Investigating Spectrum of Biological Activity of 4- and 5-Chloro-2-hydroxy-N-[2-(arylamino)-1-alkyl-2-oxoethyl]benzamides

In this study, a series of twenty-two 5-chloro-2-hydroxy-N-[2-(arylamino)-1-alkyl-2-oxoethyl]benzamides and ten 4-chloro-2-hydroxy-N-[2-(arylamino)-1-alkyl-2-oxoethyl]benzamides is described. The compounds were analyzed using RP-HPLC to determine lipophilicity. Primary in vitro screening of the synthesized compounds was performed against mycobacterial, bacterial and fungal strains. They were also evaluated for their activity related to the inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. The compounds showed biological activity comparable with or higher than the standards isoniazid, fluconazole, penicillin G or ciprofloxacin. For all the compounds, the relationships between the lipophilicity and the chemical structure of the studied compounds as well as their structure-activity relationships are discussed.


Introduction
Salicylanilides (2-hydroxy-N-phenylbenzamides) are an important class of aromatic compounds with a wide range of pharmacological activities. A number of them show antibacterial [1][2][3], antimycobacterial [4][5][6] and antifungal [4,7] as well as anti-inflammatory [8] or antineoplastic activities [9][10][11]. In spite of their promise as potential drugs, certain physico-chemical properties of salicylanilides, for example low solubility, prevented their widespread use in clinical practice. Thus, improvement of the physical-chemical properties of salicylanilides is an interesting and vital area of research.
We have focused our efforts on design and synthesis of original O-substituted derivates of salicylanilides. Promising results of antimycobacterial evaluation of some salicylanilides acetates [4] inspired us to design more sophisticated derivates of salicylanilides -amino acid esters. The design strategy of these potential antimicrobial active agents was mainly oriented towards the synthesis of a prodrug form of most antitubercular active salicylanilides by attaching an amino acid. The phenolic hydroxyl group of the most antitubercular active salicylanilides was esterified by several N-benzyloxycarbonyl α-amino acids. More lipophilic amino acids such as glycine, (R and S)-alanine, (R and S)-valine and (R and S)-phenylalanine were chosen. The prepared N-protected esters were tested for their antifungal and antimycobacterial activity; promising results were published recently [5][6][7]. Subsequent processing using standard reactions yielded unexpected products (4-chloro-and 5chloro-N-salicylamides for detailed structure see Scheme 1) that were described, including proposed reaction mechanism, and published [12][13][14].
It was expected that chloro-2-hydroxy-N-(arylalkyl)benzamides, compounds with a special skeleton originating from biologically active salicylanilides, would have interesting biological properties. A series of these compounds was prepared, and most of them were tested for their antimycobacterial, antifungal and antibacterial activity. As the presence of an amide (-NHCO-) group in acylanilides, phenylcarbamates, ureas, etc., is characteristic of a number of herbicides acting as photosynthesis inhibitors [15][16][17][18][19][20][21][22][23][24][25], the compounds were also evaluated for their photosynthesis-inhibiting activity (the inhibition of photosynthetic electron transport) in spinach chloroplasts (Spinacia oleracea L.). These compounds bind reversibly to photosystem II (PS II), a membrane-protein complex in the thylakoid membranes, which catalyses oxidation of water and reduction of plastoquinone [15], and thereby inhibit photosynthesis [26][27][28][29][30]. For example, in the presence of R' substituted salicylanilides the decreased intensity of the fluorescence emission band at 686 nm (belonging to the chlorophyll-protein complexes mainly in PS II [31]) suggested PS II as the site of action of the studied inhibitors [24].
Many low molecular weight drugs cross biological membranes through passive transport, which strongly depends on their lipophilicity, therefore this physico-chemical parameter of the prepared compounds was determined by means of the RP-HPLC. Relationships between the structure and in vitro antimicrobial activities or/and inhibitory activity related to inhibition of photosynthetic electron transport (PET) in spinach chloroplasts of the new compounds are discussed.

