (m, 2H, CH2); 3.27 (s, 6H, CH3N); 1.81 (d, 3H, J = 6.3 Hz, CH3CH@); 1.74–1.56 (m, 2H, NCH2CH2); 1.12–1.32 (m, 18H, CH2); 0.85 (t, 3H, J = 5.9 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 144.7, 138.8, 134.3, 131.1, 127.9, 115.3, 66.4, 63.9, 50.5, 32.1, 29.9, 29.8, 29.6, 29.5, 29.4, 26.4, 23.6, 22.8, 18.6, 14.3. ESI-MS m/z calcd for C22H42IN [M+H]+: 447, found = 320.1 [M I]. Elemental Analysis: calcd C, 59.05; H, 9.46; N, 3.13; found C, 59.24; H, 9.37; N, 3.09. 4.5.2. N,N-Dimethyl-N-((2E,4E,6E)-octatrien-1-yl)-tridecan-1ammonium iodide (4b) Yield: 66%. Mp 74 °C. FTIR (KBR): m = 3013, 2924, 2853, 1634, 1435, 1004, 721 cm 1. 1H NMR (200 MHz, CDCl3) d: 6.75 (dd, 1H, J = 10.6, J = 15.0 Hz, H6); 6.40 (dd, 1H, J = 10.3, J = 15.0 Hz, H4); 6.13–6.05 (m, 2H, H5, H3); 5.84 (dq, 1H, J = 6.3, J = 15.0 Hz, H2); 5.63 (dt, 1H, J = 8.0, J = 15 Hz, H7); 4.40 (d, 2H, J = 7.7 Hz, H8); 3.43 (m, 2H, NCH2); 3.27 (s, 6H, CH3N); 1.81 (d, 3H, J = 6.3 Hz, CH3CH@); 1.74–1.56 (m, 2H, NCH2CH2); 1.12–1.32 (m, 20H, CH2); 0.85 (t, 3H, J = 5.9 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 144.7, 138.8, 134.3, 131.1, 127.9, 115.3, 66.4, 63.9, 50.5, 32.1, 29.9, 29.8, 29.6, 29.5, 29.4, 26.4, 23.6, 22.8, 18.6, 14.3. ESI-MS m/z calcd for C23H44IN [M+H]+: 461, found = 334.1 [M I]. Elemental Analysis: calcd C, 59.86; H, 9.61; N, 3.03; found C, 59.63; H, 9.50; N, 3.01. 4.5.3. N,N-Dimethyl-N-((2E,4E,6E)-octatrien-1-yl)-tetradecan-1ammonium iodide (4c) Yield: 67%. Mp 79 °C. FTIR (KBr): m = 3013, 2923, 2853, 1633, 1465, 1004, 721 cm 1. 1H NMR (200 MHz, CDCl3) d: 6.76 (dd, 1H, J = 11.0, J = 15.0 Hz, H6); 6.39 (dd, 1H, J = 11.0, J = 15.0 Hz, H4); 6.17–6.11 (m, 2H, H5, H3); 5.86 (dq, 1H, J = 6.7, J = 15.0 Hz, H2); 5.65 (dt, 1H, J = 7.7, J = 15.0 Hz, H7); 4.40 (d, 2H, J = 7.7 Hz, H8); 3.45 (m, 2H, NCH2); 3.30 (s, 6H, CH3); 1.81 (d, 3H, J = 6.3 Hz, CH3CH@); 1.74–1.56 (m, 2H, NCH2CH2); 1.35–1.20 (m, 22H, CH2); 0.87 (t, 3H, J = 7 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 144.7, 138.8, 134.3, 131.1, 127.4, 115.3, 66.4, 63.9, 50.5, 32.1, 30.0, 29.9, 29.8, 29.7, 29.6, 29.5, 29.4, 26.5, 23.1, 22.9, 18.6, 14.3. ESI-MS m/z calcd for C24H46IN [M+H]+: 475.5, found = 348.3 [M I]. Elemental Analysis: calcd C, 60.62; H, 9.75; N, 2.95; found C, 60.55; H, 9.49; N, 2.81.

