2-(4- Methoxyphenyl)Ethyl]Oleamide

U.P.B. Sci. Bull., Series B, Vol. 76, Iss. 4, 2014 ISSN 1454 – 2331

SYNTHESIS AND CHARACTERIZATION OF N-[2-(4- METHOXYPHENYL)ETHYL]OLEAMIDE

Cătălina Neguţ (Cioateş)1, Eleonora-Mihaela UNGUREANU2, Florea COCU3, Constantin TĂNASE4, Constantin DRĂGHICI5, Cristian MUNTEANU6

This work is devoted to the synthesis and electrochemical characterization of N-[2-(4-methoxyphenyl)ethyl]oleamide. Its synthesis was realized by esterification of oleic acid to methyl oleate and reaction of this ester with N-2-(4- methoxyphenyl)ethylamine in anhydrous methanol, catalized by sodium methoxide. The new oleamide was fully characterized by IR, MS, 1H- and 13C-NMR spectra. Electrochemical investigations were also performed by cyclic and differential pulse voltammetry.

Keywords: N-[2-(4-methoxyphenyl)ethyl]oleamide, IR, MS, 1H- and 13C-NMR spectra, cyclic voltammetry, differential pulse voltammetry

1. Introduction

It is already known that N-acylethanolamides (NAEs) are a group of lipid mediators synthesized in the organism as a response of a many physiological and pathological stimuli, like obesity, inflammation and food intake. Between them, N-oleoylamides and also N-arachinoylethanolamides were investigated very intensively, especially in appetite and obesity regulation [1,2]. Similarly to oleylamides, N-palmitoylethanolamide (PEA) [3] and N-stearoylethanolamide (SEA) [4] were studied in relation to antiinflammatory and apoptotic properties. In the same time, from the group of macamides [5] (benzamide derivatives of saturated and unsaturated C-18 fatty acids), discovered in the Maca (Lepidium meyenii) plant, a traditional food crop of the Peruvian Andes now widely touted

1 PhD student, Faculty of Applied Chemistry and Material Sciences, University POLITEHNICA of Bucharest, Institute for Chemical – Pharmaceutical Research and Development ICCF, Bucharest, Romania, e-mail: [email protected] 2 Prof., Inorganic Chemistry, Physical Chemistry and Electrochemistry, University POLITEHNICA of Bucharest, Romania 3 Researcher, National Institute for Chemical – Pharmaceutical Research and Development ICCF, Bucharest, Romania 4 Researcher, National Institute for Chemical – Pharmaceutical Research and Development ICCF, Bucharest, Romania 5 Researcher, Organic Chemistry Center “C.D.Neniţescu”, Bucharest, Romania 6 PhD student, Romanian Academy-Institute of Biochemistry (IBAR), Bucharest, Romania 174 C. Neguţ (Cioateş), E.-M.Ungureanu, F. Cocu, C. Tănase, C. Drăghici, C. Munteanu

as a dietary supplement, five of them are oleylbenzylamides substituted in meta, or pyridylmethyl-6 substituted amides. Due to their importance, our group was involved in the synthesis of new oleamides in order to find useful properties by introducing new fragments in the amide part of the molecule. The present paper concerns the synthesis of N-[2-(4- methoxyphenyl)ethyl]oleamide 1 (Fig. 1) which was characterized physico- chemical and electrochemical methods prior to determination of its biological activity. H 1 N 1' 2' 9 C OCH3 om 10 O 17 18 1 Fig. 1. Structure of N-[2-(4-methoxyphenyl)ethyl]oleamide