Lipophilicity
Lipophilicity is a property that has a major effect on absorption, distribution, metabolism, excretion and toxicity properties as well as pharmacological activity, because drugs cross biological membranes through passive transport, which strongly depends on their lipophilicity. Lipophilicity has been studied and applied as an important drug property for decades [32].
Hydrophobicities (log P/Clog P) of compounds 8 and 9 were calculated using two commercially available programs (ChemOffice Ultra 10.0 and ACD/LogP) and measured by means of RP-HPLC determination of capacity factors k with subsequent calculation of log k. The procedure was performed under isocratic conditions with methanol as an organic modifier in the mobile phase using an endcapped non-polar C 18 stationary RP column. The results are shown in Table 1 and illustrated in Figure 1.
Both used programs did not resolve lipophilicity parameters within the series of enantiomers, as expected. The program ChemOffice did not resolve various lipophilicity values of individual positional isomers, in particular, the same log P/Clog P data were calculated for compounds 8a-8g from Group 1, 8h-8l from Group 2, 9h-9f from Group 5, 8s-8v from Group 4 and compound 4-Cl/4-Br (9g) from Group 6.
The results obtained with all the compounds show that the experimentally-determined lipophilicities (log k) of the discussed compounds are in accordance with the calculated values of compounds 8 and 9 as shown in Figure 1. Compounds series 9 substituted by chlorine in C (4) position showed higher lipophilicity than compounds series 8 with C (5) chlorine substitution, although program ACD/LogP showed an opposite trend. According to the program ACD/LogP, compounds 5a-5g substituted by chlorine in C' (3) position showed higher lipophilicity than compounds 5h-5l with C' (4) chlorine substitution, but experimental log k for C' (4) chlorine substitution is higher. As expected, the lipophilicity of the compounds substituted in the anilide part of the molecule increases as follows: OCH 3 < CH 3 < Cl < Br < CF 3 < di-Cl, although program ACD/LogP calculates higher log P for Br derivative 9g than CF 3 substituted compound 9i. The lipophilicity factor log k as well as calculated log P (ACD/LogP) and log P/Clog P values (ChemOffice) increase, depending on amino acid type (glycine, alanine, valine and phenylalanine), that is, the lipophilicity of the compounds substituted in R 3 increases as follows: H < CH 3 < CH(CH 3 ) 2 < CH 2 C 6 H 5 .
Log k data specify lipophilicity within this series of the discussed compounds, and all the chiral compounds were measured several times with the same results. The determined differences in log k parameters for individual R/S-enantiomers cannot be explained on the basis of the results presented here.

Biological activities
The compounds showed a wide range of biological activities, and some noteworthy structureactivity relationships were observed. All the results are shown in Table 2. Generally, all the discussed compounds (especially compounds 8b, 8q, 8r) exhibited lower solubility in comparison with the initial esters [5,7].

Antimycobacterial screening
The discussed amides were tested against three Mycobacteria strains. According to the results of in vitro evaluation (see Table 2), all compounds demonstrated noteworthy effects. Activities of some benzamides, especially against M. avium and M. kansasii, considerably exceeded the activity of isoniazid (INH) used as an internal standard. The discussed compounds can be divided according to the activities against individual Mycobacteria strains, and structure-activity relationships within Groups 1, 2 and 4 can be discussed. Compound 8b showed the lowest activity due to precipitation in the testing medium, contrary to its (R)-enantiomer 8c. Also compounds 8q and 8r precipitated from testing medium, therefore 8b, 8q and 8r were excluded from the below SAR discussion.
It can be concluded that compound N- showed the highest antitubercular/antimycobacterial activity, see Table 2. Comp.   Compounds 8h and 8k/l (Group 2) exhibited the highest activity against M. tuberculosis. Based on these observations, it can be concluded that chlorination in the C (4) position results in less active compounds, substitution in the position C' (4) of the anilide part is essential for high activity, and 4-Cl (Group 2) is more advantageous than 4-Br (Group 4). In general, compounds within Group 1 (3-Cl substituted) showed negligible activity. The influence of chlorine in the position C' (4) of the anilide part seems to be dominant in Group 3 (series 5-Cl/3,4-Cl), see activity in Table 2. Lipophilicity seems to be only a secondary parameter; in the same way, the use of electronic parameter σ of R 2 substituent does not improve the results of SAR discussion. High activity of 8h cannot be explained on the basis of simple pattern.