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4.5.4. N,N-Dimethyl-N-((2E,4E,6E)-octa-2,4,6-trien-1-yl)-hexdecan1-ammonium iodide (4d) Yield: 62%. Mp 82 °C. FTIR (KBR): m = 3013, 2999, 2919, 2850, 1634, 1467, 1006, 890, 721 cm 1. 1H NMR (500 MHz, CDCl3) d: 6.76 (dd, 1H, J = 11.0, J = 15.0 Hz, H6); 6.39 (dd, 1H, J = 11.0, J = 15.0 Hz, H4); 6.13 (dd, 1H, J = 11.0, J = 16.0 Hz, H5); 6.05 (dd, 1H, J = 11.0, J = 15.0 Hz, H3); 5.86 (dq, 1H, J = 6.7, J = 15.0 Hz, H2); 5.65 (dt, 1H, J = 7.7, J = 15.0 Hz, H7); 4.40 (d, 2H, J = 7.7 Hz, H8); 3.45 (m, 2H, NCH2); 3.30 (s, 6H, CH3); 1.81 (d, 3H, J = 6.3 Hz, CH3CH@); 1.79–1.65 (m, 2H, NCH2CH2); 1.75 (m, 2H, NCH2CH2); 1.35–1.20 (m, 26H, CH2); 0.87 (t, 3H, J = 7 Hz, CH3). 13C NMR (125.75 MHz, CDCl3) d: 143.8, 137.8, 133.4, 130.2, 126.5, 114.5, 65.4, 62.9, 49.6, 31.2, 28.9 (3C), 28.8, 28.7, 28.6, 28.5, 25.5, 22.1, 21.9, 17.7, 13.4. ESI-MS m/z calcd for C26H50IN [M+H]+: 503.6, found = 376.2 [M I]. Elemental Analysis: calcd C, 62.01; H, 10.01; N, 2.78; found C, 61.88; H, 9.94; N = 2.61%. 4.5.5. N,N-Dimethyl-N-((2E,4E,6E)-octatrien-1-yl)-octadecan-1ammonium iodide (4e) Yield: 62%. Mp 89 °C. FTIR (KBR): m = 2999, 2918, 2850, 1634, 1467, 1006, 721 cm 1. 1H NMR (200 MHz, CDCl3) d: 6.75 (dd, 1H, J = 10.6, J = 15.0 Hz, H6); 6.37 (dd, 1H, J = 10.3, J = 15.0 Hz, H4); 6.18–6.06 (m, 2H, H5, H3); 5.82 (dq, 1H, J = 6.6, J = 15.0 Hz, H2); 5.60 (dt, 1H, J = 7.7, J = 15.0 Hz, H7); 4.38 (d, 2H, J = 7.7 Hz, H8); 3.41 (m, 2H, NCH2); 3.27 (s, 6H, CH3); 1.79 (d, 3H, J = 6.3 Hz, CH3CH@); 1.78–1.75 (m, 2H, NCH2CH2); 1.35–1.50 (m, 26H, CH2); 0.86 (t, 3H, J = 7 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 144.8, 138.8, 134.3, 133.4, 131.2, 127.4, 126.5, 115.3, 66.3, 63.9, 50.5, 32.1, 29.9, 29.6, 29.5, 29.4, 26.4, 23.1, 22.9, 18.6, 14.3. ESI-MS m/z calcd for C28H54IN [M+H]+: 531.6, found = 404.3 [M I]. Elemental Analysis: calcd C, 63.26; H, 10.24; N, 2.63; found C, 62.97; H, 10.13; N, 2.61. 4.6. Synthesis of tertiary amines 5a–e General method: In a round bottom flask the appropriate secondary amine 3a–e (1 mmol), diisopropylenediamine (1 mmol, 0.27 mL) and benzyl chloride (1 mmol, 0.12 mL) were magnetically stirred for 3 h at 120 °C. The mixture was then cooled at room temperature, diluted with a NaHCO3 aqueous saturated solution and extracted with diethyl ether. The organic phase was washed with water, dried under Na2SO4 and evaporated to yield a yellow crude solid, which was purified by column chromatography using AcOEt as eluent. 4.6.1. N-Benzyl-N-((2E,4E,6E)-octatrien-1-yl)-dodecan-1-amine (5a) Yield: 55%. FTIR (NaCl): m = 3085, 3063, 3015, 2925, 2853, 2796, 1944, 1681, 1494, 1453, 1365, 1205, 1120, 1073, 1028, 995, 801, 735, 697 cm 1. 1H NMR (200 MHz, CDCl3) d: 7.28 (m, 5H, Harom); 6.21–6.11 (m, 4H, @CH); 5.78–5.65 (m, 2H, H2, H7); 3.54 (s, 2H, CH2-Ph); 3.09 (d, 2H, J = 6.6 Hz, H8); 2.40 (t, 2H, J = 7.0 Hz, NCH2); 1.77 (d, 3H, J = 6.6 Hz, CH3CH@); 1.46 (q, 2H, NCH2CH2); 1.25 (m, 18H, CH2); 0.88 (t, 3H, J = 6.2 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 140.2, 132.9, 132.2, 131.9, 131.07, 130.2, 129.7, 129.0, 128.9, 128.3, 126.9, 58.4, 56.0, 53.8, 32.1, 29.8, 29.7, 29.5, 27.6, 27.3, 22.8, 18.4, 14.3. 4.6.2. N-Benzyl-N-((2E,4E,6E)-octatrien-1-yl)-tridecan-1-amine (5b) Yield: 60%. FTIR (NaCl): m = 3085, 3063, 3015, 2924, 2853, 2796, 1944, 1679, 1494, 1453, 1366, 1206, 1137, 1073, 1028, 995, 800, 732, 698 cm 1. 1H NMR (200 MHz, CDCl3) d: 7.27 (m, 5H, Harom); 6.21–6.11 (m, 4H, @CH); 5.73–5.65 (m, 2H, H2, H7); 3.54 (s, 2H, CH2Ph); 3.09 (d, 2H, J = 6.6 Hz, H8); 2.39 (t, 2H, J = 7.0 Hz, NCH2); 1.77 (d, 3H, J = 6.6 Hz, CH3CH@); 1.45 (q, 2H, NCH2CH2); 1.24 (m,

