Preparation and Hydro-Lipophilic Properties of Methoxylated and Methylated 1-Hydroxynaphthalene-2-Carboxanilides †

A series of variously methoxylated and methylated N-aryl-1-hydroxynaphthalene-2carboxanilides was prepared and characterized as potential anti-invasive agents. As it is known that lipophilicity significantly influences the biological activity of compounds, the hydro-lipophilic properties of these mono-, diand tri-substituted 1-hydroxynaphthalene-2-carboxanilides are investigated in the study. All the discussed hydroxynaphthalene derivatives were analyzed using the reversed-phase, high-performance liquid chromatography method, to measure lipophilicity. The procedure was performed under isocratic conditions with methanol as an organic modifier in the mobile phase, using an end-capped, non-polar C18 stationary reversed-phase column. The present study discusses the correlations between the logarithm of the capacity factor k and log P/Clog P values, calculated in various ways, as well as the relationships between the lipophilicity and the chemical structure of the studied compounds.


Introduction
One of the major prerequisites for pharmacological screening and drug development is the prediction of absorption, e.g., the transport of a molecule through membranes.Drugs most frequently cross biological barriers by passive transport, which strongly depends on lipophilicity.Therefore, hydro-lipophilic properties are some of the most important physical characteristics of biologicallyactive compounds [1,2].The thermodynamic parameter, characterized by the partition (log P) coefficient, describes the partitioning of a compound between an aqueous and an organic phase [3].Classical methods for the determination of these constants are time-consuming and not always sufficiently reliable.Therefore, reversed-phase, high-performance liquid chromatography (RP-HPLC) methods have become popular and are widely used for lipophilicity measurements.A general procedure is the measurement of directly-accessible retention time, under isocratic conditions, with varying amounts of an organic modifier in the mobile phase, using end-capped, non-polar C18 stationary RP columns and calculating the capacity factor k [4][5][6][7][8][9].Log k, calculated from the capacity factor k, is used as the lipophilicity index, and converted to log P scale [4].
The new methoxy and methyl multisubstituted derivatives expand the spectrum of substitutions in the carboxanilide part of the molecule.As it was found that the lipophilicity of these significantly biologically-effective agents [10][11][12][13] determines their activity, this study investigated and compared the hydro-lipophilic properties of new methoxy and methyl multisubstituted 1-hydroxynaphthalene-2-carboxanilides with the hydro-lipophilic properties of recently-prepared monosubstituted Nalkoxy-1-hydroxy-naphthalene-2-carboxanilides and N-alkyl-1-hydroxy-naphthalene-2carboxanilides.This contribution is a follow-up work to the previous papers [5-9; 14-24] aimed at the investigation of the physicochemical properties of new, biologically-active agents.
The lipophilicities (log P/Clog P data) of all seventeen anilides were calculated using two commercially-available programs: ACD/Percepta, ver.2012, and ChemBioDraw Ultra 13.0.In addition, the lipophilicity of the studied compounds was investigated by means of the RP-HPLC determination of capacity factors k, with a subsequent calculation of log k.The results are shown in Table 1.
The conformity of the experimental and calculated log P (ACD) values of methoxylated derivatives are plotted in Figure 1, while the conformity of the experimental and calculated Clog P (ChemBioDraw) values of the substituted compounds are illustrated in Figure 2. Based on these results, it can be stated that log P (ACD) values for methoxy derivatives 2-7 have a good match with experimentally-determined log k (r = 0.8983, n = 6), as shown in Figure 1.In addition, log k versus Clog P of the methoxy derivatives have a poor match (r = 0.2793, n = 6), seen in Figure 2A, contrary to the dependence of log k on Clog P of methyl derivatives 8-14, that have an excellent match (r = 0.9709, n = 7), as can be seen in Figure 2B.A poor match (r = 0.6652, n = 3) can also be observed for methoxymethyl derivatives 15-17, shown in Figure 2C.In general, these differences between the experimental results of log k and the calculated log P values can denote intramolecular interactions, i.e., influences of the spatially-close phenolic moiety, the amide moiety, and substituents on the anilide ring.The experimental results of lipophilicity expressed as log k are much more interesting and probably, more realistically, plot the influence of individual substituents on the overall lipophilicity of the individually discussed derivatives.Compound 15 (R = 2-OCH3-5-CH3, log k = 1.0603) showed the highest lipophilicity, while the lowest lipophilicity was demonstrated by compound 16 (R = 2-OCH3-6-CH3, log k = 0.4574).In the middle of the log k values of these two compounds, unsubstituted derivative 1 (log k = 0.6769) can be found, as seen in Figure 3.Although slightly higher than that of compound 16, the second-lowest lipophilicity was observed for compound 17 (R = 5-OCH3-2-CH3).Based on these observations, it can be stated that the displacement of the methyl moiety from position 5 (meta) to 6 (ortho), or the preparation of "inverse" isomers (i.e., the elimination of the methoxy moiety from the ortho position; compare the compounds 15 and 17), led to a sharp decrease of lipophilicity, which supports a theory about intramolecular interactions among substituents that are spatially close (especially in the ortho position) to the amide bond (-CONH-) and the phenolic moiety.

