Dibasic derivatives of phenylcarbamic acid against mycobacterial strains: Old drugs and new tricks?

Article abstract of DOI:10.3390/molecules23102493

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 wellas1-[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 k_w; isocratic RP-HPLC) as well. The experimental log k_w 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 H₃₇R_v and ATCC 2794, respectively), M. tuberculosis H₃₇R_a 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 H₃₇R_a 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.

Full text of DOI:10.3390/molecules23102493

molecules
Article
Dibasic Derivatives of Phenylcarbamic Acid against
Mycobacterial Strains: Old Drugs and New Tricks?
Ivan Malík 1,*, Jozef Csöllei 2, Ivan Solovicˇ 3,4, Šárka Pospíšilová 1, Hana Michnová 1,
5
1
1
1
Josef Jampílek 1 , Alois Cížek , Iva Kapustíková , Jana Curillová , Mária Pechácˇová ,
ˇ
ˇ
Jirˇina Stolarˇíková 6, Daniel Pecher 7,8 and Michal Oravec 9
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Comenius University in Bratislava,
Odbojárov 10, SK-832 32 Bratislava, Slovakia; sharka.pospisilova@gmail.com (Š.P.);
michnova.hana@gmail.com (H.M.); josef.jampilek@gmail.com (J.J.); kapustikova@fpharm.uniba.sk (I.K.);
ˇ
curilova2@uniba.sk (J.C.); mariapech901@gmail.com (M.P.)
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences
in Brno, Palackého 1946/1, CZ-612 42 Brno, Czech Republic; csolleij@vfu.cz
2
3
4
5
6
7
8
9
Clinic for Tuberculosis and Lung Diseases, National Institute for Tuberculosis, Lung Diseases and Thoracic
Surgery, Vyšné Hágy, SK-059 84 Vysoké Tatry, Slovakia; solovic@hagy.sk or ivan.solovic@ku.sk
Department of Public Health, Faculty of Health, Catholic University in Ružomberok, Hrabovská cesta 1A,
SK-034 01 Ružomberok, Slovakia
Clinic for Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of
Veterinary and Pharmaceutical Sciences, Palackého 1946/1, CZ-612 42 Brno, Czech Republic; cizeka@vfu.cz
Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health,
Partyzánské námeˇstí 7, CZ-702 00 Ostrava, Czech Republic; Jirina.Stolarikova@zu.cz
Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy,
Comenius University in Bratislava, Odbojárov 10, SK-832 32 Bratislava, Slovakia; pecher1@uniba.sk
Toxicological and Antidoping Center, Faculty of Pharmacy, Comenius University in Bratislava,
Odbojárov 10, SK-832 32 Bratislava, Slovakia
Global Change Research Institute CAS, Belidla 986/4a, CZ-603 00 Brno, Czech Republic;
oravec.m@czechglobe.cz
*
Correspondence: malikivan001@gmail.com; Tel.: +421-2-501-117-227
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 5 September 2018; Accepted: 24 September 2018; Published: 28 September 2018
Abstract: 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
propylammonio)propyl]azepanium oxalates (1i
were physicochemically characterized by estimation of their surface tension (
stalagmometric method), electronic features (log ; UV/Vis spectrophotometry) and lipophilic
–
d
)/dichlorides (1e
–
h
) as well as 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-(di-
)/dichlorides (1m ; alkoxy = butoxy to heptyloxy)
; Traube’s
–
l
–p
γ
ε
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
Molecules 2018, 23, 2493; doi:10.3390/molecules23102493
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2493
2 of 37
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.
Keywords: dibasic phenylcarbamates; surface tension; electronic properties; lipophilicity;
Mycobacterium spp.
1. Introduction
The treatment of commonly encountered species of tuberculous and non-tuberculous
mycobacteria responsible for a multiplicity of different types of infections, including pulmonary,
respiratory, cutaneous, and systemic infections, by (i) brand new classes of promising compounds
preferably acting on novel targets; or (ii) ‘non-typical’ antimycobacterial drug candidates is still in very
dynamic and progressive debate [
of some -lactam antibiotics (ceftaroline or ceftazidime) in a combination with an
avibactam against Mycobacterium avium complex [ ]. Another encouraged example was in vitro and
1–
6]. The strategy was successfully used in a case of in vitro screening
β
β-lactamase inhibitor
3,4
ex vivo testing of tricyclic thioridazine (Figure 1a), an old neuroleptic phenothiazine. The molecule was
used alone or in a combinatorial therapy with anti-tuberculosis drugs (isoniazid, rifampin, linezolid or
moxifloxacin) against M. tuberculosis regardless of its antibiotic resistance phenotype and was highly
active due to its multi mechanisms of action [5,6].
Figure 1. Chemical structure of thioridazine (
); as well as effective local anesthetics ( ) lidocaine (xylocaine); and (
All these compounds showed notable in vitro efficiency against some mycobacterial strains [
a
), a neuroleptic drug containing a phenothiazine scaffold
) dibucaine (cinchocaine).
12].
(
a
b
c
5–
Structural fragments of dibucaine were color-coded as follows: a lipophilic moiety (a dark green
framework), polar group (orange), connecting hydrocarbon chain (gray) and salt-forming (basic) group
(dark red), respectively.
Antimycobacterial properties of compounds originally designed and tested as local anesthetics
(LAs) have been investigated sporadically. Moreover, their application to management of the infections
caused by a broad spectrum of species of the Mycobacterium genus has not been systematically reviewed.
Nevertheless, Schmidt and Rosenkranz [7] as well as Fujii et al. [8] found that lidocaine (xylocaine;
Figure 1b), a highly efficient LA drug [ 10], notably inhibited in vitro growth of some strains of human
9,

Molecules 2018, 23, 2493
3 of 37
M. tuberculosis, M. bovis or M. kansasii. Similarly, a quinoline core-containing dibucaine (cinchocaine;
Figure 1c) [11], was able to effectively in vitro fight rapidly growing M. phlei [12].
A common structural feature of given efficient LAs (and promising antimycobacterials) was
presence of a lipophilic moiety, polar anilido or amido group, connecting hydrocarbon chain and
salt-forming fragment. All considered moieties were color-differentiated for a molecule of dibucaine
(cinchocaine; Figure 1c).
Sequencing of Mycobacterium spp. genomes proved that these microorganisms produced a
variety of enzymes [13–15] able to hydrolyze ester, anilide or amide bonds in a chemical structure
of antimycobacterial drug candidates. Isosteric replacement of these polar groups with a carbamate
moiety would provide very good proteolytic stability, especially if the modified structural arrangement
of novel derivatives would be as follows: Aryl–NH–C(O)O–Alkyl [16]. The carbamate functionality
imposes a degree of conformational restriction due to delocalization of non-bonded electrons on
nitrogen into a carboxyl moiety. Participation of the carbamate group in hydrogen bonding through
carboxyl and backbone N–H [17] should be also taken into account. Considering the importance of the
carbamate functionality, its incorporation into a structure of novel antimycobacterial agents was one of
key tasks in a process of their design and synthesis [18–20].
Currently evaluated 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)-
propyl]pyrrolidinium/azepanium oxalates/dichlorides (1a
Scheme 1) were synthesized with intention to find effective LAs with favorable toxicity profiles [21].
The compounds contained two types of an anionic counterpart (1a and 1i versus 1e and 1m
Table 1) in order to achieve convenient aqueous solubility in biological assays [21].
–p; alkoxy = butoxy to heptyloxy; Table 1,
–d
–
l
–h
–p;
It was found that all tested derivatives showed higher indices defining their relative surface (Us) as
well as infiltration (Ui) local anesthetic efficiency than reference LA drugs cocaine (Us = 1.0, Ui = 3.6) or
procaine (Us = 0.1, Ui = 1.0) [21]. The most effective dibasic molecule in given types of local anesthesia
was 1-[2-[({[3-(pentyloxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]-pyrrolidinium
dichloride (1f) with Us = 130 and Ui = 250 (Table S1 in Supplementary Materials), respectively.
It might be expected that the carbamate moiety was sterically protected due to branching of both
connecting hydrocarbon chain and salt-forming group. Rotatable bonds of the 2-/3-alkoxy side chain
R could also contribute to the steric hindrance (Table 1). It was proved by ab initio procedures that an
intramolecular hydrogen bond between N–H of the carbamate functionality and oxygen atom of the
2-alkoxy chain was formed [22]. Such structural arrangements of compounds would provide stability
against effects of various mycobacterial enzymes and would consider them promising candidates for
antimycobacterial agents.
It was observed that surface tension (relative surface activity), electronic and lipophilic
properties of carbamate group-containing molecules notably influenced the in vitro antimycobacterial
efficiency [23–25]. However, the set 1a–p has not been satisfactorily characterized by values of relevant
physicochemical descriptors [26] (Table S1).
In a first part of this study, attention was turned on more precise physicochemical characterization
of the derivatives 1a
tension of water ( ), estimation of electronic properties described by logarithms of molar absorption
coefficients (log ) of their methanolic solutions, which were investigated in the UV/Vis region
–p. The research aimed determination of compounds’ ability to decrease surface
γ
ε
of an electromagnetic spectrum, as well as evaluation of lipophilic features defined by calculated
log kw parameters. These log kw were based on extrapolation procedures of estimated logarithms
of retention factors (log k) to elution with 100% water by reversed-phase high-performance liquid
chromatography (RP-HPLC). In addition, relationships between the log kw dataset and calculated
logarithms of partition coefficients (log P) related to the octan-1-ol/water partitioning system were
explored in order to provide a critical view on possibilities to predict lipophilic properties of the
compounds based on two-dimensional (2D) visualization of their chemical structures.
Next, a very essential objective of the research was in vitro screening of the derivatives 1a
–p
against various strains of tuberculous and non-tuberculous mycobacteria, i.e., M. tuberculosis CNCTC

Molecules 2018, 23, 2493
4 of 37
My 331/88, M. tuberculosis H37Ra ATCC 25177, M. kansasii CNCTC My 235/80, a M. kansasii 6509/96
clinical isolate, M. kansasii DSM 44162, M. avium CNCTC My 330/80, M. smegmatis ATCC 700084 and
M. marinum CAMP 5644, respectively.
A key aim of the study was to find some structural and physicochemical features of the compounds
1a–p, which might appear to be notable for their in vitro antimycobacterial efficiency.
2. Results and Discussion
2.1. Synthesis of the Compounds 1a–p
The investigated compounds 1a–p were prepared by multi step pathways using 2-aminophenol
(
10a) and 3-aminophenol (10b), respectively, as starting molecules (Scheme 1). Very briefly,
a reaction of 10a or 10b with acetanhydride led to N-(2-/3-hydroxyphenyl)ethanamide (20a or
20b). A solution of 20a or 20b in anhydrous ethanol (EtOH) was added to sodium ethanoate.
After mixing, 1-bromoalkane (alkane = butane to heptane) was added in order to prepare a series of
N-(2-/3-alk-oxyphenyl)ethanamides (30a ; alkoxy = butoxy to heptyloxy). The molecules 30a
–h –h
were suspended in 18% hydrochloric acid and heated up to reflux. The solutions were cooled,
neutralized and crude intermediates were extracted into diethyl ether (DEE). The organic layer was
dried, filtered and solutions were removed in vacuo giving 2-/3-alkoxyanilines (40a
–
h
; alkoxy = butoxy
to heptyloxy) [27]. Reulting intermediates 40a
–h were dissolved in anhydrous toluene and added
continuously into a saturated solution of phosgene [27]. The mixed solutions were refluxed, toluene
was removed in vacuo providing desired 1-alkoxy-2-/3-isocyanatobenzenes (50a–h).
Into an aqueous solution of N-propylpropanamine (60), a (
)-2-(chloromethyl)oxirane reagent
±
was added and solution was allowed to stand at room temperature (r.t.; 48h). The mixture was heated
up to 75 ◦C and treated with 38% sodium hydroxide. The cooled solution was filtered and a crude
intermediate was formed. Extraction of the filtrate with DEE, drying and removal of the organic layer
provided additional amount of the intermediate [28], which isolation and crystallization from absolute
EtOH led to (±)-N-(oxiran-2-ylmethyl)-N-propylpropanamine (70).
The addition of pyrrolidine or azepane to 70 in anhydrous propan-2-ol (2-PrOH) provided
1-(dipropylamino)-3-pyrrolidin-1-ylpropan-2-ol (80a) or 1-azepan-1-yl-3-(dipropylamino)-propan-2-ol
(80b) [29].
Oily 1-(1-azacycloalkyl)-3-(dipropylamino)propan-2-yl (2-/3-alkoxyphenyl)carbamates (90a
–p;
azacycloalkyl = pyrrolidinyl or azepanyl) were prepared by a reaction of 1-alkoxy-2-/3-isocyanato-
benzenes 50a–h with a dibasic alcohol 80a or 80b in anhydrous toluene.
Addition of a saturated solution of oxalic acid in anhydrous EtOH or ethereal hydrogen
chloride to the bases 90a
–
p
dissolved in chloroform led to solid colorless 1-[2-[({[2-/3-(alkoxy)phenyl]-
amino}carbonyl)oxy]-3-(dipropylammonio)propyl]pyrrolidinium oxalates (1a )/dichlorides (1e
and 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]azepanium
oxalates (1i )/dichlorides (1m ; Scheme 1, Table 1) as well. More detailed procedures describing
syntheses of particular intermediates and final molecules were provided in Supplementary Materials.
Chemical structure of the compounds 90a
and 1a was previously verified by their infrared
(IR) spectra, which confirmed presence of all key groups. In addition, elemental analyses results
–d
–h)
–l
–p
–p
–p
(% C, H, N) were within
±
0.40% of theoretical values for both bases 90a
–
p
and salts 1a
–
p
were
[
21].
Melting points (m.p.0s), Rf values (TLC) and acid-base pKa1 and pKa2 parameters of 1a
–p
already published [21,26] and can be found in Table S1.
Current liquid chromatography high resolution mass spectroscopy (HPLC-HR-MS) analyses,
which were performed on the LC Agilent Infinity System coupled with the Quadrupole Time-Of-Flight
Mass Spectrometer (6520 Accurate Mass Q-TOF LC/MS), confirmed structural identity of the
intermediates 90a–p (Supplementary Materials).
After re-crystallization from a mixture of acetone/ethanol (subgroups 1a
–d, 1e–h and 1m–p) or
acetone (1i ), high-resolution mass spectra (HR-MS) of these salts were measured by the Dionex
–
l

