Chromatographic and Computational Study of Hydro-lipophilic Properties of N-Alkoxyphenylhydroxy-naphthalenecarboxamides

N-Alkoxy-3-hydroxynaphthalene-2-carboxanilides, N-alkoxy-1-hydroxynaphthalene-2-carboxanilides and N-alkoxy-2-hydroxynaphthalene-1-carboxanilides were recently reported as series of compounds with antimycobacterial, antibacterial and herbicidal activity. As it was found that the lipophilicity of these significantly biologically effective agents determined their activity, in this study hydro-lipophilic properties of all three series are investigated. All fifty-seven anilides were analysed using the reversed-phase high performance liquid chromatography method for lipophilicity measurement. 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. In the present study, the correlation between the logarithm of capacity factor k and log P/Clog P values calculated in various ways is discussed 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.The drugs most frequently cross biological barriers by the passive transport, which strongly depends on the lipophilicity.Therefore hydro-lipophilic properties are one of the most important physical characteristics of biologically active compounds [1,2].The thermodynamic parameter describes the partitioning of a compound between an aqueous and an organic phases and is characterized by the partition (log P) coefficient [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 widely used for lipophilicity measurement.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 C 18 stationary RP columns and calculating the capacity factor k [4][5][6][7][8].Log k, calculated from the capacity factor k, is used as the lipophilicity index converted to log P scale [4].N-Alkoxy-3-hydroxynaphthalene-2-carboxanilides, N-alkoxy-1-hydroxynaphthalene-2carboxanilides and N-alkoxy-2-hydroxynaphthalene-1-carboxanilides were recently synthesized and tested for their antibacterial and antimycobacterial activity as well as for their activity related to the inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts [9][10][11][12][13][14].Since it was found that the lipophilicity of these significantly biologically effective agents determined their activity, in this study hydrolipophilic properties of all three series are investigated.Thus this contribution is a follow-up work to the previous papers [5][6][7][8][15][16][17][18][19][20][21][22][23][24][25] aimed at the physicochemical properties of new biologically active agents.
Lipophilicities (log P/Clog P data) of all fifty-seven 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 RP-HPLC determination of capacity factors k with a subsequent calculation of log k.The results are shown in Tables 1-3.The ChemBioDraw software does not distinguish the lipophilicity (log P and Clog P) values of neither individual anilide positional isomers within individual series nor lipophilicity among series A, B and C, and therefore these values are listed only in Tables 1-3 without other discussion.Log P values of series A and B calculated by ACD/Percepta were not distinguished as well; nevertheless, the log P values of individual positional isomers differ.Therefore, the conformity of experimental and calculated log P (ACD) values are plotted in Figure 1.Based on these results, it can be stated that log P (ACD) values have a good match with experimentally determined log k of series A (r = 0.9751, n = 19); worse match can be observed for series B (r = 0.8474, n = 19); and the worst results are given by ACD/Percepta for series C (r = 0.7939, n = 19).These differences between experimental and calculated results can denote intramolecular interactions, i.e. the influence of spatially close second benzene nucleus of the naphthalene scaffold to the phenolic moiety (series B) or the amide moiety (series C). that have the same calculated lipophilicity values as the ortho-substituted derivatives.Compounds 4a-c (R = 4-OCH 3 ) showed lower log P values than unsubstituted anilides 1a-c.Much more interesting and probably more precise are the experimental results of lipophilicity expressed as log k.These confirmed that series C possesses the lowest lipophilicity within these 3 series, see Figure 2. On the other hand, series B showed the highest log k values.ortho-Substituted derivatives of series B and C are more lipophilic than meta-and paraalkoxy substituted derivatives, while within series A, meta-substituted derivatives are slightly more lipophilic than ortho-and para-substituted anilides.Otherwise, lipophilicity logically linearly (correlation factors ranged from 0.9497 to 0.9999; n = 4) increases with the lengthening of the unbranched alkoxy tail (see Fig. 2A).Nevertheless, it should be noted that unsubstituted compounds 1a-c showed higher experimental lipophilicity than compounds 4a-c (R = 4-OCH 3 ), see Table 1.Branched alkoxy substituents, i.e. compounds 14a-c, 15a-c, 16a-c (R = OCH(CH 3 ) 2 ) and 17a-c, 18a-c, 19a-c (R = OCH(CH 3 )CH 2 CH 3 ) showed less lipophilicity than their unbranched n-alkoxy isomers 8a-c, 9a-c, 10a-c and 11a-c, 12a-c, 13a-c (see Fig. 2B), which corresponds to our previously reported results, e.g., [15,22].All these observations correspond to biological activities; e.g., lipophilic N-(alkoxyphenyl)-1-hydroxynaphthalene-2-carboxamides of series B demonstrated higher potency against nontuberculous mycobacteria Mycobacterium smegmatis and M. kansasii than compounds of B series A and C, but also stronger antiproliferative effect against the human monocytic leukemia THP-1 cell line [10,13].In addition, compounds of series B significantly affected photosystem II, which resulted in the inhibition of photosynthetic electron transport in spinach (Spinacia oleracea L.) chloroplasts [14].Thus, it can be assumed, that experimentally determined log k values specify lipophilicity within the individual series of compounds and can be used as a useful tool for other investigation of structure-activity relationships within these series of biologically effective compounds.

Lipophilicity determination by HPLC (capacity factor k/calculated log k)
The
). Isocratic elution by a mixture of MeOH p.a. (72%) and H 2 O-HPLC Mili-Q grade (28%) as a mobile phase was used.The total flow of the column was 1.0 mL/min, injection 20 μL, column temperature 40 °C and sample temperature 10 °C.The detection wavelength 210 nm was chosen.The KI methanolic solution was used for the dead time (t D ) determination.Retention times (t R ) were measured in minutes.The capacity factors k were calculated using the Empower ™ 3 Chromatography Data Software according to the formula k = (t R − t D )/t D , where t R is the retention time of the solute, whereas t D denotes 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.The log k values of individual compounds are shown in Tables1-3.Lipophilicity calculationsLog 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. USA).Clog P values (the logarithm of n-octanol/water partition coefficient based on established chemical interactions) were generated by means of ChemBioDraw Ultra 13.0 (CambridgeSoft, PerkinElmer Inc. USA) software.The results are shown in Tables1-3.

Table 2 .
Structure of N-substituted 1-hydroxynaphthalene-2-carboxanilides 1b-19b (series B), calculated lipophilicities (log P/Clog P) and determined log k of investigated compounds.Within individual series, the lipophilicity increases as follows: OCH 3 < OC 2 H 5 < OCH(CH 3 ) 2 < OC 3 H 7 < OCH(CH 3 )CH 2 CH 3 < OC 4 H As mentioned above, ACD/Percepta does not distinguish between log P values of series A and B. In general, both series are characterized by slightly higher calculated lipophilicity than series C with the exception of compound 7c (R = 4-OC 2 H 5 ) that has higher log P value than compounds 7a and 7b. 9 .The ortho-substituted derivatives showed the highest calculated log P values, while para-substituted derivatives demonstrated the lowest log P values, except 10a-c (R = 4-OC 3 H 7 ) and 13a-c (R = 4-OC 4 H 9 )