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N. Basilico et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

20H, CH2); 0.88 (t, 3H, J = 6.2 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 140.2, 133.0, 132.2, 131.9, 131.1, 130.2, 129.7, 129.0, 128.9 128.3, 126.8, 58.5, 56.1, 53.8, 32.1, 29.8, 29.7, 29.5, 27.6, 27.3, 22.8, 18.4, 14.3.

59.9, 47.4, 32.1, 29.8, 29.6, 29.5, 29.3, 26.6, 23.0, 22.9, 18.7, 14.3. ESI-MS m/z calcd for C28H46IN [M+H]+: 523.3, found = 396.2 [M I]. Elemental Analysis: calcd C, 64.23; H, 8.86; N, 2.68; found C, 64.00; H, 8.68; N, 2.47.

4.6.3. N-Benzyl-N-((2E,4E,6E)-octatrien-1-yl)-tetradecan-1-amine (5c) Yield: 62%. FTIR (NaCl): m = 3085, 3063, 3015, 2924, 2853, 2797, 1681, 1494, 1453, 1367, 1206, 1137, 1028, 996, 800, 725, 698 cm 1. 1 H NMR (200 MHz, CDCl3) d: 7.27 (m, 5H, Harom); 6.15–6.11 (m, 4H, @CH); 5.73–5.81 (m, 2H, H2, H7); 3.56 (s, 2H, CH2Ph); 3.10 (d, 2H, J = 6.6 Hz, H8); 2.41 (t, 2H, J = 7.0 Hz, NCH2); 1.78 (d, 3H, J = 6.6 Hz, CH3CH@); 1.50 (q, 2H, NCH2CH2); 1.26 (m, 22H, CH2); 0.91 (t, 3H, J = 6.2 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 140.2, 132.9, 132.2, 131.9, 131.1, 130.2, 129.7, 129.1, 128.9, 128.3, 126.9, 58.4, 56.1, 53.8, 32.1, 29.9, 29.8, 29.7, 29.6, 27.6, 27.3, 22.9, 18.4, 14.3.