Molecules 2018, 23, 2493
5 of 37
UltiMate 3000 High-Performance Liquid Chromatograph coupled with the LTQ Orbitrap XL Hybrid
Ion Trap-Orbitrap Fourier Transform Mass Spectrometer equipped with a HESI II (heated electrospray
ionization) source in a positive (1a
–d, 1i–l) or negative (1e–h, 1m–p) mode. The analyses confirmed
structural identity of given molecules.
Scheme 1. Synthesis of 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)-
propyl]pyrrolidinium/azepanium oxalates/dichlorides (1a ; alkoxy = butoxy to heptyloxy). Reagents
–
p
and conditions: (i) (CH3CO)2O, reflux (1h); (ii) Na, anhydrous EtOH, 1-bromoalkane (alkane = butane to
heptane), r.t. (12h), reflux (3h); (iii) 18% HCl, reflux (2h); (iv) phosgene, anhydrous toluene, reflux (3h);
◦
(v) (
±
)-2-(chloromethyl)oxirane, 35 C (2h), r.t. (48h), 38% NaOH; (vi) pyrrolidine/azepane, anhydrous
2-PrOH, reflux (6h); (vii) anhydrous toluene, reflux (8h); (viii) saturated solution of (a) oxalic acid in
anhydrous EtOH or (b) ethereal hydrogen chloride.
2.2. Determination and Prediction of Some Physicochemical Properties of the Compounds 1a–p
2.2.1. Surface Tension
Surface tension (relative surface activity;
γ
) of aqueous solutions of the compounds 1a
–p
(
c = 2.0 × 10−3
M) was determined by a drop count technique [30,31] using a Traube stalagmometer.
All evaluated substances were able to decrease surface tension of water (γ = 0.07259 N/m) at 21 ◦C.
Elongation of the 2-/3-alkoxy side chain R led to more surface-active derivatives within
homological sets 1a
a research paper [32]. The 2-alkoxy substituted molecules 1a
surface tension of water than their 3-alkoxy substituted isomers 1e–h or 1m–p (Table 1, Figure 2).
–d
,
1e
–
h,
1i
–l
and 1m–p. This behavior was also in agreement with conclusions of
–d
and 1i showed lower ability to reduce
–l
Relationships between the number of carbon atoms forming the chain R (nc) and
γ values
(in N/m units) was inspected using a linear function, polynomial function of 2nd order and sigmoidal
fitting, respectively.

Molecules 2018, 23, 2493
6 of 37
Equations related to the most relevant functions and values of common statistical descriptors,
namely, 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. R2),
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, can be found in Table S2.
Indication of a significance level of the F Ratio was as follows: one star symbol (*) for statistically
significant, two stars symbol (**) for statistically very significant or three stars symbol (***) for
statistically extremely significant level was used. The regression equations and their statistical
characteristics were calculated and visualized by the Origin Pro ver. 9.0.0 SR2 software (OriginLab
Corporation, Northampton, MA, USA).
The relationships between nc and
1i and 1m ) or very significant (1e
(Figure 2, Equations (S1–S4) in Table S2).
γ
were described most precisely by statistically significant
(1a
–
d,
–
l
–p
–h) models built on polynomial functions of 2nd order
Table 1. Chemical structure of evaluated compounds 1a
–
p, their surface tension
γ
(relative surface
activity; in N/m units), wavelengths of observed absorption maxima (λ1
,
λ2 (Ch-T)
, λ3) and logarithms
of molar absorption coefficients (log ε1, log ε2 (Ch-T), log ε3) of compounds’ methanolic solutions
−5
(c = 8.0 × 10 M), which were investigated in the UV/Vis region of an electromagnetic spectrum.
1
Comp.
R
X
Y
γ (N/m)
λ1
log ε1
λ2 (Ch-T)
log ε2 (Ch-T)
λ3
log ε3
1a
1b
1c
1d
1e
1f
1g
1h
1i
2-OC4H9
2-OC5H11
2-OC6H13
2-OC7H15
3-OC4H9
3-OC5H11
3-OC6H13
3-OC7H15
2-OC4H9
2-OC5H11
2-OC6H13
2-OC7H15
3-OC4H9
3-OC5H11
3-OC6H13
3-OC7H15
A
A
A
A
A
A
A
A
B
B
B
B
B
C
C
C
C
D
D
D
D
C
C
C
C
D
D
D
D
0.06464
0.06366
0.06222
0.05985
0.06316
0.06285
0.06105
0.05786
0.06302
0.06206
0.06065
0.05853
0.06298
0.06154
0.05925
0.05692
208
208
208
208
210
210
210
210
208
208
208
208
210
210
210
210
4.51
4.39
4.58
4.43
4.65
4.55
4.54
4.66
4.44
4.54
4.42
4.44
4.52
4.56
4.52
4.65
236
236
238
236
238
237
237
237
236
236
236
236
238
238
238
238
4.19
4.05
4.52
4.08
4.24
4.27
4.13
4.27
4.08
4.22
4.10
4.14
4.09
4.18
4.01
4.20
280
280
278
280
279
279
279
279
280
280
280
280
279
279
279
279
3.63
3.51
3.49
3.55
3.59
3.62
3.48
3.63
3.57
3.71
3.57
3.59
3.44
3.52
3.37
3.56
1j
1k
1l
1m
1n
1o
1p
1
B
B
B
log ε2 (Ch-T), Logarithms of molar absorption coefficients observed at the charge-transfer absorption maximum
λ2 (Ch-T) = 236–238 nm.
The derivatives containing an azepanium moiety (1i
decrease surface tension of water than their positional isomers with a pyrrolidinium salt-forming
group (1a 1e ). The most surface active were 1-[2-[({[3-(heptyloxy)phenyl]amino}carbonyl)-oxy]-3-
(dipropylammonio)propyl]azepanium dichloride (1p) with = 0.05692 N/m and 1-[2-[({[3-
–l, 1m–p) showed slightly higher ability to
–
d,
–h
γ
(heptyloxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]pyrrolidinium dichloride (1h),
which showed γ = 0.05786 N/m (Table 1, Figure 2).
ˇ
Very similar behaviors to present findings were observed when Cižmárik with co-workers [33
,34]
analyzed structurally similar compounds JC-01a , effective LAs [33], which contained only one
–
l

Molecules 2018, 23, 2493
7 of 37
centrum of basicity (Figure 3). All given molecules showed ability to decrease surface tension of water
and 3-alkoxy substituted derivatives JC-01g (alkoxy = propoxy to octyloxy) were more efficient
surfactants than their 2-alkoxy positional isomers JC-01a . Relationships between nc (nc = 3–8) and
–
l
–
f
γ
(in N/m units) of these alkoxy positional isomers were described very satisfactorily by polynomial
functions of 2nd order [34].
Concerning presently analyzed dichlorides 1m
–
p, branching of their connecting chain and
presence of a dipropylammonium moiety resulted in the
γ
values (Table 1), which were slightly
lower in comparison to those of JC-01h–k [34].
Figure 2. Relationships between number of carbon atoms forming the 2-/3-alkoxy side chain R (nc;
alkoxy = butoxy to heptyloxy) and γ values (in N/m units) of the compounds 1a–p.
Figure 3. Chemical structure of the compounds JC-01a–l, effective local anesthetics [33], which were
able to decrease surface tension of water [34].
2.2.2. Electronic Properties
Electronic properties of the molecules 1a
absorption coefficients (log ) of their methanolic solutions (c = 8.0
UV/Vis region of an electromagnetic spectrum.
–
p
(Table 1) were characterized by logarithms of molar
ε
×
10−5 M) investigated in the
The solutions showed three absorption maxima in a near ultraviolet (quarz) region of this
spectrum between 200 nm and 400 nm [35], namely, λ1 = 208–210 nm, λ2 (Ch-T) = 236–238 nm and
λ3 = 278–280 nm (Table 1), respectively. Positions of these absorption bands in the spectrum were
typical for derivatives of (substituted) phenylcarbamic acid and the bands were assigned as first
local excitation (λ3), charge-transfer (λ2 (Ch-T)) and second local excitation (λ1) absorption maxima,
respectively [36]. The λ1 and λ2 (Ch-T) maxima of 2-alkoxy substituted compounds 1a
–
d
and 1i–l were
slightly lower compared to those of their 3-alkoxy substituted positional isomers 1e
–
h
and 1m–p

Molecules 2018, 23, 2493
8 of 37
(Table 1), respectively. Chemical structures of analyzed derivatives 1a
–d
and 1i
–l
indicated that steric
inability of both carbamoyloxy and alkoxy groups to achieve coplanarity inhibited ‘proper’ resonance.
The log ε2 (Ch-T) parameters of compounds 1a , which were observed at λ2 (Ch-T), varied from
4.01 (1o) to 4.52 (1c). The methanolic solutions of 1a were characterized by higher log ε2 (Ch-T)
values than those of 1i and ranged from 4.05 (1b) to 4.52 (1c; Table 1), excluding the molecules
–
p
–
h
–
p
1j (4.22) and 1l (4.14). In addition, there was found nor linear neither quasi-parabolic relationship
between number of carbon atoms forming the side chain R (nc) and log ε2 (Ch-T) values (Figure S1 in
Supplementary Materials).
In next sections of the paper, which are aimed at relationships between structure and in vitro
activity, special consideration is devoted to the log ε2 (Ch-T) values because they could be the most
sensitive to differences in electronic environment of a phenylcarbamoyloxy moiety due to different
position and length of a substituent R [36].
2.2.3. Lipohydrophilic Properties
Lipophilicity has been the physicochemical parameter of notable importance in QSAR and SAR
studies as a predominant descriptor encoding information on a network of inter- and intramolecular
forces affecting drug transport via lipophilic compartments as well as drug’s interactions with target
effector sites [37,38].
A classical shake-flask method for a partitioning measurement of a compound in the
octan-1-ol/buffer (or water) system, thus estimation of its log Pexp value, is quite time-consuming,
it requires the solute to be pure and with adequate solubility in an aqueous phase. Insolubility often
means that highly lipophilic compounds may not be determined accurately [39]. Concerning these
issues very seriously, RP-HPLC was rather applied to estimate lipophilic properties of the molecules
1a–p than the shake-flask alternative.
Octadecyl-functionalized silica gel was currently used as a stationary phase (SPh) and a
gradient of two solvents at different volume ratios modified retention properties of the SPh [39,40].
Liquid binary mixtures of methanol (MeOH) with water were employed as mobile phases (MPhs)
in the isocratic RP-HPLC. The MeOH modifier was preferred because of similarity of both observed
and extrapolated lipophilicity parameters with the ones determined in the octan-1-ol/water partition
system, considering also sensitivity to H-bond donor properties of investigated compounds [41].
The modifier was applied in different volume concentrations, which varied from 85% to 100%
(v/v) with 5% increments. This interval was chosen due to very high lipophilicity of investigated
salts. Practically, lower volume concentration of MeOH, i.e., less than 85% (v/v) in used MPhs, caused
uncertainty of retention times (tr) due to notable peaks broadening, especially within subsets 1i
–l and
1m . In addition, their tr values were considerably prolonged with increase in water content (v/v)
–
p
of the MPhs so it was no longer possible to determine tr values in such MPhs. Taking the 85–100%
volume concentrations range, the isocratic separation was feasible and reasonable retention of the
compounds 1a–p was observed in all MPhs. The highest tr parameters were found for the molecules
1k (tr = 47.108 min) and 1l (tr = 70.012 min) in the MPh containing 85% proportion (v/v) of MeOH
(Table 2).
In addition, purity (in percentages) of the compounds 1a–p was verified by RP-HPLC. Areas of
their peaks were measured using the MPh, which contained 90% proportion (v/v) of MeOH. The purity
of given salts varied from 96.82% (1j) to 99.65% (1m; Table S3).
Estimated retention factor (capacity factor; k) values were found in an area from 0.5063 (1e
)
to 2.5096 (1l) if pure MeOH was used as the MPh, and from 3.6174 (1e) to 30.4159 (1l) if the 80:20
MeOH/water (v/v) MPh was applied (Table S3).
Regarding a group of oxalates (Scheme 1), a pyrrolidium moiety-containing derivatives 1a
showed lower log k values in all used MPhs than those with an azepanium fragment 1i . Same trends
showed lower log k outputs compared to
–d
–
l
were observed for dichlorides, i.e., the compounds 1e
those of a series 1m–p.
–h