2. Experimental

2.1. Reagents and procedure All compounds used for synthesis were purchased from Merck and used without further purification. As solvent and supporting electrolyte acetonitrile and tetrabutylammonium perchlorate from Fluka were used (as received). Melting point was determined with OptiMelt. The 1H- and 13C-NMR spectra were recorded using Varian Gemini 300 BB instrument, operating at 300 1 13 MHz for H- NMR and at 75 MHz for C-NMR, with CDCl3 as solvent and TMS as internal standard. The IR spectra (ATR) were recorded on a Vertex 70 Brucker instrument. Mass spectra were recorded using LTQ Orbitrap Velos Pro, by injecting a solution of 100 fmol/μl in 0.1 % formic acid in methanol. The spectrum was acquired in SIM scan mode (416.35 ± 10 Da). The electrochemical experiments were carried out using a PGSTAT12 AUTOLAB potentiostat coupled to a three-compartment cell. The CV curves were generally recorded at 0.1V/s or at various rates (0.1 - 1V/s) when studying the influence of the scan rate. DPV curves were recorded at 0.01V/s with a pulse height of 0.025V and a step time of 0.2 s. The working electrode was a glassy carbon disk (diameter of 3mm). The active surface was polished before each determination with diamond paste (200 μm). The Ag/10 mM AgNO3 in 0.1 M TBAP, CH3CN was used as reference electrode. The potential was referred to the potential of the ferrocene/ferricinium redox couple (Fc/Fc+) which in our experimental conditions was +0.07V. A platinum wire was used as auxiliary electrode. The electrochemical experiments were performed at 25ºC under argon atmosphere. Synthesis and characterization of N-[2-(4-methoxyphenyl)ethyl]oleamide 175

2.2. Synthesis of compound 1 a. Synthesis of methyl oleate Methyl oleate was prepared by esterification of oleic acid with methanol catalyzed by p-toluenesulfonic acid (Method A) and also by a selective method (B) which does not affect the double bond of the molecule in anhydrous mixture of THF-HMPT, when the acid is transformed in its potassium salt with KHCO3, which is then esterified with an excess of methyl iodide. Method A 28.2 g (0.1M) oleic acid in 150 mL anhydrous methanol and 1g p- toluenesulfonic acid were refluxed for 6 h, monitoring the reaction by thin layer chromatography (TLC) on Merck silica gel plates with ethyl acetate-methanol, 90:13, as eluent system; Rf oleic acid = 0.60; Rf methyl oleate = 0.80. After usual work- up, 27.5 g (yield 95%) crude methyl oleate was obtained. Method B 2.82g (10mmoles) oleic acid were dissolved in 20 mL tetrahydrofuran (THF) and 10 mL hexamethylphosphoroustriamide (HMPT), 1.4g (14 mmoles) KHCO3 were added; the mixture was stirred for 30 minutes, then 50 mL methyl iodide were added and the mixture was stirred again in the same vessel for 24 hours, monitoring the reaction by TLC as in the method A. THF was removed by distillation under vacuum, 50 mL water were added and the product was extracted with benzene (3x50 mL). Organic extracts were washed with 2x50 mL NaHCO3 saturated solution, 3x50 mL water, dried and concentrated, resulting 2.72g methyl 1 oleate as colorless liquid (yield 92%), H-NMR (CDCl3, δ ppm, J Hz): 5.36-5.33 (m, 2H, H-9, H-10); 3.67 (s, 3H, CH3O); 2.30 (t, 2H, H-2, 7.4); 2.04-1.96 (m, 3H); 1.66-1.57 (m, 3H); 1.36-1,23 (m, 20H); 0.88 (t, 3H, H-18), 13C-NMR (CDCl3, δ ppm); 174.47 (C-1); 130.14, 129.89 (C-9, C-10), 51.64 (CH3O); 34.25 (C-2), 32.05 (CH2); 29.91, 29.82, 29.67, 29.46, 29.29 (9CH2), 27.36 (CH2), 25.09 (CH2), 22.82 (C-17); 14.24 (C-18); the compound has the same characteristics with those mentioned in the literature [6].

b. Synthesis of N-[2-(4-methoxyphenyl)ethyl]oleamide 5.6g (5.4mL, 0.037 moles) of 2-(4-methoxyphenyl)ethylamine, 10g (0.033moles) methyl oleate, 100 mL anhidrous methanol and 10 mL 29% (w/w) sodium metoxide in methanol were introduced in a 250 mL round bottom flask, and refluxed under stirring for 8 hours monitoring the reaction by TLC on Merck silica gel plates, with ethyl acetate-methanol, 90:13, eluent system; Rf, methyl oleate = 0.80, Rf, amine = 0.07, Rf, compound 1 = 0.72. After cooling to the room temperature, the reaction mixture was acidulated with 35 mL 10% HCl solution, then methanol was distilled under reduced pressure. 75 mL water and 200 mL CH2Cl2 were added to the concentrate and the phases were separated; the organic phase was washed with 50 mL NaHCO3 saturated solution, dried and concentrated. The 176 C. Neguţ (Cioateş), E.-M.Ungureanu, F. Cocu, C. Tănase, C. Drăghici, C. Munteanu