4.7.2. N-Benzyl-N-methyl-N-((2E,4E,6E)-octatrien-1-yl)-tridecan1-ammonium iodide (6b) Yield: 92%. FTIR (NaCl): m = 3014, 2954, 2923, 2850, 1724, 1633, 1599, 1456, 1004, 702 cm 1. 1H NMR (200 MHz, CDCl3) d: 7.63 (m, 2H, Harom); 7.43 (m, 3H, Harom); 6.73 (dd, 1H, J = 10.3, J = 15.0 Hz, H6); 6.38 (dd, 1H, J = 10.3, J = 15.0 Hz, H4); 6.14–6.05 (m, 2H, H5, H3); 5.81 (dq, 1H, J = 6.7, J = 15.0 Hz, H2); 5.61 (dt, 1H, J = 7.7, J = 15.0 Hz, H7); 5.0 (d, 1H, J = 12.8 Hz, CH2-Ph); 4.8 (d, 1H, J = 12.8 Hz, CH2-Ph); 4.4–4.3 (m, 2H, H8); 3.22 (m, 2H, NCH2); 3.13 (s, 3H, CH3N); 1.89–1.85 (m, 2H, NCH2CH2) 1.80 (d, 3H, J = 6.7 Hz, CH3CH@); 1.42–1.23 (m, 20H, CH2); 0.87 (t, 3H, J = 5.4 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 144.6, 138.7, 134.4, 133.2, 131.1, 131.0, 129.5, 127.4, 127.3, 115.2, 64.7, 63.5, 59.9, 47.4, 32.1, 29.8, 29.6, 29.5, 29.3, 26.6, 23.0, 22.9, 18.7, 14.3. ESI-MS m/z calcd for C29H48IN [M+H]+: 537.6, found = 410.7 [M I]. Elemental Analysis: calcd C, 64.79; H, 9.00; N, 2.61; found C, 64.55; H, 8.89; N, 2.54.

4.6.4. N-Benzyl-N-((2E,4E,6E)-octatrien-1-yl)-hexadecan-1-amine (5d) Yield: 70%. FTIR (NaCl): m = 3085, 3063, 3015, 2924, 2853, 2796, 1726, 1494, 1465, 1454, 1365, 1121, 1028, 995, 734, 697 cm 1. 1H NMR (200 MHz, CDCl3) d: 7.28 (m, 5H, Harom); 6.13–6.08 (m, 4H, @CH); 5.73–5.65 (m, 2H, H2, H7); 3.54 (s, 2H, CH2Ph); 3.09 (d, 2H, J = 6.6 Hz, H8); 2.40 (t, 2H, J = 7.0 Hz, NCH2); 1.77 (d, 3H, J = 6.6 Hz, CH3CH@); 1.46 (q, 2H, NCH2CH2); 1.25 (m, 26H, CH2); 0.88 (t, 3H, J = 6.2 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 140.1, 133.0, 132.2, 131.9, 131.1, 130.2, 129.8, 129.1, 128.9, 128.3, 126.9, 58.4, 56.0, 53.8, 32.2, 29.9, 29.8, 29.7, 29.6, 27.6, 27.3, 22.9, 18.5, 14.3. 4.6.5. N-Benzyl-N-((2E,4E,6E)-octatrien-1-yl)-octadecan-1-amine (5e) Yield: 70%. FTIR (NaCl): m = 3015, 2924, 2853, 2796, 1726, 1602, 1494, 1454, 1365, 1286, 1121, 1028, 995, 734, 697 cm 1. 1H NMR (200 MHz, CDCl3) d: 7.28 (m, 5H, Harom); 6.13–6.08 (m, 4H, @CH); 5.73–5.65 (m, 2H, H2, H7); 3.54 (s, 2H, CH2Ph); 3.09 (t, 2H, J = 6.6 Hz, H8); 2.40 (t, 2H, J = 7.0 Hz, NCH2); 1.77 (d, 3H, J = 6.6 Hz, CH3CH@); 1.46 (q, 2H, NCH2CH2); 1.25 (m, 22H, CH2); 0.88 (t, 3H, J = 6.2 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 140.2, 133.2, 131.9, 131.1, 130.2, 129.8, 129.1, 128.3, 126.9, 58.4, 56.0, 53.8, 32.2, 29.9, 29.8, 29.7, 29.6, 27.6, 27.3, 22.9, 18.5, 14.3. 4.7. Synthesis of quaternary amines 6a–e General method: To solution of amines 5a–e (0.48 mmol) in dry acetone, methyl iodide (0.48 mmol, 60.4 lL) was added. The mixture was stirred for 3 h at 58 °C and then concentrated to dryness under vacuum; the residue was then suspended in AcOEt (20 mL) and washed with water. The organic phase was dried over anhydrous Na2SO4, filtered and evaporated. The crude residue was purified by column chromatography (silica gel) using AcOEt/MeOH 9:1 as eluent. 4.7.1. N-Benzyl-N-methyl-N-((2E,4E,6E)-octatrien-1-yl)-dodecan1-ammonium iodide (6a) Yield: 90%. FTIR (NaCl): m = 3014, 2954, 2924, 2853, 1724, 1633, 1597, 1456, 1004, 702 cm 1. 1H NMR (200 MHz, CDCl3) d: 7.63 (m, 2H, Harom); 7.43 (m, 3H, Harom); 6.73 (dd, 1H, J = 10.3, J = 15.0 Hz, H6); 6.38 (dd, 1H, J = 10.3, J = 15.0 Hz, H4); 6.14–6.05 (m, 2H, H5, H3); 5.81 (dq, 1H, J = 6.7, J = 15.0 Hz, H2); 5.61 (dt, 1H, J = 7.7, J = 15.0 Hz, H7); 5.0 (d, 1H, J = 12.8 Hz, CH2-Ph); 4.8 (d, 1H, J = 12.8 Hz, CH2-Ph); 4.4–4.3 (m, 2H, H8); 3.22 (m, 2H, NCH2); 3.13 (s, 3H, CH3N); 1.89–1.85 (m, 2H, NCH2CH2) 1.80 (d, 3H, J = 6.7 Hz, CH3CH@); 1.42–1.23 (m, 18H, CH2); 0.87 (t, 3H, J = 5.4 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 144.6, 138.7, 134.4, 133.2, 131.1, 131.0, 129.5, 127.4, 127.3, 115.2, 64.7, 63.5,