Molecules 2018, 23, 2493
9 of 37
Elongation of the R substituent (Table 1) led to increase in tr as well as log k values within all
evaluated subgroups 1a 1e 1i and 1m (Table 2). Increase in a volume concentration (v/v) of
MeOH caused shortening of both tr and log k for all analyzed compounds 1a–p (Table 2).
El Tayar et al. [42 43] investigated lipophilic properties of protonated basic compounds, which
–
d,
–
h,
–
l
–p
,
were therapeutically used as psychoactive agents, and inspected relationships between their log k
values and volume concentrations (v/v) of MeOH. They found that these relationships were notably
influenced by nature of solutes. The models were preferably built on linear functions and MeOH
exerted its own solvophobic effect in the MPhs, which contained more than 80% (v/v) of given organic
modifier if focusing on analyses of neutral and non-ionic compounds.
For partially and completely ionized polar compounds, the relationships appeared to be nearly
parabolic but the parts of parabolic curves, which corresponded to MeOH-rich eluents (80% or higher
volume concentration (v/v) of MeOH) were regarded as linear [42,43].
Paschke et al. [44] analyzed a series of highly lipophilic tetrachlorobenzyltoluene isomers by
isocratic RP-HPLC and found linear relationships (R > 0.9995) between their log k values and volume
concentrations of MeOH, which varied from 80% to 100% (v/v).
Table 2. Retention times tr (RP-HPLC) and lipophilicity indices log k (RP-HPLC) of the compounds
1a–p estimated in the mobile phases with a various volume ratio (v/v) of a methanol (MeOH) organic
modifier and water.
Mobile Phase MeOH/Water (v/v)
80:20
85:15
tr (min)
90:10
95:5
tr (min)
Pure MeOH
Comp.
tr (min)
log k
log k
tr (min)
log k
log k
tr (min)
log k
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
13.883
17.200
25.694
31.000
10.330
12.400
17.200
22.003
24.020
29.525
47.108
70.012
15.731
22.183
29.174
33.719
0.7165
0.8254
1.0206
1.1092
0.5584
0.6574
0.8254
0.9521
0.9881
1.0863
1.3023
1.4831
0.7805
0.9502
1.0807
1.1484
10.694
12.647
16.950
19.600
6.917
0.5756
0.6656
0.8162
0.8882
0.3183
0.4911
0.6104
0.7159
0.8153
0.8981
1.0424
1.2152
0.4922
0.6542
0.7059
0.8215
7.830
9.449
11.292
13.553
5.400
6.280
7.483
8.532
11.691
13.060
16.533
22.636
6.420
8.156
9.634
11.332
0.3958
0.5064
0.6053
0.7022
0.1478
0.2546
0.3679
0.4565
0.6240
0.6828
0.8038
0.9619
0.2695
0.4205
0.5174
0.6072
5.766
6.514
7.431
7.892
3.822
4.022
5.153
5.788
7.765
8.520
9.641
11.670
4.685
4.995
5.095
5.801
0.2008
0.2841
0.3683
0.4052
4.224
4.526
5.246
5.385
3.329
3.340
3.583
3.556
5.127
5.295
6.938
7.756
3.519
3.947
4.127
3.925
−0.0403
0.0203
0.1287
0.1573
−
0.1454
0.0941
−0.2956
−0.2913
−0.2067
−0.2153
0.1205
0.1449
0.3303
0.3996
−0.2275
−0.1047
−0.0618
−0.1102
9.200
−
11.402
13.701
16.917
20.021
27.001
38.800
9.218
12.370
13.651
17.129
0.1182
0.2092
0.3954
0.4509
0.5221
0.6271
0.0424
0.0941
0.1095
0.2051
On the other hand, observed log k data could not be used in terms of a universal scale to express
lipophilicity of compounds because the log k values were dependent on chromatographic conditions,
i.e., they depended on both MPhs and SPhs. Extrapolation of estimated solute retention (log k) to
elution with 100% water (calculation of log kw) would definitely be much more precise approach than
using of log k, because the kw parameters were independent of any effect of the organic modifier and
relied on the SPh alone [45]. It has been recognized that the log kw descriptor was very efficient for
anticipation of in vitro antimycobacterial properties of compounds [46–49].
According to the authors’ opinions based on systematic scientific literature survey, a current
research paper was the first, which aimed the log kw parameters of 2-/3-alkoxyphenylcarbamic
acid derivatives containing two centers of protonation. The log kw values were extrapolated from
intercepts of a relationship between log k and volume fraction of the MPh modifier (ϕM) using the
Snyder–Soczewin´ski linear solvent strength model [50
–52]. The relationship was justified by R > 0.9900,
Adj. R2 > 0.9750 as well as F > 180.00 for calculation of particular log kw parameters (Table 3).

Molecules 2018, 23, 2493
10 of 37
Table 3. Extrapolated log kw parameters (RP-HPLC) of analyzed molecules 1a–p and values of statistical
descriptors (χ2red, RSS, R, Adj. R2, RMSE, NR, F and Prob > F), which characterized a linear relationship
between the log k and ϕM values for a particular compound. The ϕM parameter was a volume fraction
of MeOH in the isocratic elution RP-HPLC.
1
2
3
4
5
6
7
8
9
Comp.
log kw
S
χ2
RSS
R
Adj. R2
RMSE
NR
F
Prob > F
red
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
3.7688
4.0454
4.6049
4.9487
4.0258
4.6722
4.9446
5.5384
4.4679
4.8466
5.2359
5.8966
4.7099
5.2087
5.6569
6.1749
3.7768
3.9834
4.4634
4.7736
4.3434
4.9652
5.1128
5.6830
4.3102
4.6600
4.9286
5.5102
4.9316
5.3398
5.7628
6.2672
0.0011
0.0018
0.0002
0.0011
0.0013
0.0028
0.0013
0.0042
0.0014
0.0016
0.0005
0.0007
0.0004
0.0011
0.0045
0.0022
0.0034
0.0055
0.0007
0.0033
0.0038
0.0084
0.0040
0.0126
0.0042
0.0048
0.0017
0.0020
0.0013
0.0032
0.0134
0.0067
0.9953
0.9931
0.9993
0.9971
0.9960
0.9932
0.9969
0.9923
0.9955
0.9956
0.9987
0.9988
0.9990
0.9978
0.9920
0.9966
0.9875
0.9817
0.9982
0.9923
0.9894
0.9821
0.9919
0.9795
0.9879
0.9882
0.9964
0.9965
0.9972
0.9941
0.9789
0.9909
0.0335
0.0429
0.0152
0.0332
0.0354
0.0530
0.0366
0.0648
0.0376
0.0401
0.0234
0.0259
0.0205
0.0325
0.0668
0.0474
0.0580
0.0744
0.0263
0.0574
0.0614
0.0917
0.0634
0.1122
0.0652
0.0695
0.0406
0.0449
0.0356
0.0564
0.1157
0.0820
317.64
215.17
2155.37
518.15
375.47
219.79
487.75
192.51
328.27
337.19
1106.57
1127.92
1442.73
673.05
186.23
438.042
0.0001 ***
0.0007 ***
0.0001 ***
0.0002 ***
0.0003 ***
0.0007 ***
0.0002 ***
0.0008 ***
0.0004 ***
0.0004 ***
0.0001 ***
0.0001 ***
0.0001 ***
0.0001 ***
0.0009 ***
0.0001 ***
1
S, Slope; 2 χ2red, reduced chi-square; 3 RSS, residual sum of squares; 4 R, correlation coefficient; 5 Adj. R2, adjusted
coefficient of determination; 6 RMSE, root mean squared error (standard deviation); 7 NR, norm of residuals; 8 F,
Fisher’s significance ratio (Fisher’s F-test); 9 Prob > F, probability of obtaining the F Ratio (significance of a whole
model). Indication of a significance level of the F Ratio was as follows: *** (three stars), extremely significant.
Goodness of fit for all proposed models generating log kw, in other words, quantity used to test
whether any given data were well described by a suggested function, was adjusted by χ2red values,
which varied from 0.0002 (1c) to 0.0045 (1o). As can be seen, the linear relationships minimized RSS
values, as variabilities about regression lines, which ranged from 0.0007 (1c) to 0.0134 (1o). The linearity
was also proved by calculated RMSE parameters, as standard deviations of the data about regression
lines, which were found in an interval from 0.0152 (1c) to 0.0668 (1o; Table 3).
The extrapolated log kw values of compounds 1a
their elution order and hydrophobicity and varied from 3.7688 (1a) to 6.1749 (1p). Assuming
presence of an identical azacycloalkyl moiety, oxalates (1a 1i ) were less lipophilic than
corresponding positional isomers synthesized as dichlorides (1e 1m ; Table 3). As expected,
1-[2-[({[3-(hept-yloxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]azepanium oxalate
1l) and 1-[2-[({[3-(heptyloxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]azepanium
–p (Table 3) were in accordance with
–
d,
–l
–
h,
–p
(
dichloride (1p; Table 1) were found to be the most lipophilic showing log kw of 5.8966 (1l) and 6.1749
(1p; Table 3), respectively.
Increase in nc (Table 1) led to higher log kw values in all inspected subgroups 1a
–d, 1e–h, 1i–l
and 1m . These relationships were defined by statistically significant (1e 1i ), very significant
–
p
–
h,
–l
(1a–d) and extremely significant (1m–p) linear functions and values of relevant statistical descriptors
(Equations (S5)–(S8) in Table S4).
The S index (Table 3) in RP-HPLC has been a function of molecular structure parameters. For sets
of non-polar homologues, linear relationships between their S and number of carbon atoms in a
hydrocarbon side chain was found. Increase in size and van der Waals volume of a solute led to
increased S parameter as well [53].
Results of present analyses agreed with given conclusions. Increase in length of the side chain R led
to higher S within all inspected homological subgroups. The compounds containing an azepanium moiety
showed higher S parameters, which varied from 4.3102 (1i) to 6.2672 (1p), than those with a pyrrolidium
group, i.e., from 3.7768 (1a) to 5.6830 (1h; Table 3), if same position and length of R was presumed.
The slope S values of linear regression functions used to obtain log kw were connected with
specific hydrophobic surface areas of compounds and could serve as indicative measure of uniformity
of retention mechanisms. If the uniformity was observed, a convenient model between the slope(s)
and intercept(s), i.e., log kw values, was anticipated [40]. Statistically extremely significant relationship
between the log kw and S values of 1a–p was found and described by Equation (1):

Molecules 2018, 23, 2493
11 of 37
S = 0.9449 (±0.0823) × log kw + 0.2755 (±0.4087)
(1)
n = 16, DF = 14, χ,2red = 0.0470, RSS = 0.6586, R = 0.9508, Adj. R2 = 0.8971,
RMSE = 0.2169, NR = 0.8115, F = 131.75, Prob > F = 0.0001 ***
Based on values of statistical descriptors provided above, the uniformity of a retention mechanism
was proven and suitability of chosen MPhs was confirmed for lipophilicity evaluation of the
derivatives 1a–p.
Statistical analyses indicated that both position and length of the side chain R (Equations
(S9) and (S10) in Table S5) were more important factors, which contributed to the uniformity of
retention behavior, compared to nature (and physicochemical properties) of a salt-forming fragment
(Equations (S11) and (S12) in Table S5) of inspected compounds, despite the fact that all partial models
built on linear regression analyses were statistically extremely significant.
To provide better understanding and give a critical review of relationships between the
experimental lipophilicity measures and in silico prediction of lipophilic properties, the log kw dataset
was studied together with computational logarithms of partition coefficients (log P).
Practical estimation of log Pexp for some molecules from the set 1a–p by a shake-flask method was
not possible due to their insolubility in phosphate buffer systems (pH = 7.30). Thus, the lack of enough
reliable log Pexp values would be a very limiting factor for eventual investigation of the relationships
between log Pexp and in silico log P. On the other hand, linear models between log kw and log Pexp
were already observed [39], so interchangeability of these descriptors was fully justified.
The log P parameters of non-protonated forms of analyzed compounds, i.e., 90a
–p (Scheme 1,
Table 4), were calculated for the octan-1-ol/water partitioning system. These values were generated by
the ChemBioDraw Ultra 12.0 software package (CambridgeSoft, Cambridge, MA, USA), various Java
and non-Java interactive applets as well.
The Ghose and Crippen0s log PCr 55], Viswanadhan0s log PV [56], Broto’s log PB [57] and Leo’s
[54,
CLOGP 4.0 [58] atomic and atomic/fragmental-based approaches were chosen to calculate the log p
values (Table 4) by the ChemBioDraw Ultra 12.0 software, respectively. The Virtual Computer Chemistry
Laboratory [59], a freely available web-based tool working in Java environment, was used to calculate
log P by the Wang’s XLOGP 2.0 [60], Cheng’s XLOGP 3.0 [61], Moriguchi’s MLOGP [62], Sander’s
(Actelion’s) ACLOGP [63], Molinspiration’s miLogP 2.2 [64], ALOGP [65] and Tetko’s ALOGPs 2.1 [66
]
method (Tables 4 and 5), respectively. All these methods integrated algorithms combining atomic and
fragmental principles, except for ALOGPs 2.1, which considered a molecule in the whole [66].
Finally, the SILICOS-IT hybrid method, which used proprietary fragment- and property-based
principles, was also employed to generate log P parameters (log PS-IT; Table 5). The method was
implemented in the SwissADME applet, a free web tool designed to evaluate pharmacokinetics and
drug-likeness of small molecules [67].
A common issue connected with given in silico approaches [26] was that they did not allow
to correctly predict log p values of particular salts 1a
targeted on the calculations related to non-protonated bases 90a–p.
The compounds 90i showed higher log P than their positional isomers 90a
a different salt-forming moiety. Almost all used predictor tools indicated the molecules 90a
–p. This was the reason why current research
–p
–
h, which contained
–p
highly
lipophilic with log p > 4.27 (Tables 4 and 5), excluding the MLOGP method. The calculations based on
MLOGP indicated moderate or high lipophilicity of these substances, which log P were found in a
range from 2.75 (90a and 90e) to 3.76 (90l and 90p; Table 5).
The CLOGP 4.0 predicted the highest lipophilic nature of the bases 90a
–p and their log P varied
from 6.23 (90a and 90e) to 8.94 (90l and 90p; Table 4). As might be assumed, all employed atomic and
atomic/fragmental methods did not consider a position of the side chain R when generating log P
(Tables 4 and 5). Only the whole-molecule ALOGPs 2.1 approach took into account this aspect, so the
2-alkoxy substituted compounds 90a
–d –l were less lipophilic than their 3-alkoxy substituted
and 90i
positional isomers 90e–h and 90m–p (Table 5), respectively.