crude product (13g) was purified by pressure chromatography on a silica gel column prepared in extraction gasoline and eluted with extraction gasoline-ethyl acetate (5:1), resulting 10.25 g (yield 68 %) pure compound 1. 1 was recrystallized from hexane (m.p.= 71.7-73.8°C) as yellow crystals. IR: 3009m (ν=C-H), 2958w, 2919 vs (νCH2 asim); 2850s (νCH2 sim), 1638 (νC=ONHR sec. amide), 1614w, 1584s (νCONH band II), 1548s (νC=O sec. amide, band II), 1 1514m, 1246s (ν=C-O-CH3); 1197w; 1186w; 1031w; 820w, H-NMR (CDCl3, δ ppm, J Hz), 7.09 (d, 2H, H-o, 8.5); 6.84 (d, 2H, H-m, 8.5); 5.53 (br.s., 1H, NH); ’ 5.35-5.31 (m, 2H, H-9, H-10); 3.78 (s, 3H, CH3O); 3.64 (q, 2H, H-1 , 6.9); 2.74 (t, 2H, H-2’, 6.9); 2.10 (t, 2H, H-2, 7.5); 2.05-1.94 (m, 3H); 1.63-1.52 (m, 3H); 1.56- 13 1.21 (m, 20H); 0.87 (t, 3H, H-18), C-NMR (CDCl3, δ ppm): 173.18 (C-1); 158.37 (Cq-OMe); 131.02 (Cq), 130.08, 129.83 (C-9, C-10), 129.77 (2C-o), ’’ ’’ 114.12 (2CH, C-m); 55.33 (CH3O), 40.78 (CH2, C-1 ); 36.92 (CH2, C-2 ); 34.91 (C-2); 31.97 (CH2); 29.86, 29.82, 29.61, 29.41, 29.36, 29.23, 27.32, 27.27, 25.85 (9CH2), 22.77 (C-17), 14.20 (C-18).

3. Results and Discussion 3.1. Synthesis Synthesis of amides like anandamide [7], prostamides [8], fatty acids amides [9], macamides, could be realized by several methods: direct esterification of acids with amines [10] in the presence of carbodiimide [11], chlorophormiate [12], or with carbonyldiimidazole [5], acid chloride and amines [13], esters with amines catalyzed by bases like MeONa [14]. We realized the synthesis of oleamide compound 1 according to [14], as seen in Scheme 1:

1 OH 1 OCH3 9 C 9 C 10 O 10 O 17 17 18 18 Oleic acid Methyl oleate

H 1 N 1' 2' 9 C OCH3 om 10 O 17 18 1 Scheme 1: Synthesis of compound 1

Methyl oleate was obtained by esterification according to usual methods. It was then reacted in the presence of sodium methoxide as catalyst with 2-(4- methoxyphenyl)ethylamine in anhydrous methanol at 60°C. After column chromatography purification the pure compound 1 resulted in a 68 % yield. The Synthesis and characterization of N-[2-(4-methoxyphenyl)ethyl]oleamide 177

recrystalized fraction from hexane was used for physico-chemical and electrochemical characterization of the compound.

3.2. Physico-chemical characterization 3.2.1. IR and NMR IR spectrum of the compound 1 presents a very strong band at 1638 cm-1 and two intense bands at 1584 and 1548 cm-1 characteristics for secondary amides, a strong band for phenolic methyl ether at 1246 cm-1 and also intense bands for oleyl fragment of the molecule (the entire IR spectrum is given in the experimental part). In the proton spectrum of compound 1 the signals for the linked amidic fragment at 5.53 (br.s.) are clearly distinguished for all protons: ethylene at δ 3.64 (H-1’) and 2.74 (2’) ppm, methyl ester at 3.78 ppm and aromatic group at 7.09 (H-o) and 6.84 (H-m) ppm. The same remark available for carbon atoms, both spectra confirming the structure of the molecule.