MIC [µmol/L]
Compounds 8k/l and 8g (Group 2 and Group 1) and 9e/f (Group 5) showed the highest activity against M. avium and other compounds 8i/j, 8a, 8s and 9d demonstrated medium activity. Based on these facts, it can be assumed that the substitution of R 3 , especially by benzyl moiety, as well as preferential substitution by chlorine, especially in C' (4) position of the anilide part of a molecule, is important for high activity. Chlorination of C (4) or C (5) position is only a secondary parameter. Substitution in C' (4) by bromine (Group 4) or disubstitution in C' (3,4) (Group 3) by chlorine causes activity decrease compared with Group 2. Lipophilicity seems to be also one of the parameters affecting antimycobacterial activity. The most active compounds from all groups show log k about 0.70. Stereoisomerism considerably influences antimycobacterial activity against M. avium as discussed below.
Compounds 8l, 8v and 9f (Group 2, Group 4 and Group 5) showed the highest antimycobacterial activity against M. kansasii, and other compounds, 8k, 9e, 8i/j, 8g and 8c, expressed substantial activity as well. Based on these facts, it can be concluded that chlorination of C (4) or C (5) position is only a secondary parameter, while substitution of C' (4) position is more advantageous for high activity, and substitution by chlorine is preferred. Disubstitution of C' (3,4) position is connected with activity decrease. The substitution of R 3 , particularly by benzyl or isopropyl moiety, is associated with higher activity, which can be positively influenced by lipophilicity increase. Similarly, based on Tables 1 and 2 it can be considered that antimycobacterial activity against M. kansasii is also partly affected by bulk parameters expressed as MR (reflecting bulkiness) of R 3 substituents [33]. It seems that antimycobacterial activity is increased by the increase of the bulkiness of individual R 3 substituents in the molecule.
From the set of 28 tested compounds the dependence of activity (log 1/MIC, 14 h experiment) on log k related to antimycobacterial activity against M. kansasii (see Figure 2) was linear for 24 compounds (compounds 8b/c and 8q/r were excluded from the correlation). Introduction of σ parameter did not improve the results of statistical analysis: A very important parameter influencing activity is stereoisomerism, because individual enantiomers demonstrate considerable difference in their antituberculotic/antimycobacterial activity, e.g. enantiomers 8b/c, 8f/g, 8k/l, 8o/p, 8u/v, 9c/d and 9e/f showed different antimycobacterial activity. These differences are especially evident for antimycobacterial activity against the atypical strains M. avium and M. kansasii. More often (R)-enantiomers showed higher activity. The determined differences in antimycobacterial activity for individual R/S-enantiomers cannot be explained on the basis of the results presented here. Due to these facts it can be assumed that stereospecific bond is formed between the compound and an enzyme in Mycobacterium sp. with subsequent enzyme inhibition.

In vitro antifungal susceptibility testing
The evaluation of in vitro antifungal activity of the synthesized compounds was performed against eight fungal strains, but only activity against Candida tropicalis 156 (CT), C. krusei ATCC 6258 (CK) and against Trichophyton mentagrophytes 445 (TM) is shown in Table 2. Generally, all the compounds expressed only moderate antifungal activity, which was caused by low solubility of compounds in the testing medium and their precipitation during the incubation period, therefore no definite structureactivity relationships could be established. Only four compounds from Group 1, 8f/g, 8e and 8b/c, showed very high antifungal activity comparable with or higher than the standard fluconazole (FLU) against C. krusei and against T. mentagrophytes 445. More often (R)-enantiomers showed higher activity, e.g. 8c/b, 8e/d, 9d/c, nevertheless, the determined differences in antibacterial activity for individual R/S-enantiomers cannot be explained on the basis of the results presented here.
Generally, according to the results from Table 2, it can be concluded that chloration of C (4) position or disubstitution R 2 = 3,4-Cl considerably decreases solubility, and therefore it is not advantageous. The substitution R 2 = 3-Cl is essential for high activity. The substitution of R 3 modulates lipophilicity, and it seems to be an important parameter. Based on Table 2 it can be concluded that a lipophilicity decrease results in a decrease of antifungal activity: log k = 0.70 (8f/g) > 0.61 (8e) > 0.41 (8b/c).
When the dependence of activity (log 1/MIC, 24 h experiment) on log k related to antifungal activity of tested compounds against C. krusei (which is the most inhibited fungi strain) was studied, The above discussed antimycobacterial activity against M. kansasii for the whole series of the tested compounds is mainly dependent on lipophilicity. In contrast to the antimycobacterial activity, the effect of σ parameter of R 2 substituent on the activity of evaluated compounds against C. krusei was much more expressive than that of log k:

In vitro antibacterial susceptibility testing
Twenty-eight benzamides were tested for their in vitro antibacterial activity against four Gram positive bacterial strains Staphylococcus aureus CCM 4516/08 (SA), methicilin resistant S. aureus H 5996/08 (MRSA), S. epidermidis H 6966/08 (SE) and Enterococcus sp. J 14365/08 (EF). The results are shown in Table 2. Generally, all the compounds expressed only moderate antibacterial activity caused by the low solubility of compounds in the testing medium and their precipitation during the incubation period, as it was mentioned above. In summary, five compounds 8v, 8j, 8e and 9b showed notable antibacterial activity comparable with or higher than the both standards penicillin G and ciprofloxacin against S. aureus methicilin resistant and S. epidermidis.
Based on the results from Table 2, especially halogenation of C' (4) position (R 2 = 4-Br or 4-Cl) seem to be important for antibacterial activity. Chloration of C (4) position decreased solubility compared with chloration of C (5) position. Stereoisomerism is probably also an important parameter influencing the activity, because individual enantiomers demonstrate considerable difference in their antibacterial activity, e.g., enantiomers 8u/v, 8d/e, 8i/j and 9a/b. More often (R)-enantiomers showed higher activity (the same as M. kansasii). R 3 substitution modulates physico-chemical properties of the discussed compounds. According to Table 2, a bulkier substituent is probably more advantageous. A more detailed SAR study cannot be established due to the small number of effective compounds.

Inhibition of photosynthetic electron transport (PET) in spinach chloroplasts
Seventeen compounds were tested for their PET in spinach (Spinacia oleracea L.) chloroplasts, and they showed some wide-range activity, see Table 2. Compounds 8f (IC 50 = 12.3 µmol/L), 8e (IC 50 = 13.8 µmol/L) and 9h (IC 50 = 14.2 µmol/L) were the most effective inhibitors of the tested compounds, which PET-inhibiting activity in spinach chloroplasts was measured. The activity of the rest of the studied compounds was moderate or low relative to the standard.
Due to the fact that PET-inhibiting data are available only from Group 1 and Groups 5 + 6, SAR study involves the first of these series, but a general SAR hypothesis may be proposed. Generally, it can be concluded that C (4) substitution is more advantageous then C (5) substitution. In the C (5) series C´( 3) substitution is important for PET activity. Compounds within the C (5) series with substitution in C´( 4) by Cl or Br exhibit similar activity, which is lower than that of C´( 3) substituted compounds, but solubility in the testing medium is drastically reduced in the case of C´( 4) substitution. Disubstitution in C´ ( 3,4) by Cl also causes lower solubility. Bulkiness and branching of R 3 substitution are also important. It can be noticed that mainly (S)-enantiomers are active in PET assay (compare 8d/e or 8k/l) and only 8e, 9f, 9g are slightly active (R)-enantiomers. The correlations between PET-inhibiting activity and lipophilicity could be expressed by equation:

Lipophilicity determination by HPLC (capacity factor k/calculated log k)
A Waters Alliance 2695 XE HPLC separation module and a Waters Photodiode Array Detector 2996 (Waters Corp., Milford, MA, USA) were used. A Symmetry ® C 18  Retention times (t R ) were measured in minutes. The capacity factors k were calculated using the Empower™ 2 Chromatography Data Software according to 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. Log k, calculated from the capacity factor k, is used as the lipophilicity index converted to log P scale. The log k values of the 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 CS ChemOffice Ultra ver. 10 Table 1.

In vitro antimycobacterial evaluation
The in vitro antimycobacterial activity of all prepared compounds was evaluated against Mycobacterium tuberculosis MTB CNCTC My 331/88 (identical with H37RV and ATCC 27294, dilution of the strain was 10 −3 μmol/L), Mycobacterium avium MA CNCTC My 330/88 (identical with ATCC 25291, dilution of the strain was 10 −5 μmol/L) and Mycobacterium kansasii MK CNCTC My 235/80 (identical with ATCC 12478, dilution of the strain was 10 −4 μmol/L) in the Laboratory for mycobacterial diagnostics and TB, Institute of Public Health in Ostrava, Czech Republic. All strains were obtained from the Czech National Collection of Type Cultures (CNCTC). Antimycobacterial activities were determined in the Sula semisynthetic medium (Sevac, Prague, Czech Republic). Each strain was simultaneously inoculated into a Petri plates containing the Lowenstein-Jensen medium for the control of sterility of the inoculum and its growth. The tested compounds were added to the medium as DMSO solutions. The following concentrations were used: 250, 125, 62, 32, 16, 8, 4, 2, and 1 μmol/L. Inoculated plates kept in microtone bags were incubated at 37 °C. Reading was carried out on a stand with a bottom magnifying mirror, macroscopically, with the use of magnifying glass. The growth in plates was evaluated after 14 and 21 days of the incubation. The growth of the colonies in a control well plate, corresponding to the growth into 100 colonies in the control Lowenstein-Jensen medium was considered as an optimum dilution for the evaluation of the results. In the course of the test evaluation, the MIC (μmol/L) definition was considered as the lowest substance concentration at which the inhibition of growth of mycobacteria occurred. [34] Isoniazid (INH) was used as the standard as it is a clinically used anti-mycobacterial drug. The results are shown in Table 2. The MIC for mycobacteria was defined as a 90% or greater (IC 90 ) reduction of growth in comparison with the control. The MIC/IC 90 value is routinely and widely used in bacterial assays and is a standard detection limit according to the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS, www.clsi.org/).

