Dibasic Derivatives of Phenylcarbamic Acid against Mycobacterial Strains: Old Drugs and New Tricks?

In order to provide a more detailed view on the structure–antimycobacterial activity relationship (SAR) of phenylcarbamic acid derivatives containing two centers of protonation, 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]pyrrolidinium oxalates (1a–d)/dichlorides (1e–h) as well as 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-(di-propylammonio)propyl]azepanium oxalates (1i–l)/dichlorides (1m–p; alkoxy = butoxy to heptyloxy) were physicochemically characterized by estimation of their surface tension (γ; Traube’s stalagmometric method), electronic features (log ε; UV/Vis spectrophotometry) and lipophilic properties (log kw; isocratic RP-HPLC) as well. The experimental log kw dataset was studied together with computational logarithms of partition coefficients (log P) generated by various methods based mainly on atomic or combined atomic and fragmental principles. Similarities and differences between the experimental and in silico lipophilicity descriptors were analyzed by unscaled principal component analysis (PCA). The in vitro activity of compounds 1a–p was inspected against Mycobacterium tuberculosis CNCTC My 331/88 (identical with H37Rv and ATCC 2794, respectively), M. tuberculosis H37Ra ATCC 25177, M. kansasii CNCTC My 235/80 (identical with ATCC 12478), the M. kansasii 6509/96 clinical isolate, M. kansasii DSM 44162, M. avium CNCTC My 330/80 (identical with ATCC 25291), M. smegmatis ATCC 700084 and M. marinum CAMP 5644, respectively. In vitro susceptibility of the mycobacteria to reference drugs isoniazid, ethambutol, ofloxacin or ciprofloxacin was tested as well. A very unique aspect of the research was that many compounds from the set 1a–p were highly efficient almost against all tested mycobacteria. The most promising derivatives showed MIC values varied from 1.9 μM to 8 μM, which were lower compared to those of used standards, especially if concerning ability to fight M. tuberculosis H37Ra ATCC 25177, M. kansasii DSM 44162 or M. avium CNCTC My 330/80. Current in vitro biological assays and systematic SAR studies based on PCA approach as well as fitting procedures, which were supported by relevant statistical descriptors, proved that the compounds 1a–p represented a very promising molecular framework for development of ‘non-traditional’ but effective antimycobacterial agents.

Procedures for syntheses of particular reaction intermediates 2´a, 2´b, 3´a-h, 4´a-h, 5´a-h, 7´, 8´a, 8´b and 9´a-p as well as final compounds 1a-p were provided in next sections of the supplementum.
Crude N-(2-/3-alkoxyphenyl)ethanamides (3´a-h; alkoxy = butoxy to heptyloxy) were isolated, washed with an aqueous sodium hydroxide solution and finally with water to a neutral reaction [27]. The intermediates 3´a-h (Scheme) were crystallized from a mixture of water and EtOH (3:1, (v/v)). Yields (in percentages), melting point values as well as IR spectra of the compounds 3´a-h were published in a paper [27].
Yields (in percentages), boiling point values as well as IR spectra of the compounds 4´a-h were published in a paper [27].
A partially crystallized reaction mixture was heated to 75 C and treated (15 min) with an aqueous sodium hydroxide solution (38%). After cooling to r.t., the solution was filtered and crude intermediate was formed. Continuous extraction of the filtrate with 3  250 mL DEE, collecting of all organic fractions, drying over magnesium sulfate and removal of solvent in vacuo led to a crude intermediate [28]. Isolation of this product and its crystallization from anhydrous EtOH provided (±)-N-(oxiran-2-ylmethyl)-N-propylpropanamine (7´).
Spectral (IR) and physicochemical (melting point, refractive index nD) properties as well as elemental analyses results (% C, H, N) of this intermediate confirmed its identity and were already published [28].
Final liquid compounds 8´a and 8´b were purified by vacuum distillation [29] and some of their spectral (IR) and physicochemical characteristics (boiling point values) were published [21].
Chemical structures of desired bases 9´a-p were confirmed by spectral analyses (IR). In addition, elemental analyses results (% C, H, N) were within ±0.40% of theoretical values for all proposed molecules [21]. Current liquid chromatography high resolution mass spectroscopy (HPLC-HR-MS) analyses of the compounds 9´a-p were performed on a chromatographic apparatus consisting of the LC Agilent Infinity System (Agilent Technologies, Santa Clara, CA, USA) equipped with an gradient pump (1290 Bin Pump VL), automatic injector (1260 HiPals), and column thermostat (1290 TCC). The LC system was coupled with the Quadrupole Time-Of-Flight mass spectrometer (6520 Accurate Mass Q-TOF LC/MS). Q-TOF was equipped with an electrospray ionization source operated in a positive and negative ionization mode as well.
For data acquisition and processing, a personal computer with the Mass Hunter software ver. MassHunter Workstation B 04.00 (Agilent Technologies) was used. More detailed specifications were provided in a main text of the article. The HPLC-HR-MS characterization of the compounds 9´a-p is given below.
Chemical structures of synthesized oxalates and dichlorides were verified by interpretation of their IR. In addition, elemental analyses results (% C, H, N) were within ±0.40% of theoretical values for all proposed salts [21].