3.2.2. Mass spectra For the identification of p-methoxy-phenetyl-oleylamide, MS obtained at high resolution was compared with the simulated one for a compound with the + formula C27H46NO2, in its protonated form [M+H] . This compound revealed a spectrum with peaks at m/z 416.35, m/z 417.35 and m/z 418.35, corresponding to isotope peaks at [M+H]+, [M+1+H]+ and [M+2+H]+.

416.35143 NL: z=1 6.52E5 100 COMP04_test#2819- 7674 RT: 90 33.23-48.44 AV: 54 F: FTMS + p NSI SIM 80 ms [406.35-426.35]

40 417.35465 Relative Abundance 30 z=1

10 418.35791 416.47456 416.62217 416.82087 417.03245 417.20713 417.43252 417.66780 418.03192 418.21056 z=1 418.55701 z=? z=? z=? z=? z=? z=? z=? z=1 z=? z=? 0 NL: 416.35231 7.38E5 100 C 27 H45 NO 2 +H: 90 C 27 H46 N1 O 2 pa Chrg 1 80

30 417.35566

10 418.35902 0 416.4 416.6 416.8 417.0 417.2 417.4 417.6 417.8 418.0 418.2 418.4 418.6 m/z Fig.2. Experimental MS for N-[2-(4-methoxyphenyl)ethyl]oleamide (up) and simulated isotopic + + pattern of [M+H] for a compound with the molecular formula C27H46NO2 (down) 178 C. Neguţ (Cioateş), E.-M.Ungureanu, F. Cocu, C. Tănase, C. Drăghici, C. Munteanu

Fig. 2 reveals in the upper part the MS experimentally obtained at high resolution and in the lower part the simulated isotopic pattern for the compound 1. The pattern is similar regarding m/z of the main peaks observed and the relative abundance of the [M+1+H]+ peak (~ 29 %). To further validate our identification we fragmented the precursor by using collision induced dissociation (CID). The fragmentation spectrum of m/z 416.35 reveals the presence of ions with m/z 152 and m/z 265 corresponding to fragmentation of the amide group. Also, the peaks with m/z 135 and m/z 282 corresponding to fragmentation of alkyl amide group were detected. Minor fragments of consecutive olefinic backbone fragmentation are also observed, the corresponding ions being separated by 14 Da (CH2) (Fig. 3).

x10 247 NL: 6.46E4 135.0005 398 O COMP04_test#289-1012 100 276 262 248 234 220 152 + 135 399 N RT: 1.14-2.39 AV: 546 F: ITMS + p NSI Full ms2 90 247 HH 290 [email protected] 265 282 [110.00-500.00] 80

70 OCH3 304 318 332 346 360 374 120 60 152.0009 50

Relative Abundance 30 247.1696

20 416.2566 265.2533 399.3395 178.0015 234.0860 276.1702 332.2548 10 304.2542 360.3387 388.1726 172.0847 191.0851 435.0070457.3408 483.3413 0 135.0796 NL: 4.12E5 R=231755 COMP04_test#1443-2813 100 RT: 10.58-32.59 AV: 21 F: FTMS + p NSI Full ms2 90 [email protected] [50.00-500.00] 80

40 152.1060 30 R=227731 161.0950 416.3498 20 R=224117 R=135349 178.0851 247.2405 265.2510 345.9739 10 R=208576 220.1318 R=177737 327.9635 371.3287 399.3234 R=172443 290.2097 R=145765 448.2345 478.2851 R=191403 R=146064 R=147169 R=141358 R=134350 R=98135 R=123668 0 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 m/z Fig. 3. MS/MS CID fragmentation (up) and HCD fragmentation (down) spectra of ion m/z 416.35

The isotopic pattern obtained for compound 1 corresponds to the empirical formula (C27H46NO2). Fragmentation of this precursor yields a series of cluster ions (spaced 14 Da apart) specific for the fragmentation at the oleyl chain. Major peaks are found in the MS/MS fragmentation, corresponding to the cleaveage at the amide group.

Synthesis and characterization of N-[2-(4-methoxyphenyl)ethyl]oleamide 179

3.3. Electrochemical characterization

The electrochemical behavior of 1 was studied on a stationary electrode in acetonitrile (CH3CN) containing tetrabutylammonium perchlorate (TBAP) as supporting electrolyte, using a glassy carbon electrode. The electrochemical experiments were carried out by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Anodic and cathodic curves were recorded individually, starting from the stationary potential. CV and DPV curves were recorded for various concentrations (0-3mM) of the studied compound in 0.1M TBAP in CH3CN. The DPV curves obtained for different concentrations of compound 1 are presented in figure 4. Three anodic (1a-3a) processes are observed, denoted in the order in which they appear in the voltammograms. No cathodic process can be evidenced. The CV curves for increasing concentrations of compound 1 are shown in Fig. 4.