4.7.3. N-Benzyl-N-methyl-N-((2E,4E,6E)-octatrien-1-yl)-tetradecan1-ammonium iodide (6c) Yield: 74%. FTIR (NaCl): m = 3014, 2956, 2923, 2855, 1729, 1704, 1633, 1599, 1463, 1455, 1006, 890, 702 cm 1. 1H NMR (200 MHz, CDCl3) d: 7.61 (m, 2H, Harom); 7.42 (m, 3H, Harom); 6.75 (dd, 1H, J = 10.6, J = 15.0 Hz, H6); 6.34 (dd, 1H, J = 10.6, J = 15.0 Hz, H4); 6.18–6.06 (m, 2H, H5, H3); 5.81 (dq, 1H, J = 6.6, J = 15.0 Hz, H2); 5.65 (dt, 1H, J = 7.7, J = 15.0 Hz, H7); 5.01 (d, 1H, J = 12.8 Hz, CH2Ph); 4.86 (d, 1H, J = 12.8 Hz, CH2-Ph); 4.4–4.3 (d, 2H, J = 7.7, H8); 3.19 (m, 2H, NCH2); 3.10 (s, 3H, CH3); 1.90–1.87 (m, 2H, NCH2CH2); 1.86 (d, 3H, J = 6.6 Hz, CH3); 1.29–1.22 (m, 22H, CH2); 0.85 (t, 3H, J = 7.0 Hz, CH3). 13C NMR (53 MHz, CDCl3) d: 144.4, 138.6, 134.2, 133.3, 131.1, 130.9, 129.5, 127.5, 127.3, 115.3, 64.7, 63.5, 60.1, 47.2, 32.1, 29.8, 29.7, 29.6, 29.5, 29.3, 26.6, 23.1, 22.9, 18.6, 14.3. ESI-MS m/z calcd for C30H50IN [M+H]+: 551.7, found = 424.3 [M I]. Elemental Analysis: calcd C, 65.32; H, 9.14; N, 2.54; found C, 65.17; H, 8.98; N, 2.36. 4.7.4. N-Benzyl-N-methyl-N-((2E,4E,6E)-octatrien-1-yl)-hexadecan1-ammonium iodide (6d) Yield: 60%. Mp >300 °C. FTIR (NaCl): m = 3014, 2956, 2918, 2855, 1735, 1704, 1633, 1585, 1454, 1009, 702 cm 1. 1H NMR (500 MHz, CDCl3) d: 7.67 (m, 2H, Harom); 7.42 (m, 3H, Harom); 6.72 (dd, 1H, J = 10.7, J = 14.8 Hz, H6); 6.38 (dd, 1H, J = 10.7, J = 14.9 Hz, H4); 6.15 (dd, 1H, J = 10.8, J = 14.9 Hz, H5); 6.01 (m, 1H, H3); 5.85 (dq, 1H, J = 6.8, J = 14.7 Hz, H2); 5.85 (dt, 1H, J = 7.7, J = 15.0 Hz, H7); 5.01 (d, 1H, J = 12.8 Hz, CH2-Ph); 4.86 (d, 1H, J = 12.8 Hz, CH2-Ph); 4.38–4.32 (m, 2H, H8); 3.25 (m, 2H, NCH2); 3.12 (s, 3H, CH3CH); 1.90–1.86 (m, 2H, CH2); 1.85 (d, 3H, J = 6.8 Hz, CH3); 1.31–1.23 (m, 24H, CH2); 0.88 (t, 3H, J = 6.8 Hz, CH3). 13C NMR (125.75 MHz, CDCl3) d: 143.5, 137.7, 133.3, 132.4, 130.2, 130.0, 128.6, 126.5, 126.4, 114.4, 63.8, 62.6, 60.1, 46.3, 31.2, 29.0, 28.9, 28.7, 28.6, 28.5, 28.4, 28.3, 28.2, 25.7, 22.1, 21.9, 17.8, 13.4. ESIMS m/z calcd for C32H54IN [M+H]+: 579.6, found = 452.2 [M I]. Elemental Analysis: calcd C, 66.30; H, 9.39; N, 2.42; found C, 66.08; H, 9.21; N, 2.25. 4.7.5. N-Benzyl-N-methyl-N-((2E,4E,6E)-octatrien-1-yl)-octadecan1-ammonium iodide (6e) Yield: 50%. Mp >300 °C. FTIR (NaCl): m = 3014, 2956, 2918, 2855, 1735, 1704, 1633, 1585, 1454, 1009, 702 cm 1. 1H NMR (500 MHz, CDCl3) d: 7.65 (m, 2H, Harom); 7.44 (m, 3H, Harom); 6.72 (dd, 1H, J = 10.7, J = 14.8 Hz, H6); 6.38 (dd, 1H, J = 10.7, J = 14.9 Hz, H4);