Molecules 2018, 23, 2493
12 of 37
It was found that linear relationships between the extrapolated log kw values of compounds
(Table 3) and particular in silico log P parameters of non-protonated derivatives 90a
, namely,
1a
–
p
–p
log PCr, log PV, log PB, CLOGP 4.0, XLOGP 2.0, XLOGP 3.0, MLOGP, ACLOGP, miLogP 2.2, ALOGP,
log PS-IT and ALOGPs 2.1 (Tables 4 and 5), respectively, were statistically extremely significant (Prob >
F = 0.0001) and were defined by R > 0.9300, Adj. R2 > 0.8650, F > 99.00 and other common statistical
descriptors (Equations (S13)–(S24) in Table S6).
0
Table 4. Values of logarithms of partition coefficients of basic (non-protonated) compounds 9 a
–
p
predicted for the o0ctan-1-ol/water partitioni0ng system by the Ghose and Crippen0s approach (log
P
Cr), Viswanadhan s principle (log PV), Broto s algorithm (log PB) and CLOGP method (CLOGP 4.0),
respectively, as well as by both XLOGP approaches (XLOGP 2.0 and XLOGP 3.0).
CLOGP
4.0
XLOGP
2.0
XLOGP
3.0
Comp.
R
X
log PCr
log PV
log PB
0
9 a
2-OC4H9
2-OC5H11
2-OC6H13
2-OC7H15
3-OC4H9
3-OC5H11
3-OC6H13
3-OC7H15
2-OC4H9
2-OC5H11
2-OC6H13
2-OC7H15
3-OC4H9
3-OC5H11
3-OC6H13
3-OC7H15
A
A
A
A
A
A
A
A
B
B
B
B
B
4.59
5.00
5.42
5.84
4.59
5.00
5.42
5.84
5.42
5.84
6.26
6.67
5.42
5.84
6.26
6.67
4.45
4.85
5.24
5.64
4.45
4.85
5.24
5.64
5.24
5.64
6.04
6.43
5.24
5.64
6.04
6.43
4.51
4.97
5.42
5.88
4.51
4.97
5.42
5.88
5.42
5.88
6.33
6.79
5.42
5.88
6.33
6.79
6.23
6.76
7.29
7.82
6.23
6.76
7.29
7.82
7.35
7.88
8.41
8.94
7.35
7.88
8.41
8.94
4.52
5.09
5.66
6.23
4.52
5.09
5.66
6.23
5.24
5.81
6.38
6.95
5.24
5.81
6.38
6.95
5.14
5.68
6.22
6.77
5.14
5.68
6.22
6.77
5.86
6.40
6.84
7.48
5.86
6.40
6.84
7.48
0
9 b
0
9 c
0
9 d
0
9 e
0
9 f
0
9 g
0
9 h
0
9 i
0
9 j
0
9 k
0
9 l
0
9 m
0
9 n
B
B
B
0
9 o
0
9 p
0
Table 5. Values of logarithms of partition coefficients of basic (non-protonated) compounds 9 a
–
p
predicted for the octan-1-ol/water partitioning system by the MLOGP, ACLOGP, miLogP 2.2, ALOGP,
SILICOS-IT (log PS-IT) and ALOGPs 2.1 method, respectively.
miLogP
2.2
ALOGPs
2.1
Comp.
MLOGP
ACLOGP
ALOGP
log PS-IT
0
9 a
2.75
2.96
3.16
3.36
2.75
2.96
3.16
3.36
3.16
3.36
3.56
3.76
3.16
3.36
3.56
3.76
5.05
5.52
5.98
6.45
5.05
5.52
5.98
6.45
5.69
6.16
6.62
7.08
5.69
6.16
6.62
7.08
5.30
5.80
6.32
6.81
5.32
5.83
6.33
6.84
6.31
6.81
7.32
7.82
6.33
6.84
7.34
7.85
5.42
5.87
6.33
6.79
5.42
5.87
6.33
6.79
6.33
6.79
7.24
7.70
6.33
6.79
7.24
7.70
4.27
4.68
5.10
5.53
4.27
4.68
5.10
5.53
4.78
5.20
5.62
6.04
4.78
5.20
5.62
6.04
5.18
5.57
5.93
6.25
5.21
5.61
5.95
6.32
5.93
6.23
6.54
6.77
5.97
6.27
6.59
6.83
0
9 b
0
9 c
0
9 d
0
9 e
0
9 f
0
9 g
0
9 h
0
9 i
0
9 j
0
9 k
0
9 l
0
9 m
0
9 n
0
9 o
0
9 p

Molecules 2018, 23, 2493
13 of 37
In an effort to characterize these relationships more precisely, similarities and differences between
given descriptors were analyzed by using unscaled principal component analysis (PCA), a powerful
multivariate statistical technique, that analyzed a set of values, in which observations were described
by several inter-correlated quantitative dependent variables. A set of new orthogonal variables, called
principal components (PCs), and pattern of similarity of observations was a result the analysis [68].
Current PCAs were performed by the Origin Pro ver. 9.0.0 SR2 software. Number of concerned PCs
was determined by visual evaluation of a scree plot, as a relationship between calculated eigenvalues
(
λe) and number of PCs. The λe descriptor measured amount of variation retained by each PC [68].
The first two PCs, i.e., Principal Component 1 (PC 1) and Principal Component 2 (PC 2), of the analysis
accounted for 99.26% of the total variance in the data. It meant that other PCs might be ignored
without losing any substantial information. The PCs did not prove existence of ‘real’ parameters,
they only indicated that existence of these descriptors was mathematically possible within the set of
analyzed values.
A relationship between PC 1 and PC 2 resulted in division of examined compounds into
characteristic groups. Differences in lipophilic properties of the bases 90a
–
p
were reflected in their PC 1
values. One group, defined by negative PC 1 values, included derivatives containing a pyrrolidin-1-yl
moiety (90a 90e ) as well as molecules with both azepan-1-yl fragment and butoxy side chain (90i
90m). Conversely, second group, which was characterized by positive PC 1 values, encompassed
compounds containing an azepan-1-yl moiety (90j 90n
) as well as derivatives with both sterically
smaller azacycloalkyl ring and heptyloxy substituent (90d 90h). The most negative PC 1 value
–
c,
–
g
,
–l
,
–p
,
was generated for the least lipophilic derivative 90a, which salt 1a showed the lowest experimental
lipophilicity (log kw = 3.7688). The most positive PC 1 output was registered for the most lipophilic
compound 90p, a base of the most lipophilic salt 1p (log kw = 6.1749; Figure 4).
It was found that PC 2 took into consideration a type of an azacycloalkyl moiety as well as position
–h –p. Higher PC 2
and length of R. The derivatives 90a showed higher PC 2 than the compounds 90i
were assigned to the 3-alkoxy substituted molecules compared to their 2-alkoxy positional isomers,
when assuming their identical salt-forming moiety.
Increase in nc of the side chain R led to higher PC 1 as well as PC 2. There were found no
compounds (points), which could be regarded as outliers to the remaining ones along PC 1 or PC 2
(Figure 4).
The PCA-based 2D biplot showed both PC scores (PC 1, PC 2) of evaluated compounds (green
dots) and loadings of variables indicated as orange straight lines [68]. The lines characterizing log
kw, log PI-IT, XLOGP 2.0, XLOGP 3.0 and ACLOGP variables were located on upper right side of this
biplot. The line related to log kw formed the smallest angles with those of log PI-IT, XLOGP 2.0 or
XLOGP 3.0, which meant that these descriptors correlated each other most positively. Conversely,
the largest angle was observed between the lines connected with log kw and CLOGP 4.0, so these
variables were unlikely to correlate each other (Figure 4).
The PC 1 was influenced almost equally by loadings of all inspected variables, as indicated
particular scores on top axis. The scores varied from 0.27 (log kw) to 0.28 (log PCr, log PV, log PB,
miLogP 2.2 and ALOGP, respectively). The most positive impact on PC 2 showed a loading of the
log kw and log PI-IT variable, which scored approximately 0.45 and 0.37, respectively, on right axis.
Conversely, the strongest inverse relationship was found between PC 2 and loading of the CLOGP
4.0 variable (−0.28; Figure 4).
Following visualization, the in silico log PI-IT, XLOGP 2.0 and XLOGP 3.0 descriptors might be
used to characterize lipophilic properties of analyzed salts and bases, especially if they contained
highly lipophilic side chain R. Those molecules were defined by positive values of both PC 1 and PC 2.
In addition, very close grouping was observed for 90l with 90p (Figure 4).

Molecules 2018, 23, 2493
14 of 37
Figure 4. Two-dimensional biplot (mapping) showing both Principal Component 1 and 2 scores
0
of the compounds 9 a–p and loadings of extrapolated chromatographic (log kw; 1a–p) and in silico
lipophilicity variables, i.e., log PS-IT, XLOGP 2.0, XLOGP 3.0, ACLOGP, ALOGPs 2.1, miLogP 2.2,
CLOGP 4.0, log PCr, log PV, log PB, MLOGP and ALOGP, respectively.
Considering observed physicochemical characteristics of the compounds 1a
–p, there was found
statistically extremely significant linear relationship between the and log kw values. This dependence
γ
was expressed by Equation (2) and values of relevant statistical descriptors as follows:
γ = −0.0031 (±0.0003) × log kw + 0.0765 (±0.0015)
(2)
n = 16, DF = 14, χ,2red = 6.436 × 10−7, RSS = 9.010 × 10−6, R = 0.9383, Adj. R2 = 0.8718,
RMSE = 8.022 × 10−4, NR = 0.0030, F = 103.02, Prob > F = 0.0001 ***
Linearity of this model was adjusted by relatively very low χ2red, RSS and RMSE values. The
and log kw parameters of 1i showed the most positive impact to final statistical analysis data
γ
–
p
(Equation (S28) in Table S7) connected with Equation (2) compared to those of a subset 1a–h (Equation
(S27) in Table S7).
One of the major tasks to consider in drug design is the ability of a compound to cross biological
membranes. The blood–brain barrier (BBB) is a critical biological structure for maintaining homeostasis
of central nervous system and preventing damage to the brain. Compounds primarily designed as
neuroactive agents are required to cross the BBB to provide their therapeutic effect. Conversely,
molecules that target other compartments of a body ideally should not cross this barrier to avoid
possible psychotropic side effects. Thus, the task of predicting the BBB permeability of new compounds
is of great importance [69].
Norinder and Haeberlein [70] suggested some simple rules to preliminary answer a question
whether a drug candidate would be able to cross the BBB. Very briefly, if a sum of nitrogen and oxygen
atoms (nN + nO) in a molecule was
≤
5 or if [CLOGP
−
(nN + nO)] > 0 then the compound would
have a good chance to enter the brain by passive diffusion. The originally employed CLOGP was a
lipophilicity descriptor generated in silico. In addition, molecular weight (MW) of such compound
should be ≤ 450 to facilitate brain permeation [71].

Molecules 2018, 23, 2493
15 of 37
Each of the oxalates 1a–d and 1i–l contained (nN + nO) = 10, each of the chlorides 1e–h and 1m–p
contained (nN + nO) = 6 (Table 1), respectively. If presently calculated CLOGP 4.0 parameter would be
replaced with log PS-IT (Table 5), which was closer to a ‘real’ log kw lipophilicity descriptor (Figure 4),
only basic forms of the most lipophilic compounds 1l and 1p, i.e., the substances 90l and 90p, might
be considered to possess very limited brain permeation. On the other hand, calculated MW > 450 for
these derivatives [21] indicated that they might not be able to passively cross the BBB.
2.3. In Vitro Antimycobacterial Assays
The in vitro activity of compounds 1a–p was inspected against Mycobacterium tuberculosis CNCTC
My 331/88 (identical with H37Rv and ATCC 2794, respectively; abbreviation used: MTv H37Rv),
M. tuberculosis H37Ra ATCC 25177 (MTa H37Ra), M. kansasii CNCTC My 235/80 (identical with
ATCC 12478; MK 235/80), a M. kansasii 6509/96 clinical isolate (MK 6509/96), M. kansasii DSM 44162
(MK DSM), M. avium CNCTC My 330/80 (identical with ATCC 25291; MA 330/80), M. smegmatis
ATCC 700084 (MS) and M. marinum CAMP 5644 (MM), respectively, by the methods described
earlier [20,72–75]. In vitro susceptibitity of given mycobacteria to reference drugs isoniazid (INH),
ethambutol (EMB), ofloxacin (OFLX) or ciprofloxacin (CPX) was tested as well (Tables 6 and 7).
A value of a minimum inhibitory concentration (MIC) was the lowest concentration of a tested
compound, (i) which inhibited growth of MTv H37Rv, MK 235/80, MK 6509/96 and MA 330/80,
respectively; (ii) at which no visible growth of MTa H37Ra, MS, MK DSM as well as MM was
observed [20,72–75].
The in vitro screening procedures related to particular mycobacterial strains were repeated
three times and the MIC values, reported in Tables 6 and 7 (in
µM units), were average values
of these determinations.
A very unique aspect of a present research was that many compounds from the set 1a–p
were
in vitro efficient against almost all tested mycobacteria. The most promising molecules with observed
MIC ≤ 8 µM were indicated in gray (Tables 6 and 7).
The 3-alkoxy substituted derivatives 1e–h and 1m–p were slightly more effective against all
screened strains than their 2-alkoxy substituted isomers 1a
(Heptyloxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]azepanium dichloride (1p
was the most active against almost complete spectrum of tested microorganisms (MIC M),
–d and 1i–l, respectively. 1-[2-[({[3-
)
≤
8 µ
excluding MA 330/80 only. Comparable efficiency to 1p was observed for 1-[2-[({[3-(heptyl-
oxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]pyrrolidinium dichloride (1h), which
also showed MIC
MS. In addition, antimycobacterial activity of 1-[2-[({[3-(pentyloxy)phenyl]amino}carbonyl)oxy]-3-(di-
propylammonio)propyl]azepanium dichloride 1n and 1-[2-[({[3-(hexyloxy)phenyl]amino}-
≤
8 µM against almost all tested mycobacteria, excluding MA 330/80 as well as
(
)
carbonyl)oxy]-3-(dipropylammonio)propyl]azepanium dichloride (1o), respectively, was only slightly
lower compared to activity of the substances 1p and 1h (Tables 6 and 7).
It was found that MTv H37Rv was not so sensitive to the compounds 1a
0.5 µM and 1 M). The most promising substances 1d 1h 1l 1n or 1p showed MIC = 4
(Table 6), respectively.
Similar trends were observed when evaluating susceptibility of MK 235/80. The molecules 1h
and 1p showed MIC = 8 M, however, the OFLX reference drug was more active (MIC = 0.5 M and
M; Table 6). Comparable ability to fight MK 6509/96 was found for the derivative 1h, series 1n
(MIC = 4 µM and 8 µM) and INH (MIC = 2–8 µM; Table 6), respectively.
All 3-alkoxy substituted molecules 1e–h and 1m–p were more active against MTa H37Ra (MIC =
3.7–8.1 M) than INH (MIC = 36.5 M) or CPX (MIC = 48.3 M). The dibasic compounds were also
able to fight MK DSM more effectively, their MIC values ranged from 1.9 M (1h) to 16.2 M (1e),
than INH (MIC = 29.2 M; Table 6). Moreover, the derivatives 1g 1h and 1n were almost equally
efficient against MA 330/80 as EMB (MIC = 16 µM; Table 7).
–
p
as to INH (MIC =
µ
,
,
,
µM and 8
µM
,
1n
µ
µ
1
µ
–p
µ
µ
µ
µ
µ
µ
,
–p