Physicochemical Properties of Analyzed Compounds
Purity of the molecules 1a-p was verified by thin-layer chromatography (TLC) using ethanol/benzene/diethyl amine eluant (10:3:0.2, v/v) as a mobile phase. Spots were observed under iodine vapors/UV light at a wavelength (λ) of 254 nm [21]. Elongation of an alkoxy side chain R led to higher Rf values within particular subsets 1a-d, 1e-h, 1i-l and 1m-p, as expected (Table S1).
All investigated salts 1a-p were freely soluble in distilled water, soluble in anhydrous EtOH and practically insoluble in chloroform [26]. Their uncorrected melting point values were published in [21] and are provided in Table S1.
Acid-base dissociation constant (pKa1, pKa2) values of analyzed substances 1i-p were estimated by alkalimetric titration with potentiometric indication of a titration (equivalence) point at 21 °C in an investigated pH range from 3.50 to 11.50 [26].
In accordance with knowledge about physicochemical properties of these derivatives, firstly protonization of an aliphatic amine (dipropylamino group) proceeded followed by protonization of a cyclic amine (azepan-1-yl fragment). Conversely, the dissociation constants were assigned to centers of protonation as follows: pKa1 for an azepanium moiety and pKa2 for a dipropylammonium fragment, respectively. Both pKa1 and pKa2 values of the derivatives 1i-l were lower than those of 1m-p. On the other hand, the pKa1 and pKa2 constants have not been observed for a subset 1a-h.

Local Anesthetic Activity of Analyzed Compounds
Relative surface local anesthetic activity (RLAAs; rabbit cornea; 0.01 M cocaine as a standard drug) and infiltration local anesthetic activity (RLAAi; guinea pig; an intradermal application; 0.02 M procaine as a standard drug) of investigated compounds 1a-p was already published [21].
Descriptors, which defined their relative surface (Us) as well as infiltration (Ui) local anesthetic efficiency, are listed in Table S1. These parameters were calculated from observed molar concentrations, which provided same local anesthetic effect as a standard, i.e., cocaine or procaine.
As can be seen, the biological screening aimed especially estimation of the Us indices. In fact, the Ui parameters were observed only for the compound 1b, 1f, 1j and 1n, respectively. Other molecules have not been tested due to capacity reasons.

Acute Toxicity of Analyzed Compounds
Acute toxicity of all compounds 1a-p was defined by LD50 values (in mg/kg units). The LD50 descriptor (lethal dose) was the amount of a substance, given all at once, which led to death of 50% (one half) of a group of tested animals (white mice; subcutaneous application). The LD50 was considered one way to measure the short-term poisoning potential (acute toxicity) of analyzed derivatives.
The LD50 values of the molecules 1a-p [21] were higher compared to those of cocaine (LD50 = 125 mg/kg) indicating lower toxicity of 1a-p (Table S1). In addition, the molecule 1c showed identical acute toxicity [21] than procaine (LD50 = 600 mg/kg). Table S1. Chemical structure of presently evaluated compounds 1a-p, their yields (in percentages), molecular formula, molecular weight (MW), melting point (m.p.) values, Rf parameters (TLC) and dissociation constants (pKa1, pKa2) as well as indices describing their relative surface (Us) and infiltration (Ui) local anesthetic efficiency, respectively. The compounds 1a-p showed relatively low acute toxicity, which was proven by estimated LD50 parameters (in mg/kg units).     Table S4. Relationships between number of carbon atoms forming the alkoxy side chain R (nc; alkoxy = butoxy to heptyloxy) and log kw values (RP-HPLC) of evaluated compounds 1a-p. The relationships were expressed by linear functions (Equations (S5)-(S8); Eqs.) and characterized by values of relevant statistical descriptors, i.e., number of points (number of cases; n), degrees of freedom (DF), reduced chi-square (χ, 2 red), residual sum of squares (RSS), correlation coefficient (R), adjusted coefficient of determination (Adj. R 2 ), root mean squared error (standard deviation; RMSE), norm of residuals (NR), Fisher´s significance ratio (Fisher´s F-test; F) and probability of obtaining the F Ratio (significance of a whole model; Prob  F), respectively.  Table S6. Relationships between the log kw values (RP-HPLC) and in silico log P parameters of evaluated compounds 1a-p and non-protonated bases 9´a-p. The relationships were expressed by linear functions (Equations (S13)-(S24); Eqs.) and characterized by values of common statistical descriptors, i.e., number of points (number of cases; n), degrees of freedom (DF), reduced chi-square (χ, 2 red), residual sum of squares (RSS), correlation coefficient (R), adjusted coefficient of determination (Adj. R 2 ), root mean squared error (standard deviation; RMSE), norm of residuals (NR), Fisher´s significance ratio (Fisher´s F-test; F) and probability of obtaining the F Ratio (significance of a whole model; Prob  F), respectively.