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6.15 (dd, 1H, J = 10.8, J = 14.9 Hz, H5); 6.01 (m, 1H, H3); 5.85 (dq, 1H, J = 6.8, J = 14.7 Hz, H2); 5.85 (dt, 1H, J = 7.7, J = 15.0 Hz, H7); 5.01 (d, 1H, J = 12.8 Hz, CH2-Ph); 4.86 (d, 1H, J = 12.8 Hz, CH2-Ph); 4.38–4.32 (m, 2H, H8); 3.25 (m, 2H, NCH2); 3.12 (s, 3H, CH3CH); 1.90–1.86 (m, 2H, CH2); 1.85 (d, 3H, J = 6.8 Hz, CH3); 1.31–1.23 (m, 24H, CH2); 0.88 (t, 3H, J = 6.8 Hz, CH3). 13C NMR (125.75 MHz, CDCl3) d: 143.5, 137.7, 133.3, 132.4, 130.2, 130.0, 128.6, 126.5, 126.4, 114.4, 63.8, 62.6, 60.1, 46.3, 31.2, 29.0, 28.9, 28.7, 28.6 (2C), 28.5, 28.4, 28.3, 28.2, 25.7, 22.1, 21.9, 17.8, 13.4. ESI-MS m/z calcd for C34H58IN [M+H]+: 607.6, found = 482.7 [M I]. Elemental Analysis: calcd C, 67.19; H, 9.62; N, 2.30; found C, 67.07; H, 9.45; N, 2.14. 4.8. Synthesis of N,N-dimethyl-N-octyldodecan-1-ammonium bromide21 (7) N,N-Dimethyldodecyl bromide (2.34 mmol) and 1-bromooctane (4.68 mmol) were magnetically stirred for 72 h at 50 °C. The reaction product was triturated with cyclohexane. Yield: 83%. Mp 73 °C. FTIR (KBR): m = 2955, 2924, 2854, 1486, 1467, 722 cm 1. 1H NMR (200 MHz, CDCl3) d: 3.47 (m, 4H, CH2N); 3.38 (s, 6H, CH3N); 1.67 (m, 4H, NCH2CH2); 1.30–1.22 (m, 28H, CH2); 0.84 (t, 6H, J = 5.9 Hz, CH3). 13C NMR (50.3 MHz, CDCl3) d: 64.04, 51.5, 32.8, 31.8, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 27.1, 26.5, 23.0, 22.8, 22.7, 14.3, 14.2. ESI-MS m/z calcd for C22H48BrN [M+H]+: 406, found = 326.1 [M Br]. Elemental Analysis: calcd C, 65.00; H, 11.90; N, 3.45; found C, 64.24; H, 11.65; N, 3.27. 5. Biological evaluation