Molecules 2018, 23, 2493
16 of 37
If attention was paid to the 2-alkoxy substituted compounds 1a
–
d
and 1i
–
l, 1-[2-[({[2-(heptyl-
oxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]pyrrolidinium dichloride (1d) was
more active against both MTa H37Ra (MIC = 12.5 µM) and MK DSM (MIC = 6.2 µM) than INH
(Table 6).
Waisser et al. [76] in vitro tested two series of structurally similar molecules JC-01a–l (Figure 3)
and JC-02a (Figure 5) against MTv H37Rv, MK 235/80, MK 6509/96 and MA 330/80, respectively.
–
l
The authors found that the compounds with an azepanium group were antimycobacterially more
promising agents than the ones containing a pyrrolidinium heterocycle.
Current in vitro assays revealed that both branching of a connecting hydrocarbon chain and
presence of ‘an additional’ dipropylammonium group within a salt-forming part of tested compounds
1a–p were favorable for their antimycobacterial activity and they were more effective than the sets
JC-01a–l and JC-02a–l.
The INH standard was less effective against MS (MIC = 117
than almost all screened phenylcarbamic acid-based substances, exluding the derivatives 1a
µ
M) as well as MM (MIC = 467
1i 1k or 1l
(Table 7). 1-[2-[({[2-(Pentyloxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]-azepanium
oxalate (1j) was the most active against MS (MIC = 6.2 M), the molecule 1h fight MM most efficiently
(MIC < 3.7 M). On the other hand, these strains were sensitive to lower MICs of CPX (MIC = 0.4 µM
and 0.8 M; Table 7). It should be also mentioned that both sets JC-01a (Figure 3) and JC-02a
(Figure 5) have not been in vitro tested against MTa H37Ra, MK DSM, MS or MM.
µM)
,
,
µ
µ
µ
–
l
–l
Table 6. The in vitro activity (MIC values in µM units) of investigated compounds 1a–p and reference
drugs isoniazid (INH), ethambutol (EMB), ofloxacin (OFLX) and ciprofloxacin (CPX) against M.
tuberculosis CNCTC My 331/88 (M. tuberculosis H37Rv; MTv H37Rv), M. tuberculosis H37Ra ATCC
25177 (MTa H37Ra), M. kansasii CNCTC My 235/80 (MK 235/80), M. kansasii 6509/96 (MK 6509/96)
and M. kansasii DSM 44162 (MK DSM), respectively.
MIC [µM]
MTv
H37Rv
MTa
H37Ra
MK
DSM
MK 235/80
MK 6509/96
Comp.
1
2
3
14-d
21-d
7-d
7-d
14-d
21-d
7-d
14-d
21-d
7-d
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
INH
EMB
OFLX
CPX
32
16
16
8
16
16
8
32
32
16
8
32
16
16
8
32
32
16
8
16
8
213
52
25
12.5
8.1
<3.9
<3.8
<3.7
203
199
195
96
15.4
7.5
7.3
62.5
32
32
16
32
16
16
8
32
16
16
16
16
8
62.5
32
32
16
32
16
16
8
62.5
32
32
32
16
8
16
8
>250
–
1
–
62.5
62.5
32
32
32
32
16
8
62.5
62.5
62.5
32
32
8
16
8
>250
–
1
32
32
16
8
16
8
62.5
32
16
16
16
16
8
62.5
62.5
16
16
32
16
16
8
32
32
32
32
16
8
8
8
8
2
1
–
53
26.1
12.7
6.2
16.2
7.9
3.8
1.9
51
8
4
4
4
16
16
8
4
8
4
8
4
0.5
1
16
16
16
16
8
4
4
4
2
32
32
32
16
16
8
8
8
4
2
50
98
382
7.7
7.5
3.6
3.6
29.2
–
16
8
1
2
2
8
8
7.1
36.5
–
–
48.3
>250
–
0.5
–
1
0.5
–
1
–
0.5
–
–
3.0
–
–
1
2
3
14-d, 14-Day cultivation; 21-d, 21-day cultivation; 7-d, 7-day cultivation. The most promising values of
compounds´ in vitro antimycobacterial activity (MIC ≤ 8 µM) were indicated in gray cells of Table 6.
The chemometric PCA tool was applied to study activity–activity relationships of the compounds
1a–p to explore similarities or differences in their impact on particular mycobacterial strains.
The analysis was carried out by the XLSTAT ver. 2016.02.28451 software (Addinsoft, New York,

Molecules 2018, 23, 2493
17 of 37
NY, USA), a statistical application for Microsoft Excel ver. 2013 (Microsoft Corp., Redmont, WA, USA).
Number of PCs was determined using visual evaluation of a scree plot, as a relationship between
calculated λe and number of PCs. A present selection of PCs was based on a Kaiser–Guttman rule, as
the most common stopping rule in PCA [77], which aimed an average value of λe > 1.0. The first two
interpreted PCs of the analysis (with λe > 1.0) accounted for 89.59% of the total variance in the data as
follows: 77.22% (PC 1) and 12.37% (PC 2), respectively.
Table 7. The in vitro activity (MIC values in µM units) of investigated compounds 1a–p and reference
drugs isoniazid (INH), ethambutol (EMB), ofloxacin (OFLX) and ciprofloxacin (CPX) against M. avium
CNCTC My 330/80 (MA 330/80), M. smegmatis ATCC 700084 (MS) and M. marinum CAMP 5644
(MM), respectively.
MIC [µM]
Comp.
MA 330/80
MS
MM
1
2
3
14-d
21-d
3-d
21-d
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
125
62.5
32
32
62.5
32
16
8
62.5
62.5
32
125
125
62.5
32
62.5
32
213
104
51
24.9
16.2
15.8
15.4
15.0
203
6.2
390
382
30.7
15.0
7.3
106
52
25.5
12.5
32.5
<3.9
<3.8
<3.7
51
50
98
191
15.4
15.0
7.3
16
16
125
125
62.5
62.5
32
32
16
16
16
16
16
1o
1p
16
> 250
16
32
–
16
> 250
16
62.5
–
7.1
117
–
–
0.4
7.1
467
–
–
0.8
INH
EMB
OFLX
CPX
2
1
3
14-d, 14-Day cultivation; 21-d, 21-day cultivation; 3-d, 3-day cultivation. The most promising values of
compounds in vitro antimycobacterial activity (MIC ≤ 8 µM) were indicated in gray cells of Table 7.
Figure 5. Chemical structure of the derivatives of 2-/3-alkoxyphenylcarbamic acids JC-02a–l (alkoxy =
propoxy to octyloxy), which were in vitro screened against MTv H37Rv, MK 235/80, MK 6509/96 and
MA 330/80 [76], respectively.
The quality of 2D representation of a variable was visualized by a distance between the projected
variable onto a plane and circle of correlation. The loadings of variables were indicated as variously
colored vectors, connected with particular mycobacterial strains, and numbered according to their
position in the circle of radius 1 in an absolute value (Figure 6). In more detail, the digit 1 was assigned
to the vector built on the log (1/MIC [M]) values, which were connected with 14-d in vitro testing of
the compounds 1a–p against MTv H37Rv (MTv H37Rv, 14-d). Analogously, numbering of other vectors

Molecules 2018, 23, 2493
18 of 37
was as follows: 2 (MTv H37Rv, 21-d), 3 (MK 235/80, 7-d), 4 (MA 330/80, 14-d), 5 (MK 235/80, 21-d),
6 (MK 235/80, 14-d), 7 (MK 6509/96, 14-d), 8 (MK 6509/96, 7-d), 9 (MA 330/80, 21-d), 10 (MK 6509/96,
21-d), 11 (MTa H37Ra, 7-d), 12 (MM, 21-d), 13 (MK DSM, 7-d) and 14 (MS, 3-d), respectively.
A position of the variable in mapping could be defined by values of square cosines (cos2), which
estimated quality of representation. The sum of cos2 for variables on PCs is equal to one. The higher
cos2, the more perfect representation of a variable by chosen PCs [68]. The highest sum of cos2 on PC 1
and PC 2 was found for the vector 1 (MTv H37Rv, 14-d). This vector was characterized by cos2 = 0.961
(Table S8) indicating its almost exclusive representation by both PC 1 and PC 2. In addition, its position
was the closest to a circle of correlation (Figure 6) compared to positions of the vectors 2–14. The cos2
values on particular PCs related to concerned vectors are listed in Table S8.
The vectors 1–3 were defined by PC 1 > 0.75 and PC 2 > 0.25, the vectors 4–10 were described
by PC 1 > 0.75 and PC 2, which could be found in an interval from 0.25 to -0.25. Furthermore, PC 1 >
0.75 together with PC 2 between
−
0.35 and
−
0.53 were used to characterize the vectors 11–13. Finally,
the vector 14 was described by PC 1 < 0.75 and PC 2 < −0.50 (Figure 6), respectively.
Visual assessment indicated the smallest angles between the vectors 5–10. A relatively sharp angle
was also observed between 1 and 2 as well as 12 and 13, respectively. Conversely, there was found ‘almost
orthogonal’ arrangement of the vectors 1 and 14. Regarding this visualization, it could be assumed that
the compounds 1a–p showed (i) similar mechanisms of action against MK 235/80, MK 6509/96 and MA
330/80, respectively; (ii) different mechanisms of action against MTv H37Rv and MS.
Figure 6. Two-dimensional mapping of the loadings of variables (variously colored vectors) indicating
their (i) positions towards a circle of correlation; and (ii) relationships to both Principal Component 1
and 2. Numbering of the vectors was as follows: 1 (the vector built on the log (1/MIC [M]) values, which
were observed after 14-d in vitro cultivation against MTv H37Rv), 2 (MTv H37Rv, 21-d), 3 (MK 235/80,
7-d), 4 (MA 330/80, 14-d), 5 (MK 235/80, 21-d), 6 (MK 235/80, 14-d), 7 (MK 6509/96, 14-d), 8 (MK
6509/96, 7-d), 9 (MA 330/80, 21-d), 10 (MK 6509/96, 21-d), 11 (MTa H37Ra, 7-d), 12 (MM, 21-d),
13 (MK DSM, 7-d) and 14 (MS, 3-d), respectively.

Molecules 2018, 23, 2493
19 of 37
2.4. Structure–Activity Relationships
As suggested in previous sections of the paper, surface tension (relative surface activity γ;
in N/m units), electronic (log ε2 (Ch-T)) and lipophilic (log kw) properties of the compounds 1a–p
might notably affect their in vitro efficiency (in log (1/MIC [M]) units) against tested mycobacterial
strains. The PCA approach was applied for preliminary SAR studies of these derivatives by the
XLSTAT ver. 2016.02.28451 software. Number of PCs was determined using visual evaluation of a scree
plot, as a relationship between calculated λe and number of PCs. The first three PCs of the analysis
with λe > 1.0 [77] accounted for 90.01% of the total variance in the data as follows: 69.59% (PC 1),
14.35% (PC 2) and 6.07% (PC 3), respectively.
The 2D visualization of relationships between the vectors describing physicochemical parameters
and in vitro antimycobacterial activities of the compounds 1a–p on axes (i) PC 1 and PC 2 (Figure S2),
as well as (ii) PC 1 and PC 3 (Figure S3) revealed the closest connection between lipophilicity (log kw;
vector A) and efficiency against MTv H37Rv (14-d; 1). In addition, there were found notably negatively
correlated variables (vectors), which were positioned on opposed quadrants of the loading plots
(Figures S2 and S3). These correlations aimed
γ (C) versus log kw (A) or C versus the vectors built
on in vitro activity against MTv H37Rv (14-d; 1), MTv H37Rv (21-d; 2), MA 330/80 (14-d; 3) as well as
MK 235/80 (7-d; 4). It seemed that ‘almost orthogonal’ arrangement of the vector characterizing log
ε2 (Ch-T) (B) to those of antimycobacterial activities (5–14; Figure S3) indicated no correlation.
A linear, quasi-parabolic (polynomial function of 2nd order) and sigmoidal function, respectively,
was also employed in order to investigate SAR in more detailed manner. The research focused on
relationships between the independent variables (
γ
, log ε2 (Ch-T), log kw) and dependent variable(s),
i.e., biological values (in log (1/MIC [M]) units).
Resulting equations and values of common statistical descriptors, namely, n, DF, χ 2
,
red, RSS,
R, Adj. R2, RMSE, NR, F and Prob > F, respectively, were calculated by using the Origin Pro ver.
9.0.0 SR2 software. In addition, only statistically significant (Prob > F values varied from 0.0100 to
<0.0500; one-star indication), very significant (Prob > F from 0.0010 to <0.0100; two stars indication) or
extremely significant (Prob > F from 0.0000 to <0.0010; three stars indication) relationships between the
independent and dependent variables were presented in next paragraphs. These models were also
characterized by R ≥ 0.9000, Adj. R2 ≥ 0.7750 and F ≥ 25.00, respectively.
Respecting given criteria, the following discussion considered relationships connected with MTv
H37Rv, MK 235/80, MK 6509/96, MK DSM, MA 330/80 and MS as well. Conversely, there were found
no statistically significant, very significant neither extremely significant models related to MTa H37Ra
or MM. It should be also noted that suggested models meeting those requirements were connected
mostly with the subgroups containing a limited number of compounds (n = 8). However, it was not
possible to perform the specific PCA for such subsets due to this restriction, i.e., number of effective
observations was lower (n = 8) than number of examined variables (n = 17). This was a reason why
next sections were focused mainly on linear, quasi-parabolic or sigmoidal fitting procedures and their
statistical descriptions.
2.4.1. Models Related to Mycobacterium tuberculosis CNCTC My 331/88
Notable linear relationships between the
on both 14-d and 21-d in vitro screenings against MTv H37Rv were observed for the series 1a
models were defined by Equations (3)–(6) and found to be statistically very significant or extremely
γ
or log kw parameters and log (1/MIC [M]) values based
–
h. These
significant. Decrease in
antitubercular profile.
γ and increase in log kw of these derivatives led to their more promising
MTv H37Rv (14-d)/1a-h: log (1/MIC [M]) = -120.2060 (±12.9578) × γ + 12.3509 (±0.8027) (3)
n = 8, DF = 6, χ,2red = 0.0058, RSS = 0.0347, R = 0.9669, Adj. R2 = 0.9240,
RMSE = 0.0761, NR = 0.1863, F = 86.06, Prob > F = 0.0001 ***