0mM DPV -4 1mM 1a 1.0x10 3a 2mM

i(A) 3mM

0.0

-2 0 2 2a -4 3a 4.0x10 0mM CV 1mM 2mM 1a i(A) 3mM

1c 0.0

-2 0 2 + E(V) vs. Fc/Fc Fig. 4. DPV and CV (0.1 V/s) curves for different concentrations of 1 in 0.1 M TBAP, CH3CN on glassy carbon electrode (3 mm diameter)

Both DPV and CV curves show peak currents linearly increasing with the concentration (plots up and down on the left of Fig. 5). The peak potentials linearly increase with the logarithm of concentration (plots up and down on the right of Fig. 5). 180 C. Neguţ (Cioateş), E.-M.Ungureanu, F. Cocu, C. Tănase, C. Drăghici, C. Munteanu

Ep1a ip1a 1,510 Ep2a ip2a 1,505 2 -4 ip1a = 1,293E-5+2,629E-5C (r =0,983) 1,5x10 1,500 2 2 Ep1a = 1,130+0,0453lgC (r =0,926) ip2a = 5,991E-5+3,239E-5C (r =0,998) 1,495 2 1,490 Ep2a = 1,47+0,0623lgC (r =0,599) 1,485 1,480 1,0x10-4 1,475 1,470 i (A) E (V)

1,14 5,0x10-5 DPV DPV ip vs C 1,12 E vs lgC

1,10 0,0 ip1a 0,5 1,0 1,5 2,0 2,5 3,0 0,0 0,2 0,4 5,0x10-4 ip2a c (mM) 1,8 lg c ip3a ip1a = -3,287E-6+6,085E-5C (r2 =0,999) 2 CV 4,0x10-4 ip2a = 1,690E-5+1,470E-4C (r =0,994) ip3a = 3,360E-5+1,392E-4C CV E vs lgC ip vs C 1,6 3,0x10-4

2 i(A) E(V) Ep1a Ep1a = 1,203+0,079lgC (r =0,984) 2,0x10-4 Ep2a 2 1,4 Ep2a = 1,542+0,095lgC (r =0,989) Ep3a Ep3a = 1,790-0,166lgC

1,0x10-4

1,2 1,0 1,5 2,0 2,5 3,0 0,0 0,2 0,4 C(mM) lg C

Fig. 5. Dependences on concentration of the currents (up and down on the left) and potentials (up and down on the right) of the peak shown in Fig.4 The influences of the scan rate and scan domain on the CV curves are presented in Fig. 6 (A and B, respectively). All processes are irreversible. The first peak current linearly increases with the square root of the scan rate showing a diffusion controlled process. The diffusion coefficient can be estimated.

5.0x10-4 1V/s D 0.5V/s Polynomial Fit of Sheet1 D 4.0x10-4 4.0x10-4 0.3V/s

0.2V/s -4 3.0x10 y = Intercept + Equation B1*x^1 ipa1(A) 0.1V/s Weight No Weighting Residual Sum 4.40724E-11 i(A) of Squares -4 -4 2.0x10 Adj. R-Square 0.99925 2.0x10 Value Standard Error D Intercept -2.70094E-5 4.71105E-6 D B1 5.31896E-4 7.26932E-6 1.0x10-4 0.4 0.6 0.8 1.0 A v1/2 (V/s)1/2 0.0 -2 0 2

4.0x10-4

-4 i(A) 2.0x10

B 0.0

-2 0 2 + E(V) vs. Fc/Fc Fig. 6. CV curves at different scan rates (A): 0.1; 0.2; 0.3; 0.5; 1V/s in the domain of the peak 1a, and for various scan domains (B) in the domains of the peaks 1c and 1a at 0.1V/s obtained on glassy carbon (3 mm diameter) in (3 mM) solution of 1 in 0.1 M TBAP, CH3CN Synthesis and characterization of N-[2-(4-methoxyphenyl)ethyl]oleamide 181

By cycling the potential in the range of the first anodic peak a decrease of the current can be seen (Fig.7), showing the formation of a film or accumulation of insoluble products on the electrode surface. The cycling led to a modified electrode.