in MCDB 131 medium (Invitrogen, Milan, Italy) supplemented with 10% fetal calf serum (HyClone, Celbio, Milan, Italy), 10 ng/mL of epidermal growth factor (Chemicon), 1 lg/mL of hydrocortisone, 2 mM glutamine, 100 U/mL of penicillin, 100 lg/mL of streptomycin, and 20 mM Hepes buffer (EuroClone). Unless stated otherwise, all reagents were from Sigma Italia, Milan, Italy. For the cytotoxicity assays, cells were treated with serial dilutions of test compounds and cell proliferation evaluated using the MTT assay already described.27 Plates were incubated for 72 h at 37 °C in 5% CO2, then 20 lL of a 5 mg/mL solution of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) (M-2128 Sigma) in PBS was added for an additional 3 h at 37 °C. The plates were then centrifuged, the supernatants discarded and the dark blue formazan crystals dissolved using 100 lL of lysing buffer consisting of 20% (w/v) of a solution of SDS (Sigma), 40% of N,N dimethylformamide (Merck) in H2O, at pH 4.7 adjusted with 80% acetic acid. The plates were then read on a microplate reader (Synergy 4 BioTek Instruments) at a test wavelength of 550 nm and a reference wavelength of 650 nm. The results are expressed as IC50, which is the dose of compound necessary to inhibit cell growth by 50%. All the tests were performed in triplicate at least three times. References and notes 1. 2. 3. 4. 5. 6.

5.1. Parasites cultures Biological tests have been done on in vitro cultured P. falciparum parasites according to Trager and Jensen22 with slight modifications.23 The CQ-sensitive (D10) and the CQ-resistant (W2) strains were maintained at 5% haematocrit (human type A-positive red blood cells) in RPMI 1640 (EuroClone) medium supplemented with 1% AlbuMaxII (lipid-rich bovine serum albumin) (Invitrogen), 0.01% hypoxantine (Sigma), 20 mM Hepes (EuroClone), 2 mM glutamine (EuroClone). All the cultures were maintained at 37 °C in a standard gas mixture consisting of 1% O2, 5% CO2, 94% N2. The tested compounds and the reference drug (CQ) were dissolved in either water or DMSO to a concentration of 10 mg/mL and then diluted with complete medium to achieve the required concentrations (final DMSO concentration 61%, which is non-toxic to the parasite). Drugs were placed in 96-wells flat-bottomed microplates (Costar) and serial dilutions made. Asynchronous cultures with parasitaemia of 1–1.5% and 1% final haematocrit were aliquoted into the plates and incubated for 72 h at 37 °C. The parasites growth was determined spectrophotometrically (OD650) by measuring the activity of the parasite lactate dehydrogenase (pLDH), according to a modified version of Makler’s method in control and drug-treated cultures.24 Antimalarial activity was determined as concentration of drugs inducing 50% of growth inhibition (IC50). Each IC50 value is the mean and standard deviation of at least three independent experiments performed in duplicate.25 5.2. Cell cytotoxicity assays

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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

26. 27.

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The long-term human microvascular endothelial cell line (HMEC-1) immortalized by SV 40 large T antigen26 was maintained

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