Molecules 2018, 23, 2493
20 of 37
MTv H37Rv (14-d)/1a-h: log (1/MIC [M]) = 0.4301 (±0.0737) × log kw + 2.9438 (±0.3393) (4)
n = 8, DF = 6, χ,2 = 0.0133, RSS = 0.0798, R = 0.9220, Adj. R2 = 0.8251,
red
RMSE = 0.1153, NR = 0.2825, F = 34.03, Prob > F = 0.0011 **
MTv H37Rv (21-d)/1a-h: log (1/MIC [M]) = -103.4692 (±18.7975) × γ + 11.1642 (±1.1644) (5)
n = 8, DF = 6, χ,2 = 0.0122, RSS = 0.0730, R = 0.9136, Adj. R2 = 0.8072,
red
RMSE = 0.1103, NR = 0.2702, F = 30.30, Prob > F = 0.0015 **
MTv H37Rv (21-d)/1a-h: log (1/MIC [M]) = 0.3957 (±0.0633) × log kw + 2.9505 (±0.2911) (6)
n = 8, DF = 6, χ,2 = 0.0098, RSS = 0.0587, R = 0.9312, Adj. R2 = 0.8449,
red
RMSE = 0.0989, NR = 0.2424, F = 39.13, Prob > F = 0.0008 ***
It seemed that ability to decrease surface tension of water was slightly more important than
increase in lipophilicity of inspected compounds 1a–h with the intention to improve their antitubercular
properties, especially when concerning 14-d in vitro biological evaluation.
It would not be reasonable to suggest some biparametric linear models containing γ, log kw
and log (1/MIC [M]) parameters with an attempt to provide more satisfactory values of statistical
descriptors related to the set 1a . The reason not to apply bilinear reggresion analyses would be
–
h
that statistically extremely significant relationships between
Table S7) were already observed.
γ and log kw (Equations (S25)–(S28) in
ˇ
Current findings were only in partial agreement with a research of Cižmárik et al. [38,78,79],
who studied derivatives of 2-/3-alkoxyphenylcarbamic acid (alkoxy = propoxy do decyloxy), which
basic part was formed by only one azacycloalkyl moiety (pyrrolidinium or piperidinium). The authors
employed a linear, quasi-parabolic and sigmoidal model, respectively, to provide comprehensive
QSAR investigation. They found that simultaneous increase in lipophilicity and in vitro antitubercular
activity of these molecules was quite limited [78,79]. In addition, this behavior was also observed
when inspecting relationships between the in silico log p values (CLOGP 4.0) and in vitro efficiency
of these monobasic compounds against non-tuberculous MK 235/80, MK 6509/96 and MA 330/80,
respectively [38].
The antitubercular activities of derivatives 1a–p might theoretically indicate that comparable
efficiency of homologues containing a pyrrolidinium group would be observed, if length of their side
chain R would be beyond a certain border, i.e., if R = 2-/3-OC8H17 or 2-/3-OC9H19.
A cell envelope of MTv H37Rv is composed of three major segments, namely, a plasma membrane,
cell wall core and outermost layer. The cell wall core, which is essential for viability, consists of
peptidoglycan (PG) in covalent attachment via phosphoryl-N-acetylglucosaminosyl-rhamnosyl linkage
units with heteropolysaccharide arabinogalactan (AG). The AG is in turn esterified at its non-reducing
ends to long-chain (C70–C90) mycolic acids. The latter form the bulk of the inner leaflet of the outer
membrane, with the outer layer consisting of a variety of non-covalently attached (glyco)lipids,
polysaccharides, lipoglycans, and proteins [80].
Results of present statistical analyses indicated that not only lipophilic nature of the compounds
1a–p, possible interactions of their side chain R with some components of the cell wall or ability do
decrease surface tension of water in order to ‘non-specifically’ disrupt the cell envelope could be
decisive for their in vitro efficiency. In fact, suggested mechanisms of antitubercular action might
become more relevant for the subgroup 1a–h compared to 1i–p.
A closer look at the relationships between inspected physichochemical and biological descriptors
among particular positional isomers proved that there were calculated more convenient statistical
values for the 2-alkoxy substituted compounds 1a
–d and 1i–l (Equations (S29)–(S32) in Table S9)
compared to those of the 3-alkoxy substituted isomers 1e–h and 1m–p, respectively.

Molecules 2018, 23, 2493
21 of 37
2.4.2. Models Related to the Mycobacterium kansasii Species
Statistically significant linear relationships between the log kw parameters and log (1/MIC [M])
values connected with in vitro testing of the series 1a against MK 235/80 were found. The MIC
–
h
values observed after 7-d and 14-d cultivation were the same (Table 6), so both proposed models were
defined by Equation (7). More prospective anti-MK 235/80 compounds showed higher log kw values.
MK 235/80 (14-d)/1a-h: log (1/MIC [M]) = 0.4395 (±0.0650) × log kw + 2.6388 (±0.2990)
(7)
n = 8, DF = 6, χ,2 = 0.0103, RSS = 0.0620, R = 0.9403, Adj. R2 = 0.8648,
red
RMSE = 0.1016, NR = 0.2489, F = 45.76, Prob > F = 0.0001 ***
Statistically extremely significant linear models between the log kw and log (1/MIC [M]) values
resulting from 7-d and 21-d in vitro screening of the subgroup 1a against MK 6509/96 were also set.
–
h
The observed dependences were defined by Equations (8) and (9). Higher log kw values were related
to more promising anti-MK 6509/96 agents.
MK 6509/96 (7-d)/1a-h: log (1/MIC [M]) = 0.5072 (±0.0757) × log kw + 2.5914 (±0.3485)
(8)
n = 8, DF = 6, χ,2 = 0.0140, RSS = 0.0842, R = 0.9392, Adj. R2 = 0.8624,
red
RMSE = 0.1185, NR = 0.2901, F = 44.86, Prob > F = 0.0001 ***
MK 6509/96 (21-d)/1a-h: log (1/MIC [M]) = 0.5061 (±0.0737) × log kw + 2.3356 (±0.3393) (9)
n = 8, DF = 6, χ,2 = 0.0133, RSS = 0.0798, R = 0.9418, Adj. R2 = 0.8682,
red
RMSE = 0.1153, NR = 0.2825, F = 47.12, Prob > F = 0.0005 ***
Increase in lipophilicity of the derivatives 1a–h was more important factor to positively influence
their efficiency compared to surface properties. However, it could be assumed that constant increase
in lipophilicity due to elongation of the side chain R would not led to more promising derivatives,
as observed for a subgroup 1i–p (Table 6).
Statistically very significant and extremely significant linear relationships between the
γ or
log kw and log (1/MIC [M]) descriptors were also found when concerning the molecules 1a
–
h
and their 7-d in vitro efficiency against MK DSM. The models were characterized by Equations (10)
and (11), respectively.
Highly lipophilic compounds with ability to decrease surface tension of water more markedly
showed higher potential to fight given mycobacterium. Conversely, increase in lipophilicity due to
increase in number of carbons of the azacycloalkyl fragment (1i
improvement in anti-MK DSM activity (Table 6).
–p) would not be a guaranty of
MK DSM (7-d)/1a-h: log (1/MIC [M]) = -192.0357 (±33.5781) × γ + 16.8889 (±2.0800)
(10)
(11)
n = 8, DF = 6, χ,2red = 0.0388, RSS = 0.2330, R = 0.9192, Adj. R2 = 0.8192,
RMSE = 0.1971, NR = 0.4827, F = 32.71, Prob > F = 0.0012 **
MK DSM (7-d)/1a-h: log (1/MIC [M]) = 0.7579 (±0.0817) × log kw + 1.5371 (±0.3758)
n = 8, DF = 6, χ,2red = 0.0163, RSS = 0.0979, R = 0.9670, Adj. R2 = 0.9240,
RMSE = 0.1277, NR = 0.3129, F = 86.15, Prob > F = 0.0001 ***
2.4.3. Models Related to Mycobacterium avium CNCTC My 330/80
Different models were suggested when exploring relationships between
γ
and log (1/MIC [M])
values resulting from 14-d and 21-d in vitro screening of the compounds 1a–p against MA 330/80.
Concerning well-known differences in their salt-forming fragment, a statistically very significant linear
model was found for a subgroup 1a–h (14-d) and defined by Equation (12).

Molecules 2018, 23, 2493
22 of 37
MA 330/80 (14-d)/1a-h: log (1/MIC [M]) = -150.5250 (±29.7405) × γ + 13.7803 (±1.8423)
(12)
n = 8, DF = 6, χ,2red = 0.0305, RSS = 0.1828, R = 0.9001, Adj. R2 = 0.7790,
RMSE = 0.1745, NR = 0.4275, F = 25.61, Prob > F = 0.0032 **
If a main criterion to consider was a position of compounds’ side chain R, a bilinear model between
γ and log (1/MIC [M]) was regarded as the most convenient for two subsets 1e–h and 1m–p (Figure 7).
A bilinear model proposed by Kubinyi [81] was generally applicable and presented a smooth
synthesis of both linear and non-linear parts of SAR as an effort to simulate a complex process in a
rather simplistic way.
The compounds with
capability to act against MA 330/80 (21-d). Further increase in
γ
varying from 0.05692 N/m (1p) to 0.06154 N/m (1n) showed a comparable
, i.e., minor ability to decrease surface
γ
tension of water, resulted in sharp decrease in their antimycobacterial efficiency. The observed
behavior could indicate a non-specific mechanism of action of these derivatives, which might be
based on their ability to disrupt an outermost layer of a cell envelope of MA 330/80. The layer,
which consists of diverse amphiphilic glycolipids, namely, mycosides C, glycolipids, peptidolipids,
and phospholipids, respectively, hinders diffusion of chemotherapeutic agents via the wall thus causing
multiple drug-resistance by exclusion [82].
Statistically very significant or extremely significant linear relationships between log kw and log
(1/MIC [M]) resulting from 14-d and 21-d in vitro screening of the compounds 1a–h were also observed.
The suggested models were defined by Equations (13) and (14). As expected, higher log kw values
were connected with more effective agents.
MA 330/80 (14-d)/1a-h: log (1/MIC [M]) = 1.6968 (±0.3122) × log kw + 0.6051 (±0.0678)
(13)
(14)
n = 8, DF = 6, χ,2 = 0.0113, RSS = 0.0675, R = 0.9643, Adj. R2 = 0.9182,
red
RMSE = 0.1061, NR = 0.2599, F = 79.57, Prob > F = 0.0001 ***
MA 330/80 (21-d)/1a-h: log (1/MIC [M]) = 1.8560 (±0.4695) × log kw + 0.5458 (±0.1020)
n = 8, DF = 6, χ,2 = 0.0255, RSS = 0.1528, R = 0.9092, Adj. R2 = 0.7978,
red
RMSE = 0.1596, NR = 0.3909, F = 28.62, Prob > F = 0.0081 **
Statistical characteristics given above might support a hypothesis, which was based on a
non-specific mechanism of action of the molecules 1a against MA 330/80. However, the model
–
h
related to 21-d in vitro biological evaluation (Equation (14)) provided clearly worse values of the
statistical descriptors than those connected with 14-d in vitro screening (Equation (13)).
If attention was turned to the 3-alkoxy substituted derivatives 1e–h and 1m–p, increase in their
log kw up to approximately 5.1000 (Table 3) meant sharp increase in activity against MA 330/80 (21-d).
Further increase in lipophilicity beyond this border was not reflected in higher antimycobacterial
potential of the compounds 1n, 1h, 1o and 1p showing log kw of 5.2087, 5.5384, 5.6569 and 6.1749
(Table 3, Figure 8), respectively.
2.4.4. Models Related to Mycobacterium smegmatis ATCC 700084
There was observed a very significant quasi-parabolic relationship between the log ε2 (Ch-T)
values and log (1/MIC [M]) parameters connected with 3-d in vitro evaluation of the pyrrolidium
moiety-containing compounds 1c–h against MS (Equation (S33) in Table S9). The possibility to exclude
the molecules 1a and 1b from a current analysis was based on the observation that they showed the
lowest efficiency against given mycobacterium (Table 7).
It was found that anti-MS activity increased with increasing log ε2 (Ch-T), reached a maximum
if this independent variable was between 4.24 and 4.27 (1e, 1f and 1h) and then decreased with

Molecules 2018, 23, 2493
23 of 37
further increase in the log ε2 (Ch-T) values (Figure S4, Table 1). This behavior, defined as a cut-off effect,
was originally reviewed and rationalized in number of mechanistic ways by Hansch and Clayton a
few decades ago [83].
It was also found that lower
γ and higher log kw did not lead to increase in anti-MS activity of the
compounds 1i , as explored by some models, which were not provided because of their statistical
–
p
insignificance (Prob > F ≥ 0.0500).
Figure 7. Bilinear relationship between the
γ (in N/m units) and log (1/MIC [M]) parameters resulting
from 21-d in vitro screening of the 3-alkoxy substituted compounds 1e–h and 1m–p (alkoxy = butoxy
to heptyloxy) against MA 330/80.
Figure 8. Bilinear relationship between the log kw and log (1/MIC [M]) parameters resulting from
21-d in vitro screening of the 3-alkoxy substituted compounds 1e
heptyloxy) against MA 330/80.
–h and 1m–p (alkoxy = butoxy to