General
All reagents were purchased from Merck (Sigma-Aldrich, St. Louis, MO, USA) and Alfa (Alfa-Aesar, Ward Hill, MA, USA).Reactions were performed using a CEM Discover SP microwave reactor (CEM, Matthews, NC, USA).Thin layer chromatography was performed on alumina-backed silica gel 40 F254 plates (Merck, Darmstadt, Germany).The plates were illuminated under UV (254 nm) light and evaluated in iodine vapor.Melting points were determined on a Kofler hot-plate apparatus (HMK Franz Kustner Nacht BG, Dresden, Germany) and are uncorrected.The purity of the final compounds was checked by a Waters Alliance 2695 XE HPLC separation module (Waters Corp., Milford, MA, USA).A detection wavelength of 210 nm was used.The peaks in the chromatogram of the solvent (blank) were deducted from the peaks in the chromatogram of the sample solution.The purity of the individual compounds was determined from the area peaks in the chromatogram of the sample solution.Infrared (IR) spectra were recorded on a Smart MIRacle™ ATR ZnSe for a Nicolet™ Impact 410 FT-IR spectrometer (Thermo Scientific, West Palm Beach, FL, USA).The spectra were obtained by the accumulation of 64 scans with 2 cm −1 resolution in the region of 4000-650 cm −1 .All 1 H and 13 C NMR spectra were recorded on an Agilent VNMRS 600 MHz system (600 MHz for 1 H and 151 MHz for 13 C; Agilent Technologies, Santa Clara, CA, USA) in DMSO-d6.Chemical shifts are reported in ppm (δ) using the signal of the solvent (DMSO-d6) as the reference (2.500, resp.39.50) against the internal standard, Si(CH3)4.High-resolution mass spectra were measured using a Dionex UltiMate ® 3000 high-performance liquid chromatograph (Thermo Scientific) coupled with a LTQ Orbitrap XL TM Hybrid Ion Trap-Orbitrap Fourier Transform Mass Spectrometer (Thermo Scientific), with an injection into a HESI-II Probe in the positive or negative mode.

Synthesis
General Procedure: 1-Hydroxynaphthalene-2-carboxylic acid (5.3 mmol), and the corresponding substituted aniline (5.3 mmol), were suspended in 30 mL of dry chlorobenzene.Phosphorous trichloride (2.65 mmol) was added dropwise, and the reacting mixture was heated using infrared flask-surface control of temperature at maximal allowed power 500 W and 130 °C in the microwave reactor for 15 min.The solvent was evaporated under reduced pressure.Subsequently, the solid residue was washed with 2 M HCl and the crude product was recrystallized from aqueous ethanol.All of the studied compounds are presented in Table 1.

Lipophilicity Determination by HPLC (Capacity Factor k/Calculated log k)
The HPLC separation module Waters ® e2695 equipped with a Waters 2996 PDA Detector (Waters Corp., Milford, MA, USA) was used.A chromatographic column Symmetry ® C18 5 μm, 4.6 × 250 mm, Part No. W21751W016 (Waters Corp.) was used.The HPLC separation process was monitored by the Empower ™ 3 Chromatography Data Software (Waters Corp.).Isocratic elution, by a mixture of MeOH p.a. (72%) and H2O-HPLC Mili-Q grade (28%), was used as a mobile phase.The total flow of the column was 1.0 mL/min, the injection was 20 μL, the column temperature was 40 °C, and the sample temperature was 10 °C.A detection wavelength of 210 nm was chosen.A KI methanolic solution was used for the dead time (tD) determination.Retention times (tR) were measured in minutes.The capacity factors k were calculated using the Empower ™ 3 Chromatography Data Software, according to the formula k = (tR − tD)/tD, where tR is the retention time of the solute and tD is the dead time obtained using an unretained analyte.Each experiment was repeated three times.Log k, calculated from the capacity factor k, is used as the lipophilicity index converted to log P scale [4].The log k values of individual compounds are shown in Table 1.

Lipophilicity Calculations
Log P, i.e., the logarithm of the partition coefficient for n-octanol/water, was calculated using the programs ACD/Percepta 2012 (Advanced Chemistry Development, Inc., Toronto, ON, Canada, 2012) and ChemBioDraw Ultra 13.0 (CambridgeSoft, PerkinElmer Inc., Waltham, MA, USA).Clog P values, i.e., the logarithm of the n-octanol/water partition coefficient based on established chemical interactions, were calculated by means of the ChemBioDraw Ultra 13.0 (CambridgeSoft) software.The results are shown in Table 1.

Figure 3 .
Figure 3.Comparison of experimentally-determined log k values of N-methoxy and N-methyl substituted compounds.