3.0x10-4 0.1 (V/s)

2.0x10-4 i (A)

1.0x10-4

0.0

-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 + E(V) vs Fc/Fc

Fig. 7. Successive scanning (20 cycles) with 0.1 V/s The transfer of this modified electrode in pure electrolyte shows altered CV curves for a redox probe (ferrocene). The anodic peak current for Fc/Fc+ couple is less influenced in comparison with the cathodic peak current which is smaller than that for the bare electrode (Fig. 8). The difference between the peak potentials of ferrocene is bigger (0.144V) for the modified electrode than for the bare electrode (0.083V), confirming the electrode covering with a insulating layer (film or insoluble products). The electrode modification can be performed also by controlled potential electrolysis.

bare electrode modified electrode

2.0x10-5 i (A) 0.0

-0.2 0.0 0.2 E(V) vs Fc/Fc+ Fig. 8. CV curves in 1mM ferrocene solution in 1 (3mM) in 0.1M TBAP, CH3CN for the modified electrode prepared by scanning (20 cycles) as shown in Fig. 7 182 C. Neguţ (Cioateş), E.-M.Ungureanu, F. Cocu, C. Tănase, C. Drăghici, C. Munteanu

4. Conclusions

The new oleylamide compound 1 was synthesized from oleic acid in a sequence of two reactions. The compound was purified by pressure chromatography and recrystallized from hexane. The physico-chemical characterization by IR, MS and 1H- and 13C-NMR spectra confirmed the structure of the compound. Three anodic irreversible processes were identified by cyclic and differential pulse voltammetry. Scanning the potential in the range of the first anodic peak led to the coverage of the electrode with an insulating layer.

Acknowledgements The work has been funded by the Sectoral Operational Programme Human Resources Development 2007-2013 of the Ministry of European Funds through the Financial Agreement POSDRU/159/1.5/S/132395.

R E F E R E N C E S

1. R. Capasso, A. Izzo, Journal of Neuroendocrinology, 20 (SUPPL. 1), 2008, 39-46. 2. H.S. Hansen, T.A. Diep, Biochemical Pharmacology, 78 (6), 2009, 553-560. 3. S. Cerrato, P. Brazis, M.F. Miolo, A. Puigdemont, Veterinary Immunology and Immunopathology, 133 (1), 2010, 9-15. 4. M. Maccarrone, R. Pauselli, M. Di Rienzo, A. Finazzi-Agrò, Biochemical Journal, 366 (1), 2002, 137-144. 5. H. Wu, C. J. Kelley, A. Pino-Figueroa, H. D. Vu, T. J. Mahe, Bioorganic & Medicinal Chemistry 21 (2013), 5188–5197. 6. G. Knothe, Lipid library.aocs.org., 2006, 1-11. 7. S. P. Nikas, M. D’Souza, A. Makriyannis, Tetrahedron 68 (2012), 6329-6337. 8. Kudriashova VV, Dikovskaia KI, VKalnin'sh AP, Kropivets LS, Freĭmanis IaF., Bioorg Khim. Feb; 14 (2), 1988, 216-221. 9. E. T. Roe , J. T. Scanlan , D. Swern, J. Am. Chem. Soc., 1949, 71 (6), 2215–2218. 10. M. Hosseini-Sarvari, E. Sodagar, M. M. Doroodmand, J. Org. Chem., 2011, 76, 2853-2859. 11. N. Nakajma, Y. Ikada, Bioconjugate Chemistry, 6 (1), 1995, 123-130. 12. B. Weiss, J.Org. Chem. 1959, 24 (9), 1367-1367. 13. T. Ohshima, Y. Hayashi, K. Agura, Y. Fujii, A. Yoshiyama, K. Mashima, Chem. Commun., 48, 2012, 5434-5436. 14. B. R. Kim, H.-G. Lee, S.-B. Kang, G. H. Sung, J.-J. Kim, J. K. Park, S.-G. Lee, Y.-J. Yoon, Synthesis, 44, 2012, 42-50.