Molecules 2018, 23, 2493
24 of 37
3. Materials and Methods
3.1. General Information
Liquid chromatography high resolution mass spectroscopy (HPLC-HR-MS) analyses of the
compounds 90a
(Supplementary Materials) were performed on a chromatographic apparatus
–
p
consisting of the LC Agilent Infinity System (Agilent Technologies, Santa Clara, CA, USA) equipped
with a 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.
Each of the compounds 90a
1 mg/L. Particular solutions were filtered via 0.22
–
p
was dissolved in 50% (v/v) MeOH to reach a concentration of
m nylon syringe filter and 1.0 L was used for the
50 mm, 1.7 µm
µ
µ
HPLC-HR-MS analyses at 35 ◦C using the RP-C18 column Zorbax Extend-C18, 2.1
×
(Agilent Technologies).
The mobile phases consisted of 0.1% aqueous solution of formic acid in demineralized water
(mobile phase A) and acetonitrile (mobile phase B). Gradient elution was used with linear gradient
from 5% to 95% of acetonitrile per 8 min. The flow rate of a mobile phase was set to 400 µL/min.
The MS spectrometer was operated in a positive and negative ionization mode, respectively,
◦
keeping particular specifications as follows: drying gas temperature 360 C, drying gas flow 12 L/min,
nebulizing gas pressure 60 psi, ESI source voltage 4000 V, fragmentor voltage 125 V, skimmer voltage
65 V and collision gas N2, respectively. The m/z ratios of both [M + H]+ and [M
recorded in an interval from 50 m/z to 1500 m/z. Observed m/z values of the adducts were compared to
the theoretical ones. The difference (in ppm units; Supplementary Materials) was calculated according
to Equation (15):
−
H]− ions were
Difference = [(m/z theoretical − m/z obtained)/m/z theoretical] × 106
(15)
High-resolution mass spectra of the compounds 1a–p were measured using the Dionex UltiMate
3000 high-performance liquid chromatograph (Thermo Scientific, West Palm Beach, FL, USA) coupled
with the LTQ Orbitrap XL Hybrid Ion Trap-Orbitrap Fourier Transform Mass Spectrometer (Thermo
Scientific) equipped with a HESI II (heated electrospray ionization) source operating in a positive
(
1a
Chromatography Data System ver. 7.2 (Thermo Scientific). The separation was performed on a
C18-Hypersil Gold (3 m, 50 mm 2.1 mm) column (Thermo Scientific). A mobile phase consisted of
–d, 1i–l) or negative (1e–h, 1m–p) mode. The HPLC system was controlled through the Chromeleon
µ
×
water Purelab Classic (ELGA LabWater, High Wycombe, Bucks, UK) and acetonitrile hypergrade for
LC-MS LiChrosolv (Merck KGaA, Darmstadt, Germany) in 80:20 volume ratio (v/v). Total flow rate
was 0.3 mL/min, injection volume was 1 µL, and column temperature was set to 30 ◦C, respectively.
The UV/Vis spectra of methanolic solutions of analyzed compounds 1a
–
p
(c = 8.0
×
10−5 M)
were observed on◦the 8452A Diode Array spectrophotometer HP-8452A (Hewlett-Packard, Palo Alto,
CA, USA) at 21 C. Methanol for UV-spectroscopy (Merck, Darmstadt, Germany) was used for
preparation of these solutions. Results of the UV/Vis analyses were collected and stored digitally using
the ChemStation controller software (Agilent Technologies, Waldbronn, Germany). The HP-8452A
apparatus measured a complete range of compounds’ spectrum from 190 nm to 820 nm.
The HPLC separation module Waters e2695 equipped with a Waters 2996 PDA Detector (Waters
Corp., Milford, MA, USA) and chromatographic column Symmetry C18
W21751W016 (Waters Corp.) were used for estimation of lipophilic properties of the compounds 1a–p
at 21 ◦C.
×
5
µ
m, 4.6
×
250 mm, Part No.

Molecules 2018, 23, 2493
25 of 37
The HPLC separation process was monitored by the Empower 3 Chromatography Data Software
(Waters Corp.) working on a personal computer HP Compaq (Hewlett-Packard, Palo Alto, CA, USA)
equipped with Intel® CoreTM i5 processor (Intel Corp., Santa Clara, CA, USA), 2400 CPU, 3.10 GHz,
and 4.00 GB of RAM, respectively. The computer operated on the Windows 7 Professional system,
a 64-bit version (Microsoft Corp., Redmont, WA, USA).
Isocratic elution was carried out using mobile phases consisting of methanol p.a. (MeOH;
Honeywell, Paris, France) and purified water in various volume ratios (80:20, 85:15, 90:10, 95:5
(v/v) and pure MeOH, respectively). Distilled water (CentralChem, Bratislava, Slovak Republic) was
purified by the Aquinity2 P10 ultra-pure water system for production of ultra-pure water with an
integrated 10 L permeate tank (membraPure, Hennigsdorf, Germany).
Total flow of the column was 1.0 mL/min, injection volume was 4 µL and column temperature
was set to 40 ◦C. The detection wavelengths varying from 236 nm to 238 nm were chosen for current
experimental procedures.
3.2. Synthesis of Compounds
Presently analyzed 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-(dipropylammonio)propyl]azepanium oxalates (1i
–l
)/dichlorides (1m
–p;
alkoxy = butoxy to heptyloxy) were prepared by multi step pathways (Scheme 1) using 2-aminophenol
(1a) and 3-aminophenol (1b), respectively, as starting compounds [21,27–29].
Procedures for preparation of reaction intermediates 20a
,
20b
,
30a
–
h,
40a
–
h
,
50a
27
–
h
–
,
70
, , ,
80a 80b
90a
–
p
and final molecules 1a
–
p
were originally published in research papers [21
,
29] and are
provided in Supplementary Materials. In addition, HPLC-HR-MS spectral characterizations of the
compounds 90a–p are included as well.
Spectral data (IR), elemental analyses results (% C, H, N), melting point (m.p.) values, Rf
parameters (TLC) and acid-base dissociation constants (pKa1, pKa2), respectively, of solid colorless
compounds 1a
Supplementary Materials.
The molecules 1a–p were re-crystallized from mixture of acetone/ethanol (1a–d, 1e–h, 1m–p) or
acetone (1i ) before being spectrally, physicochemically and biologically investigated. Present HR-MS
–p can be found in the papers [21,26] and some of given characteristics are also listed in
–
l
spectral description of these compounds was given below.
The compound 1a: HR-MS for C24H42N3O3 [M + H]+ calculated 420.32207 m/z, found 420.32343
m/z; 1b: HR-MS for C25H44N3O3 [M + H]+ calculated 434.33772 m/z, found 434.33920 m/z; 1c: HR-MS
for C25H46N3O3 [M + H]+ calculated 448.35337 m/z, found 448.35471 m/z. 1d: HR-MS for C27H47N3O3
[M + H]+ calculated 462.36902 m/z, found 462.37048 m/z; 1e: HR-MS for C24H40N3O3 [M
−
H]−
H]− calculated
H]− calculated 446.33881
H]− calculated 460.35446 m/z, found
calculated 418.30751 m/z, found 418.30746 m/z; 1f: HR-MS for C25H42N3O3 [M
432.32316 m/z, found 432.32294 m/z; 1g: HR-MS for C26H44N3O3 [M
m/z, found 446.33912 m/z; 1h: HR-MS for C27H46N3O3 [M
−
−
−
460.35458 m/z; 1i: HR-MS for C26H46N3O3 [M + H]+ calculated 448.35337 m/z, found 448.35458 m/z;
1j: HR-MS for C27H48N3O3 [M + H]+ calculated 462.36902 m/z, found 462.37045 m/z; 1k: HR-MS for
C28H50N3O3 [M + H]+ calculated 476.38567 m/z, found 476.38608 m/z; 1l: HR-MS for C29H52N3O3
[M + H]+ calculated 490.40192 m/z, found 490.40210 m/z; 1m: HR-MS for C26H44N3O3 [M
−
H]−
H]− calculated
H]− calculated 474.37011
H]− calculated 488.38576 m/z, found
calculated 446.33881 m/z, found 446.33908 m/z; 1n: HR-MS for C27H46N3O3 [M
460.35446 m/z, found 460.35474 m/z; 1o: HR-MS for C28H48N3O3 [M
−
−
m/z, found 474.36996 m/z; 1p: HR-MS for C29H50N3O3 [M
488.38596 m/z.
−

Molecules 2018, 23, 2493
26 of 37
3.3. Determination and Prediction of Physicochemical Properties
3.3.1. Estimation of Surface Tension
Surface tension (relative surface activity;
γ
) of the derivatives 1a
–
p
was determined by a drop
count method using a glassy Traube stalagmometer (Kavalier Glass, Prague, Czech Republic). Number
of drops of compounds’ and reference solutions were counted between upper and lower marks of
the stalagmometer [30
deionized water (c = 2.0
which showed = 0.07259 N/m. The drops were allowed to form at constant temperature 21 C.
,
31]. Aqueous solutions of the molecules 1a
×
–p were prepared with distilled,
10−3 M). Distilled deionized water was also used as a reference solution,
◦
γ
The experimental procedure was described in a paper [31]. Current measurements were done in six
replicates and average γ values (in N/m units) were reported (Table 1).
3.3.2. Estimation of Electronic Properties
The log
ε
values characterizing methanolic solutions of the compounds 1a
–
p
(c = 8.0
×
10−5
M) were observed at λ1 = 208–210 nm, λ2 (Ch-T) = 236–238 nm and λ3 = 278–280 nm (Table 1),
respectively, in a near ultraviolet (quarz) region of the electromagnetic spectrum between 200 nm and
400 nm [35]. The log ε values for presently observed absorption maxima were calculated following the
Lambert-Beer’s law, which was discussed in [35], for example, and expressed by Equation (16):
A = ε × c × l
(16)
where the A parameter represented absorbance of a compound’s solution, the
ε descriptor was a molar
absorption coefficient (in L/mol/cm units) and l was path length (in cm units).
3.3.3. Estimation and In Silico Investigation of Lipohydrophilic Properties
Lipophilicity of the compounds 1a–p was determined by reversed-phase high-performance liquid
chromatography (RP-HPLC). Methanol (MeOH)/water mobile phases with varying volume ratio of the
organic modifier and water (80:20, 85:15, 90:10, 95:5 (v/v) and pure MeOH, respectively) were chosen.
A methanolic solution of potassium iodine was used for dead time (td) determination. Retention
factors (capacity factors; k) were calculated according to Equation (17):
k = (tr − td)/td
(17)
where tr was retention time of a solute (in min), the td parameter denoted dead time of potassium
iodine, an unretained analyte (in min).
The observed retention (tr) and dead (td) times were means of three independent determinations.
Average td values of potassium iodine in used MPhs were as follows: 2.237 min (MeOH/water
ratio (v/v) was 80:20), 2.245 min (85:15), 2.245 min (90:10), 2.228 min (95:5) and 2.210 min (pure
MeOH), respectively.
The log kw values, i.e., the logarithms of extrapolated retention (capacity) factors for 100% water
in the isocratic RP-HPLC, were determined from intercepts of linear plots between the log k and φM
(a volume fraction of an organic modifier in the isocratic elution RP-HPLC) according to Equation (18):
log k = log kw − S × φM
(18)
where the S parameter represented the slope of a regression curve, which was related to solvent
strength of a pure organic solvent [51,52].
Purity (in percentages) of the compounds 1a–p was also verified by RP-HPLC. Areas of their
peaks were measured using the MPh, which contained 90% proportion (v/v) of MeOH. The purity of
given salts is provided in Table S3.

Molecules 2018, 23, 2493
27 of 37
The log p values of non-protonated bases 9a0
–
p
(Scheme 1, Table 4) for the octan-1-ol/water
partitioning system were calculated by the Ghose and Crippen0s log PCr
[ ,
55], Viswanadhan0s
54
[56], Broto’s log PB [57] and Leo’s CLOGP 4.0 [58] atomic as well as combined atomic and
log PV
fragmental methods using the ChemBioDraw Ultra 12.0 software package (CambridgeSoft, Cambridge,
MA, USA).
The Virtual Computer Chemistry Laboratory [59], a freely available web-based tool working
in Java environment, was taken to generate log P by the Wang’s XLOGP 2.0 [60], Cheng’s XLOGP
3.0 [61], Moriguchi’s MLOGP [62], Sander’s (Actelion’s) ACLOGP [63], Molinspiration’s miLogP
2.2 [64], ALOGP [65] and Tetko’s ALOGPs 2.1 [66] method (Tables 4 and 5), respectively. All these
approaches integrated algorithms based on atomic/fragmental principles, excluding ALOGPs 2.1,
which considered a molecule in the whole [66].
The SILICOS-IT hybrid method, which used a proprietary fragment- and property-based
principles, was also employed to generate log P parameters (log PS-IT; Table 5). The method was
implemented in the SwissADME applet, a free web tool designed to evaluate pharmacokinetics and
drug-likeness of small molecules [67].
3.4. In Vitro Antimycobacterial Assays
The in vitro activity of compounds 1a–p was inspected against Mycobacterium tuberculosis CNCTC
My 331/88 (identical with H37Rv and ATCC 2794, respectively; abbreviation used: MTv H37Rv),
M. kansasii CNCTC My 235/80 (identical with ATCC 12478; MK 235/80), the M. kansasii 6509/96 clinical
isolate (MK 6509/96) and M. avium CNCTC My 330/80 (identical with ATCC 25291; MA 330/80),
respectively, in the Laboratory for Mycobacterial Diagnosis and Tuberculosis (Institute of Public
Health in Ostrava, Czech Republic). These strains were purchased from the National Reference
Laboratory—Czech National Collection of Type Cultures (CNCTC; The National Institute of Public
Health, Prague, Czech Republic), excluding MK 6509/96, which was clinically isolated because the
INH-resistant M. kansasii strains have not been found in Czech Republic or Slovak Republic. In the
experiments, dilution of the strains was as follows: 10−3 M (MTv H37Rv), 10−4 M (MK 235/80,
MK 6509/96) and 10−5 M (MA 330/80), respectively.
The molecules 1a–p were also in vitro screened against an avirulent M. tuberculosis H37Ra
ATCC 25177 (MTa H37Ra), M. kansasii DSM 44162 (MK DSM), M. smegmatis ATCC 700084 (MS)
and M. marinum CAMP 5644 (MM), respectively, in the Department of Infectious Diseases and
Microbiology (Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences
in Brno, Czech Republic).
Standard drugs. Isoniazid (INH), ethambutol (EMB), ofloxacin (OFLX) and ciprofloxacin (CPX)
reference drugs were purchased from Sigma-Aldrich (Darmstadt, Germany), respectively, showing
purity of analytical grade.
Determination of a minimum inhibitory concentration (MIC) against MTv H37Rv, MK 235/80,
MK 6509/96 and MA 330/80. Efficiency of the compounds 1a–p and standard drugs (INH, EMB and
OFLX, respectively) against given mycobacteria were determined in lyophilized Šula’s semisynthetic
medium (Sevac, Prague, Czech Republic) by a dilution-micromethod [20,72,84].
Each tested mycobacterial strain was simultaneously inoculated into Petri plates containing the
Šula’s medium for sterility control and growth of the inoculum [84]. All screened molecules were
added to this medium as solutions in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Irvine, UK). In the
in vitro assays, following concentrations of the solutions were used: 1000, 500, 250, 125, 62.5, 32, 16, 8,
4, 2, 1, 0.5 and 0.25
µ
M, respectively. The inoculated plates kept in microtone bags were incubated at
◦
37 C. Particular reading was carried out macroscopically on a stand with a bottom magnifying mirror
using a magnifying glass.
Growth in the plates [20,72] was evaluated after 7, 14 and 21 days (MK 235/80, MK 6509/96) or
after 14 and 21 days (MTv H37Rv, MA 330/80).