In vitro antifungal susceptibility testing
The broth microdilution test [35][36][37] was used for the assessment of in vitro antifungal activity of the synthesized compounds against Candida albicans ATCC 44859, C. tropicalis 156, C. krusei E28, C. glabrata 20/I, Trichosporon asahii 1188, Trichophyton mentagrophytes 445, Aspergillus fumigantus 231 and Absidia corymbifera 272. Fluconazole (FLU) was used as a standard, since it is a clinically used antimycotic drug. The procedure was performed with a two-fold dilution of the compounds in RPMI 1640 (Sevapharma a.s., Prague, Czech Republic) buffered to pH 7.0 with 0.165 mol of 3-morpholino-propane-1-sulfonic acid (MOPS, Sigma, Germany). The final concentrations of the compounds ranged from 500 to 0.975 μmol. Drug-free controls were included. The MIC determination was performed according to the CLSI reference protocol M27-A2 for yeasts (IC 80 value) and M38-A for moulds (IC 50 value). IC 50 values were defined as a 50% reduction of growth in comparison with the control, and IC 80 values were defined as a 80% reduction of growth in comparison with the control. The trays were incubated at 35 °C, and MICs were read visually for filamentous fungi and photometrically for yeasts as absorbance at 540 nm after 24 h and 48 h. The MIC values for the dermatophytic strain (T. mentagrophytes) were determined after 72 h and 120 h. MICs were determined twice and in duplicate. The results are summarized in Table 2.

In vitro antibacterial susceptibility testing
The synthesized compounds were evaluated for in vitro antibacterial activity against Staphylococcus aureus CCM 4516/08, methicilin resistant Staphylococcus aureus H 5996/08, Staphylococcus epidermidis H 6966/08 and Enterococcus sp. J 14365/08. Penicillin G (PEN) and ciprofloxacin (CPF) were used as standards since they are clinically used antibacterial drugs. All strains were sub-cultured on nutrient agar (HiMedia) and maintained on the same medium at 4 °C. Prior to testing, each strain was passaged onto nutrient agar and bacterial inocula were prepared by suspending a small portion of bacterial colony in sterile 0.85% saline. The cell density was adjusted to 0.5 McFarland units using a densitometer (Densi-La-Meter, PLIVA Lachema Diagnostika, Czech Republic). The final inoculum was made by 1:20 dilution of the suspension with the test medium (Mueller-Hinton broth). The compounds were dissolved in DMSO and the anti-bacterial activity was determined using Mueller-Hinton broth (MH broth, HiMedia, pH 7.0 ± 0.2). Controls consisted of MH broth and DMSO alone. The final concentration of DMSO in the MH broth did not exceed 1% (v/v) of the total solution composition. The activity of the studied compounds was determined as the minimal inhibition concentration (MIC) according to NCCLS guidelines [38] using broth microdilution test. The MICs were defined as 90% inhibition of bacterial growth compared to the control and were determined after 24 and 48 h of static incubation at 37 °C. After incubation MICs were read visually as an absorbance at 540 nm. The results are shown in Table 2.

Study of inhibition photosynthetic electron transport (PET) in spinach chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and Kralova [39]. The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was determined spectrophotometrically (Genesys 6, Thermo Scientific, USA), using an artificial electron acceptor 2,6-dichlorophenol-indophenol (DCIPP) according to Kralova et al. [40], and the rate of photosynthetic electron transport was monitored as a photoreduction of DCPIP. The measurements were carried out in phosphate buffer (0.02 mol/L, pH 7.2) containing sucrose (0.4 mol/L), MgCl 2 (0.005 mol/L) and NaCl (0.015 mol/L). The chlorophyll content was 30 mg/L in these experiments and the samples were irradiated (~100 W/m 2 ) from 10 cm distance with a halogen lamp (250 W) using a 4 cm water filter to prevent warming of the samples. The studied compounds were dissolved in DMSO due to their limited water solubility. The applied DMSO concentration (up to 4%) did not affect the photochemical activity in spinach chloroplasts. The inhibitory efficiency of the studied compounds was expressed by IC 50 values, i.e. by molar concentration of the compounds causing 50% decrease in the oxygen evolution rate relative to the untreated control. The comparable IC 50 value for a selective herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU (Diurone ® ) was about 1.9 μmol/L [41]. The results are summarized in Table 2.