Molecules 2018, 23, 2493
28 of 37
The value of a minimum inhibitory concentration (MIC) was the lowest concentration (on the
above concentration scale) of a tested compound, which inhibited growth of the mycobacteria [20 72].
M units), reported in Tables 6 and 7,
,
The screening was repeated three times and the MIC values (in
µ
were the same.
Determination of a minimum inhibitory concentration (MIC) against MTa H37Ra. The mycobacterium
was grown on Middlebrook broth (MB; MiddleBrook Pharmaceuticals, Inc., Westlake, TX, USA),
supplemented with Oleic-Albumin-Dextrose-Catalase (OADC) supplement (Difco, Lawrence, KS, USA)
and salicylate-derived mycobactin J (2
siderophore [85,86].
µg/mL; Allied Monitor Inc., Fayette, MO, USA), an iron-binding
At log phase growth, a culture sample (10 mL) was centrifuged at 15,000 rpm/20 min using
a bench top centrifuge MPW-65R (MPW Med Instruments, Warsaw, Poland). Following removal
of the supernatant, a pellet was washed in fresh liquid Middlebrook 7H9GC broth (MiddleBrook
Pharmaceuticals, Inc.) and re-suspended in a fresh OADC-supplemented MB (10 mL).
Turbidity was adjusted to match the McFarland standard No. 1 [87] containing approximately
3 × 108 Colony Forming Units (CFU) with MB broth. Further 1:20 dilution of the culture was performed
in MB broth.
Susceptibility of MTa H37Ra was investigated in a 96-well plate format. In the experiments, sterile
deionized water (300
of the medium in test wells during incubation. Each evaluated compound (100
MTa H37Ra (100 L). Dilutions of each compound were prepared in triplicate and final concentrations
varied from 1000 g/mL to 8 g/mL. All tested molecules were dissolved in DMSO (Sigma-Aldrich,
µL) was added to all outer-perimeter wells of the plates to minimize evaporation
µL) was incubated with
µ
µ
µ
Irvine, UK), and subsequent dilutions were made in supplemented MB broth. The plates were sealed
with parafilm and incubated at 37 ◦C for 7 days.
Following incubation, 10% addition a water-soluble dye, the alamarBlue reagent (AbD
Serotec, Kidlington, UK) was mixed into each well. This resaurin-based reagent served as an
oxidation-reduction indicator of metabolic function and cellular health in cell viability bioassays [88].
Absorbance readings at 570 nm and 600 nm were taken, initially for background subtraction and
after 24h re-incubation. The subtraction is necessary for strongly colored compounds, where the color
may interfere with the interpretation of any color change. For non-interfering compounds, a blue color
in a well was interpreted as absence of growth and pink color was scored as growth [73,74].
The minimum inhibitory concentration (MIC) was defined as the lowest concentration of a
compound, at which no visible bacterial growth was observed. In other words, the MIC was the lowest
concentration that prevented visual color change from blue to pink. The MIC value has been routinely
and widely used in bacterial assays and it has been a standard detection limit according to the Clinical
and Laboratory Standards Institute [73,74]. Clinically used antimycobacterials INH and CPX were
applied as reference drugs. The estimated MIC values (in µM units) were provided in Table 6.
Determination of a minimum inhibitory concentration (MIC) against MK DSM, MS and MM. A broth
dilution micromethod in Middlebrook 7H9 medium (Difco, Lawrence, MO, USA) supplemented with
BD BBL Middlebrook OADC Enrichment medium (Becton, Dickinson & Company, Franklin Lakes,
NJ, USA) containing 8.5 g NaCl, 50.0 g bovine albumin (fraction V), 20.0 g dextrose, 0.03 g catalase,
and 0.6 mL oleic acid [89], respectively, was used to determine the MIC values for given strains, as
described [75].
Tested phenylcarbamic acid-based molecules 1a–p as well as standard drugs INH and CPX were
dissolved in DMSO (Sigma-Aldrich, Irvine, UK) and final concentration of DMSO did not exceed 2.5%
of the total solution composition. The final concentrations, varying from 256 µg/mL to 0.125 µg/mL,
were obtained by a two-fold serial dilution of a stock solution in a microtiter plate with sterile medium.
Bacterial inocula were prepared by transferring colonies from culture into sterile water.
Cell density was adjusted to the 0.5 McFarland units [87] using the cell density meter Densi-La-Meter
(LIAP, Riga, Latvia). Final inoculum was made by 1:1000 dilution of the suspension with sterile
water. Drug-free controls, sterility controls and controls consisted of medium and DMSO alone were

Molecules 2018, 23, 2493
29 of 37
included. Results were determined visually after static incubation in darkness in aerobic atmosphere
◦
◦
◦
for: (i) 3 days at 37 C in a case of MS; (ii) 7 days at 37 C (MK DSM) and (iii) 21 days at 28 C
(MM), respectively.
The MIC parameter was defined as the lowest concentration of a compound, at which no visible
bacterial growth was observed. The MIC value has been routinely and widely used in bacterial
assays considering it a standard detection limit according to the Clinical and Laboratory Standards
Institute [73,74]. Presently observed MIC values (in µM units) are provided in Tables 6 and 7.
3.5. Calculations and Statistical Analyses
Regression equations of relevant fitting procedures and their statistical characteristics were
calculated and visualized by the Origin Pro ver. 9.0.0 SR2 software (OriginLab Corporation,
Northampton, MA, USA).
In a current research, those statistical parameters were calculated: 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. R2), root mean squared error
(standard deviation; RMSE), norm of residuals (NR), Fisher’s significance ratio (Fisher’s F-test; F) and
probability of obtaining F Ratio (significance of a whole model; Prob > F), respectively.
The analyses were also focused on indication of a significance level of calculated F Ratio as
follows: * (one star)—statistically significant; ** (two stars)—statistically very significant; *** (three
stars)—statistically extremely significant. Detailed information regarding given statistical descriptors
could be found in a research paper [47].
The chemometric principal component analysis (PCA) tool was used to explore relationships
between (i) experimental and in silico lipophilicity descriptors, (ii) in vitro efficiency against all tested
mycobacterial strains as well as (iii) physicochemical descriptors and in vitro biological activities of
the compounds 1a–p. The PCA is a mathematical algorithm that reduces the dimensionality of the
data while retaining most of variation in the data set. It accomplishes this reduction by identifying
directions, called principal components (PCs), along which the variation in the data is maximal.
By using a few components, each sample can be represented by relatively few numbers instead of by
values for thousands of variables. Samples can then be plotted, making it possible to visually assess
similarities and differences between samples and determine whether samples can be grouped [90].
The PCA was performed by the XLSTAT ver. 2016.02.28451 software (Addinsoft, New York, NY,
USA), a cloud-based statistical application for statistics and data analyses, which worked as an add-on
to the Microsoft Excel ver. 2013 software (Microsoft Corp., Redmont, WA, USA).
4. Conclusions
In summary, determination of physicochemical properties and in vitro antimymycobacterial
screening in connection with SAR analyses of 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-
(dipropylammonio)propyl]pyrrolidinium oxalates (1a
oxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]azepanium oxalates (1i
(1m–p; alkoxy = butoxy to heptyloxy) were considered main objectives of the research.
All compounds 1a in aqueous solutions (c = 2.0
10−3 M) were able to decrease surface tension
of water ( varied from 0.05692 N/m (1p) to 0.06464 N/m (1a).
= 0.07259 N/m) at 21 ◦C and their
Relationships between number of carbon atoms forming their side chain R (nc) and values (in N/m
–
d)/dichlorides (1e
–
h) and 1-[2-[({[2-/3-(alk-
–l
)/di- chlorides
–p
×
γ
γ
γ
units) were described most precisely by statistically significant or very significant models based on
polynomial functions of 2nd order within all homological subsets 1a–d, 1e–h, 1i–l and 1m–p.
Electronic properties of the molecules 1a
–
p
were characterized by logarithms of molar absorption
10−5 M) investigated in the UV/Vis
coefficients (log ) of their methanolic solutions (c = 8.0
ε
×
region of an electromagnetic spectrum. The solutions showed three absorption maxima in a near
ultraviolet (quarz) region of the spectrum between 200 nm and 400 nm. The maxima were found
at λ1 = 208–210 nm (second local excitation band), λ2 (Ch-T) = 236–238 nm (charge-transfer band) and

Molecules 2018, 23, 2493
30 of 37
λ3 = 278–280 nm (first local excitation band), respectively. There was observed nor linear neither
quasi-parabolic relationship between nc and log ε2 (Ch-T), as the maximum being the most sensitive to
differences in electronic environment of a phenylcarbamoyloxy moiety due to different position and
length of a substituent R.
The log kw values, as descriptors characterizing lipophilic properties of 1a–p, were extrapolated
from intercepts of statistically extremely significant linear relationships between log k parameters and
volume fractions of MeOH modifier (φM) using the Snyder–Soczewin´ski solvent strength model. These
log kw values were in accordance with an elution order and hydrophobicity of the molecules 1a–p
and varied from 3.7688 (1a) to 6.1749 (1p). Increase in length of their side chain R led to higher S, as a
function of molecular structure parameters, within all homological sets. The compounds containing an
azepanium moiety showed higher S than those with a pyrrolidium group presuming same position
and length of R. The calculated S parameters varied from 3.7788 (1a) to 6.2672 (1p). A statistically
extremely significant relationship between the log kw and S values was found.
The experimental log kw dataset was studied together with computational logarithms of partition
coefficients (log P). The log P parameters of non-protonated bases 90a
–p were calculated for the
octan-1-ol/water partitioning system using the Ghose and Crippen0s log PCr, Viswanadhan0s log
PV, Broto’s log PB, Leo’s CLOGP 4.0, Wang’s XLOGP 2.0, Cheng’s XLOGP 3.0, Moriguchi’s MLOGP,
Sander’s (Actelion’s) ACLOGP, Molinspiration’s miLogP 2.2, ALOGP, SILICOS-IT and Tetko´s ALOGPs
2.1 method as well. Similarities and differences between log kw and in silico descriptors were analyzed
by unscaled principal component analysis (PCA). There was confirmed high sensitivity of PCA for
detecting of small differerences between analyzed positional isomers, which given in silico methods
could not perform with satisfactory visibility. The log PI-IT, XLOGP 2.0 and XLOGP 3.0 descriptors
might be used to characterize lipophilic properties of investigated salts and bases, especially if they
contained highly lipophilic side chain R.
The compounds 1a–p were in vitro screened against Mycobacterium tuberculosis CNCTC My
331/88 (identical with H37Rv; abbreviation used: MTv H37Rv), M. tuberculosis H37Ra ATCC 25177
(MTa H37Ra), M. kansasii CNCTC My 235/80 (MK 235/80), the M. kansasii 6509/96 clinical isolate
(MK 6509/96), M. kansasii DSM 44162 (MK DSM), M. avium CNCTC My 330/80 (MA 330/80),
M. smegmatis ATCC 700084 (MS) and M. marinum CAMP 5644 (MM), respectively. In vitro susceptibility
of given mycobacteria to clinically used reference drugs isoniazid (INH), ethambutol (EMB), ofloxacin
(OFLX) or ciprofloxacin (CPX) was tested as well.
A very unique aspect of a present research was that many compounds from the set 1a
–p were
in vitro efficient against almost all tested mycobacterial strains. The compounds 1h and 1p were the
most active practically against the whole spectrum of tested mycobacteria (MIC
MA 330/80.
≤
8 µM), excluding
All 3-alkoxy substituted molecules 1e
MIC = 3.7–8.1 µM) than INH or CPX. The dibasic compounds also inhibited growth of MK DSM
more effectively (MIC = 1.9–16.2 M) compared to INH. Moreover, the derivatives 1g 1h and 1n
showed approximately equal activity against MA 330/80 (MIC = 16 M) as EMB. Regarding MTv
H37Rv, however, none of the molecules 1a showed similar ability to fight given mycobacterium as
INH. Comparable activity against MK 6509/96 was found for the substances 1h 1n (MIC = 4
and 8 M) and INH. If concerning the 2-alkoxy substituted compounds 1a and 1i , the derivative
1d was more promising against MTa H37Ra (MIC = 12.5 M) and MK DSM (MIC = 6.2 M) than INH.
The INH standard was also less effective against MS or MM than practically all tested phenylcarbamic
–h and 1m–p were more efficient against MTa H37Ra
(
µ
,
–p
µ
–
p
,
–
p
µM
µ
–d
–l
µ
µ
acid-based substances, from which were 1j (MIC = 6.2 µM; anti-MS) and 1h (MIC < 3.7 µM; anti-MM)
the most efficient.
The chemometric PCA approach as well as fitting procedures, supported by relevant equations
and adequate statistical analyses, were used to comprehensively investigate SAR of the derivatives
1a
–
p. It seemed that ability to decrease surface tension of water was slightly more important than
increase in lipophilicity of a pyrrolidinium moiety-containing compounds 1a
–h
in order to improve